DISEASES

OF THE BLOOD
and
Atlas of Hematology
With Clinical and Hematologic Descriptions of the Blood
Diseases Including a Section on TeChnic and Terminology

hy

Roy R. ,Kracke, M.D.

Professor of Bacteriology, Pathology and Laboratory Diagnosis, Emory
UnIversity &hool of Medicine. Pathologist to the Emory University
HospItal Consultant 10 Hematology to the Grady HOSpital a.nd Eggleston
Hospital for Children, AtlllIlta. Ga.. Formerly. Director of the Hema·
tologleal Reli$tty, Amenca.n Sac.iety of Clltlica.l Pathologists

SECOND EDITION
THOROUGHLY REVISED, RESET, AND ENLARGED

Including f 4 color plates and 46 other illustrations

PHILADELPHIA LONDON MONTREAL

J. B. LIPPINCOTT COMPANY-
 PREFACE TO SECOND EDITION
The warm reception accorded the first edition of this book has proved the
n~ed for an American atlas of hematology, and also has necessitated a thorough
revision after four years. lIIany new developments in hematology have taken
place during this time, and we have endeavored to incorporate these in this edi-
tion. Thus, there is included new material on fractionation of liver extract, a
new chapter on hemolytic anemias, a new chapter on hemoglobinuria, new ma-
terial on 'the action of drugs on the blood, and a new section on hemoglobin and
its derivatives as well as the porphyrin compounds. The reader will find a com-
prehensive section on blood transfusion, the operation of a blood bank, and the
use of- blood plasma. Recent work on bone marrow is incorporated in the chapter
prepared by Dr. R. P. Custer. '
Certain omission, of the fIrSt edition have now been corrected, including mao
terial on osteosclerotic anemia, achrestic anemia, ovalocytosis, Hodgkin's disease,
and histoplasmosis. Because of the rapid advances in this field, there is a com·
plete section on the development of vitamin K. A valuable addition to this vol-
ume will be found in the chapter prepared by a new contributor, Dr. Lloyd
,Craver, of Memorial Hospital, New York City. Dr. Craver's chapter on the
treatment of leukemia includes recent advances in the use of radiation and radio-
active isotopes.
We have endeavored to bring the bibliography up-to-date, so the reader can
consult recent articles dealing with important phases of blood diseases. There
have also been added ten new color plates, most of these drawn by Frances Baker
at Emory University, and in addition there are 29 other new illustrations.
In revising this volume, we have used freely the current and authoritative
sources of information in this field, including the annual review of Diseases of
the Blood by Sturgis and his associates at the University of Michigan. Downey's
masterful four-volume work iu hematology has provided a great deal of readily
available information. The University of Wisconsin Symposium in blood dis·
eases held in '939 has summarized the recent advances in many important phases
of the subject. Forkner's book on leukemia has provided a summary in that
field better than it has ever been done before. All of this has made the revision
of this book a comparatively easy and pleasant task.
This revision could hardly have been accomplished without the loyal and
efficient aid of my associates at Emory University. Among these I am indebted
to Dr. Byron Hoffman for preparation of material on Hodgkin's disease and
hemolytic anemias, and to Dr. William Riser, Jr., for preparation of a summary
of hematologic findings in the various diseases. Blanche Lockard has revised
the technical procedures in the section on technic and Elizabeth Korst has corre-
lated the data pertaining to preparations of liver extract and iron. I am indebted
particularly to my graduate students Gere Jenkins and Mary Margaret Price for
their unremitting toil in the preparation of manuscript.
Roy R. KRACKE
March 28, '94'
Emory University, Ga.
iii
COPYRIGHT, 1941, BY J. B. LIPPINCOTT COMPANY

FOURTH IMPRESSION
To WhICh Has Been Added
"Tbe Clrnrcai SIgnificance 0/ Ihe
Rb J..:tClor wtth Comments Concerntng
the Laboratory Problems"

FIRST EDITION BY
ROY R. KRACKE AND nORTENSE ELTON GARVER
COPYRIGHT, 1937, BY J. D. -r;PPINCOTT COMPANY

THIS BOOK IS FULLY PROTECTED BY COPYRIGHT

AND NO PART OF ITS CONTENTS, EXCEPT SHORT
POFTIQNS FOR REVIEW, MAY 'BE REPRODUCED
WITHOUT \VRITTEN PERMISSION FROM THE
PUBLISHERS

THERE IS ALSO AN EDITION OF, THIS BOOK IN

PORTUGUESE, PUBLISHED BY EDITORA GUANA·
BARA, RIO DE JANEIl\O, BRAZIL, AND AN EDI-
TION IN SPANISH, PUBLISHED BY EDITORIAL
ANICETO LOPEZ, BUENOS AIRES, ARGENTINA

MADE IN THE UNITED STATES OP' A:MEIUCA

 PREFACE TO FIRST EDITION
We have been impressed with the need for a volume that includes both the
tlinical and laboratory phases of diseases of the blood, and especially the need for
an American Atlas of Hematology.
The recent increased interest in blood diseases justil.es the preparation of
5u<h a volume since most book~ of this type have been prepared largely in' Euro-
pean countries. It is hoped tbat the inclusion of colored plates and a section on
terminology will do mnch to clarify the confusion that has existed relath'e to the
identification of cells and the use of hematologic terms.
In the preparation of the plates incorporated in this volume, 1ields have been
drawn from actual preparations under the microscope and all of the cells shown
are from verified cases of the various diseases represented. The drawings have
been made from preparations stained with Wrights stain since we feel that prac-
tically all workers in the l'nited States usc that stain. For the most, part the
magnification is approximately twelve hundred diameters in the dra\\ ings of
microscopic fields. The blood films from which the drawings were made were
from patients observed in our own experience oyer the past ten years, and also
from cases on me in the Hematological Registry of the American Soci~ty of
Clinicall'athologists. A few of the drawings represent composite blood pictures,
based on the well recognized principle in teaching that all diagnostic points can
not be brought out in a single microscopic field. In a careful study of the colored
plates it has been demonstrated to our complete satisfaction that they are faithful
and accurate reproductions of the original drawings.
We believe that it is desirable to include a comprehensive section of hema-
tologic technic in which we have tried· to present at least one method that has
been satisfactory in our own laboratory. In this we have taken into account the
desirability of a simplified technic that can be used in most laboratories. Abo,
we have incorporated a chapter on the normal blood picture of laboratory animab
because this information is Yery dif11cult to fmd in medical literature.
It is our wish that thi; work will fmd a useful place at the bedside, in the
clinical laboratory, and in the research laboratory of hematology. It has been
organized with the view of presenting the c",ential facts of hematolo~y with con-
siderable emphasis placed on the fundamental \'lewpoint. Based upon years of
teaching hematology to medical students, we believe that it should be useful to
this group.
We are indebted to our contributors for the various ,ections under their
names: To Dr. James J. Clark, Emory University School of :-'Iedicine, for the
section on Roentgenological Treatment of the Leukemic States; to Dr. Francis r.
Parker, Emory University School of ~Iedicine, for the chapter on Blood Group,
and Blood Transfusion; to Dr. Elizabeth Gambrell, Emory University School of
~Iedicine, for the chapter on :\falaria; and to Dr. R. P. Custer, University of
Pennsylvania School of :\Iedicine, for the section on Bone :\Iarrow.
v
vi P"":P'AC:t

In the preparation of this manuscript we have consulted and used freely the
more important sources of authoritative material in hematology, this inchlding,
the annual review of'th\s field by ::;turgis:and jlsaacs and their a~sociates; tile re-
view of :\1inot and Castle in the Year Books of General ;\fedicine; the works of
Piney in Great Ilritain; and the comprehensive textbook of Naegeli, whic)l has
been completely translated for this purpose by our graouate student, :\Iiss Gail
Nelson. Therefore, we fecl that the material incorporated in this volume repre-
sents the composite opinion of American hematologists and the European aothor-
ities. For some of the definitions in terminology we are grateful for the use of the
unpublished monograph on agranulocytosis by Dr. Regena Beck of Richmond.
Virginia. For untiring interest and much work we are indebted to ~!iss Herm"
Barmettler, Secretary in the Department of Pathology, Emory Unh'ersity School
of ;lIedicine.
Roy R. KRACKt
HORTENSE GARVtR
September I, 1937
Emory University, Ga.
 TABLE OF CONTENTS

SECTION ONE
HEMATOLOGIC TERMINOLOGY
CHAPTER PAGE
I. Principles of Terminology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
GENERAL CONSIDERATIONS. . . . . . . • . • . . . • . . . . . . . • . . . . . • . . . . . . . 3
ORIGIN OF HEMATOLOGIC TERMS ••••.•••••••••• :............. 4
DESCRIPTIVE TERMS. . . . . . . . . . • . . . . • . . . . • . . . . . . . . . . . • . . . . . . . . . 6
LEUIWCYTES. . . . . . . . . . • . . . . • • . . . . . • . . . • • • •. ••••••••.••.. 6
NORMAL BLOOD CELLS •........•••........................
SIZE AND SHAPE OF RED CELLS ...... ..................... 9
MORPHOLOGY OF ERYTHROCYTES .................•....••.... 9
VARIATION IN RED CELL PICTURES.......................... 10
HEMOGLOBIN CONTENT... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II
BLOOD PLATELETS. . . •• ....•...•.•.•.•••..••••...•••..•..•• 12
IMMATURE BLOOD CELLS. . . . • . . • . . . . . • • • . . . . • • . . . • • • . • • . • • . . 12
DEVJ;:LOPMENT OF A NEUTROPHIL. . • • • .••.•..•••........••... "3

2. Definitions of Hematologic Terms. . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . "S

SECTION TWO
THE DEVELOPMENT AND MORPHOLOGY OF tn:OOD
CELLS
3. Origin and Development of Blood Cells .................. ' . . . . . . . .. 43
THE MONOPHYLETIC THEORY . . . . . . . . . . . . . . . . . . . . . . . ,........ 43
THE POLYPHYLETIC THEORY................................ •• 44
HEMATOPOIESIS IN THE EMBRYO. . • . . • • . .• ....•.....••.•....•. 46
DISTRIBUTION OF BONE MARROW, . . . . . . . . • . • • • • • • . • • .. ...•... 46
PHYSIOLOGY OF BONE MARROW..... . . . . . . . . . . . . . . . • . •• . . . . . . .. 47
MATURATION OF ERYTHROCYTES ............. '. . •. . . . . • . • • • . . . • .. 47
MATURATION OF GRANULOCYTES. . . • . • . • • . . . • . . . . . • . • . . . . . • . . .. 49
MATURATION OF THROMBOCYTES , ..•.•...•..••••.•.•.......•. 51
MATURATION OF LYMPHOCYTES •.....•....•.....••.. 5"
vii
",iii CONTENTS

CHAPTER PAGE
MATURATION OF MmWCYTES .•••..•.•.•••.••••..•••.••••..... 52
SUMMARY ..... " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4. Morphology of Blood Cells ................. . 55
GRANULOCYTES. . . . . .. .• . ............. . 55
MYELOBLAST ... " . . . . . . • . • . . •. . ..... . 55
PREMYELOCYTE.. .. .. .. .. .. .. .. .. .. .. .... ....... ..... .. 5S
NEUTROPHILIC MYELOCYTE. . . • . . . . .. .... .......... . ..... . 56
JUVENILE NEUTROPHIL .•..........•..•.......•.•...•.•.•... 56
BAND NEUTROPHIL • . . . . . . . . • . • • • . . . . . . . . . . • . . . . . • . . . • . . . . S6
SEGMENTED NEUTROPHIL .•.................. • •............ 56
EOSINOPHILIC MYELOCYTE ...•...........................•... 56
JUVENILE EOSINOPHIL............................... . ...... . 56
SEGMENTED EOSINOPHIL . . . . . . . . . . . . • • • . . • • . . . . . . . . . . . . . . • • 57
BASOPHILIC MYELOCYTE •....... , .................••...•..•. 57
JUVENILE BASOPHIL ...............•.•••...•.•..........••••• 57
SEGMENTED BASOPHIL... . . . . . . . . .......................... . 57
LYMPHOCYTES, MONOCYTES AND ERYTHROCYTES .... ' ...........•. 57
LYMPHOBLAST. . . . . . . . . . . • . . . . . ' • • . . . . . . . . . • . .. . •..•.... 57
YOUNG LYMPHOCYTE . . • • . . . . . • . . . . . . . . • . . . . . . . . . . . . . . • • • . . 57
MATURE LYMPHOCYTE .....• " ." .............. . .......•.. 60
MONOBLAST .........•.•....................•...........•...• 60
INTERMEDIATE MONOCYTE •.•.••.......•..................... 60
MATURE MONOCYTE ...•....•........•....•..........••.... 60
PLASMA CELL. . .....•.••.••••.•.....•..•.....••.........•• 61
PRIMITIYE CELL ...•••....••.....•..•.•.....••..........•.•. 61
MEGAKARYOCYTE" .... " " " . . . . ........ " ............ . 6,
MEGALOBLAST .....•••......................•......•.••.•.•. 61
NORMOBLAST .. " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ..... . 61
RETICULOCYTE .............. : •. '........ '.' . . . . . . . . . . . . . . . . . .. 6'2

5. Myeloblasts and Myelocytes ..................................... . 63

MYELOBLASTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
GENERAL DISCUSSION .......... : .........•.•..•..••..•...... 63
ATYPICAT. TYPES • •••••••••.•• . •.•..••••••.•••••••••••••• 63
OCCURRENCE AND DISTRIBUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
MYELOCYTES.. ..... .. .. .. .. . .. . .. .. • .. . .. ... ..... .. ..... .. 67
DESCRIPTION. . . . . . . • • • • . . . . . . . . . . . . . . • . • . . . . • . . . .. . .... :... 67
OCCURRENCE" " ........... " " .... " .. " . . . . 68
DEVELOPMENT OF A MYELOCYTE INTO A SEGMENTED NEUTROPHIL. 69

6. Lymphocytes, Monocytes and Plasma Cells... ..................... 72,

LYMPHOCYTES. . .•.............. •...............••... •...... 72
DEVELOPMENT AND MORPHOLOGY .. ....•..•.••.•.••........ 72
ATYPICAL FORMS ... '........... " . . . . . . . . . . . . . . . . . . . . . . . . . . 72
 CONTENTS ix
CHAPTER PAGE
FUNCTION .......................................... ' . • .. . • . 73
MONOCYTES.................................................. 73
HISTORY............ ....•.•..•.......•...................• 73
THEORIES OF ORIGIN •...... '. . . . • • . . . . . . . . • • • .. ............ 77
FUNCTION AND OCCURRENCE.......... . . • • . . • . . . . . . . . . . . • . . . . 78
MORPHOLOGY ..................................... , ....... 78
PLASMA CELLS ........ , ..................................... 79
DESCRIPTION. . . . . . • . .. . . . . . • . . . . . • . . . . • . . .. . . . . . . . . . . . . . . 79
ORIGIN AND FUNCTION .........•....•••.......... : .• .....•. 79
DEGENERATIVE LEUKOCYTES. : ... . • . . . . • • . . . • • . . . . . . . . . . . . . .. .. 83

7. Erythroblasts, Erythrocytes and Thrombocytes. . . . . . . . . . . . . . . . . . . .. 87

ERYTHROBLASTS. ......... .........•....•... •...••.••... .. 87
ORIGIN AND DEVELOPMENT. . . . • . • • . . . . . . . • • . . . . • . . . • . . . . . . .. 87
DESCRIPTION. . .• •.....••....•....•.. ..•...........•...... 87
OCCURltENCE ............................................... 92
ERYTHROCYTES. . . . . . . . • . . . . . .. ......•.• .•... .............. 92
DESCRIPTION .....•............ " ........ . .......... ". 92
CHEMICAL CONTENT.... ..... ........ .................... 92
FACTORS INFLUENCING NUMBER OF RED CELLS .....•...•...• 93
ROULEAUX FORMATION OF RED CELLS.. . .............•. '.. . 94
VARIATIONS IN SUE, SHAPE AND COLOR.. .. .. ....•.•..••.. 95
RETICULOCYTES. . . . . . . . . . • . . . . . . . . • . . . . . . • . . . . . . . . . • • . .. .., 96
BASOPHILIC STIPPLING.... . . . . . . . . .. .....•...•...•.•........ 96
THROMBOCYTES (BLOOD PLATELETS). . . . . . . . . • . . • . . . . . . • . . . . . ... 97
ORIGIN, MORPHOLOGY AND FUNCTION .....•...•....••....... , 97
OC=NCE. , , , , .... , •....•• , , . , , , , •• , , , .. , , , , .• , , . . .. .., 99

8. Supravital Staining of Leukocytes .. , ... , ... , ....... , , , .... , . .. . . .. 103

SECTION THREE
LEUKOCYTOSIS AND LEUKOPENIA
9. Normal Blood ..... " .. " .. ,., ... ".,.,"', .. , .. " ... , .. " .. , .... III
THE ERYTHRON, , • ' •• ','" ••• " , ' •• , •••• " •• " ••• , ' . " . , ' , . , '
III
'!'HE LEUKON . . . . . . " •.. " .... , ....•..... , ...•... " ...... , .. ,
III
THE THROMBON •••• " , . , ' , . , " ' , ••• , ••• , •••• ,."." •• ,.,"'"
U3
BLOOD PLASMA AND SERUM .... , •.. ,', •....... ,........... . • . .. II3
HEMOCONIA. . ..................... ,', ...•...• ,.............. 113
BLOOD VOLUME ........• " ......... " ............ " ....... , .. II4
EXAMINATION OF BLOOD . . . • , ....• , ... , ..... , ........ , ....... 114
NORMAL HEMATOLOGIC STANDARDS ..... " ..........•...... , •... 116
ADULTS ...••..••• ",., ..... " . , . , " " , . , ' , . " ... " .... , .... II6
x CONTENTS

CHAPTER PAGE
CHILDREN ...........•.•...•...•..•.•••..•......•.•........• II7
CHEMICAL CONSTITUENTS .••...••••....••••..••..•.•..•••.•...•• II9

10. Neutrophilic Leukocytosis (Neutrocytosis) ......... '...•............ 121

NORMAL NUMBER OF LEUKOCYTES ....•..............•...•..••. In
PHYSIOLOGIC LEUKOCyTOSIS.... . . . . . . . . . . . . . . . . . • • . . . • . • . • . • • .• 121
BONE MARROW DELIVERY.. . • . .• . ...........•••.. ' .......... , In
EFFECT OF FOOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
EFFECT OF PREGNANCY . . . . . . :.. ....... ....... .. .... .'.... 123
IN THE NEW BORN .......................................... 123
MUSCULAR ACTNITY . . . . • • . . . • . • . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
TEMPERATURE. . .. .. .......................•..... . ....•.. 124
ALTITUDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
PATHOLOGIC LEUKOCYTOSIS (NEUTROPlULIC) ................... ~. 125
GENERAL DISCUSSION....................................... 125
DIFFERENTIAL COUNT ...... ........•........ ...•........... 129
SCHILLING'S CLASSIFICATION .•........•....... '. . . . . . . . . . . . . .. 130
DEGENERATIVE INDEX .........•........•.....•.......•...•.. 131

II. Lymphocytosis, Monocytosis, Eosinophilia, and Basophilia ........... , '33

LYMPHOCYTOSIS ..............•..... , ....•..... ,............... '33
MONOCYTOSIS . . . . . . . . . . . . . . . . . ..............•...••..•...... "37
EOSINOPHILIA EOSINOPHILIC LEUKOCYTOSIS .. . . • . •• ......•..... 139
INCREASED BASOPHILS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

12. The Leukopenic Diseases .................................... , ... , 144

THE BONE MARROW IN LEUKOPENIA . . ........•.. .'........... 144
FACTORS DEPRESSING GRANULOPOIESIS. . . • . . . . . . • . • . . . . . . .. . .. 145
INFECTIONS. .. ........ ..............................•.• • 145
CHEMICALS . •......••... ....•..................•......... 146
RADIATION .•.. ...•....•..•... ....... ......•............ 147
DIET............... ...•.................• ...... 148
DRUGS .................. 149
HORMONES ..............•....••..••...•...•.•.........•..• 152
MISCELLANEOUS.. ................. .......... • . ....... 152
MALIGNANT NEUTROPENIA . . . . • . . . • . . . . . . . . . • . . . • . . . . . . . . • . . . 153
,HISTORY .•••••••••••••••••••••••••••.•••••••••.•••..••..•• 153
ETIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . .. '53
INCIDENCE. •. ••.••.•••• ., • . • • • • • • • • . • • • • • • • • . • • • . • • • • . •• 154
DESCRIPTION ..•................. : .. '" . . . . . . . . . . . . . . . . . . . . 154
HEMATOLOGIC FINDINGS..................................... ISS
PATHOLOGY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
COURSE ..............•..•..••.•...•...•••...•...••....... 156
TREATMENT .......•.....••••.••.••...••••.•••••••.••.••••• 156
CHRONIC NEUTROPENIA .••...•••.•.•.•.•••......•.......•.•.. 158
 CONTENTS xi

CHAPTER PAGE
BLOOD DYSCRASIAS CHARACTERIZED BY NEUTROPENIA.. . • • • . . . . .• 159
APLASTIC ANEMIA. . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . • . . • • . . . •. 159
ALEUKEMIC (HYPOCYTIC) LEUKEMIA. . .. . . • • . • . . . . • . • • • • . • . • . .• 159
PERNICIOUS ANEMIA ..•..••......••...••..••..••.•••••••..•• 160

SECTION FOUR
THE ANEMIAS
13. Factors Influencing Erythropoiesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 165
AGE......... •••..•.••••• . •••••••••••••••••••••••.••••••• 165
SEX. .. . .......•.........•.......•....••.••..••..••..••..••. 165
INDIVIDUAL REQUIREMENTS ........................... '.' ....... 166
BLOOD Loss .... , ....•...................•............••.... , 166
BLOOD DESTRUCTION ...........•..••......•...•...••...•••.• 166
ROLE OF THE SPLEEN ..................................... I ... 167
IRON ...•.................•...•..•...........•........•.... 168
COPPER ..............•....•..••...•..•...............•..... 170
PROTEIN.. ....••..•...••..•......... •. '.' •......•.. ' ..•...•. , 1'72
GASTRIC FACTORS ..............••...•..•....•..........•.•,' .... 172
LIVER STORAGE. . . . • . . . • . . . . . • . . . . • . .• ..•...•....... ......• 177
YEAST AND VITAMIN B, ......•........•..•.•.........•....•.. 180
VITAMIN C. . . . . .......................... ...•............ 181
THYROID SECRETION ............... ..•.........•.•.......•.. I8r
PITUITARY SECRETION. . . . . • . . . • . . . . . . . . . . . . . • • . . . . . . . . . • • . . . .• 181
PREGNANCY ...........•..............•.....•.........•....... 182
OXYGEN TENSIOl'I' . . . . . . :.................................... 184
TEMPERATURE. . . . . • • . . • • . . • • . . . • . . . . . . . . . . • . . . • . • • . . . • . . . . . .• 184

14. Classification of the Anemias .................................... , 191

IS. Hemoglobin and Its Derivatives and the Porphyrin Compounds ....... 194
HEMOGLOBIN. . . . . . .. '" ...... ........ ......•.....••...... 194
STRUCTURE OF THE HEMOGLOBIN MOLECULE .......•...•..•.... 194
DERIVATIVES •..•...•••..•..••..........•.•.....•.•.•.•..•... 195
OXYHEMOGLOBIN. . • . . • . .• .•..............•.............. .... 195
:M1>THEMOGLOBIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '.'... 196
CYANOSIS .....•..•...•...••••••..................•....•.... 196
SULFHEMOGLOBIN . . . . • . . . . . •. •..•...•..........•.•....... 197
CARBON MONOXIDE HEMOGLOBIN. . .. .....•.......•....... 197
'THE PORPHYRINS ........•...'. .. . • . . . . . . . . • .. ..•... . .......• 198
PROTOPORPHYRIN . . . . . . . . . . ' •. . .....•. ,..... . . . . . . . . . . . 198
COPROPORPHYRIN I. ....... ..................... ......... 199
COPROPORPHYRIN III. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 199
xii CONTENTS

CHAPTER ~AGE
UROPORPHYRIN I , . ' . , ' . , . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . ,. 200
DEUTEROPORPHYRIN '.' .•..•.• " . . . . . . . . . ........•.•.•. . 200
HEME AND HEMOCHROMOGEN .•. , ',' . • . . . . . ." . . . • .• ..•..•.• 200

16. Hypochromic Anemia. . ...................................... . 202

GENERAL CONSIDERATIONS. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
SYMPTOMS OF ANEMIA . . . . . . . . : .•.• , '.' • . • . . . . . . . . .. .• . ...•.. 203
ANEMIA OF ACUTE HEMORRHAGE...... .... . . . . . . . . . . . . . . . . . . 204
ANEMIA OF CHRONIC BLOOD Loss.... ............ . ........ . 20 5
ANEMIA OF ACUTE INFECTIONS . . . . . . . . . . . . . . • . . . . . . . . . . . . . . • . . 20
5
ANEMIA OF CHRONIC INFECTIONS... . . . . . . . . . . . . . , .......... . 206
ANEMIA OF MALIGNANCY ............••..•........•.•.••..•••.. 206
ANEMIA OF PARASITIC INFESTATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0 7
ANEMIA OF MALARIA. . . . . . . . .. . .....•..•...........•.•...•.. 2"9
ANEMIAS OF PREGNANCY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , •.. 20 9
ANEMIA OF HYPOTHYROIDISM ......................••.•.••..•.. 212
ANEMIA OF INADEQUATE IRON INTAKE ......••.••.............. 212
ANEMIA OF VITAMIN C DEFICIENCY ..•.......... . ....•....... 21 3
ANEMIA OF GASTRO-lliTESTINAL DISEASE.. . . . . . . . . . •••....... 214
ANEMIA OF CHLOROSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
TREATMENT OF HYPOCHROMIC (SECONDARY) ANEMIAS . . . . . . . . • . . 215
USE OF TRANSFUSIONS . . . . . . . . . . . . . . • . . . . . . . . . . • • • . . . . . .• 2 I 7
USE OF IRON ... . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • • • 218
SUPPLEMENTARY MEASURES TO IRON TrIERAPY. . . • . • . . . . . . . . .. 223
RESPONSE TO ADEQUATE TREATMENT ....•........•....••..•.. 225

17. Idiopathic Hypochromic Anemia .................................... 229

HISTORY. .• •......... . .......•..•....•.......•... , ........ 229
INCIDENCE AND DISTRIBUTION.. .........•. . . . . . . . . • . . . . . .. 229
SYMPTOMS AND PHYSICAL FINDINGS .............. :. . • . • . . . . . . . .. 230
HEMATOLOGIC FLNDINGs.... . • . . • • • • • . . • .. • . . . . . .• .......... . 233
TREATMENT. . . . . . . . . . . . . • . . . . . . • . . . . . . . . . . . . . . . . . . . . • • . . . . . .. 233
COURSE AND PROGNOSIS . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . 234

IS. The Hemolytic Anemias .... , .................................... 236

THE BLOOD FINDINGS... . . • . . • . . • . . . • . . . . . . . . . • . . • . . • . 237
MECltANISM OF HEMOLYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
CLASSIFICATION.. . . • . . . . . . . .. . . . . . . . . . . . . . . . . . . . . ........... 238
HEMOLYTIC ANEMIAS CAUSED BY INFECTIONS. . .. . ...•••.•..• 239
HEMOLYTIC ANEMIAS FROM CHEMICALS, DRUGS AND POISONS .• 240
HEMOLYTIC ANEMIA FROM THE SULFONAMIDE GROUP (SULFANILA-
MIDE, SULFAPYRIDINE, SULFATHIAZOLE, SULFADIAZINE) .... " ... 241
HEMOLYTIC ANEMIAS CAUSED BY HEMOLYSINS AND RELATED
BODIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
ACQUIRED HEMOLYTIC ANEMIA ..........•.....•.•.••.•.. ,...• 244
ACUTE HEMOLYTIC ANEMIA OF LEDERER. • . . . • • • • • • • • • • • • • • • .. 245
 CONTENTS xiii

CHAPTER PAGE
I9. Hemoglobinuria...... . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. 248
CLASSIFICATION. . . . . • . . . . . . . .. .' ...... . .....• 249
HE1.<OGLOBINUlUA. . . . • . . . •. .... ........ ..... ." ........ 249
EXOGENOUS TypE................... .. . . . . . . . . . . . . . . . . . . 250
ENDOGENOUS TYPE ....... ....... ...... 251
PAROXYSMAL HEMOGLOBINURIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

20. Hemolytic Jaundice .................... '. . . . . . .. ...... ...... 259

HISTORY. . . . . . . . . . . • . . . . . 259
. . . . .. ...... .
FAMILIAL TRANSMISSION.. . . . . . 259
. . . . . . .. .......
INCIDENCE. . . . . . • • • . • . . . . . . . . . . .. . ........ .. 260
ETIOLOGY AND PATHOGENE~IS .. .•••••. 260
SYMPTOMS AND PHYSICAL FINDINGS ... .. ..... . 262
LABORATORY FINDINGS....................... . . . . . . . . . . . . . 263
TREATMENT. ......•..•.......• ...... .............•... .... 264
INDICATIONS FOR SPLENECTOMY .'. ......... ........... .... 268
RESULTS OF SPLENECTOMY. . .. .•.................. ..... .... 268

21. Hypo~hromic Anemia of Lead Poisoning. . .. " ...... ............ 272

SYMPTOMS. . . . . . • . . . . . . . . . . . . . • . . . . . . . . . . . . .. ....... ..... 272
HEMATOLOGIC FINDINGS. . . . . . • . . . . • . . . . . . . • . . . . .. . .....•.. 273
TID;; BASOPHILIC AGGREGATION TEST. . . . . . .. ..... . ........... 275

22. PerniciOUS Anemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .............. 276

PRESENT CONCEPT OF PATHOGENESIS . • . . . • . . . . • . . . . . . . . . . . . • 277
DISTRIBUTION AND INCIDENCE . . . . : .....'. .. ....... ........... 279
SYMPTOMS AND PHYSICAL FINDINGS. . • . . . . . . . .. ....... 280
LABORATORY FINDINGs .........•.... , . • . . . . . . . . . . . . . . . •. . 283
BLOOD . . . . . . . . . . . . . . . . . . . . . ; . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
URINE ................................................. · ... 286
STOOLS......................... ....... .. ............... 286
GASTRIC ANALYSIS. . . . . . . . . . . . . . . . . • . . . . . . • . . . . • . . . . • . . . . . .. 286
DIAGNOSIS ..•......... , ••....... . .....•.. : . . . . . . . . . . . . • . .. 287
TREATMENT . . . . . . . . . . . . . . . . . . . . . . '. . . . .• ..... ..•.......•...• 288
DEVELOPMENT OF LIVER AND STOMACH PREPARATIONS ......... 288
TREATMENT OF THE PATIENT ............•..•••••.•••••...•• 293
ESTABLISHMENT OF THE MAINTENANCE DOSE.. .•......•••..• 297

23. Diseases Characterized by Macrocytic Anemia. : .. , . . . . . . . . . . . . . . . .. 301

SPRUE .......•.....••..••..••.•...........••••....•.......•• 301
PELLAGRA. . . . . . • . . . . . . . . . . . • . . . . • • • . . . . . . . . . . . . . • • . • . .. •...• 302
PREGNANCY .... ' ....•.• '. . . . . . .. . . . . . . • • . . . • • . . . . . . . . .• •..•..• 303
GASTRO-INTESTINAL DYSFUNCTIONS: ....'. • . . . . . . . . . . . .. • ..••.• 305
ACHRESTIC ANEMIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 307
LIVER DAMAGE .....••.•.....•••••••••.....•.....•••.•.••••... 308
xiv CONTENTS

CHAPTER PAGE
TROPICAL MACROCYTIC ANEMIA ........................... '. ,.. • .. 308
LEUKEMIA ...• . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . .. 309

2~. Aplastic Anemia ................................................. 31 I

SECONDARY APLASTIC ANEMIA .. ............ ... ..... ..... 314
BENZENE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
ARSENIC PREPARATIONS. . . . . . . . . . . .. ..... . .........•...... 316
RADIATION.... ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
IN OTHER DISEASES. . . . .. .. ........•. . ........••..... 319
'PRIMARY IDIOPATHIC APLASTIC ANEMIA ......................... . 3 20
SYMPTOMS AND PHYSICAL FINDINGs. •....•. . ....•....•••.... 3 20
LABORATORY FINDINGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 21
TREATMENT AND PROGNOSIS . . . . . . . . . . . . . . . : ...........•.... 3 21
OSTEOSCLEROTIC ANEMIA . .. .'. . . . .. . ..............• : ..•..••.... 3 22

25. Sickle Cell Anemia .............................................. 325

HISTORY. . . . . . . . . . . . • . . • . . . . . . . . . . . • • . . . . .. • •.•.••....•.•... 325
DISTRIBUTION. ..•.....• . . . . . . . . . . . . . . . . .......•..•......•. 325
FACTORS PRODUCING SICKLING......... . .......•••.......••..• 32q
SYMPTOMS ANI;j PHYSICAL FINDINGS .•.....•....•.•.......•.••.. 327
LABORATORY FINDINGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
PATIIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
TREATMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
PROGNOSIS ....•........••....•..........•...........•..•.•... 331
OVALOCYTOSIS. • . . • • . . • • • • • . • • • • • • . • • . • • . . • . • . . . • • . . • . . • • • • • .• 33 I

.6. The Anemias of Childhood ....................................... 334

PHYSIOLOGIC ANEMIA OF FIRST MONTH ...••.....•..........•••• 334
SYMPTOMS OF CHILDHOOD ANEMIA.. . . . . • . . . . . • . . • • . . • • . . . • • • . .. 335
RELATION OF ANEMIA TO AGE LEVEL ..................••..•••.• 335
BIRTII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
FIRST MONTH. . .....•................•....•• : ...••....•... 335
FROM FOURTII MONTH TO FOURTH YEAR ...•.•..•.....••....• 336
SCHOOL AGE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '.' • '. .. 336
EFFECTS OF IRON DEFICIENCY.·......................... ••...• 336
CLASSIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
ANEMIA OF PREMATURITY. . . . . . . .. • .. . . . • . . . .. • • . . . . • • .. . • . . .• 338
NUTRITIONAL ANEMIA OF INFANTS •....•....•.••...•....••.••.• 339
PROPHYLAXIS AND TREATMENT ................................. 340
HypOCHROMIC ANEMIA OF COELIAC DISEASE ....•....... " ••.••• 341
IRON DEFICIENCY ANEMIAS IN OLDER CHILDREN .....•...•..•• " 341
SUMMARY AND TREATMENT OF IRON DEFICIENCY ANEMIAS. . • • • •• 342
ANEMIA OF VITAMIN DEFICIENCY (SCllRVY)' ..•••••••...•...•• " 343
ANEMIA OF THYROID DEFICIENCY. . • . . . . • • . . . • • . . . . • • . .. • • . . . .• 343
IDIOPATHIC MACROCYTIC ANEMIA OF THE NEWBORN .•..••...•• " 343
 CONTENTS xv

CHAPTER PAGE

MACROCYTIC ANEMIA OF COELIAC D,SEASE.,.. •..... ,'144

HEMOLYTIC ANEMIAS OF CHILDHOOD (GENERAL DISCUSSION) .... ;45
HEMOLYTIC A~'EMIAS OF THE NEWBORN. . .. , . . . . . .. .. j46
ERYTHROBLASTOSIS OF TIlE NEWBORN (ICTERUS GRAVIS NEONA-
TORUM) ........ , ............... .
HEMOLYTIC ANEMIA WITHOUT ICTERUS GRAVIS .....•........•.
HEMOLYTIC ANEMIA LATER IN THE NEONATAL PERIOD . . . . . . . . . . .
ACUTE HEMOLYTIC ANEMIA (LEDERER TYPE) .............. .
CHRoNIC HEMOLYTIC ANEMIA (FAMILIAL HEMOLYTIC ICTERUS) •..•
S,CKLE CELL ANEMIA . . . . . . . . . . . , .. , ........................ .
VON }AKSCH'S ANEMIA. ........•... ...... . ........•••.. '.' •. ' ~49
CLINICAL FEATURES. . ...........•...•..•...•........•..... j50
HEMATOLOGIC FINDINGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :!5 1
TREATMENT ., . . . . . .. ........•.....•...•..•.•.• ..••.... ;'5 1
PROGNOSIS . . . . . . . . . . . , ...........•..•.... . .•...•...... ".. ,5 1
DISCUSSION ..........................•...•...•....•...•.••.. j 51
COOLEY'S ERYTHROBLASTIC M'EMIA. . . . . . . . . • • •. ........... jS3
CLINICAL FINDINGS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '.' ... j53
HEMATOLOGIC FINDINGs. . . • .. • . . . . . . . . . . . . . . . . . . . . . . . . . .., ;'54
BONE CHANGES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,: .• :!54
PATI'{OLOGY... • • •• • . • • • • • • • •• • • . • • • • • • • • • • • • . •• • • • • . • • • .. .• :!54
THERAPY. . . . .. .. .. .. .. .. .. .. . .. . . .. .. .. . .. .. .. .. .. .. . ... . jS5
ETIOLOGY •...•............ , .... . . . . . . . . . . . . . . . . . . . • . . . • . . ,55
SPLENIC ANEMIA (BANTI'S DISEASE) . . . . . . • . . . . ! . . . . . . . . . ' ..... , ~56
ANEMIAS ASSOCIATED WIm INFECTION •......•.................. ;5 6
'tREATMENT. . • . . . .. .. ......... ....... . . . . .. ...•.... . ~57
ANEMIAS ASSOCIATED WITI'{ HEMORRHAGIC DISEASES . . . . . . . . . . . . . 158
ANEMIAS ASSOCIATED WITI'{ LEUKEMIA ..................••....• , 158
ANEMIA AND SPLENOMEGALY IN CHILDREN ... , ••..•••••.••.•... , 55 8
SECTION FIVE
THE LEUKEMIAS
27. The Leukemic State ............................................ .
HISTORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :. '.' " ., ..... '.' .. .
DISTRIBUTION AND INCIDENCE . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . ,..
NATURE OF LEUKEMIA. .. ..... .. .•...•.•...•..••. . ........ .
EXPERIMENTAL PRODUCTION OF LEUKEMIA. '.' .. .' .......•..•.....
LEUKEMIA IN FOWLS . . . . . . . . . . . . . . . . • . . • : ......•.........•...
LEUKEMIA OF MICE . . . . . . . • . . '.' .•.........•.•..•...•.........
CLASSIFICATION OF LEUKEMIA .••.... .' .•.....••..•..•.. " .•......
xvi CONTENTS

CHAPTER PAGE
28. Chronic Myelogenous Leukemia., , "',',"' .................. , ... 37'
SYMPTOMS AND PHYSICAL F,NDINGS, " . . . . . . . . . . . . . . . . . . . . ,'.'. 37'
LABORATORY FINDINGs, .. " . , ...••••... " •.••••••• , " " , .....• 375
DIAGNOSIS .........•..••.... , ....• , , .... , .. " . , .........•. '. 377
TREATMENT, , , . . . . . . . • . • • . . . .• ........ ' ........ ,',.,....... 377
PROGNOSIS, . , ....•••••••.••...... , ' . ' ........• ' . , ..... ,' ••. , .. 379

29. Chronic Lymphatic Leukemia ................ "" ................. 38r

SYMPTOMS AND PHYSICAL FINDINGS, .•...... , ........ ,., ....... 38r
LABORATORY FINDINGs .•...... , . . . .• ., ......... ,', ........•. ,. 382
PATHOLOGY, ..........', , ......... , .......... , ........ , ,.. .. . .. 383
DIAGNOSIS. . . . . . • . • • . . .. . . • .. . • . . . . .. ' •. , ....• , " , .......... 383
TREATMENT, , •••...•. , ...•...... , •.....•...•• , . , .....• ' . . . . .• 384
COURSE AND PROGNOSIS ......••••.•.... , .... , ....• ' ....... ,.,. 384

30. The Acute Leukemias.", , .......... , ...... " ......... , ...... ,' 385
SYMPTOMS AND PHYSICAL FINDINGS, ., ........ , " , ...... " . , .. 391
HEMATOLOGIC FINDINGS. , ............... " ........ '.......... 392
DIAGNOSIS, , , , ..................... , , , ........•...... , . • .. . .. 394
PATHOLOGY .•..•. , • , .........••........•......... , ' .......... 395
TREATMENT AND COURSE .••...•••...• , .......••..............• 395

31. Monocytic Leukemia, ........................................ ' .. 396

INCIDENCE ..•..•• , ' ......••. , . . . . . . . . . . • • . . . . . . . • • • . . • . . . . . .. 397
SYMPTOMS AND PHYSICAL FINDINGS ...•....... , , , ...... , ' , . . . .• 397
LABORATORY FINDINGS .................. " •....... ,.".. ..... 400
PATHOLOGY",." .......••••.•..•...•. , ...•.•. , ...•.. , •. ' ....• 40r
DIAGNOSIS. ' ............................. , , . . . . . . . . . . . . . . . .. . .• 401
TREATMENT AND PROGNOSIS ....••.••.•...' ..•• , ................ , 40r

32. Atypical Types of Leukemia. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 405

ALEUKEMIC LEUKEMIA, .' .••.•..••••...... , •....••..••.....•.. 405
PLASMA CELL LEUKEMIA .....•.••••...•••••..... , . , • . • . • • • . . .. 406
CmoRoMA, .'." ........................... '................ 406
M,XED CELL LEUKEMIA .............. , . , ........ , , , .......... " 407
ERYTHROLEUKEMIA ...••••......•.••••.....• , ..•..•.. , . .. . ...• 407
LEUKEMOID REACTIONS •••••........•••...•• '.' . '.' ..•... '. . . . . .. 407

33. Treatment of Leukemia ...... , ' ....... , .. ' ...... ,', ... ' 4'3
CLINICAL AND PATHOLOGICAL TYPES OF LEUKEMIA. , ." ••. ,' .•.• 4r3
METHODS AVAILABLE FOR TREATMENT .•••••....•••.•..•... , .•.. 4r5
IRRADIATION METHODS ...•..••...•••••.••••••••.••.•• ," •.. 4r5
EXTERNAL IRRADIATION .....•.•...•..••.....•••••...•.• , .• 415
INTRACAVITARY IRRADIATION ............................... 4r7
INTERSTITIAL IRRADIATION .... , . . . . . . . . . . . . . . . . . . . . . . . . . . . 4'7
 CONTENTS xvii
CHAPTER PAGE
THORIUM, ETC. . ..•..•.••••.•...•.....•...••.••••.•..•• '. 418
RADIOACTIVE ISOTOPES .....•••..•........••.•......•.•••.. 418
ARSENIC AND BENZOL ••.....•....•... '.... ,..••......•.•... " 420
ARSENIC. . . . . • • • • • • • • • . . • . . • • • • . . . . . . . • . • • . . . • . . • . . • • . .. 420
BENZOL ......•..•..•.•..•..•..•..••.•••.••••••••••..•.. 420
ADJUVANT AND PALLIATIVE MEASURES .....................' . . 420
TREATMENT OF COMMON FORMS ......•••••.•...•..•.....•••••.. 421
EXTERNAL RADIATION ................. : .. ' .................... " 421
CHRONIC LYMPHATIC LEUKEMIA •.....•....•....•.•..•••.. 421
CHRONIC MYELOGENOUS LEUKEMIA .....••.........•.•.•... 424
ACUTE LEUKEMIA... . . . . • . . . .. •...•.•..........•........ 426
TREATMENT OF SOME COMPLICATIONS, ........................... 427
THE HEMORRHAGIC TENDENCY . • . . . . . . . . . . . . . . . ' .... , ......... 427
INFECTIONS. . .. . . . . . . . . . . ........... 427
PREGNANCY IN LEUKEMIA . • . . . . • . . . . . . . • . • . . . . . . . . . . . . . . . . . . 428
HERPES ZOSTER. . .. . .. ,.. .. ',' . . . . . . . . . . . .. . ....•.•....... 428
GROSS LESIONS' OF BONES.. . •••.••.•.....•...•......•...•.. 429
PRIAPISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 429

~ECTION SIX
HEMORRHAGIC DISEASES
34. Mechanism and Classification of the Hemorrhagic Diseases ......... " 433
COAGULATION OF THE BLOOD ..... . 433
CLASSIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
35. Essential Thrombocytopenic Purpura ............................. 440
INCIDENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . • . • . . . . . . . . . . . . . " 440
ETIOLOGY. . . • . ....•....... , . . . . . . . . . . . . . . . . . . " 440
SYMPTOMS AND PHYSICAL FIJ'lDINGS . . . . . . . . . . . . . . . . . . . . . . . . . . " 441
LABORATORY FINDINGs. . . . . . . . . . • • . . . . • . . . . . •. ....•••...• '" 442
DIAGNOSIS . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 442
TREATMENT. . . . . . . • • . • . . • • . • . .. . • •• •.•••.... •.•.. ......... 443

36. Hemophilia ...................................................... 447

HEREDITY ........................................... " 447
NATURE OF THE DEFECT ... .. . ................ 448
INCIDENCE. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .• 450
SYMPTOMS AND PHYSICAL FINDINGS . . . . . . , . . •..•....••.••••. , 450
HEMATOLOGIC FINDINGS . . . . . . . . . . . . . . . . . o' • • • • • • • • • • • • • • • • • • • • • 451
PATHOLOGY. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . ......... 451
DIAGNOSIS. . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . •. . .......•• 452
TREATMENT .•••.•...................••••....................• 452
PROGNOSIS.............................. . . . • . . . . . . . • • .•. ,. 454
 xviii CONl"ENTS

CHAPTER PAGE
37. Non-Thrombopenic Purpura........ . .. . .. ... . .. . . . . 456
SYMPTOMS AND PHYSICAL FINDINGS .....•...........•.. '. . . . . 457
HEMATOLOGIC FINDINGS ...•..•.••....•.................. " • 458
DIAGNOSIS.. •.•••.•....•..••••.••.••.•......•••.......•. . 458
TREATMENT .......•..•••.••••.•..•••....•..•........•..... 458
COURSE AND PROGNOSIS. . . . . . . • . . . . . • • • . . . . . . • • . . . . . . . . . .. . 460
OTHER NON-THROMBOPENIC PURPURAS . . . . . • • . . . . . . . . • . . . . . . 460
HEMORRHAGIC DISEASE OF THE NEWBORN . . . . . . . . . . . . . . . . . . . . • . 46I

38. Vitamin K ...................................................... 463

DISTRIBUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' " 463
PHYSIOLOGY AND MECHANISM OF ACTION. . . . . . . . . . . . . . . . . . . . . 464
CONDITIONS IN 'VHICH VITAMIN K DEFICIENCY MAY OCCUR .. '" 465
HEMORRHAGIC DISEASE OF THE NEWBORN . . . . . . . . . . . . . . . • . . . 466
OBSTRUCTIVE JAUNDICE. . . . . . . . . . .. . . . . . . . . . ...•.•.... 467
HEMORRHAGIC DISEASE OF LIVER DAMAGE ... . • • . . . . •• . ••... 467
HEMORRHAGIC DISEASES FROll OTHER CAUSES. . . . . . . . . •• . •.. 467
PREPARATIONS OF VITAMIN K .....................' .......... 467

SECTION SEVEN
MISCELLANEOUS
39. Infectious Mononucleosis ....... " ......... , ..............•....... 473
HISTORV. . . . . . . . . . . . . . • • . . . . . . . . . . . . ........••.•.........•. 473
ETIOLOGY . . . . . . . . . . . . . . . . .......••••.......••........•.•. 474
SYMPTOMS AND PHYSICAL FINDINGS . . . . . . . . . . . . . . . . . . . . . . . • • • . 476
HEMATOLOGIC FINDINGS ...... " . . . . . . .. . •........•••.....•... 480
DIAGNOSIS. .. . ...... . . . . . . . . . . . . • . . . . . . . • • • . . . . . • . . • • . .. 48.t
THE HETEROPHILE ANTIBODY TEST ...••••..................•..• 485
PROGNOSIS ...... .....••..•........••..........••••.....•... 487
TREATMENT .........••......••••.......••..•.....•..•••.•... 487

40. Polycythemia Vera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 490

ETIOLOGY. . . . . . . . . • . . . . . • . . . . . . . . . • . . . . • . • • • . • • . • . . . . . . . . .. 490
SYMPTOMS AND PHYSICAL FINDINGS .•....••......••••••.... " 49I
LABORATORY FINDINGS . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . 492
TREATMENT. . . • . . . . . . . . . . . .. .• . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 ..
PROGNOSIS. . . . .. • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ."': . . . . . . . . . . 495
SYMPTOMATIC ,AND COMPENSATORY ERYTHROCYTOSIS.' ...•.... '. " 496

41. The Bone Marrow ................................ .......... .... 500

TERMINOLOGY AND CELL MORPHOLOGY ............••....••••. " 500
UNDIFFERENTIATED CELLS.. . • . . . . .. •........ . ...•••.••.•.• 501
 CONTENTS xix

CHAPTER PAGE
THE GRANULOCYTE SERIES .. ',' . . . . . . . . . . . . . .. . . . . . . . . . . 502
THE THROMBOCYTE SERIES. . . . . . . ...... ....•. 502
THE PLASMOCYTE SERIES. . . . . .. • ..... . . . . . . . . • . . . . . . . ' .... 502
THE LYMPHOCYTE SERIES. .. ...... ..... 503
THE MONOCYTE-MACROPHAGE SERIES. .. . . . . . . . • . . . . .. ... 503
ORIGIN AND DEVELOPMENT.. . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
STATUS AS AN ORGAN............... . • . . . . . . . . . . . . . . . . . . . . . 503
STRUCTURE........ ..... . . . . . . . . . . . . . . . . . . . . . . . . 505
FORMA1'lON AND DELIVERY OF BLOOD CELLS. .............. 506
ERYTHROCYTES . . . . • . . . . . . . • . " ........ .... 506
GRANULOCYTES. . . . . . . . . • . . . . . . . . . . . 507
PLATELETS. . . .. . . . . . . . . . . . . . . . . . 507
MONOCYTES AND MACROPHAGES... . . . . 507
LYMPHOCYTES AND PLASMOCYTES . ...... . . .. " .. 507
DESTRUCTION OF BLOOD CELLS. . . . . . . . . . . . . . .. . 507
MEANS OF STUDY BY BIOPSY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
THE TREPHINE METHOD. .. ....... ... ...... '" . 508
THE ASPIRATION METHOD. . . . . . .. ..... .... . . . . . . . . . . . . .. 509
THE DRiLL METHOD.......... . . . . . . . . . . . . . . . . . . . . . . . . . . 510
EXAMINATION OF BIOPSY MATERIAL . . . . . . . . . . , '" 510
MEANS OF STUDY AT AUTOPSY. . . . . . . . .... ..... .. ........... 5"
ApPEARANCES IN ANEMIA. 512
WITH HYPERPLASTIC MARROW..................... 512
WITII HYPOPLASTIC OR APLASTIC MARROW . . . . . . . • . . . . . . . . . '1" 514
WITH DISPLACED MARROW. . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . 514
APPEARANCES IN HEMORRHAGIC DISEASES... .........•.. 514
APPEARANCES IN POLYCYTHEMIA . . . . . . . . • . . . . . . . . . . . . . • . . . . . . SIS
APPEARANCES IN SIMPLE NEUTROCYTOSIS . . . • . . . . . . . . . . . . . • . .
APPEARANCES IN NEUTROPENIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
APPEARANCES IN TIIE LEUKOSES (LEUKEMIAS) ......•.....•.....•
MYELOSIS (MYELOGENOUS LEUKEMIA) . . . . . . . . . . . . . . . . . . . . .
LYMPHADENOSIS (LYMPHATIC LEUKEMIA) . . . . • . . . . . • . . . . . . . . . .
RETICULOSIS (MONOCYTIC LEUK'EMIA) .. " ......•............
PLASMOCYTOSIS (PLASMA CELL LEUKEMIA) . . . . . . . . . . . . . . .', " .
MEGAKARYOCYTOSIS (MEGAKARYOCYTIC LEUKEMIA) . . . . . . . . . . .
APPEARANCES IN STORAGE DISEASES ... .,.... ..... . ... .
TUMORS . . . . . . . . . • . . . • . • . . . . . . . . . .
PRIMARY TUMORS. .. . . . . . . • • . . . . . . . . . . . . . . . . ........ I •

SECONDARY TUMORS. ................. .......•.. . ..... .

42. Malaria. . . . . . . . . . . . . . . . . . . . . . . . . .. ............................ 522

HISTORY. '.' . . . . . . . . . . . . . . . . . . '.' .. .", ..... ',' ..........•••.. " 52~
CLASSIFICATION AND LIFE CYCLE OF THE MALARIA PLASMODIA .• 523
THE SEXUAL OR SPOROGENOUS CYCLE IN THE MOSQUITO .•....•.. 525
 CONTENTS

CHAPTER PAGE
THE ASEXUAL OR SCIIIZOGENOUS CYCLE IN MAN" .. " ••.•••••..• 525
EXO-ERYTHROCYTIC STAGES""" .... , ....•.....•.•••.•..••.•. 526
MORPHOLOGY OF THE PARASITES" .....••.•• " •. , ••..••••••.. ,. 527
PLASMODIUM VIVAX. , ..•.. , , , , , ••.......• '. . , . . . . . . . . . . . . . . 527
PLASMODIUM MALARIAE. .. . .....•....•...•••.• , ..••.• ,.... 528
PLASMODIUM FALCIPARUM .•.........••.•.. ,., .. ,',"" •.•..• 52S
PLASMODIUM OVALE ..•....•••...•...•......•• , .......••••.. 530
CLINICAL SYMPTOMS ... , ....•..•.•••••.•. , ....•..• ',' •• , • . . • . •. 530
INCUBATION PERIOD . . . . . . . . . . . . . . . . . . . . . . . . . . . ' • . . .. .. .. • . . .• 531
THE DEVELOPED INFECTION .... , ' , .. , ' ........•...• , • • • . . . . . •. 53'
ESTIVo-AUTUMNAL FEVER, . " .•......... , ......••. , .•...•••.. 53 2
TYPE OF FEVER. AND DEVELOPMENT OF THE PARASITE., •..•.•..• 533
THE BLOOD PICTURE IN MALARIA, " " , , " ' , , " ' , ' " , " ' , ' , ' 534
PATHOLOGIC CHANGES, , " " " .. , , " " " , ' " , , , , , , , ' , , , , , , , , , 53 6
THE SPLEEN, , , , , , , ' , , , ' , ... , ......... , , , . , ," ..... , ... , •... 53 6
THE LIVER ... , .... " •..•. , .•.•....• '....•..............•.. , 53 6
THE KIDNEYS, _____ " ' , •....... , ... , _ ... , ......••...•... ,. 537
MORBID PATHOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " •• '" ..
537
BLACK WATER FEVER, ..•. , •.....•........••• , ••...••.• , •... 539
TREATAIENT OF MALARIA ....... , ., .•• ' ...•••..•.•..• , , ••.... , . 539
QUININE"",., . . . . . . . . . . . . . . . . . . . . . . . ,', ............. : ••.. , 540
PLASMOCHIN ....•...•...•• , ......... , . . . . . . . . . . . . . . . . . . . . . . 54 2
ATABRUIE, ..•... , •.•.••...••....••.•••.••....••..•...•••.•• 542

43. Hodgkin's Disease: Histoplasmosis .... " ................... ',' ..... .

HODGKIN'S DISEASE, •.. , . . . . . . . . . . . . . . . . . . . . . . . , • : . . . . . . . . . . .
HISTORY ... , . . . . . . . . . . . . . . . . . . . . . . ,' ... , .. , .... , ........... .
ETIOLOGY •....•............ , .....•..... " .....•...•...••..•
AN ATYPICAL FORM OF TUBERCULOSIS " ..•. ,' ....•.•.••..•
AN INFECTIOUS GRANULOMA OF UNKNOWN ETIOLOGY. • ••• ,.
A VIRUS DISEASE, . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . .
PATHOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , •••..
HEMATOLOGICAL FINDINGS . . . . . . . . . . . . . . . . . . " •.•.•••.....•.. 550
THE GORDON TEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,.,." ...... 550
CLINICAL FEATURES ..•.•...••.••....••.. , .... , •..•...., •.••• 551
DIAGNOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ..... , .. , , , , , • . . . ... 552
TREATMENT AND PROGNOSIS ....•.. , . , . . . . . . • . . . . . . • . • . . . • . •• 552
HIsTOPLASMOSIS, . . . . . . . . . . . . . . . . . . . . , , ... , . • • . . . • . . • . . • .. . • .. 553
CLINICAL FEATURES - , .. ' , . , .... ' , . ' ...... ' . , • , ........ , .. ,. 553
PATHOLOGY, .. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . , .. , . . . . . . . . . . ,. 553
TREATMENT . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , . . • • .. 554

44. Blood Groups and Blood Transfusions. . . . . . . . . . . . . . • . . . . . . . . . . . . .• 556

HISTORY . . . . . . . . . . . . . . , . , , .. ' .......... , .. , .... , • • • . . . . • • . • •• 556
THE FOUR BLOOD GROUPS, • .. .. .. . . . . . . .. .. .. • • . • . . . • . . . .. . .. 556
 CONTENTS xxi

CHAPTER tAGE
RACIAL AND GEOGRAPHIC", , , , , , , ' , , , , , , , , , , , , , , , " ",""'" 557
BLOOD GROUPS AT BIRTH, , , " ' , ' " "",'," ,',""""""'" 557
ANmIALIES IN BLOOD GROUPS " ' , ' , " " " ' , ' , ' ~ , ' , ' " , ' , ' , " 55 8
HEMAGGLUTINATION , , ' , " , , " " " ' , " " "" " ' , " " , ' , " , " 55 8
TECIDUC OF BLOOD GROUPING, , , , , , , , , •.. , .... , , ... , " ..••. ,. 5 60
CROSS MATCHING, .. ' , . , .•... , • .. • .••••....•.... , ' , ' , . , " , . , ' 562
SOURCES OF ERROR, " , ... , ........ , ........ , " " ' , . " . , ' '." 563
OTHER AGGLUTINABLE BODIES IN RED CELLS: THE BLOOD TYPES 565
BLOOD TRANSFUSION" .. , . . . . . ., •.... , ... , . . . . . . , ... " ... ' 566
TRANSFUSION OF FRESH WHOLE BLPOD, . . , .• , .... , 566
TRANSFUSION OF STORED WHOLE BLOOD. ,. .. "." .. 567
TRANSFUSIO~ OF STORED PLACENTAL BLOOD .' 57 0
TRANSFUSION OF BLOOD PLASMA,. . .. " , . ' . ,... . ..... , ... 57 0
INDICATIONS, . , . '. , , .•.•..••...•. , , ......• ' .. 572
DOSAGE .. , ..•..•••..•••.... ,' •.. ,', ... ,.. , '" .. ,." 577
REACTIONS. , ...•••••• , .•••. '... ,. , ... ',... . . ' , . , . , " , ... ' 57 8
MEDICo-LEGAL APPLICATIONS OF BLOOD GROUPS. . , •. , . " .' 580
INHERITANCE .. , . . . • . . • •• ' •.. , ..... , ... , , ..••... , .. " ••• ,. 581
LEGAL APPLICATION ..... , ., , ....•.•• , . . . . . . . . . .. " . , ..•. , 582
ATTITUDE OF COURTS ... , ...•.• , •.. ' , ..... , ...••....... ,. 583

45. The Blood Picture of N onnal Laboratory Animals ... ' . , ........ , . . .. 589
THE RABBIT.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
THE GUINEA PIG, .. , . • .. , ... ,.',....... . ..• ,., ... , ' . . . . . . . . 59 0
THE MOUSE" , ...••.•....•......•.. ,' .... , ...• " ..... '" . 59 1
THE DOG ..• , .....•.• ' . . •• . . . • . .• . .••.. , . , . " . . . .......•... 59 2
THE MONKEY, . , . , .. ' . . . . . . . . . . • . . . . . . . • . .. . .. , .... , .. , ' . . . . 59 2
THE RAT., ...... " . . . . . . . . . . . . " .....•..• , .... ,.,.,., ......• 593
THE CHICKEN ....• , • . . . • , .••.....•.. , ••..... , . " . , ... " ..... 594
THE FROG .•....••......• " .....••• , " , •.. , ...•.. , •.•.••.••.• 594

SECTION EIGHT
HEMATOLOGic TECHNIC
46. Hematologic Technic ... , ................ , ...... , ...........•.... 59 6
OBTAINING BLOOD FROM ADULTS .. , ... , . , , ' .... , ....•••...• 597
FINGER PUNCTURE, ...•..•••.•...••..••.•.••..•.•..••.••.••• 597
VENEPUNCTURE... . ..• , .. ,.... . .. ' .•. ' . .. ' ... , .....•.••. 59 8
OBTAINING BLOOD FROM INFANTS ...•.••••..••••.•.• , ........ .. 599
FROM THE EXTERNAL JUGULAR VEIN', •. , , . . .. . ....••. ,' ... . 599
FROM LONGITUDINAL SINUS •.. , ..... " ......• , ... , , ' .. , . 600
ANTICOAGULANTS. ,. . . " , . , ' . , .... ,.,', .. 600
USE OF OXALATED BLOOD . . . . . . . . . . ' .. 601
xxii CONTENTS

CHAPTER FAGE
OBTAINING BLOOD SERUM. . . • . . . . . • • . . . . . • . . . • • . • . .. 602
DETERMINATION OF fuMOGLOBIN (DIRECT METHODS) ..•.....•... 604
TALLQUIST HEMOGLOBIN SCALE. " . . . . . . . . . . . . . . . . . . . . . . • . . . 604
DARE HEMOGLOBINOMETER. . . .. . . • . . . . . . . . . . . . . . . . . . . . . 604
DETERMINATION OF HEMOGLOBIN (INDIRECT METHODS) ........... 605
SAHLI HEMOGLOBINOMETER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
SAHLI-HELLIGE HEMOMETER ...... . . . . . . . . . . . . . . . . • . . . . . . 606
NEWCOMER HEMOGLOBINOMETER. . . . . . . . . . . . . . . • . . . .... 606
SHEARD-SANFORD PHOTELOMETER . . . . . . . . . . • . . . . . . . . . . . . . . . . . . 607
ENUMERATION OF ERYTHROCYTES. ........... 608
CALCULATION. . . • • . . . . . . . . .. ..................... ....... . 6rr
EXAMPLES OF CALCULATION . . . . . . . . . . .. . 6rr
SOURCES OF ERROR.. . . . . . . . . . . • • . . . . . . . . . . . . .. . . . . . . . . . 612
COLOR INDEX . . . . • . . . . . . . . . . . . . . . • . . . . . . . . . . • . . . . . . . . . . . . . . 6r2
ENUMERATION OF LEUKOCYTES . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . 6r3
CALCULATION.... . .• . . . . . . . . • . . • . . .. ...•.... ...... ...... 6r3
EXAMPLE OF CALCULATION OF WHITE CORPUSCLES...... 6r3
CORRECTION OF THE LEUKOCYTE COUNT FOR NUCLEATED RED CELLS 614
EXAMPLE................ •..•...•.•......... 6r4
EXAMINATION OF FRESH BLOOD. . . . . . . . . . .. .... . . . . . . . . . . . . . 6I4
PREPARATION OF BLOOD SMEARS................... 615
COVER GLASS METHOD..... 6,6
SLIDE METHOD. . . . . .. . . . . . .. ...•... . . . . . . . . . . . . . . . . 6,6
CONCENTRATION METHOD FOR LEUKOCYTES. . ................• 617
FIXATION OF BLOOD FILMS. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 6r8
STAINING OF BLOOD SMEARS .....•.. ~ . • . . . . . . . . . . . . . . . . . . . . . . 6r8
WRIGHT'S STAIN . . . • . . . . . . . . • . • . . . . . . . . . . . . . . . . . . . . . . . . . . . 6r9
GIEMSA'S STAIN . . . . . . . . . . . . . . . . . . . . . '. •........... 620
PEROXIDASE STAINS.... . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . 62I
INDOPHENOL BLUE SYNTHESIS .•...... • . . . . . . . . . . . . . . . . . . . . . 622
DIFFERENTIAL LEUKOCYTE COUNT......... ........ 622
RELATIVE AND ABSOLUTE VALUES . . . . . . . . . . . . . . . . . . . . . . . 623
SCHILLING'S NUCLEAR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
FIT.AMENT AND NON-FILAMENT COUNT . . . . . . . . . . . . . . . . . • • . . . . 6 24
VOLUME INDEX.. ...•.....•.•...•.. . . . . . . . . . . . . . . . . . . . . .... 62 5
CENTRIFUGE TUBE METHOD . . . • . • • . . . . . . . . . . . . . . . . . . . . . . . 625
CALCULATION.... . . • • . . • . . . .. . . . . . . . . . . .. . . . . . . . ...... . 62 5
EXAMPLE ...•••••........ ...... . . . . . . . . . . . . . . . . . • . . . . ~25
WINTROBE'S HEMATOCRIT METHOD......... . . . . . . . . . . . . . . 626
CALCULATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
EXAMPLE .•..•. " . . • • • • • • . . . . . . . . . . . . . . . . . 626
THE MEASUREMENT OF RED CELL DLAMETER ...........••...... 626
PRICE-JONES CELL DLAMETER CURVE... ..•...............• . 626
HADEN-HAUSSER ERYTHROCYTOMETER... . ..•...••••.•......• 62 7
SUBSTITUTE FOR HALOMETER. . . . . . . . . . . • . . • . .• . . . . . . . . . . . . . 628
 CONTENTS xxiii

CHAPTER PAGE
MEAN CORPUSCULAR VOLUME (WINTROBE) .......••...•.••••... 62 9
MEAN CORPUSCULAR HEMOGLOBIN (WINTROBE) .•.......•••••.••• 62 9
MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (WINTROBE) .. 62 9
SATURATION INDEX (OSGOOD) ....•••••.....•...........•..•.•.• 63 0
RETICULOCYTE COUNTS ........ ' ....•••••.•. , .•. , •. , .. , . , .••.•.• 63 0
ENUMERATION OF PLATELETS . . . . . , ..••...•••••....•..••. ••••·• 63 1
FONIO'S SMEAR METHOD .. , ... " •. ,', .....••..•...... , •••.. 63 1
OLEF'S METHOD,. • . . . . • . . . . . . ,., .......• , . . . . . . . . . . . . . . . 63 2
DIRECT METHOD FOR COUNTING PLATELETS ...........•.•.•• " 633
COAGULATION TIME. . . . . . • • • • • . • .. • ....•..•.•....•••.•••.•... 634
SLIDE METHOD . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . • . . • . • . . • . . . . 634
CAPILLARY TUBE METHOD..... ..... .. . ..•..•.••.......•.• 634
VENEPUNCTURE METHOD (LEE AND WHITE) ...••......•...... 635
HOWELL'S METHOD. . . . . . . . . . . . . . • . . . . . . .. ........•.•.••... 635
BLEEDING TIME .......••.••.'. ..•••..........••........••.•. " 63 6
DUKE's METHOD ...••.•....••••.•........•.....•....•.••.. 63 6
THE IVY METHOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 6
CAPILLARY RESISTANCE TEST OF RUMPEL-LEEDE ....••••..••.. " 63 6
CLOT RETRACTION TIME . . . . . . . . . . . . . . _.................•••.... 637
PROTHROMBIN TIME .......................................... 637
HOWELL'S METHOD . . . . . . . . . . . . . . . . . . . '. . . . . . . . . . . . . . . . . . . . 637
THE QUANTITATIVE PROTHROMBIN METHOD OF QUICK ......... 63 8
PROTHROMBIN METHOD OF SMITH, ZIFFREN, OWEN AND HOFFMAN 639
A MICRO TEST OF PLASMA PROTHROMBIN. .. . . . . . . . . • • . . • .. 639
CALCIUM TIME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
FRAGILITY TIME .....•••••••••••...••........•.•••.••.•• •••••• 640
SANFORD'S METHOD .••.••.••••.••.••.....•..••.....••.••••.. 640
PIPETTE METHOD .....•.•••••• , •..•.•••.•........•...•••. " 64 1
SEDIMENTATION RATE. . . . . . . . . . . . . . . . . . . . . . . • • . . .. 64 2
WINTROBE AND LANDSBERG METHOD ......••••........•.••.• 643
USE OF THE CHART.... . . . . . . .. .. ... . . . . . . . . . . . . . . . . . 644
WESTERGREN'S METHOD ......• . .......... " 645
A MICRO-METHOD FOR DETERMINING SEDIMENTATION ON CHIL-
DREN. . .......... 645
TEST FOR BILE PIGMENT IN BLOOD SERUM.. . ...•••.•.•...... 64 6
IcTERUS INDEX. • . . • . • . . . . . . . . • • . . • . . . ....•••..••.•.•.•. " 646
VAN DEN BERGH REACTION....... .. . . . . . . . . . . . . . . . . . . 647
MODIFICATION OF 1'HANNHAUSER AND ANDERSON ......•••... '. 647
ELTON'S RING TEST. ...... .•.. 648
QUANTITATIVE DETERMINATION OF UROBILINOGEN IN FECES 649
SPARKMAN'S METHOD . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . • , .••... 649
QUANTITATIVE DETERMINATION OF UROBILINOGEN IN URINE ... , . 65 0
STERNAL PUNCTURE ..•....•••••....••...•••.•..••. , ........ . 65 1
REICH'S METHOD ..•.. , •.••.... , .•• , ....•......... , ... ···· . 65 1
YOUNG AND OSGOOD'S METHOD, .......••..•..••.• , ......•..• 65 2
xxiv CONTENTS

CHAPTER PRGE
COMPLETE EXAMINATION OF BONE MARROW .•...•...••..•..••.. ii53
SURGICAL BONE MARROW BIOPSY. . . . . . . . . . . . ........•••..• (555
GROSS EXAMINATION .....•....... : . . . . . . . . . . . . . . . . : . . . . . . . " (555
COUNTING BONE MARROW CELLS. . ....•.....••.. (555
PREPARATION OF SERUM SPREADS . . . . . . ' . . . . . . . . . . . . . . . . . . . • . . (55 6
PREPARATION OF HISTOLOGIC SECTION . . . . • . . . . . . . . . . . . . . . . . . . (>5 6
CULTURE OF BONE MARROW CELLS. . . . . . . . . . . .. . . . . . . . . .. (557
SUPRAVITAL STAINING OF BLOOD CELLS.... .. . . . . . . . ... , .... (55 8
EXAMINATION FOR MALARIA PARASITES.. ....... . .....•..... e60
FRESH BLOOD. . . . . . • • • • • .. . . . . . . . . . . . . . .. . '" . . . . . . . . . . (560
THIN SMEAR METHOD ............ :...... ..... . " ......... (560
THICK SMEAR METHOD. '" ..... . ' ...... . ...•......••... (56,
TESTS FOR INFECTIOUS MONONUCLEO",S. . . .. . . . . . . . . . . . . . . . . . . e 62
THE PRESUMPTIVE TEST. . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . .. (562
THE DIFFERENTIAL TEST... . . . . . . . . . . . . . . . ...•.... (563
TEST FOR HODGKIN'S D,SEASE (GORDON) . . . . . . . . . . . . . . . . . . . . . (566
DETERMINATION OF THE SPECIFIC GRAVITY AND TOTAL .PROTEIN OF
BLOOD OR PLASMA BY TIlE FALLING DROP METHOD . . . . . . . . . (567
V,SCOSIMETRY (BIRCHER). . .. . . . . . . . . . . . .. . . .• . ................ (57 2
BASOPHILIC AGGREGATION TEST. . . . . . . . . . . . .. . . . . . . . . . .. (57 2
AUTOHEMAGGLUTINATION..... . . . . . . . . . . . .. . .. ••...... .....• . (573
SPECTROSCOPIC EXAMINATION OF BLOOD. . . . . . . . . . . . . . . . . . . . . . . e74
METHODS OF DEMONSTRATING SICKLE CELLS . . . • . . . . . . . . • . • • . . (;7 6
TUllE METHOD ..••...••...... , .....•.•......•••.•..••..•... (;7 6
SEALED MOIST PREPARATIONS............ . ................ 677
 SECTION ONE
PRINCIPLES OF TERMINOLOGY
DEFINITIONS OF HEMATOLOGIC TERMS
 SECTION ONE
HEMATOLOGIC TERMINOLOGY

CHAPTER 1
PRINCIPLES OF TERMINOLOGY
GENERAL COl\SIDERATIONS

The question of terminology in the field of blood diseases remains in a state

of confusion with many meaningless and conflicting terms, to such an extent that
one hematologist often refers to a condition identical with that seen by "'lOche,
with au entirely different name. Thl> confusion has also resulted in different
dbea~e5 being given the ~a~1c de<;ignation. For this reuson, we fecI that COIl-
,iderable space should be devoted to the question of nomenclature.
Some years ago an organization of pathologists took steps to attempt to correcl
a chaotic situation relative to recognition of hlood cells when it was suggested
that a number of hematologists from various parts of the United States 'gather
together with their microscopes and study the various types of typical and
atypical leukocytes and determine whether or not it were possible for all of
them to agree on the identity of these various cells. It was felt that if such
could be done, there would be some unanimity of thought relative to the im-
portant problem of all workers "seeing with the sanle eye." This plan could
well be applied to the chaos.of terminology.
It is small comfort to realize that the same confusion exists throughout the
entire field of medicine, but this should not prevent attempts to clarify the situa-
tion, and hence, we present this discussion of this important question.
It is not diffIcult to understand how such terms worm their way into the
literature. It seems that most of the undesiraOle terminology arises fro';' the. care-
lessness of those who rcport disca,"s and new clinical entities with but little
thought to the proper derivation of medical terms. Other terms arise from the
desire of a few individuals to establish a new name with the fOl\d hope that the
author's name may by chance become associated with it. The suspicion 111ay
be well grounded thac an occasional author in describing,a,new disease may at·
tempt to make the name so complex and cumbersome that the medical rearler
will be forced to apply the author's name to it.
~Iany of our hematologic terms date from hundreds of years ago when
medical literature was in its infancy and no person 'conld foresee the future im-
portance of the subject. In those days it mattered little what a disease was <oilied
because recognizable diseases were few, and the literature was not voluminom;
3
4 IhMA!OLOGIC T~ItMINOLOGY

for example, when the word "leukemia" was introduced, meaning white blood.
no one could tell that in the I ulure there would be many kinds of leukemia and
that actually the patient did not hine white blood, and that some t} pes of
leukemia would be characr~fi~~tby a'~,\k·pf·'lh!~rb,l~ .. To m~ke bad m(ltters
'''lorse, then came the deSIgnation of .1 aleukc11ttc leukentta" whIch has become
establisl)ed in the liter!lt..ure,. which .could ,be .strictly defined, a~ "Ilif state of
leukemia it,lthbutl !euke;Il'ia,v'or "white blood wiih'out 1anyl white }bloo,!": For-
tunately the term pemicious allcmia has come through the test of time unsullied
by the addition of the name of the original author who described the discdsc, at-
though we still see efforts to designate' the'disease as "Addisonian Anemia" or
"Biermer Allemia" or ".1ddis01z·Bicrmer" disease.
All of the fault for our hematologic terminology should not rest with the
ancients, since in modern times we still observe the introduction of ill-considered
terminology, and if one writer is not pleased with a term he will u5ually attempt
to introduce and establish another until the situation is chaotic to the point of
absurdity. We have only to look at the most recent blood dyscrasia known to
medicine as an excellent example. Thus, agranulucytosis, although only Ij
years old, now has over 20 different names, including agra1!u/ocytic a"/illw.
mucositis necroticans aglamtioc)'tica, sepsis with granulopenia, monocytic allg;IIa,
agrallUlosis, agranulocytic leukopenia, agra1lU/ocytopcnia, agranulcmia, etc., to
mention only part of them, Thus, it can readily be seen tbat the modern physician
is as little or less versed in correct usage of medical terms as the ancient authors
in'medicine.
This is no small problem to the student of medicine, whether he he graduate
or undergraduate, since it is difficult enough to keep abreast of medical advances
without the path littered with tenninologic obstacles. It is a discouraging out-
look to witness a class 'of second year medical students attempting to stumble
through a maze of unrelated and meaningless terms in hematology.
How~ver, the situation is not hopeless as evidenced by the recent clarification
of bacteriologic nomenclature, and also the new classification of diseases that now
is in wide use.

ORIGIN OF HE~iATOLOGIC TERMS

It is obvious that medical terminology should he based on a dead and un-
changeable language if possible. This has been done to a considerable extent in
the utilization of the Latin roots and stems but certainly the Greek terms are
more widely used than any other. In Table I is shown a list of ba,ic terms from
which most of the hematologic terminology is derived. It will be noted that only
a few Latin words arc used. It is our hope that the reader will study this list
Lefore attempting to evaluate our terminology that we suggest as being more
appropriate, more correct, equally descriptive and much less confusing, which is
set forth in the following pages with a list of definitions.
 PRINCIPLES OF TERMINOLOGY

TABLE I
Tnt ORIGIN OF MOST HlmATOLOGIC TERMS
DERIVATION MEANINGS HEMATOLOGIC USAG~
Aor An without AorAn
Acidum (L) acid acid
Ampho both ampho
Anisos unequal aniso
Basis
Blastos
m.... mso
germ blast
Chroma color chrome
Crena (L) a notch crenation
Eidos a resemblance oid
Endon within enda
Eos dawn eosin
Erythros red erythro
Fragilitas (L) fragile fragile
Fuscus (1) brown fucsin
Generare (L) to beget genesis
Globus (L) globe globin
Granulum (L) granule granulo
Grapho I record (write) graph
Haima (Haimat) blood hem-haem-hemat
Histion tissue histio
Hyper above--over hyper
Hypo under hypo
Juvenia (L) young juvenile
Karyon nut (nucleus) karyo
Klasis a breaking clasia
Konia dust conia
.1E.riW' .{,ep3alCe erre
Kylos hollow (a cell) cyte
Leukos white leuk
Logia astudy ology
Lympha (L) clear (spring water) Iympho
Lysis solution lysin
Makros large, great, long maCro
Medulla(L) marrow medullary
Megas (megalo) large mega
Memskos crescent meniscus
Mesos middle meso
Meta change meta
Mikros small micro
Monos single mono
Morphe, form morpho
Myelo. marrow myelo
Neos new neo
Nucleus (L) dim. of l'Irux nut (meaning of nux) nucleus
Oligos few oligo
Orthos correct ortho
6 HEMATOLOGIC TERMINOLOGY:

TABLE I (Concluded)
DERIVATION MEANINGS lIEMATOLOGIC USAGE
Osis (state of, condition of, osis
increase of)
Ouron urine uria
Ovum(L) egg oval
Oxys acid oxy
Pathos suffermg pathology
Pas all pan
Penia poverty, paucity penia
Phago I eat ph ago
Philos fond phile
Phthisis a wasting away phthisis
Plasis a forming or inolding plasia
PllIsso I form plastic
Plastikos formative plastic
Poieo I make poiesis
Poikilos manifold, varied poikilo
Polys much, many poly
Pro before pro
Reticulum (L) a net reticulo
Rouleau (Fr.) a roll Rouleaux
Skaios left skeo
Skopeo I view scope
Sphaira sphere sphero
Thele nipple thelio
Thrombos clot thrombo
Tonos strain, tension tonia
(L) Refers to Latin derivation
(Fr.) Refers to French derivation.
All others are of Greek derivation.

DESCRIPTIVE TERMS

LEUKOCYTES
Some of the changes that we propose are worthy of comment, and these in-
clude changes in the more basic terms, such as the names of cells. For example,
we prefer the word neutrocyte instead of neutrophil, believing that the cellular
constituents should carry the suffix "eyte" to identify them as being cellular bodies.
Also the use of the terms neutrocyte, basocyte, acidocyte is consistent with well
accepted terms, lymphocyte, monocyte, erythrocyte, thrombocyte, reticulocyte,
etc. Then it is obvious that increased or decreased numbers of these cells may be
more correctly and concisely expressed by the suffix "osis" and "penia," resulting
in lymphocytosis, monocytosis, reticulocytosis, neutrocytosis, etc. (see Table II).
 PRINCIPLES OF TERMINOLOGY

TABLE II
INCREASES AND DECREASES IN NUMBER OF CELLS
INCREASED NUMBER IN ELOOD DECREASED NUMBER IN :BLOOD
Acidocytosis Acidocytopenia (Acidopenia)
Basocytosis Basocytopenia (Basopenia)
'Erythrocytosis 'Erythrocytopenia *CErythrppenia)
Granulocytosis 'Granulocytopenia '(Granulopenia)
Hematocytosis Hematocytopenia (Hematopenia)
'Leukocytosis Leukocytopenia '(Leukopenia)
'Lymphocytosis Lymphocytopenia '(Lymphopenia)
*Monocytosis Monocytopenia '(Monopenia)
Neutrocytosis Neutrocytopenia '(Neutropenia)
'Reticulocytosis Reticulocytopenia '(Reticulopenia)
'Thrombocytosis "Thrombocytopenia '(Thrombopenia)
Terms in current use.

NORMAL BLOOD CELLS

At once it can he seen that the simple "neutrocytosis" is more desirable than
"neutrophilic leukocytosis" or "polymorphonuclear neutrophile leukocytosis."
Likewise, the term "neutropenia" is consistent with such widely used terms as
granulopenia, thrombopenia, erythro penia, etc. If the neutrocytes are decreased
it is expressed simply by neutropenia. If all granulocytes are decreased it is
granulopenia, and if this should represent the essential feature of a disease as in
the so-called agranulocytosis, it can be aptly designated as primary, essential,
idiopathic, or pernicious granulope;';a. If one prefers the inclusion of the word
"cyte," the\ term 'can be enlarged to "granulocytopenia," consistent with "neutro-
cytopenia," and "thrombocytopenia." In any event when due consideration is
given to proper building of terms the terminology stands on a sound basis, is
easily understandable, tends toward simplicity, and even more important, it be-
comes a descriptive term for the disease in question (see Table III).
TABLE III
NORMAL CELLS OF THE PERIPHERAL BLOOD
'HEMATOCYTE-Includes all cellular elements of the blood; erythrocytes, leukocytes
and thrombocytes.
*LEUKOCYTE-A white blood cell.
*ERYTHROCYTE-A red blood cell.
'THROMBOCYTE-A blood platelet.
*GRANULOCYTE-A white blood cell containing cytoplasmic granules; neutrocytes,
acidocytes and basocytes. •
NEUTROCYTE-(NEUTROPIllL) (POLYMORPHONUCLEAR) (POLYMORPH) (POLYNUCLEAR)
(POLYMORPHONUCLEAR LEUKOCYTE). A white blood cell of bone marrow origin
having a diffuse neutrophilic, cytoplasmi(_: granulation with an irregular nucleus
which may be segmented into two or more lobes.
ACIDOCYTE-(EoSINOPlllL). A granulocytic cell of bone marrow origin with large
acidophilic cytoplasmic granules with an irregular multilobed nucleus.
BASOCYTE-(BASOPHIL). A granular cell with basophilic granules.
*LYMPHOCYTE-A rounded mononuclear cell from the lymphoid tissues; may be either
large or small.
8 HEMATOLOGIC TERMINOLOGY

*MONOCYTE-(MoNONUCLEAR) (TRANSITIONAL), (ENDOTHELIAL CELL) (ENDOTHEliO-

cYTE). A large mononuclear cell from the reticulo-endothelial tissues.
*l<ETICULOCYTE-A young immature red blood cell containing a skein, or network, of
chromatin material.
,.. Terms widely used in current literature. For detaiJed definitions see Section on Definitions.

If the term "agranulocytosis" is analyzed it is seen to refer to "an increase of

agranulocytes, and the term, agranulocyte;is defined in standard dictionaries as
being a cell without granules. Presumably such cells in the blood are lymphocytes.
Therefore the term "agranulocytosis" strictly means an increase in non-granular
cells or lymphocytes, while in that disease no such condition exists. If the coiner
of that word had given more thought to proper derivations, much confusion
could have been avoided. This is only one example of many and is pointed out
because it is the most recently introduced term.
The terms neutrophil, basophil, and eosinophil, had their origin from the
fact that they have an affinity for neutral, basic and acid dyes when stained with
the Romanowsky stains (Wright's stain as an example). The suffix "phil," from
the Greek "philos," meaning "fond of," was added because the granules of tbese
cells were "fond of" these various dyes. Why is it ,necessary to indicate that
these cells are fond of these dyes when the same fondness is expressed once by
the neutro:, baso-, and eosino-, prefixes? Also the dye eosin derives its name
f;om the fact that its color is similar to that of the golden rising sun, hence its
origin from "eos," meaning the dawn. At least consistency should be considered
and the cell designated as an acidophil instead of an eosinophil or better still,
as an acidocyte, corresponding with neutrocyte and basocyte. This group of cells
therefore are named on a basis of their staining reaction. The lymphocyte is so
called because of the sky blue clearness of its cytoplasm, from the Latin "lympha"
meaning clear (spring water), and the monocyte named thus because of its single
nucleus, from the Greek "monos," meaning single. Still another cell, the erythro-
cyte, is so called because of its red color, and the reticulocyte because of its skein-
like reticulum of chromatin, etc. Thus, it can be seen that the various cells bear
their names because of many different characteristics and for different reasons.
Hematologic literature is filled with terms that are meaningless, with double
negative or double positive components, for ex;tmple, hypereosinophilia, and
hyperleukocytosis and even hypoleukocytosis. Also one condition is given many
designations such as leukocytosis being called hyperle1Jkocytosis, hyperleukocy-
tltemia, hypercytosis, hypernormocytosis, hyperorthocytosis, and neutrocytosis
being called neutrophilia, neutrophilic leukocytosis, polymorphonuclear leukocy-
tosis, hyperneocytosis, polyneucleosis, hyperleukocythemia. One standard diction-
ary defines the word hyperhypocytosis as "leukopenia with a rela~ive increase, in
neutrophiles" and even defines hyperhypercytosis. Thus, we see hematologic
terminology carried to absurdity. '
We realize well that the introduction of new' terminology does not receive
a welcome from one who is versed in the older terms, and we are further cognizant
of the fact that radical departures from accepted terminology oftentimes fail of
adoption because of attempts to completely change fronl time worn terms at one
 PRINCIPLES OF TERMINOLOCY 9
time. Certain terms are so firmly ingrained into medical thought that it would
be impossible to abandon ~hem. After all, the chief purpose of this discussion is to
stimulate the use of better terminology among those who write about these dis·
eases, so that. at least, we may all try to speak the same hematologic language,
and to attempt to abandon some of the terms that are glaring in their errors and
deficiencies.

SIZE AND SHAPE OF RED CELLS

The terms normocyte, microcyte, and macrocyte, which are descriptive of
changes in the size of red cells are so well grounded in medical literature that
changes would hardly be advisable. A more accurate designation would be nor·
moerythrocyte, microerythrocyte, etc., but when terms are constructed properly
with a view of correctness being the sole end to be attained, the terminology will
become cumbersome. Therefore terminologic accuracy has to be tempered with
brevity and simplicity (see Table IV).

TABLE IV
MORPHOLOGY OF ERYTHROCYTES (RED BLGOD CELLS)
*NORMOCYTE-A red blood cell of normal size.
*MICROCYTE-A red blood cell with a diameter smaller than normal.
*MACROCYTE-A red blood cell with a diameter larger than normal.
*POIKILOCYTE-A red blood cell with an atypical shape.
*ANISOCYTE-Term referring to variation in size of red cells; the condition, anisocytosis.
'SPHEROCYTE-A red blood cell with a globular or rounded form.
FRAGILOCYTE-A red cell that is unusually fragile when SUbjected to a hypotonic salt
solution.
'OVALOCYTE-An oval shaped red blood cell.
MENISCOCYTE (SICKLE CELL)-A sickle or crescent shaped red blood cell.
llASOERYTHROCYTE-A red blood cell showing the changes of basopbilic degeneration.
POLYCHROMATOCYTE (POLYCHROMATOPHIL)-A red blood cell showing a variation from
the normal staining reaction.
CRENOCYTE-A red cell with serrated, notched edges.
* Terms in current use.

The terms poikilocyte and anisocyte are widely used and are consistent with
others referring to red cell changes. A condition that is now appreciated more
than ever before is that state of the red cells where they become rounded or
globular in shape, as seen in hemolytic icterus. Instead of being flattened bi·
concave discs their shape tends to become spheroidal. The term spherocyte aptly
describes this, and this is well worth inclusion in the nomenclature. The word,
ovalocyte, describing the oval red cell seen in that rare disease, ovalocytosis, is
equally descriptive.
We would suggest the addition of a term that would be descriptive of the
sickle cell seen in sickle cell anemia. Although this disease was described in
America and the term sickle cell is !,ppropriate, it lacks the quality of consistency
with other terms. Here again, it would be .desirable to resort to the older lan-
10 HEMATOLOGIC TERMINOLOGY

guages and find a term descriptive of the sickle cell. If the Latin is used, the
word S,(el, meaning sickle, could be used and the term, seculocyte, would describe
the sickle cell. If the Greek is preferred, the term, meniskos, meaning curt'e
could be applied as meniscocyte. For reasons previously stated the Greek is
preferable, and so we have incorporated this word instead of sickle cell and
the condition of sickle cell anemia as meniscocytosis (see Table V). This term
has been used by Piney.

TABLE V
VAEIATION IN THE RED CELL PICTURES
*ANrSOCYTOs1s-Referring to variation in size of red cells.
BASOERYTHROCY'IOSIS-As seen in chronic malaria, and severe hypochromic anemia.
(Basophilic stippling, etc.)
CRENOCYTOSIS (CRENATWN)-As seen when red cells are subjected to the action of
hyper-tonic salt solution.
FRAGILOCYTOSIS-Red cells excessively fragile.
'*MACROCYTOSIS-Average cell size larger than normaL
MENISCOCYTOSIS-Sickle shaped .r~d cells.
*MICROCYTOSIS-Average cell size smaller than normal.
N ORMOCYTOSIS-Normal size of red cells.
*OVALOCYTOSIS-Oval shaped red cells as seen in some lower animals, namely the
camel. Occasionally hereditary in man.
*POIKILOCYTOSIS-Referring to variation in shape of red cells.
POLYCHROMATOCYTOSIS (POLYCHRO"ASIA) (POLYCHROMATOPHILIA) (POLYCHROMA-
'IOsls)-Variation in staining reaction.
'SPHEROCYTOSIS-Referring to a condition when a considerable number of the cells
are globular in shape .
• Terms in current use.

The spherocyte, as stated before, is a rounded globular ,cell characteristic of

hemolytic icterus. It is also excessively fragile and is hemolyzed more easily than
the normal cell. This increased fragility could easily be expressed by fragilocyte,
from tbe Latin "fragilitas," and the state of increased fragility by fragilocytosis,
instead of stating, as is usually done, that there "is an increased fragility of red
cells." Therefore, briefly stated, hemolytic icterus is a disease characterized by
spherocytosis, microcytosis, and fragilocytosis.
Over a period of years there has arisen a wide variety of terms to describe
morphological alterations in red cells characterized by basophilic changes, these
including such as basophilic stippling, punctate basophilia, basophilte degenera-
tion, with the atypical types being designated as Cabot ring bodies, named after
the man who described them, and Howell-folly bodies, similarly named. It seems
quite obvious now that all of these basophilic changes are a parLof the same fun-
damental process. Furthermore, none of these changes is diagnostic of or sig~
nifical)~ of any particular disease. Any or all of them may be seen in a number
of diseases characterized by prolonged hypochromic anemia. Thus, they are seen
in chronic lead poisoning, malaria, ,and other states of anemia characterized by
chronicity. It would appear advisable to clarify tbis phase bf terminology by in-
 PRINCIPLES OF TERMINOLOGY II

elusion of such basophilic changes under a single, term, as "basoerylhrocytosis,"

and a cell that exhibits such as a "basoerythrocyte." The'word "basocyte" could
be used but as pointed out before, this should apply to the basophil in the
leukocyte series.
There is a well'recognized state of the red cells known as "polychromato-'
philia" in which there are atypical variations in the staining reaction of the vari·,
ous cells. Any red cell that exhibits such changes could well be designated as a
"polychromatocyte" and the condition as "polychromatocytosis."
Finally; that state of red cells whereby they become shrunken when placed in
hypertonic salt solution, although not characteristic of any disease, is referred
to as crenation from the Latin, Crena, or notch, because of the notched, serrated
spicules that project from the periphery. This term is well used, but We would
propose a slight modification in the'form of "crenocytosis" and the cell exhibiting
such change as a "crenocyte." This again would fit the pattern of consistency
with the terms stated before (see Table V).

HEMOGLOBIN CONTENT
TAllLE VI
VARIATlONS IN COLOR CONTENT OF STAINED RED BLOOD CELLS
"ACHROMIA-A term meaning a condition in which the r~d cells are without hemoglobin.
"HYPERCHROMIA-Referring to that state in which the red blood cells contain more than
their normal amonnt of hemoglobin. •
"HYPoCHROMIA-Referring to that condition in which most of the red blood cell~ contain
a decreased hemoglobin conten.t. ',
'NORMOCHROMIA-Referring to the normal hemoglobin content of red cells as a group.
* Terms in current use.
A red cell that contains its normal amount of hemoglobin is a normochromo-
cyte, and strictly speaking, is a normo-chromo-erythro-cyte. However, this term
is too cumbersome for practical usage,.so the term "normocyte," describes the cell
with respect to size, shape and color. It is necessary to have terminology that will
adequately express variations in color content due to increased or d~creased
amounts of hemoglobin, and since it is a matter of little importance what a single
cell contains, but rather emphasis 'should. be placed 'on the hemoglobin content
of the average cell, then the suffix cyte, Can well be omitted from consideration
in this phase of the, terminology. To 'express these variations have arisen the
terms normochromia, hypochromia, and hyperchromia, referring to those condi'
tions in which the red cells as' a group show the normal hemoglobin content, de-
creased hemoglobin, or increased hemoglobin, respectively. These terms 'ICe good
and should be used. Also the term "color index" which is a simple math"matical
expression of the average hemoglobin content of red cells, is worthy'of continued
use.
There has come about however, the incorrect usage of the word "achromia,"
which means, literally, without hemoglohin. This condition does not exi~t in the
red cell, for if the cells were entirely depleted of hemoglobin, this would be in-
compatible with' 'life, and the cells in question would not be erythrocytes. In
I2 HEMATOLOGIC TERMINOLOGY

describing blood'pictures, the term "central achromia oj the red cells" has a wide
use, It is conceivable that the central and thinnest part of the r~d cell could be
entirely without hemoglobin and the term retained for descriptive purposes, but
the term "hypochromia" more correctly expresses the true sta,te of the cells. Fr(,ID
this word achromia has arisen such words as achroacyte, achromatocyte, achroma-
cyte, and many others. IIi like manner we speak of "aplastic anemia" which may
or may not actually exist, but no doubt "hypoplastic anemia" does exist in vary-
ing degrees. All of this discussion may appear to be quibbling over minutiae, but
we wish to emphasize that the more thought that is given to proper usage of terms,
the better the terms will finally become.

BLOOD PLATELETS
Blood platelets are known correctly as thrombocytes, from the Greek,
"thrombos," meaning clot, and hence, derive their name on a functional basis,
pecause of their role in the process of blood clotting. These cells have been'
given many names including blood plaques, blood plates, platelets, etc., and have
also been named after various men who described them and discussed them, in-
cluding such as Hayem's hematoblasts, Zimmerman's corpuscles, etc. It is ob-
vious that the ter,m thrombocyte is correct and adequate. Increases are then
designated as "thrombocytosis" and decreases as "thrombopenia" or "thrombo-
cytopenia." The latter is preferred for consistency with similar terms (see
Table II). •

THE IMMATURE BLOOD CELLS

It is in this field of hematology that most confusion exists relative to termi-
nology, but this is because of the confusion and uncertainty of the origin of many
of the cells, in particular the monocytes. In this discussion, however, we shall
attempt to set forth only some fundamental principles and not attempt to name
the various types of immature cells. For a discussion of the origin of cells see
Chapter III. All blood cells are hematocytes, and likewise all immature or early
forms of cells should be designated as hematoblasts, from the Greek, haima
(blood)-blastos (germ). Therefore, the term hematoblast includes all blast
f~rms, as leukoblasts, erythroblasts, and thromboblasts, which develop into leuko-
cytes, erythrocytes, and thrombocytes, respectively. The term leukoblasts
should be applied to the immat~re cells of all white hlood cells, and therefore,
would include the myeloblasts,lymphoblasts, and monoblasts, which develop into
the granulocytes, lymphocytes, and monocytes. Some writers use the term
"leukoblast" incorrectly when it is designated as the stem cell of the granulocytes,
erythrocytes, and thromhocytes, and hence, would use it to designate the stem
cell of the entire bone marrow seri"". The term thromboblast refers to. the pre-
cursor of the thrombocyte, although this cell is more often referred to as a
megakaryocyte because of the size of its nucleus mega (large)-karyon (nucleus).
However, it must be stated that if one believes that all blood cells arise from a
single stem cell, whatever its location or tissue types, then, the term "leukoblast"
is correct.
Some of the cells arising in the bone marrow are called mveloblasts from the
 PRINCIPLES OF TERMINOLOGY

Greek, myelos (marrow). From this standpoint the erythroblast is a myeloblast,

but since it is recognizable as a cell that will develop into a red cell, the erythros
component is prefixed to the worn. The suffix "blast" should be added to the
earliest recognizable form of the various cells. If earlier forms are encountered,
as they often are, it is sufficient for the present to refer to such as "stem cells;"
primitive blood cells, etc. in light of the uncertainty of blood cell origins.* Thus,
all early forms usually encountered in blood diseases can be well named as shown
in Table VII. For more detailed definitions, see section on Definition of Terms.
TABLE VII
ERYTHROBLAST-Precursor of all red blood cells.
GRANULOBLAsT-Precursor of all granulocytes. (Same as myeloblast.)
HEMATOBLAST-Precursor of all blood cells.
LEUKOBLAST-PreCursor of all white blood cells.
LYMPHOBLAST-Precursor of lymphocytes.
MACROBLAST-Precursor of large or macrocytic erythrocytes.
MEGALOBLAST-Precursor of megalocytes, if such exist.
MICROBLAST-Precursor of microcytic or small erythrocytes.
MONOBLAST-Precursor of monocytes.
MYELOBLAST-Precursor of all granulocytes (neutrocytes, acidocytes, basocytes).
N ORMOBLAST-Precursor of all normal erythrocytes.
THROMBOBLAST (MEGAKARYOCYTE)-Precursor of thrombocytes.
Most of the blast types develop into their respective adult cells without the
interposition of many descriptive terms, except the myeloblast. This cell de-
velops into the premyelocyte, myelocyte A, myelocyte B, myelocyte C, metamyelo-
cyte (the juvenile of Schilling), the band neutrocyte and finally the segmented
neutrocyte. Thus, these terms, as shown in Table VIII, are applied to the various
levels of development of the segmented neutrocyte.
TABLE VIII
TERMINOLOGY IN USE PERTAINING TO THE DEVELOPMENT OF A NEUTROPHIL

PREMYELOBLAST-The earliest recognizable precursor of the granulocyte.

MYELOBLAST-Definitely recognizable. (See description, page 55.)
PREMYELOCYTE-An occasional granule added in cytoplasm.
MYELOCYTE A-Differentiated into three types.
MYELOCYTE B-Further growth.
MYELOCYTE C-Further growth.
METAMYELOCYTE-(The juvenile of Schilling) Nuclear constriction. Occasionally in
blood stream.
BAND NEUTROCYTE-About five per cent in blood stream.
SEGMENTED NEUTROCYTE-The adult cell, delivered, active, functional.
HYPERSEGMENTED NEUTROCYTE-The aged neutrocyte.
For illustrations see Plate 2.
For descriptions see Chapter 4.
* We have recently studied a patient with leukemia in which the predominating (en was
at a level of development even earlier than the myeloblast, and" we deSignated this as pre-myelo~
blastic leukemia. It could have been called acute stem cell leukemia, primitive cell leukemia, or
acute leukosis.
14 HEMATOLOGIC XERMINOLOGY

It would be a decided improvement if s~me consistency and order in termi-

nology: could be established in the development of a neutrocyte. The myelocytic
levels A, 'B, and C were introduced hy Sahin for descriptive purposes in her de-
tailed studies of !ione marrow cells, and the terms, juvenile, stab, band, and seg-
menter, were introduced hy Schilling in describing cell development at about the
stage of maturity of neutrocytes. These have become firmly ingrained into the
literature and their. removal, would appear almost impossible.
Tbere are other misused and impractical terms in use in hematology, but we
are more concerned here with the proper application of terminology as applied
to basic names of cells and individual changes tbat occur in cell development, as
well as alterations in morphology. When this is done, terms to express increases
or decreases can be easily formulated, and it becomes easier for all hematologists
to "speak the same language," and this is equally important as "seeing with the
same eye."
Within recent years Osgood has proposed a new set of terms for the various
types of mature and immature blood cells. These terms, however, present. such
a radical departure from those in wide use that they would appear practically
impossible of adoption. Furthermore, as pointed out earlier in. this chapter, it is
desirable that the entire field of terminology in blood diseases be overhauled and
this can best be accomplished by a concerted effort by a considerable number of
nematologists. It is hoped that in the near future someone wili initiate such a
movement. I ~is)l to emphasize that· the. changes. in terminology. suggested in
this chapter are made with the hope that they will stimulate more. thought on the
part of those who propose· new hematologic terms and not with the idea that
such changes suggested should receive widespread acceptance.
 CHAPTER 2
DEFINITIONS OF HEMATOLOGIC TERMS
Absolute Increase of Cells
This term usually refers to au increase of one of the leukocyte cell types.
If the total number of cells in anyone type is above normal this is an 'ab-
solute increase, but if the total number of a cell type is at the normal level
and another type of leukocyte is decreased, there would then be a relative
increase of the former. With a count of 10,000 cells and 70 per cent neutro-
phils, the absolute number would be 7,000 which would be the absolute
increase of neutrophils. Thus, there is no relative increase but an absolute
increase of neutrophils.
Absolute Number of Neutrophils
The absolute number of any variety: of leukocytes is determined by multi-
plyi~g the total number of leukocytes by the percentage of the variety of
cell in question. For example: if the toial leukocyte count is 2,000 and
the percentage of neutrophils is 20, the absolute number of neutrophils would
be 400. With a total leukocyte count of r ,000 and 40 per cent neutrophils,
the absolute number of neutrophils would still be 400. In this latter case
there is a relative increase of neutrophils but not an absolute increas~. In
all cases of neutropenia, the ahsolute numbers of the various types of cells
as well as their percentages should be recorded.
Achroiocythemia
A state of deficiency of hemoglobin of the red blood cells.
Achromacyte (Achroacyte) (Achromatocyte)
A degenerated erythrocyte without color. A shadow corpuscle.
Achromasia
Absence of the ordinary staining reaction in a cell.
Achromatophil
A cell that shows no affinity for stains.
Achromatophilia
A condition of being refractory to staining processes.
Achromia
A term incorrectly used, meaning a condition in which the red cells are
without hemoglobin. Such a condition would be incompatible with life.
Hypochromia should be used.
Achromic Erythrocytes
Erythrocytes characterized by a reduction in, or absence of, the coloring
matter (hemoglobin).
Acidocyte (Eosinophile) (Acidophile) (Eosinocyte)
Susceptible of imbibing acid stains. Having an affinity for acid stains.
Suggested as being more appropriate than eosinophile.
Many definitions included in this list are taken from standard medical dictionaries, including
Gould's Medlcal Dictionary, 211d Ed., and Stedman's MediC.ll Dlctionary, IIth Ed.
IS
16 HEMATOLOGIC TERMINOLOGY

Addisonian Anemia
See pernicious anemia (Chap. 22).
Agonal Leukocytosis
An increase in the total number of leukocytes in the blood stream just pre-
ceding death. This may be due to an actual increase in anyone of the types
of leukocytes,-bllt usually all types are involved.
Agranulocyte
A non-granular leukocyte or one without cytoplasmic granulation. A_bad term.
Agranulocytosis (Malignant Leukopenia) (Essential Granl'lopenia)
A disease characterized by a deficiency of polymorphonuclear, leukocytes in
the blood stream. Practically all granular leukocytes disappear from the
blood. The lymphocytes and monocytes also are decreased in number.
Agranuloplastic
Capable of forming non-granular cells.
Aletocyte
A wandering cell of uncertain origin.
Aleukemia (Aleuchaemia)
A leukemic state characterized by leukopenia. A morbid condition in which
the histological changes in the blood forming organs are characteristic of
a leukosis (myelosis or lymphadenosis) but there is no leukocytic increase
in the blood, although there is the typical qualitative alteration of the blood-
picture.
Aleukemic Leukemia (Aleukemic Myelosis) (Aleukemic Lymphadenosis)
(Hypocytic Leukemia)
In the usual form of leukemia there is an enormous increase of leukocytes in
the peripheral circulation. The aleukemic form probably has the same under-
lying pathology but for some reason the leukocytes do not appear in the
peripheral circulation and the total leukocyte count is normal or decreased,
occasionally greatly' decreased. The leukocytes are deposited in the internal
organs in great numbers as in the usual form of leukemia. This form of leu-
kemia is said to be caused by the retention of all pathologic leukocytes in the
organs. Basically it does not differ from high cell count leukemia.
Aleukia
Absence or marked decrease of white cells and blood platelets. A. Hemor-
rhagica. Aplastic anemia. Synonym: myelophthisis; commonly used, but
not satisfactory because all blood-forming tissues atrophy, not just bone
marrow.
Aleukocytic
Marked by an absence of leukocytosis or by leukopenia.
Aleukocytosis
Leukopenia, a diminution, relative or absolute, in the number of white blood
cells. Not a good term in view of the term leukocytosis being used to de-
scribe an increase of leukocytes.
Aiexocyte
A leukocyte which is thought to secrete alexin or complement. Patients with
absent granulocytes do not have decreased amounts of complement.
 DEFINITIONS OF HEMATOLOGIC TERMS

Ameboid Motility
Motility resembling that of an ameba, i.e., moving about by extending from
its circumference processes of protoplasm called pseudopodia.
Amphileukemic
A leukemic state in which the leukocytosis corresponds to the organ changes.
Amphophil (Amphophile) (Amphochromophil) (Amphochromatophil)
(Amphocyte)
Having equal affinity for acid and basic dyes and a eel! which stains readily
with e1ther. The neutrophil of the rabbit. .
Amyelonic
Without bone marrow. Without participation of bone marrow, therefore
purely lymphollc or perhaps monocytic.
Anemia
A condition in which the blood is reduced in amount (oligemia) or is deficient
in red blood cells (oligocythemia) or in hemoglobin (oligochromemia), mani-
fested clinically by pallor, shortness of breath .and palpitation. Deficiency
of blood as a whole, or deficiency of the number of red corpuscles or of the
hemoglobin. Reduction in the number of red corpuscles. A clinical term.
Anerythrocyte
A non·nucleated red cell without hemoglobin. Therefore, one with marked
central pallor.
Anerythroplasia .
A condition in whi~h there is no formation of red blood cells.
Anerythroregenerative
A condition in which regeneration of red blood cells does not take place.
Angina, Agranulocytic (Agranulocytosis)
Malignant leukopenia. See agranulocytosis.
Anisocyte
See anisocytosis.
Anisocytosis
A condition in which the red blood cells are not uniform in size. Abnormal
inequality in the size of the red blood corpuscles. As seen in permclOus
anemia .. Excessive variation in the size of red corpuscles, as seen in films of
blood.
Aplasia
Incomplete or defective development. A cessation of regeneration.
Aplastic
Marked by defective or arrested development. Having no tendency to
develop new tissue.
Aplastic Anemia
A rare and rapidly fatal anemia of obscure etiology which is apparently the
result of more or less complete failure of blood formation. The red bone
marrow is found at autopsy to have almost wholly disappeared, even from
the flat bones and the bodies of the vertebrae. The usual thing is for the
erythropoietic tissue to be involved first, then the granulopoietic, and finally
the thrombopoietic tissue.
18 HEMATOLOGIC TERMINOLOGY

Arneth Index
The arrangement of neutrophiles into classes according to their nuclear con-
figuration and number of nuclear lobes. There are five classes of neutro-
philes. The Arneth index is seldom used.
Ayerza's Disease
Polycythemia associated with stenosis of the pulmonary artery.
Azurophilia
The property of staining with the "azur" component of such stains as
Wright's and other Romanowsky stains.
Azurophilic Granules
Granules staining well with blue anilin dyes. These granules are regarded
by some authors as specific for the lymphocyte. With the Romanowsky
stains they appear as rounded, discrete, reddish purple granules, larger than
the granules of neutrophilic leukocytes. The number of these granules is
variable; usually 'five to ten appear in the cytoplasm of the lymphocytes,
especially large lymphocytes.
Band Forms
Neutrophilic granular leukocytes with indentation of the nucleus in the
early stage of nuclear lobulation. The stage before the segmented type.
Banti's Disease
A syndrome characterized by splenomegaly, hepatic cirrhosis, hemorrhages
from the upper gastrointestinal tract, and oftentimes a leukopenia of, variable
degree.
Basket Cen (Smudge Cell) (Degenerated Leukocyte)
A cell characterized by a network of fibrils irregular in shape, non-nucleated.
Said to be the old, degenerated leukocyte. (See Plate No. 10.)
Basocyte
See basophil.
Basoerythrocyte
A red blood cell showing the changes of basophilic degeneration including
basophilic stippling, punctate basophilia, Cabot ring bodies, Howell-Jolly
bodies, etc.
Basoerythrocytosis
As seen in chronic malaria and severe hypochronic anemia.
Basometachromophil
Staining a color different from that of the surrounding substance with basic
dyes.
Basophil (Basophile) (Basocyte)
Having an affinity for basic or nuclear stains. A cell, especially a wbite
blood cell, which stains reailily with hasic dyes. Also termed basophilic
leukocyte or mast cell. The granules of the cytoplasm stain blue black with
bask stains. The nucleus stains light blue and is lobulated. One of the
granulocytes.
Basophilia (Basophilic Granulation) (Diffuse Basophilia)
A condition in which basophils or mast cells are present in undue number.
A degenerative condition of the erythrocyte in which they present a number
 DEFINITIONS OF HEMATOLOGIC 'tii"MS 19
of minute granules taking a basic stain. Increase in the number of basophils
in the circulating blood. (GRANULAR DEGENERATION) (BASO-
PHILIC DEGENERATION) (PUNCTATE BASOPHILIA) (BASO-
PHILIC STIPPLING.) In reference to the myeloblasts and myelocytes it
refers to the blue staining Droperties of the cytoplasm of these 'cells. The
term would also indicate an accumulation of an unusual number of basophils
in the blood stream. Basoerythrocytosis, when seen in red cells.
Basoplasm
T1mt part of the cytoplasm whicb stains readily with basic dyes.
Biermer's Anemia
'See pernicioils anemia, definition, page 225.
Blood Crisis· '
Tne appearance of large intmDers of nucleated red cefls in the peripheraf
blood. Also accompanied by reticulocytosis. Seen in the "exhausted" min-
row, in pernicious anemia, alld in hemolytic' icterus.
Blood Dyscrasia
A diseased state of the blood. It usually refers to abnormal cellular com-
position of more or less permanent character.
Blood Islet
The primitive blood-forming tissue of tbe embryonic yolk sac.
Blood Platelet
See tbrombocyte.
Cabot Ring
A ring-like or figure-of-eight structure found in some red corpuscles in
severe anemias; probably a 'remnant of the nuclear membrane. A form of
basophilic degener'ative pro~esses.
Chemotactic Factor
An agent which exerts a chemical influence on certain living cells attracting or
repelling them, i.e., positive or negative chemotaxis, respectively. An illus-
tration is the accumulation of leukocytes at the site of inflammation owing
to the positively chemotactic influence of bacteria and their products or of
tissue products.
Chemotaxis
The phenomenon shown by certain living cells of moving toward (positive
c.) or away from (negative c.) certain other 'cells or substaoceS wbich exert
a chemical influence. -
Chloro-Leukemia
A'leukemic blood-picture, almost invariably of myeloid type, associated with
tumor growths of bright green calor.
Chloroma
Green infiltrations of tumor-like character associated with a leukemic bloodo
picture and a well marked tendel/c}' to il/vasiol/ of the orbital tissl!es bJ'
the leukotic cells. J
Chlorosis (The Green Sicknes~)
A severe anemia of puberty in girls, characterized by low hemoglobin, low
color index and iron deficiency. Seldom seen in recent years.
20 HEMATOLOGIC TERMINOLOGY

Color-Index
The ratio between the amount of hemoglobin and the number of red cor-
puscles. Obtained by dividing the per cent of red cells into the per ceut of
hemoglobin.
Corpuscle (Erythrocyte)
The non-nucleated derivative of any form of nucleated red cell.
Crenate
Notched; indented, scalloped; noting the outline of a shriveled red plood
cell.
Crenation
A mulberry-like appearance of the red corpuscles of the blood. The altera-
tion seen in red corpuscles when exposed to the actiou of hypertouic solution:
prickles are seen on the surface of the affected cells.
Crenocyte
A red cell with serrated, notched edges.
Crenocytosis (Crenation)
As,seen when red cells are subjected to the action of hypertonic salt sohltion.
Cytopoiesis
Formation of cells.
Degenerative Index
A term used to indicate the proportion of granulocytes that show toxic
granules in the cytoplasm.
Depressed Bone ]\farrow
Bone marrow with lowered vitality, lessened functional activity, and impaired
maturation. There may be a cessation of maturation. Bone marrow may
be depressed at any stage or level and remain indefinitely at that level.
Dohle Bodies
Cytoplasmic inc1usioJls found in neutrophilic leukocytes, often seen in scarla-
tina, bULalso in other infections.
Dorothy Reed Cells (Sternberg Cells)
The large, multi-nucleated, acidophilic giant celIs seen in Hodgkin's dHease.
Dualism
Referring to the concept that all blood cells have two origins; one lyn1pho-
cytic and one myeloid.
Endothelial Leukocyte (Endotheliocyte) (Monocyte) (Transitional)
They are the largest whit~ cells of the normal blood and are characterized
by variability of size; large, various shaped, eccentrically placed nuclei and
an abundant reticulated cytoplasm. The ,single nucleus may be lobulated,
deeply indented, horseshoe shaped, round or oval. The mononuclear9 and
transitionals are merely different torms or ages of the same cell. According
to Sabin, these cells are derived from the reticulo,endothelial system. (See
Origin of Blood Celis.)
Endotheliocyte (Endothelial LeUkocyte)
A cell arising from the reticulo-endothelial system.
Eosinoh1ast (Eosinophilic Myeloblast)
:'f)eJoblast that later develops into an,eosinophile.
 DEFINITIONS OF HEMATOLOGIC TERMS 21

Eosinocyte (Eosinophil)
See eosinophil.
Eosinopenia
The presence of eosinophil cells in abnormally small number in the periph-
eral blood.
Ehsinophil (Eosinophile) (Eosinocyte) (Eosinophilic Leukocyte)
A cell or other element, especially a leukocyte, which stains readily with
eosin. The granulocyte in which the cytoplasmic granules are large, stained
red, shiny and refractile.
Eosinophilia (Acidophilia) (Oxyphilia) (Acidocytosis)
A relative leukocytosis in which the main increase is in t!!e codnop::ils.
Erythremia (Polycythemia Vera)
Polycythemia rubra, a disease characterized by increase of the red blood
corpuscles with cyanosis (Vaquez's disease). Not simply erythrocytosis.
Erythroblast (Erythrocytoblast)
A nucleated red blood cell.
Erythroblastemia
The presence of nucleated red cells in the peripheral blood. To be dis-
tinguished from the disease, erythroblastosis.
Erythroblastosis
The presence in considerable number of erythroblasts in tbe blood .
• Erythroblastosis, Fetal
A condition of congenital general edema, enlargement of liver and spleen,
a large number of erythroblasts in the blood, liver and spleen.
Erythroca talysis
Excessive destruction of the red blood cells by phagocytosis;
Erythroclasis
Fragmentation and breaking up of the red blood. cells.
Erythroclastic
Relating to erythroclasis.
Erythrocyte
A red blood corpuscle. The mature hemoglobin carrying, non-nucleated cell
of the blood, including all varieties of shape, color and size.
Erythrocythemia (Polycythemia) (Erythremia)
Abnormal increase in the number of red blood cells.
Erythrocytoblast
See erythroblast.
Erythrocytolysis (Hemolysis)
Dissolution or destruction of the red blood corpuscles with escape of the
hemoglobin into the blood plasma.
Erythrocytometer (Hemocytometer)
An instrument for counting the red blood cells. Haden uses this term for an
instrument to measure red cell diameter.
Erythrocytopenia
See erythropenia.
 HEMATOLOGIC TllllMINOLOGY

Erythrocytopoiesis (Erythropoiesis)
The process of production of red blood corpuscles.
'Erythrocytorrhexis (Plasmorrhexis)
A partial erythrocytolysis, ,in which particles of protoplasm escape from the
cells which become crenated and deformed.
Erythrocytosis (Polycythemia) (Erythremia)
An increase in, the number of red blood corpuscles, usually marked by' more
or less cyanosis. It may be merely compensatory for lack of oxygen.
Erythrodegenerative
Relating to or marked by a degeneration of the red blood cells.
Erythrogenic
Producing red blood corpuscles.
Erythrogonium
The precursor of an erythrocyte. Sometimes used to designate the erythro-
poietic tissue in its entirety.
Erythroid Cells
Cells of the erythrocytic series.
Erythroleukosis (Leukanemia) (Panmyelosis)
A condition resembling "leukemia," in which the erythropoietic tissue is
affected in addition to the involvement of the leukopoietic one.
Erythron
A term used to designate all of the circulating red~ cells and the erythro-
poietic tissues from which they are, derived.
Erythroneocytosis
Presence in the peripheral circulation of regenerative forms of red bi()od
cells. The presence of regenerative forms of red blood corpuscles in the
circulating blood.
Erythropenia (Erythrocytopenia)
Deficiency in the number of red blood corpuscles.
Erythrophage
A phagocyte which englobes and destroys red blood corpuscles ..
Erythrophil (Erythrophile) (Fuchsinophile)
A cell or histologic element which'stains promptly with red dyes.
Erythropoiesis (Erythrocytopoiesis)
The production of erythrocytes.
Erythropoietic
Pertaining to the tissue which takes part in the formation of erythrocytes.
Normally, in the adult, this is located in the red ,bone marrow.
Erythropycnosis (Erythropyknosis)
Alteration of the red blood cells to the condition ,called "brassy bodies,"
under the influence of the malarial parasite. It, consists ,in Jhe development
of a brassy appearance of the blood cells, together' with distinct crenation.
Filament-Non-Filament Count
A differential count of the number of neutrophiles showing nuclear division
and those that do not show such division. A divided nucleus is one tllat
 DEFINITIONS OF HEMATOLOGIC TERMS 23

contains two or more lobes connected with slender strands of chromatic

material (filament).
Fixed Blood Film
A drop of blood spread thinly on a cover ,lip' or slide, dried quickly and
fixed either by chemicals or by heat. Stains which are dissolved in methyl
alcohol combine fixation with the staining process.
Fragilocyte
A red cell that is unus~ually fragile when subjected to a hypotonic salt solu'
tion; as seen in hemolytic icterus. A red cell of excessive fragility.
Fragilocytosis
, Increase in fragilocytes in the peripheral blood, as seen in hemolytic icterus.
Gaisboek's Disease
Polycythemia associated with hypertension.
Glandular Fever (Infectious Mononucleosis) (Benign Lymphadenosis)
A disease (or perhaps a variety of diseases) in which widespread glandular
enlargement is associated with a monocytic or lymphocytic blood picture witli
leukocytosis.
Globin
A proteid derived from hemoglobin.
Globular Valve (Color Index)
It is aJraction of which the numerator is the .percentage of hemoglobin and
the denominator the percentage of corpuscles.
Globulin
-A simple protein insoluble in water, but soluble in one·half to one per cent
solution of a neutral salt, and coagulable by heat. Varieties present in
milk, muscle and blood (hemoglobulin).
Gordon Test
A test designed to establish or rule out the diagnosis of Hodgkin's disease.
~erformed by injecting intracerebrally into rabbits the macerated sterile
biopsied glands from the patient. See section on technic.
Granuloblast
The mother cell of a granulocyte.
Granuloeyte
A white blood cell containing cytoplasmic granules, presumably having its
origin from the bone marrow. Includes neutrocytes, eosinocytes aud baso·
cytes.
Granulocytosis
The accumulation of an unusual number of granulocytes in the blood stream.
Granulopenia (Granulocytopenia)
A decrease of granulocytes in the blood stream.
Granulopoiesis
The production of granulocytes.
Granulopoietic
Pertaining to the tissue which takes part in the formation of granulocytes.
In the adult this is located in the red bone marrow of flat bones.
24 HEMATOLOGIC TERMINOLOGY

Gumprecht's Shadows (Shadow Cells) (Smudges) (Basket Cells)

The name applied to the crushed and deformed cells so often found in leu-
kemic lymphadenosis.
Hem-, Hema-, Hemato-, Haem-
Prefixes denoting blood.
Hemachrome
The coloring matter of the blood. Hemoglobin or hematin.
Hemachrosis
An intensified redness of the blood.
Hemacytometer (Hemocytometer) (Hematometer) (Hemometer)
An instrument for estimating the number. of corpuscles in'the blood.
Hemafacient (Hematopoietic)
An agent that increases the cellular output from blood forming tissue.
Hemagglutinin
An agent that causes agglutination or clumping of red blood corpuscles.
Hemanalysis
Analysis of the blood; a blood examination.
Hematoblast
The immature form of all blood cells.
Hematocrit
A centrifuge for separating the solid elements of the blood from the plasma.
Hematocyte
Includes all cellular elements of the blood: erythrocytes, leukocytes and
thrombocytes.
Hematocytoblast
Same as hematoblast.
Hematogenesis (Hematopoiesis)
The formation of blood.
Hematoglobin (Hematoglobulin) (Hemoglobin)
See hemoglobin.
Hematohistioblast
An indifferent polymorphous white blood cell of large size and irregular shape,
having a single round or oval nucleus, with a characteristic reticulated inter-
nal structure likened to that of a sponge; it has distinct nucleoli and baso-
philic cytoplasm in varying amount, with or without granules. Probably
a monocyte of reticulo-endothelial origin.
Hematologist
One who makes a special study of the blood and is skilled in the technic of
blood examinations and in th~ treatment of blood diseases.
Hematology (Hematopathology)
The branch of medicine wbich has to do with the blood in all its relations;
anatomy, physiology, pathology, semiology, and therapeutics. '
Hematophagocyte
A leukocyte which destroys the red blood cells.
Hematoplastic
Relating to blood formation.
 DEFINITIONS OF HE"'-ATOLOGIC TERMS

Hematopoiesis
The formation or production of blood.
Hematopoietic
Pertaining to the tissue which takes part in the formation of blood. Nor-
mally, in the adult, the red bone marrow, reticulo-endothelial sysiem and
lymphatic tissues comprise the hematopoietic tissue.
Hematopoietin
A hypothetical substance of the nature of a hormone, causing regeneration
of the red blood cells. Supposed to be elaborated in tbe stomach and stm eJ
in the liver.
Hemoblastosis
Pertaining to proliferative conditions of hematopoietic tissues in general.
Hemoclasia (Hemoclasis) (Hemolysis)
Lysis or breaking up of the red blood corpuscles. Destruction of the erythro-
cytes.
Hemoconia (Blood Dust) (Blood Motes)
Minute, colorless, highly refractive, spheroidal shaped bodies constantly
present in normal or pathological blood. Probably extruded cytoplasmic
granules from granulocytes. They apparently have no significance.
Hemoconiosis
A condition in which there is an increased amount of blood dust in the blood,
as in leukemias with high white cell counts.
Hemocyte (Haemocyte)
Same as hematocyte.
Hemocytoblast (Lymphoidocyte) (Primitive Blood Cell)
The primitive polyvalent stem-cell of the monophyletists.
Hemoeytogenesis
The production of blood corpuscles.
Hemocytopoiesis
See hematopoiesis.
Hemodiagnosis
Diagnosis by means of examination of the blood.
Hemoglobin (HemoglobuJin)
The coloring matter·of the red corpuscles, a conjugated protein, yielding a
simple protein and hematin on hydrolysis.
Hemoglobinemia
A condition in which the hemoglobin is dissolved out of the red corpuscles
and is held in solution in the serum. The blood is "lake" colored and is
said to be "laked."
Hemoglobiniferous
Cells yielding or carrying hemoglobin.
Hemoglobinometer
An instrument for estimating the amount of hemoglobin, indicated in- per
cent of the normal or grams per 100 cc. of blood.
Hemogram
A systematic description of the findings in a blood examination.
26 HEMATOLOGIC TERMINOLOGY

Hemohistioblast
Same as hematohistioblast.
Hemoleukocyte
A white blood corpuscle.
Hemolysis
Destruction of red blood cells by dissolution.
Hemonormoblast
Same as erythroblast.
Hemopathy
A disease of the blood.
Hemopoiesis
See hematopoiesis.
Hemorrhagic Diathesis
Name for the syndrome showing tendency to spontaneous hemorrhages.
Hemosiderosis '
Infiltration of tissues with iron pigment, derived from hemoglobin.
Histiocyte" ,
See reticulo-endothelial cells.
Howell-Jolly Bodies
Small basophilic particles, probably nuclear remnants, sometimes found in-
side red corpuscles. '
Hyperchromemia
Presence of high color index of the blood because of an increase in the aver-
age amount of hemoglobin per corpuscie.
Hyperchromia
Referring to that state in which the red blood cells contain more than their
normal amount of hemoglobin. The color index is above one.
Hypercythemia
The presence of an excessive number of red blood corpuscles.
Hyperneocytosis (Hyperskeocytosis)
The presence of many immature forms, that is, with a deviation to the left,
or a "shift to the left."
Hyperorthocytosis
Leukocytosis in which the relative percentages of the different forms ar,e
normal and no immature forms are present.
Hyperplasia
The abnormal mUltiplication or increase in number of tissue cells. In hyper-
plasia of the hematopoietic, tissue, instead of the normal primary blood cells,
there may be less developed earlier stages with a great increase in their
numhers.
Hyperplastic , '
Hyperplastic tissue is tissue that shows an abnormal multiplication of the
cells.
Hyperskeocytosis
See hyperneocytosis.
 DEFINITIONS OF HEMATOLOGIC TERMS

Hypochromia (Hypochromemia)
Referring to that condition in which most of the red blood .cells cOlt .
decreased hemoglobin content. On a stained smearrthis is seen.as a tam a
pallor in the cell. The color index is below one. ~ntral
Hypocytosis (Cytopenia) (Oligocythemia) (Hematopenia)
Poverty of the blood in cellular elements. Diminution in' the nu,"" I
blood corpuscles. oer of
Hypoeosinophilia
See eosinopenia.
Infectious Mononueleosis
A dis!,ase with symptoms resembling those_ of glandular fever and. ~
terized by increase in the monocytes or/and lymphocytes of the blood. harac-
Immature Granulocyte
See immature neutrophil. -The same cell as described
phil except that it may be neutrophilic, eosinophilic
Immatnre Neutrophil
A young neutrophil. The neutrophilic metamyelocyte which
showing beginning constriction and slight ameboid movement.
runner of the polymorphonuclear neutrophil (neutrocyte).
Juvenile Form (Schilling) .
The same as a metamyelocyte.
Karyorrhexis
Breaking of nuclear chromatin into fragments inside the cell.
Large Hyaline Leukocyte
See monocyte.
Length of Survival of Blood Cells
Tne granular leUkocytes are said· to survive irom three to nve .daYs l,
blood stream; the erythrocytes survive approximately seventy days !n the
blood stream. Old concept .thirty days. .Length of survival of thrombin the
and lymphocytes has not been determined. lcytes
Leukanemia
Leukemia associated with a rapid fall in bemoglobin percentage and (
number of red cells; fever, hemorrhages, increasing pallor, -and progr'n ?,e
muscular weakness. eSSlve
Leukemia (Leucemia) (Leu~hemta' lLeUKocytnemia) (Myelosis)
(Lympbadenosis )
A disease of the blood marked by persistent leukocytosis, associated
changes in the spleen and bone marrow.or·the lymphoid system: with
Leukemoid Reaction
A blood picture resembling that of leukemia.
-Leukoblast (Myeloblast) (Lymphoblast) (Monoblast)
The stem cell 'of all leukocvtes.
Leukoblastosis
Any type of leukocytic proliferation of leukemic ,character.
Leukocidin
A substance destructive to leukocytes. Some bacteria produce thi; J
cidin; for example, streptococci and staphylococci. [euko-
 HEMATOLOGIC TERMINOLOGY

Leukocyte
A white blood corpuscle.
Leukocyte-Endothelial,
See monocyte.
Leukocythemia
See leukemia.
Leukocytogenesis
The formation of leukocytes.
Leukocytoid
Resembling a leukocyte.
Leukocytomctcr (Hemocytometer)
A device for estimating the number of leukocytes.
Leukocytopenia (Leukopenia)
A condition in which the number of leukocytes in the circulating blood is
reduced below the normal.
LeUkocytopoiesis
Tbe process of production of leukocytes.
'Leukocytosis
Increase in the number of circulating leukocytes.
Leukon
A term refercing to all circulating leukocytes and the leUkopoietic tissues
from which they are derived. Aoalogous to the erythron of red cells.
Leukopenia (Leukocytopenia)
Deficiency in the number of circulating leukocytes:
Leukopoiesis
The formation or production of leUkocytes.
LeUkopoietic
Pertaining to the tissue which takes part in the formation of leukocytes.
The red bone marrow, reticulo-endothelial tissue and the lymphatic tissue.
Leukosis
Abnormal proliferation of leukopoietic tissues.
Leukotoxin
A cytotoxin destructive to the leukocytes. Any substance that has a toxic
effect on leukocytes. Some normal sera contain leukotoxin for normal leu-
kocytes of other patients even, though the. bloods are of the same type.
(Experiments of Doan.)
J,evels of Bone Marrow
A comparison of the levels or layers of the bone marrow with the epithelial
layer of the skin will give a clear picture of the meaning. The epithelial
layer of the skin is made up of four layers (or levels) from within outward,
as follows: (1) The stratum mucosum (malpighian or basal layer) which has
very active regenerating cells that are constantly replacing worn out cells,
this layer may be compared to level I of the bone marrow which has the
same function; (2) the stratum granulosum, consisting of flattened, glandular,
more matured cells; this may be compared to level II of the bone marrow;
(3) the stratum Iucidum, the layer next to or succeeding the functioning layer,
 DEFINITIONS OF HEMATOLOGIC TERMS

may be compared to level III of the bone marrow; the stratum corneum (the
horny layer), consisting of layers of flattened horny cells, the function of
which is protective, may be compared to the bone marrow cells in the blood
stream, the function of which is to protect the body. (Beck.)
Lymphadenosis (Lymphatic Leukemia)
The tissue proliferation underlying the leukemic process.
Lymphatic Leukemia
A fatal disease characterized by proliferation of the lymphatic tissues with
a marked increase in the number of lymphocytes in the peripheral blood,
together with an enlargement of the lymphoid tissue of the spleen, lymphatic
glands and bone marrow. Lymphoblasts may appear in the blood stream.
Lymphatic Reaction
See glandular feveL
Lymphoblast
A lymphocyte in its germinative stage; a developing lymphocyte. When
fully developed it becomes a lymphocyte.
Lymphoblastosis
The presence of Iymphoblasts in the peripheral blood. Acute lymphatic
leukemia.
Lymphocyte
A rounded mononuclear cell from the lymphoid tissues; may be either large
or small. The large form is probably the immature variety and the small
form the adult cell. Occasionally there will be azurophilic granuies in the
cytoplasm of lymphocytes.
Lymphocythemia (Lymphocytosis)
An excess of lymphocytes in the blood.
Lymphocytoblast (Lymphoblast)
The mother cell of a lymphocyte.
Lymphocytoma
A tumor-like growth composed of lymphocytes.
Lymphocytopenia
A decrease below the normal number of lymphocytes in the peripheral blood.
Lymphocytopoiesis
The process of production of lymphocytes.
LymphocytosiS
Increase in the number of lymphocytes in the blood.
Lymphoidocyte
See hemocytoblast.
Lympholeukocyte
A lymphocyte.
Lymphopenia (Lymphocytopenia)
A decrease in the number of lymphocytes in the blood stream.
Lymphopoiesis
The formation of lymphocytes.
30 HEMATOLOGIC TERMINOLOGY

Macroblast
A large erythroblast. It differs from a megaloblast, which is the youngest
recognizable red blood eeli.
Macrocyte
A large blood cell of any kind. A red blood cell with a diameter larger than
normal.
Macrocythemia
The presence of macrocytes ~in the blood.
Macrocytic ,Anemia,
Anemia marked by the presence of macrocytes (giant red blood corpuscles)
in the blood stream.
Macrocytosis (Macrocythemia)
The presence of macrocytes (large red cells) in the blood.
Macro-erythroblast
1\ large erythroblast.
Macroleukoblast
A large leukoblast.
Macromonocyte
An eXl:e"ively large monocyte.
Macrlfnormoblast
Usually defined as a large I)ormoblast. A large, incompletely hemoglobin-
iferous, nucleated red cell with "cart-wheeF' nucleus.
Macrophage
The large phagocytic cells. Usually the endothelial leukocytes are termed
the large phagocytic cells. They are cells which phagocytose foreign mate-
rial, i.e., they envelop and destroy it.
Macropolycyte
Cooke's name for abnormally large neutrophilic 'leukocytes.
Macropromyclocyte
A large promyelocyte.
Mast Cell (Basophile) (Basocyte)
See basophil.
Maturation
The stage or process of becoming mature, Le" the process of ripening. In
biology, a process of cell division during which the number of chromosomes
in the germ cells is reduced to one-half th~ numher characteristic of the
species.
Maturation Factor
A substance which will cause cells to rij)en and come to maturity.
l\lature Neutrophil
A polymorphonuclear neutrophil with two or more distinct lobes.
Medulla .
Marrow. Any soft center structure of a part: I, the bone marrow; ~, the
spinal cord; 3, the medulla oblongata. Aoything resembling, marrow in
structure.
 DEFINITIONS OF HEMATOLOGIC TERMS JI
Megakaryoblast (Thromboblast) (Thrombocytoblast)
The precursor of a megakaryocyte. A cell with a large nucleus, a~ the
giant cells of the bone' marrow Which are the mother cells of the l,lood'
platelets. Blood platelets are called thrombocyt~s. According to W tight,
platelets are detached portions of the cytoplasm of the megakaryocytes.
Megaloblast
A large nucleated red blood corpuscle with a "cart-whee!" and reticular
nucleus when stained. To be distinguished from macroblast.
Megalocyte
An abnormally large red blood corpuscle; a pathological red cell.
Megalocytosis
The presence o( large numbers of megalocytes in the blood.
lIIegalokaryocyte
Same as megakaryocyte, the precursor of blood platelets. 'See page 93.
Meniscocyte (Sickle Cell)
A sickle or crescent shaped red blood cell as seen in sickle cell anemia.
Meniscocytosis (Sickle Cell Anemia)
The presence of meniscocytes (sickle cells) in'the'blood.
IIIctachromaphil (The ccII); Metachromasia (The condition)
Not staining true with a given dye.
Metamyelocyte
A transitional,form of myelocyte intermediate between the mature myel~cyte
(myclocyte C of Sabin) and thc two-lobed granular leukocyte. Also Called
immature granulocyte' and by Schilling a juvenile form.
Microblast
A small nucleated red blood COt;Duscle.
Microcyte
A red blood, cell with a diameter smaller than normal.
Microcythemia (Microcytosis)
The presence in the blood of many microcytes,
Microcytic Anemia
Anemia marked by the presente of'microcytes in the blood stream.
Microcytosis
See microcythemia.
Micromyeloblast
A small myeloblast; often the predominating cell in myeloblastic leukemia,
Micromyeloblastic Leukemia
Leukemia with the micromyeloblast the predominating cell in the blood.
Microspherocytosis
A state of blood characterized by excessive numbers of red cells that are
smaller than normal (microcytes) and also more globular than nOrmal
(spherocytes). Seen in hemolytic icterus. See that section.
Monoblast
The parent cell of the monocyte,
Monochromatic (Monochromic) (Monochromatism)
0f a single color.
32 HEMATOLOGIC TERMINOLOGY

Monochromatophil (Monochromophil)
A cell that takes only one stain.
Monocyte (Large Mononuclear) (Endotheliocyte) (Transitional)
(Endothelial Leukocyte) (Large Hyaline Leukocyte)
A large mononuclear cell with pale blue cytoplasm containing a fme dust· like
granulation with a large irregular nucleus. There is much controvprsy
concerning its origin. See section on "Development of Blood Cells." •
Monocytic Angina
That clinical phase of glandular fever showing evidence of infection in the
buccal cavity.
Monocytopenia (Monopenia)
Diminution in the number of monocytes in the blood.
Monocytosis (Mononucleosis)
Increase in the number of monocytes in the peripheral blood.
Mononuclear
Any cell having. a single nucleus. Usually refers to the endothelial mono·
cyte.
Mononucleosis (Monocytosis)
See monocytosis.
Monophyletism
The belief that all blood cells arise from a single ancestor, of multi'potential
capacity. Also called Unitarianism. Those who believe it are "Unitarians."
Motile Leukocyte
A leukocyte having the power of spontaneous motion, ameboid motilitYJ i.e,
moving by finger-like extensions of its substance. This is seen principally
in the mature granular leUkocytes. The eosinophils are the most active,
the neutrophils next and the basophils show the least activity of the three.
Very feeble motility has been ascribed to the endothelial leukocytes.
Myelemia (Myelocytosis) (Myeloid Leukemia) (Myelocythemia)
Strictly, the presence of myelocytes in the blood. See myelogenous leukemia.
Myeloblast
One of the large mononuclear nongranular cells of the bone marrow which
develop into myelocytes. The nucleus contains from one to three nucleoli.
The cytoplasm stains dark blue, Le., is very basophilic. Mitochondria are
present in the cytoplasm.
Myeloblastemia
The presence of myeloblasts in the peripheral blood.
Myeloblastic Leukemia
A leukemic process with myeloblasts predominating in the blood.
Myeloblastoma
A tumor-like mass of myeloblasts. Seen in acute myeloblastic leukemia or
acute leukosis.
Myeloblastosis
A disease (myeloblastic leukemia) characterized by myeloblasts in the
circulating blood, their excessive proliferation in the bone marrow and de-
position in the various fixed tissues.
 DEFINITIONS OF HEMATOLOGIC TERMS 33
Myelocyte
The parent cell of granulocytes found in bone marrow. From the myelocytes
are developed the granular leukocytes of the blood. They are not normally
found in the peripheral circulation.
Myelocytes A, Band C (Sabin, Austrian, Cunningham and Doan)
To provide means of statistically expressing the severity of the bone marrow
activity, it has been suggested that the myelocyte period be arbitrarily
divided into three stages; myelocyte A (youngest) contains ten or less
specific granules by actual count; myelocyte B (intermediate) one-half of
the cytoplasm is filled with specific granules, mitochondria and basophilic
material being plentiful; myelocyte C (oldest) just before the nucleus
elongates preparatory to formation of two lobes and before any evidence
of motility is apparent, but with cytoplasm filled with granules. A still
further "shift to the left," and its degree, may be ascertained by partitioning
the myelocytes according to this cytoplasmic criteria, the study of the
;"hole cycle thus providing data upon which to estimate quite accurately
the state of myeloid activity in the bone marrow.
Myelocythemia
The presence in the blood of large numbers of myelocytes. Sec myclemia.
Myelocytic Crisis
A temporary but marked and sudden increase in the blood stream of cells of
the myelocytic series. A shower of myelocytes of all ages being thrown into
the blood stream.
Myelocytoma
A tumor-like mass, composed of myelocytes.
Myelocytosis (Myelemia) (Myelocythemia)
"The presence of myelocytes in abnormal numher in the blood. Tbe presence
of an excess of myelocytes in the blood.
lIfyeloid
Relating or pertaining to cells of the myelogenous series.
Myeloid Hyperplasia
Marked increase in tbe cells of the myeloid tissue. Undue or excessive
growth of the myeloid tissue.
lIIyeloid Metaplasia
The appearance of myeloid centers of growth in sites other than the uone
marrow.
lIlyeloid Tissue
The portion of red bone marrow devoted to the production of granular cells.
Myelogenetic (Myelogenic) (Myelogenous)
Originating in the bone marrow.
Myelogenous Leukemia (Myelosis) (Myelocythemia) (Myeloid Leukemia)
Also called medullary or myeJogenic'leukemia. A fatal disease with a marked
increase in the number of mature granulocytes, myelocytes and myeloblasts
in the peripheral blood, together with a great deposition of these cells in the
spleen, liver and other internal organs and marked proliferation in the bone
34 HEMATOLOGIC TERMINOLOGY

marrow. Myeloid foci may be established in the liver and other tissues
(extramedullary foci).
".jelolymphocyte
A small lymphocyte arising 'in and formed in the bone marrow.
Myeloma
A tumor-like mass composed of cells similar to those of marrow hematopoietic
tissue. I1Iultiple 1\1.. A diseas~ characterized by many such tumor-like
masses, particularly in bony structures.
Myelomatosis (Kahler's Disease)
The appearance of numerous myelomas in the same' person, often associated
with the excretion of Bence Jones' proteose in the urine.
Myelomonocyte
A monocyte arisi~g in the bone marrow.
Myelopathy
. Any disease of the spinal cord or myeloid tissue.
Myelophthisis (Aplastic Anemia) (Panmye10phthisis)
Atrophy of all cellular elements of the bone marrow. See aplastic anemia.
Myelopoiesis
Formation of blood cells in bone marrow_
Myelosis (Myelocythemia)
See myelogenous leukemia.
Neutrocyte (Neutrophil) (Polymorphonuclear) (Polymorph)
(Polynuclear) (Polymorphonuclear Leukocyte)
A white blood cell 01 bone marrow origin having a diffuse neutrophilic,
cytoplasmic granulation with an irregular nucleus 1"hich may be segmented
into two or more lobes.
Neutropenia (Neutrocytopenia)
Deficiency in the number of neutrophilic granulocytes in the. blood stream.
Neutrophil (Neutrocyte)
See neutrocyte.
Nentrophilia (Neutrocytosis)
Increase in the number of neutrophils in the blood.
Neutrophilic Granulocyte
A leukocyte containing neutrophilic (neutral stained) granules in its cyto-
plasm. (Neutrophilic leukocyte.)
Non-Granular Leukocyte
This term refers to the lymphocytes, endothelial leukocytes (monocytes
and transitionals), plasma cells and Tiirk's ,irritation forms. They are leuko-
cytes without granules in their cytoplasm.,
Non-l\Iotile Leukocyte .
A leukocyte which does not. have ameboid movement.
Normoblast
The nucleated precursor of the erythrocyte.
Normochromia
Referring to the normal hemoglobin content of red cells.
 DEFINITIONS OF HAMATOLOGIC, TERMS

Normocyte
A red blood cell of normal size, from seven to eight micra in diameter.
Normocytosis
Referring to the normal state of red. cells.
Oligemia
A state in which the total quantity of blood is diminished.
Oligochromemia
Deficiency of hemoglobin in the blood.
Oligocythemia,
Deficiency' of all blood cellular elements.
Oligocrythrocythemia (Erythrocytopenia) (Erythropcnia)
Deficiency of red cells in the blood.
Oligoleukocythemia (Leukopenia)
A deficiency of white corpuscles in the blood.
Orthocytosis
Condition of the blood in which only: normal blood cells are present.
Ovalocyte
An oval shaped red blood cell.
Ovalocytosis
• As seen in some lower animals, 'particularly the camel. Occasionally heredi-
tary in man.
Oxydase
See peroxidase.
Oxyphil
Acidophil, eosinophil: having an'affinity for acid dyes, such as .eosin. His-
tologic elements that attract acid dyes.
PanmyelQphthisis (Myelophthisis)
See myelophthisis.
PanmYelosis
Proliferation of all cellular elements of the bone marrow.
Pcciloeyte
See poikilocyte.
Peripheral Granulopenia
Decrease of granular leukocytes (eosinophilic, basophilic and neutroPhilic)
in the blood stream. This .condition may occur with a normal supply in the
granulopoietic tissue because of interference of- "deliveryH into the blood
stream.
Peripheral Neutropenia
Decrease of neutrophils in the circulating blood. This condition maY'bcc_ur
with a normal supply in the granulopoietic tissue.
Peroxidase Reaction
Many cells possess an oxidizing ferment, which may be disclosed b' the
formation synthetically of naphthol blue when such cells .are treatel first
with a-naphthol and then with dimethyl-phenylendiamin., ,The method is
particularly useful for differentiating myelocytes from cells of theJymphocyte
series; the myeloid cells give a positive reaction, while the lymphocytes !dve a
 HEMATOLOGIC TERMINOLOGY

negative reaction. The endothelialleukocytes are variable in their reaction to

this stain. Some give a very faintly positive reaction. This has caused some
authors to feel that these cells originate in the bone marrow. Probably they
give a positive reaction if they have phagocytosed the debris of myeloid cells.
Pessary Corpuscle
An elongated red corpuscle in which lhe hemoglobin is collecled mainly at
the edge.
Plasma Cen
A cell thought to arise from lymphocytes and often seen in subacute and
pathologic processes. It is characterized by cytoplasm which stains a deep
blue and a round nucleus at the periphery of the cytoplasm, i.e., is eccen-
trically placed. There may be one or two nucleoli in the nucleus. The
chromatin has a "wheel-like" appearance. They are rare in the circulating
blood and bave no diagnostic significance except in the rare plasIl1a cell
leukemias.
Platelet (Hayem's Hematoblast) (Zimmermann's Corpuscle) (Bizz(jzero's
Corpuscles) (Neumann's Corpuscles) (Charcot's Crystals)
A blood plate; a round or oval disc. See thrombocyte. .
Poikiloblast
A nucleated red corpuscle of irregular shape.
Poikilocyte (Pecilocyte)
A red blood cell with an atypical shape. It may be elongated, tailed, dumb-
bell shaped, tennis racquet shaped or constricted, etc.
Poikilocytosis (Pecilocytosis) (Poikilocythemia)
The presence in the blood of red blood corpuscles of irregular shape.
Poikilothrombocyte
A blood platelet of abnormal shape.
Polychromasia (Polychromatophilia) (Polychromatosis) (PolychrOlnatia)
(Polychromophilia) (Polychromatocytosis)
A tendency of certain cells, such as the red blood cells in pernicious anemia,
to stain with both acid and basic dyes. The presence in the blood Of poly-
chromatophils (polychromatocytes).
Polychromatocyte (Polychromatophil)
A red blood cell showing variation from the' normal staining reactior, in so
far as color change is concerned. The cell may have a blue ting', since
it stains easily with acid or basic dyes.
Polychromatocytosis
See polychromasia.
Polychromatophile (Polychromophil)
See polychrumatocyte.
Polychromemia
An increase in the amount of hemoglobin in the blood.
Polycythemia (Hyperglobulism) (Erythrocytosis) (Erythremia)
(Polyglobulia) (Hyperglobulia)
A state of the blood characterized by an excess of corpuscles, usually the
red corpuscles.
 DEFINITIONS OF HEMATOLOGIC TERMS

Polycythemia Hypertonica
, See Gaisbock's disease.
Polyeytosis
A condition in which the red and white hlood corpuscles are increased in
numbe'r, and the plasma is reduced in volume.
Polyemia
Abnormal increase of the total quantity of the blood.
Polymorph (Polymorphonuclear Leukocyte) (Polymorphonuclear)
(Polynuclear) (Neutrocyte)
See neutrocyte.
Polynucleosis (Neutrophilia) (Neutrocytosis) (Neutrophilic Leukocytosis)
The presence of numbers of polynuclear cells in the peripheral blood.
Polyphyletism
The hematologic theory that assumes that there are several stem cells.
Premyelocyte
The precursor of the myelocyte.
Proerythroblast
The precursor of the erythroblast. An immature nucleated red cell.
Proleukocyte
, The parent cell of a leukocyte.
Promegaloblast
Precursor of a megaloblast.
Promyelocyte
The stage of development just before the myelocyte.
Pseudoleukemia (Pseudoleukoeythemia)
A state of the blood resembling leukemia.
Punctate Basophilia (Stippling)
See basophilia.
Pyknosis
Condensation of nuclear chromatin. A stage of cell death.
Relative Increase in Number of Cens
It refers to an increase in percentage of cells only.
Reticulation
The presence or formation of a reticulum or network, such as is seen in red
cells during active blood regeneration.
Reticulocyte (Reticulated Erythrocyte)
A red blood cell (erythrocyte) showing a reticulum or network under vital
staining. With brilliant cresyl blue this reticulum stains dark blue. Oc-
casionally instead of the reticulum there will only be a number of discrete
coarse granules scattered irregularly through the cell. It is only the "cry
young erythrocytes that show thi~ reticulum. An increase ·in these cells is
an index of the activity of blood regeneration.
Reticulocytosis
Condition in which the normal percentage of reticulocytes in the blood is
exceeded.
 HEMATOLOGIC TERMINOLOGY

Reticulo-Endothelial Cell (Histiocyte)

The reticulo-endothelial tissues include Kupffer's cells in the liver, the ,pleen,
hemolymph nodes and bone marrow. By reticulo-endothelial system' we
mean 'all of the cells of the body that are' "vital stainable" with carmine,
i.e., especially the body cells which lie between the endothelial cells of the
liver capillaries and related elements in the splenic pulp, in the bone mar-
row, in the glands and in the connective tissue. Some believe that the endo-
thelialleukocytes are derived from these histiocytes.
Reticulo-Endothelial System
The cells that function as a macrophage system.
Reticulo-Endotheliosis
A progressive and excessive proliferation of reticulo-endothelial cells.
ReticulosLq
See reticulocytosis.
Rhythmic Delivery of Granulocytes
Relating to the periodicity or paroxysmal appearance of these cells in the
blood stream due to their rhythmic delivery from the bone ,marrow.
Rouleaux Formation
The stacked arrangement of red blood corpuscles in shed blood, forming
figures resembling stacks of coins.
Schilling Index
A method of classifying neutrocytes, based on their age.
Schilling's Juvenile Forms
See metamyelocyte.
Senile Leukocyte Stage
In reference to the neutrophils this means the time at which they have
reached their maximum lobulation, have completed their functions, and are
ready for physiologic degeneration and ultimate destruction.
Shift to the Left
(In reference to the blood picture.) We must visualize the bone marrow
on the left with its immature myeloid cells and the circulating blood on
the right with its mature neutrophils. When the percentage of the imI1lature
cells increases in the circulating blood we say that the blood picture is
shifting to the left, i.e., shifting towards the bone marrow types. We
thus estimate the state of myeloid activity of the bone marrow. In a
regenerative shift to the left the bone marrow is responding to the need
of a rapid increase of cells. There is a high total white cell count vyith
myeiocytes ani:! juvenile forms appearing in the blood stream and there
is an increase in stab cells. In some cases there will be an occasional
myeloblast.
Sickle Cell Anemia
A familial disease affecting only Negroes, characterized by blood ,showing
the presence of large numbers of sickle shaped corpuscles. (Meniscocytes.)
Sicklemia
See sickle cell anemia.
 DEFINITIONS OF HEMATOLOGIC TERMS 39
Spherocyte
A red blood cell with a tendency toward a globular or rounded form. The
normal red cell is a bi-concave disc. A spherocyte would be bi-convex and
conceivably could be completely rounded.
Spherocytic Anemia (Globe cell anemia)
An anemia characterized by the presence of large numbers of red cells
that are more globular than normal (bi-convex in shape). As in hemolytic
icterus.
Spherocytosis
The presence in the blood of numerous spherocytes.
Stab o~ Rod Nuclear Forms
They are neutrophils in which the nucleus does not become segmented but
instead appears sausage shaped. This nucleus becomes elongated and as-
sumes different forms, S, T, V, W, U, etc., to accommodate itself to the size
of the ccli. They arc usually without ameboid movement. Four per cent
is considered normal in the circulating blood.
Stippling
See basophilia.
Supravital Stain _
A non-toxic stain which may be used to stain cells while they are still alive
so that their vital processes may be studied. A microslide incubator or
warmed stage is used in this technic. The cells remain alive from one to
three or four hours with this method.
Thromboblast
The precursor of the thrombocyte (blood platelet). Probably the same as
megakaryocyte.
Thrombocyte (Blood Platelet)
Circular or oval disc, from two to three micra in diameter, forming one of
the constituents of the blood. They are believed to assist -in producing
coagulation. They are derived from the cytoplasm of the megakaryocyte
in the bone marrow and several types have been described, classified and
named.
Thrombocytopenia (Thrombopenia)
Decrease in the number of blood platelets below normal.
Thrombocytosis
Increase in the number of platelets.
Thrombon
A term to designate all of the circulating thrombocytes (blood platelets)
and the tissue from which they arise (thrombohlasts or megakaryocytes).
Analogous to erythron and leukon of the red cells and white cells, respec-
tively.
Thrombopenia (Thromhocytopenia)
See thrombocytopenia.
Thrombopoiesis
Pertaining to the production of thrombocytes.
 HEMATOLOGIC TERMINOLOGY

Thrombopoietic
Pertaining to the tissue )vhkh takes part in the formation of throlllbocytes
or blood platelets. Normally in the adult this is located in the red bone
marrow.
Transitional Cell (Endothelial Leukocyte)
See monocyte.
Turk Cell
An atypical type of lymphocyte, characterized by dark blue cytoplasm in the
center of which is the nucleus.
Viability of Granulocytes
Their capacity to maintain life. The normal time for the granulocytes to re-
main motile and viable outside of the body, ·i.e., on a microslide incubator or
warmed stage, has been determined by Sabin and others. Normally they re-
main viable for from one to three or four hours. In certain conditions thei:
viability may be decreased.
Von Jaksch's Anemia (Luzet's Anemia)
An anemia of childhoorl, characterized by leukocytosis with marked im-
maturity and large numbers 'of normoblasts.
Young Forms
The immature types of neutrophils.
 SECTION TWO
ORIGIN AND DEVELOPMENT OF BLOOD
CELLS
MORPHOLOGY OF BLOOD CELLS
MYELOBLASTS AND MYELOCYTES
LYMPHOCYTES, MONOCYTES AND
PLASMA CELLS
ERYTHROBLASTS, ERYTHROCYTES AN])
THROMBOCYTES
SUPRAVITAL STAINING OF LEUKOCYTES
 SECTION TWO
THE DEVELOPMENT AND MOR-
PHOLOGY OF BLOOD CELLS

CHAPTER 3
ORIGIN AND DEVELOPMENT OF BLOOD CELLS
A discussion of the development of blood cells necessitates the presentation
of a number of controversial issues which, if given in too much detail, tend to
confuse rather tban enlighten the student of hematology. We present, therefore,
only the essential points of the various theories pertaining to hematopoiesis and
purposely omit many details.
Investigators are in accord concerning the well-known origin of blood cells
from the mesenchymal tissue of the embrya and also agree that, in adult life, the
reticula-endothelium is the potential site of blood formation, and that the myeloid
tissue of the bone marrow, the lymphoid tissue, and even the general connective
tissues are the major sites of hematopoiesis. The most controversial point cen-
ters around the question of whether, in post-natal life, the cells of the blood are
derived from a single primitive stem cell or whether the various blood cells have
individual precursors and separate cycles of maturation. This controversy is
represented by various groups of investigators who are divided into the so-calJed
monophyletic or polyphyletic schools, according to their beliefs on this question.
Although we do not underestimate the importance of this problem in hematology,
we are cognizant of the fact that the origin of blood celJs is of little practical
importance far the clinical hematologist. For this reason also we present only
the essential points relative to this question.

THE MONOPHYLETIC THEORY

The members of the monophyletic school, or the "monophyletists," also

called "unitarians," including such notable figures as Pappenheim and Maximow,
adhere to the idea that a single polyvalent cell, the "Iy~p~oidocyte" or "hemato-
cytoblast," is present in all adult hematopoietic tissues, and that this cell is
capable of giving rise to the various types of blood cells. For example, this
"lymphocyte-like cell" gives rise directly to the granular leukocytes, to the megalo-
blasts, to the lymphocytes, and to the monocytes as shown in the following din·
gram. (This cell is also called "hemocytoblast" and "haemocytoblast.")
44 , THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

~Lymphocyte5
Reticulo-endothelitun -+ Haemocytoblast ,,---Granulocytes
+
Megaloblast
Monocytes

.:t(. ,
.Erythroblast'
+
Erythrocytes
Downey modifies this ~oncept!and a~s,}m~~ '~he' presence iif two stem cells, one
for granulocytes and ·one for iympnocytes, .both of'which, h~wever, are derived
from a lymphocyte-like ltaemocytoblast. His theory is illustrated below in 11 simi-
lar diagram.
#' .. "r.ymphoblast---Lymphocytes
Retjculo~endothelitun -+ Haemocytoblast ( MonocytO$ .
. -t. . , ,Myeloblast Granulocytes
Erythroblast
+
Erythr~cyte
To summarize, these authors believe that tile four cellular elements of the
blood, the granulocytes, lymphocytes, monocytes, and ery,throeytes are derived
from a single cell, the hematocytoblast. This, in brief, is the concept of the mono-
phyletic school.

THE POLYPHYLETIC THEORY

The "polyphyletists" deny the existence of this hypothetical hematocytoblast

but here again the sponsors of this. school disagree among themselves concerning
the number 01 leukoblastic stem cells. 'The chie! point 01 disagreement con-
cerns the presence of either two or three blast cells from which the three type,
of leukocytes may· arise, this divergence of opinion dividi;'g the polyphylelists
into the 50~called "dualists" and Htrialists." Thus, if one believes that the granulo.
cytes, lymphocytes, and monocytes, arise frqI]1 .onl):' two slem. cells, then he is
classified as a "dualist," and if he hold, that each cell hits a separate stem cell, he
is a "trialist." As will be seen later, the divergerice of opinion concerns largely
the origin of the monocyte. ,.
The dual origin of blood cells has been championed by Ehrlich, Piney; and
Naegeli who believe that there are only two po.stnatal stem cells or leukocytes,
these being nrst the myeloblast, which gives rise to the 'granulocytes and mono-
cytes, and secondly, the lymphoblast, which is a precursor of the Iymph?cyte.
Furthermore, Naegeli denies the existence of'megaloblasts in postnatal life ex-
cept in certaiu diseases such as pernicious anemia. This theory is shown in tht>'
following diagram.
rLymPhoblast--LymPhocytes
RetiCulo-endothelitun~
t -cGranUIOCytes
Erythroblast . Myeloblast
1 Monocytes
Erythrocyte
 ORIGIN AND DEVELOPMENT OF "BLOOD CELLS 45

(I) The first blood cells'in the embryo are megaloblasts of endothelial origin
and are not present normally in 'postnatal life; (2) later hematopoiesis occurs
by the differentiation of reticulo-endothelial cells which, depending upon their
environment, are capable of'producing myeloid and lymphatic stem cells; (3) the
myeloid stem cell is capable of generating nucleated red 'cells intravascularly and
myeloblasts extravascularly and the !ymphoid stem cell giv.es rise to the lymphatic
series of cells; (4) the reticulo-endothelial system 'i~ capable of producing all
types of blood cells under different conditions but no common parent cell such as
the hemocytoblast can be found.
A summary of the Dualist concept involves the following points: First, the
hemocytoblast is regarded somewhat as a mythical cell type. Secondly, in adults
two precursors for blood cells are recognized to exist, these being the lymphoblast
and the myeloblast, both of which are derived from reticulo-endothelium. The
lymphoblast in turn becomes the precursor for all types of lymphocytes and the
myeloblast is the precursor for all.of the, three ,types of granulocytes as well as the
monocytes. However, just as in the Trialistic viewpoint, it is believed that all
of these cells arise from reticulo-endothelium and the difference between the
DualIst and the Trialist school is that! in the, latter there is postulated the ex-
istence of the monoblast, which in turn is a precursor of the monocyte. This is
shown in the diagram below.
The most widely accepted theory is that' of the trialist school which pro-
poses a separate phylogony for every type of blood cell, which means, an indi-
'vidual stem cell, and a different site of origin in normal postnatal life. For ex-
ample, in the bone marrow the' myeloblast 'acts as a progenitor for the granular
'leukocytes; in lymphoid tissue'the lymphoblast is the parent of the lymphocytes,
and in the connective tissue the monoblast is the specific ancestor of the mature
monocyte. Thus, for the first' time, there are recognized three respective stem
cells for the three types of leukocytes. According to Sabin 'and associates these
three leukoblasts arise from a primitive free cell which is present in all leuko-
poietic tissue and which in turn arises from the reticular cell of the reticulo·
endothelial system. The trialists further separate the development of erythro-
cytes by tracing their origin from the endo'thelium lining the vascular system of
the bone marrow. This .concept, is summarized in the following diagram.

/Lymphoblast--LymPhOCytes

J
Reticulo-endothelium'-" Myeloblast --Granulocytes
"-Monoblast --Monocytes
Megaloblast

~
Erythroblast

~
Erythrocyte
46 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

~In a summary of the trialist theory, the following points are of practical
importance: (I) there are three stem cells for the three types of circulating leuko-
cytes, the myeloblast, the lymphoblast, and the monoblast; (2) these leukoblasts
arise from a fixed reticular cell through an intermediate primitive free cell i (3)
under normal conditions·the type of cell produced depends upon the site of odgin,
that is, the reticulum in the bone marrow produces only granular leukocytes, but
in lymphoid tissue produces lymphocytes, and in connective tissue produces mono-
cytes; (4) under abnormal conditions, however, the reticulum in any tissue may
give rise to various types of cells; for example, Doan and Sabin have shown
that monocytes may be stimulated to arise in the bone marrow by the injection
of large numbers of tubercle bacilli. We believe that monocytes are produced
in the bone marrow in some cases of monocytic leukemia. \Ve base this on a
study of over one hundred cases of verified monocytic leukemia, and at least
six of these patients -who at one time had typical monocytic leukemia, later
eventuated into typical myelogenous leukemia.* Under norma] conditions, bow-
ever, each cell seems to have its own home, its own parent and its own prOcess
of maturation.

HEMATOPOIESIS IN THE EMBRYO

In the embryo, blood cells have their origin from mesenchymal cells. The
megaloblast, which is the progenitor of the erythrocyte, exists a long time before
leukocytes are differentiated. During the last few months of fetal life the liver
and spleen are the active sites of hematopoiesis. By the time of birth the flat
and long bones have become centers of blood formation, although during carly
infancy the liver and spleen may retain in part, their pre-natal function so that
when an extra demand lor blood formation arises, these extramedullary cellters
become active and flush the peripheral blood with immature, embryonic cell
types. Extramedullary hematopoiesis is often found in adults in the various
leukemic states where there is a reversion to the embryonic type of cell produc-
tion.

DISTRIBUTION OF THE BONE MARROW

Piney gives an excellent description of the distribution of post-natal or "dult

bone marrow. At birth a pink-red marrow is distributed in all bones of the Ilody
and the fatty marrow does not appear until about the age of seven. At early
puberty, there appears a considerable section of fat in the mid-region of the long
bones. In healthy adults, active red marrow is limited to the sternum, ribs,
vertebrae, bones of the skull, and the flat bones of the pelvis. The yellow, fatty
marrow fills the long bones with the exception of one small area near the superior
end of the diaphysis which provides a potential reserve center for hematopoiesis.
Doan has shown experimentally how fatty marrow is transformed into active
red marrow when there is a need for increased hematogenesis. There is, first, a
replacement of fatty tissue with gelatinous marrow. In cases of extreme Jlee,d,
* Cases on file in the Hematological Registry of the American Society of Clinical PatholOgists.
 ORIGIN AND DEVELOPMENT OF BLOOD CELLS 47
some of the spicules of bone may be removed to enlarge the marrow cavity
(Drinker, et al.). The next step is erythropoiesis followed by leukopoiesis, and
in regenerating marrow the former invariably precedes the latter.

PHYSIOLOGY OF THE BONE MARROW

For a thorough knowledge, of the development of each type of hematocyte

as shown in Plate I, it becomes necessary to consider the mechanism of hema-
topoiesis with emphasis upon the bone marrow pattern since it is here that
granulocytes, erythrocytes and thrombocytes are produced.
Evidence has accumulated to show that the vascular system of the bone
marrow is of the closed type. This fact is important since erythrocytes are pro-
duced intravascularly and the leukocytes extravascularly and the hone marrow
has a vascular pattern that is of paramount significance in hematopoiesis (Doan).
It consists of central arteries and of transitional capillaries leading to tufts of
sinusoids which are partly open to the circulation and partly collapsed. This
mechanism insures a sluggish flow of blood. These sinusoids finally lead into
the venous system. They resemble arteries in their histology and function hut
have the size of veins when dilated. They function at one time as a collapsed
capillary and at another time as a dilated sinus open to the circulation. During
collapse, with a partial stagnation of blood and low oxygen tension, the erythro-
poietic production is at a maximum. In addition, the collapsed bed acts as a store-
house for newly formed granulocytes which have worked their way by their mo-
tility from the outside marrow into the vascular system. In contrast to this,
granulogenesis is an extravascular phenomenon and is at its height during
sinusoidal dilatation which results in a flushing of the mature granulocytes into
the peripheral circulation. Thus, there are two processes occurring in the bone
marroW the production of erythrocytes during vasomotor constrictioll 7 and the
j

maturation of granulocytes during the stage of dilatation (Sabin, et a1.).

Vasomotor control, however, is not the primary factor in hematopoiesis be-
cause a substance which initiates the orderly maturation of erythrocytes has been
discovered to be produced in the stomach and stored in the liver (Minot, Castle,
et al.). No maturation factor for leukocytes has been isolated. Stimulants such
as bacterial toxins and nucleo-proteins have been shown to evoke a leukocytosis
but these are considered chemotactic factors which draw the mature leukocytes
into the peripheral blood from the store houses in the spleen and the bone marrow.
This creates a need for the production of new cells and presumably some un-
known substance, resident in the body and normally regulating leukogenesis, is
responsible for the speeding up of maturation. The search for the leukopoietic
factor comparable to the erythropoietic lactor for red cells represents one of the
most intriguing and important problems in hematology.

MATURATION OF ERYTHROCYTES

As previously stated, red blood cells arise intravascularly from the endothelial
cells lining the ·capillary vessels of the bone marrow. When the sinusoidal bed is
 PLATE I

ORIGIN AND DEVELOPMENT OF BLOOD CELLS

I. Lymphoblast. 16. Juvenile neutrophil.

2. Large lymphocyte.
3. Intermediate lymphocyte.
4. SmaD lympbocyte.
s. Monoblast.
6. Large monocyte without azure granules.
7. Monocyte with a few granules.
8. Mature monocyte with azure granules.
g. Myeloblast.
10. Premyelocyte.
I r. Eosinophilic myelocyte.
12. Juvenile eosinophil.
13. Band eosinophil.
14. Segmented eosinophil.
IS. Neutrophilic myelocyte.
17. Band neutrophil.
18. Segmented neutrophil.
Ig. Basophilic myelocyte.
20. Juvenile basophil.
2 r. Band basophil.
22. Segmented basophil.
2.1. Megakaryocyte (bone marrow).
24. Later megakaryocyte (bone marrow).
25. IMcg~karyocyte (peripheral blood) ..
26. Thrombocytes (platelets).
27. Megaloblast.
28. Normoblast.
29. Reticulocyte.
30. Normocyte (erythrocyte).
 Plate I.

I
I

1 1

From koy R. Kracke's Disuses of the Dl()O(l

and Atlas of Hematology. COl)yriJ,;Ju, 19t',
by J. B. Lippincott COmJl3ny
 ORIGIN AND DEVELOPMENT OF BLOOD CELLS 49

collapsed, tl1e blood flow is sluggish, and the oxygen tension low. The endothelial
cells swell and divide and the outside daughter cell continues to be the wall of the
vessel; the inside daughter cell becomes a megaloblast (Plate I, cell 27) with a
large vesicular nucleus and a zone of milky blue cytoplasm. 'l'hese cells are the
ancestors of the erythroblasts, anyone of which may divide at any stage. The
next cell in the series is the macroblast. Its nuclear chromatin assumes a dense
spoke-wheel arrangement and its cytoplasm retains most of the basophilic color.
This cell is not shown in the developmental cycle in Plate I, bnt is illustrated
in detail in Plate 11. The nucleus becomes more dense and the cytoplasm more
acidophilic as hemoglobin is incorporated until finally the typical normoblast
stage is attained as shown in Plate I, cell 28. These cells are clustered around
the lumen of the capillary which dilates to permit increased cell production
while the megaloblasts remain close to the vessel wall (Doan, et al.). Normoblasts
lose their nuclei, probably by a process of solution, and finally reach the reticulo-
cyte stage (Plate I, cell 29) in which there appears a skein-like mesh or a fine
reticulum. Supravital stains are required to demonstrate this network. This
stage is soon passed and the resulting normocyte (Plate I, cell 30) is mature and
then ready to be carried into the peripheral blood.

MATURATION OF GRANULOCYTES

Sabin has shown in the following chart the development of granulocytes

in detail, with emphasis upon their reaction to supravital dyes;

Reticular cell

Primitive free cell

Myeloblast

l\lyclocyte A
J\lyclocytc B
Myelocyte C
Metamyelocyte

Leukocyte
(Arneth range)
Senile 1eukocyte
Non-motile leukocyte
50 THE DEV1!;LOPlIENT AND MORPHOLOGY OF BLOOD CELLS

She separates th,e granular cells and their precursors into three levels. Level
I represents a stage of the development of mitochondria and basophilic cyto-
plasm. The first cell is the fixed reticular cell which has a faintly basophilic cyto-
plasm but no mitochondria nor granules. This primitive cell divides and produces
a primitive free cell which is indistinguishable from myeloblasts in fixed smears
but with supravital stains differs in its small size, its fewer number of mito-
chondria, and its more indistinct nuclear differentiation. She believes that 'this
cell is found in large numbers in normal bone marrow rather than the myeloblast.
but, under stimulation, is converted very quickly into the myeloblast. The
primitive free cell often resembles the small lymphocyte in fixed preparations
and accounts for the confusion of earlier authors in believing that myeloblasts
evolved from lymphocytes. The cells are differentiated by supravital stains. The
third cell in level 1 is the myeloblast in which basophilia and mitochondria reach
their highest development. After this stage these elements gradually disappear.
Level 2 is characterized by a decrease in basophilia of cytoplasm and a simul-
taneous increase in granulation followed by indentation of the nucleus. Myelo-
cytes, therefore, develop from the myeloblast and are divided into types A,
B, and C. Type A contains the fewest and type C the largest number of granules.
Nuclear indentation begins here and culminates in the metamyelocyte which
represents the last stage before segmentation. Division may occur at any stage
up to and including the myelocyte. Succeeding the metamyelocyte there is a
process of maturation into the adult form. The three types of granulocytes,
eosinophils, basophils and neutrophils, a~e differentiated at the myelocyte stage.
For their individual reaction to supravital stains, see Plate 14.
Level 3 represents a stage of maturation and segmentation of the nucleus,
the development of ameboid movement which gives the mature granulocyte the
power to gain entrance into the blood stream, and finally, in the blood stream,
the cell loses its motility and function with the onset of senility.
In Plate I is shown the development of granulocytes as they appear in fixed
preparations stained with polychrome dyes. Earlier primitive cells preceding
the myeloblast are purposely omitted since their differentiation is difficult if not
impossible with ordinary staining methods. This classification is limited to
those cells which may be identified with polychrome dyes, thereby providing a
practical list of types easily recognized and classified, For the detailed morpho-
logical description of these cells and their abnormal variants see the succeeding
plates.
A typical, non-granular, deeply staining myeloid stem cell, the myeloblast is
shown in cell 9. The next recognizable stage is the premyelocyte (cell 10) in
which a few wine colored granules appear. At this stage differentiation of the
three types of granular cells begins, each following a similar maturation cycle.
The development of the eosinophilic series is shown in cells II through 14.
The granules of the eosinophilic myelocyte are more intense than in the more
mature forms. There occurs, frequently, a cell, as shown in cell II, in which
occasional basophilic granules are scattered among the eosinophilic types. The
nuclei of eosinophils are less intensely basophilic than in neutrophils, but undergo
a similar process of indentation and segmentation. Although the eosinophilic
 ORIGIN AND DEVELOPMENT OF BLOOD CELLS

juvenile (cell 12) and band forms (cell 13) are shown for the sake of complete-
ness, they are infrequently seen except in chronic myeloid leukemia and are
of little significance as compared to the same stages in the neutrophilic series.
A large majority of the premyelocytes develop into the neutrophilic 'series
where their classification into the stages of myelocyte, juvenile, band, and seg-
menter becomes of practical importance in interpreting the cellular response to
certain infectious processes, since these cells form the classes of Schilling's
nuclear index. Cells 15 through IS represent the development of neutrophils
from the myelocyte to the mature form: first, the highly granular myelocyte
(cell IS) with a round or oval and somewhat indistinct nucleus: second, the
indented, horseshoe-shaped juvenile (cell 16); third, the elongated band (cell
17); and fourth, the mature segmenter (cell IS) which is actively motile and
is present in the peripheral blood where it acts as a phagocyte in defense of
the body against certain bacteria.
The development of the basophilic series (cells 19 through 22) is similar,
although the interm~diate forms occur rarely.

MATURATION OF THROMBOCYTES

Various theories have been proposed to explain the origin of thrombocytes.

Some investigators assume their derivation from endothelial cells, others from
plasma thrombogen, and still others believe that they originate from leuko-
cytes. None of these theories has received credence. The most widely accepted
idea has been advanced by Wright. According to his concept, the megakaryocyte,
a giant cell found in extravascular bone marrow, is the precursor of the small
pseudo-cellular platelets of the peripheral blood. A typical megakaryocyte (cell
23) is an extremely large cell, about 40 micra in diameter, with loosely woven
nuclear chromatin, blue cytoplasm and numerous azurophilic granules. It is
actively ameboid but because of its size does not pass into the circulation except
under abnormal conditions. According to Wright's theory, thrombocytes (26)
are pinched-off pseudopodia which presumably protrude between the endothelial
cells of bone marrow capillaries. These bodies are non-motHe and are swept into
the circulation by the force of the blood flow. Cells 23 and 24 are megakaryocytes
and were drawn from bone marrow preparations. Cell 25 represents a fragment
of a megakaryocyte found in the peripheral blood from a case of myeloid leukemia.
In Plate 13, cells J and 2 are shown as large non-granular cells which we believe'
to be earlier forms than megakaryocytes and which we prefer to call thrombo-
blasts. This cell type is not shown in Plate I in conjunction with the develop-
ment of thrombocytes since its identity is not definitely established.

MATURATION OF LYMPHOCYTES

The developmental cycle of lymphocytes appears less complex than that of

the granulocytic series. Normally these cells originate from a reticular cell
in the follicles of lymphoid tissue through an intermediate stage, the primitive
free cell, identical with that described as the precursor of the myeloid series
52 THE DEVELOP1\fENT AND MORPHOLOGY OF BLOOD CELLS

in the bone marrow. The first cell o,f the series is the large mononuclear lympho-
blast (cell I). These cells divide and develop through intermediate stages
called large lymphocytes (cells 2 and 3) until finally the mature lymphocyte
(cell 4) is a small cell consisting almost entirely of nucleus with a narrow zone
of cytoplasm. Naegeli does not believe that the small form represents the mature
stage and this question is not entirely settled. However, evidence is against
Naegeli's belief, if analogy to the development of other cell types from large
to small forms is of any value. At the present time, it is of little importance
to separate tl,e intermediate large lymphocytes from the small ones since
both occur normally in the peripheral blood and little significance is attached to
an increase of one Over the other.
It is still a current practice among some workers, in doing different ceIl
connts, to separate large from small lymphocytes. This seems entirely unneces-
sary, since there probably is no disease characterized by an increased number of
one in which the other is not also increased.

MATURATION OF MONOCYTES

If the trialist view for the origin of blood cells is accepted, an individual
cycle for the development of monocytes must be admitted, probably taking
place in the following order: (r) primitive reticular cell in connective tissue:
(2) primitive free cell; (3) monoblast: (4)· intermediate monocyte: and (s)
mature monocyte. We feel certain that monoblasts occur, since we have seen a
well established case of mono blastic leukemia from ·which the monoblast shown
in cell S, was selected for reproduction. Admitting the existence of monoblasts
favors the acceptance of Sabin's theory for an individual cycle of development
from connective tissue in the normal state. We believe that in some cases of
monocytic leukemia the monocyte is produced in the hone marrow as well as
connective tissue but in that disease there is an abnormal hematopoiesis with a
reversion to embryonic processes. We have also studied cases of monocytic
leukemia that later become myeloid leukemia. Such a state, however, should
not detract from the trialistic concept of the normal maturation process. In
lymphatic leukemia lymphocytes are frequently produced in the bone marrow in
the late stages and yet no one contends that the marrow is their normal site
of origin.
In regard to the development from the monoblast (cell 5) stage to maturity,
as shown in cells S through 8, the salient features are a decrease in basophilia, an
increase in the delicacy of staining reactions, a progressive indentation and
elongation of the nUcleus of intermediate monocytes (cells 6 and 7) until the
mature monocyte (cell 8) contains a typical sausage-shaped, convoluted nucleus
and numerous fine azurophilic granules in the cytoplasm. The similarity of
these granules to those of neutrophils and of the nucleus to the juvenile neutro-
phil is a strong morphological argument in favor of the myeloid origin of mimo-
cyies. However, Illorphologic differentiations are always unceriajn criteria
and it appears more consistent that each cell type in adult life has its own site
of origin and a cycle of evolution peculiar to its species.
 ORIGIN AND DEVELOPMENT OF BLOOD CELLS S3

SUMMARY

A brief summary of the various ideas concerning the origin of blood cells
can best be shown as follows in a comhination of two charts given by Sabin.
Spleen and Connective
Bone Marrow Lymph Glands Tissue
/
EndotheHum "-
Reticulum
I
Reticulum
I
Reticulum

. PrimitiJe
I
Primitive
I
Primitive

I
Megaloblast
free cell
I
Myeloblast
free cell
I
. Lymphoblast
free cell
I
Monoblast
I
Myelpcytes A, B, C.
I
Metamyelocytes
I
Erythrocytes' Granulocytes Lymphocytes Monocyte"
It is our belief that granulocytes, lymphocytes, and monocytes arise from
myelohlasts, lymphoblasts, and monoblasts, respectively. The evidence seems
more ~onvincing that the monocyte arises from reticulo-endothelium under
normal conditions, but in leukemic states may arise elsewhere, but only from
reticular tissue, whatever its location. At least, it is the most satisfactory and
workable concept for the clinical hematologist in correlating cellular disturbances
with clinical findings. If the clinical hematologist keeps in mind two funda-
mental points he is assured of a sound working basis for the study of blood.
These are as follows: First, that granulocytes arise in the bone marrow from
myeloblasts; that lymphocytes arise from lymphoid tissue; and that monocytes
arise from reticula-endothelium which is a widespread tissue found in many
places; and second, that granulocytes, erythrocytes, and thrombocytes are formed
in the red marrow of the flat bones.

BIBLIOGRAPHY
ORIGIN AND DEVELOPMENT

CASTLE~ W. B.: "The etiology of pernicious aBemia and related macrocytic anemias." Ann. Int.
Med., 7, 2, 1933.
CASTLE, W. B., and LOCKE, E. A.: "Observations on the etiological relationship of achylia gastrica
to pernicious anemia." Jour. Clin. lnvestigation~ 6, 2, I92S.
COHN 1 E. J., MJ.Nor, G. R., A.Lu:s, G. A., and SALTER, W. T.: "The nature of the material in liver
effective in pernicious anemia." Jour. BioI. Chern., 77, 325, I9ZS.
CUNNINGHAM, R. S., SADIN, F. R., and DOAN, C. A.: "Development of leukocytes, lymphocyte.s~
and monocytes from a specific stem cell in adult· tissue.1I Contrb. Embryol., Carnegie [nst.,·
lVash.~ 16, 221, ,192S.
DOAN, C. A.: "The capillaries of the bone marrow." Bull. Johns Hopkins Hosp., 33, 222, Ig22.
DOAN, C. A.: "Current views on the origin and maturation of the cells of the blood." J. Lab. and
CUn ..Med., 17, 881, 193Z.
54 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

DOAN, C. A., CUNNING:1IAM, R. S., and SABIN, F. R.: "Experimental studies on the origin and
maturation of avi.an and mammalian red blood cells." Conlrb. Embryol., Carnegie Inst.,
TVash., 16, 163, 1925.
DOAN, C. A., and SABIN, F. R.: uLocal progression with spontaneous regression of tuberculosis in
the bone marrow of rabbits, correlated with transitory anemia and leukopenia after intravenous
inoculation." Jour. Exper. Med., 46, 315. 1927.
DOWNEY, H.: "The occurrence and significance of the 'myeloblast' under normal and pathologic
conditions." Arch. Int. Med., 33, 301, ]924.
DRINKER, -C. K., DRINKER, K. R., and LUND, C. C.; "CircUlation in mammalian bone marrow!'
Am. J. PlIysiol., 62, :I, 1922.
MAXIMOW, A. A.: "Relation of blood cells to connective tissue and l:ndotbelium." Physiol. Rev.,
4, 533, 1924.
MINOT, G. R., and MURPHY, W. P.: "Tr~atmcnt of pernicious anemia by special dieLIt Jour. Amer.
M ed. Assoc., 87 ~ 470, 1926.
NAEGELI, 0.: Blutkrankheiten una B11~tdiagnostik. ed. 5, J. Springer, Berlin, 1931.
PARSUNS, L. G.: "Deficiency anemias of childhood." Brit. Med. Jour .• 2, 631, 1933.
PINEY, A.: "Recent advances in hematology," ed. 3, P. Blakiston)s Son & Co., Philadelphia, 1931.
PINEY, A.: "The anatomy of the bone marrow j with special reference to the distribution of red
marrow." Brit. Med. Jour., 2, 792, 1922.
SABIN, F. R.: uDone marrow." Physiol. Rev., 8, 191, 1928.
SABIN IF. R. t DOAN J C. A., and CUNNINGHAM, R. S.: "The discrimination of two types of phago-
cytic cells in connective tissue by the supravital technique:' Contrb. Embryol., Carnegie inst.,
Wash., 16, 125, 1925.
SCHILl.INO, V.: "The monocyte frolU the trialistic standpoint and its clinical significance." Med.
Klin., 22, 563, 1926.
SHARP, E. A.: "An antianemic factor in desiccated hog stomach." Jour. Amer. Med. Assoc., 93,
749, I9 29·
STURGIS, C. C., and ISAACS, R.: "Desiccated stomach in the treatment of pernicious anemia."
Jour. Amer . .!tIed. Assoc., 93, 147, I929.
WRIGHT, J. H.: "The origin and nature of the blood plates." Boston Med. and Surg. Jour., 154,
643, 1906. .
 CHAPTER 4
MORPHOLOGY OF BLOOD CELLS
THE GRANULOCYTES

r. MYELOBLAST (15-20 micra in diameter). See Plate 2.

Granulocytes have their origin from a large mononuclear deeply stained cell,
the myeloblast. Under abnormal conditions this cell may become extremely large,
(macromyeloblast) or it may appear no larger than the average lympho-
cyte (micromyeloblast). It is the only cell of the granular series that does not
contain granules in the cytoplasm and for this reason bears a close resemblance
to other leukoblasts, notably the lymphoblasts. With a polychrome dye, the
nucleus is red-purple in color, round or oval in shape, eccentric in position and
contains, as a rule, from three to five nucleoli. The finely woven chromatin
strands lend a homogeneous appearance to the nuclear framework and serve as a
morphologic contrast to the coarse chromatin arrangement of the lymphoblast.
There is no definite nuclear membrane. The cytoplasm varies in color from a pale
to a light blue without the clear perinuclear zone which is typical of the lympho-
blast.
The myeloblast, reacting negatively to the peroxidase stain, is easily confused
with other leukoblasts, and even with the megaloblast. Frequently morphologic
differentiations fail and the distinction rests upon the presence of the more mature
cells which belong to the same series. When there is a preponderance of imma-
ture granulocytes, it is likely that the questionable leukoblast is a myeloblast.
Morphologic distinctions between leukoblasts are at all times difficult but their
successors in the series are found generally in sufficient numbers to make a cor-
rect identification possible. The old adage, "A cell is known by the company it
keeps," is the safest rule to follow in identifying leukoblasts.

2. PREMYELOCYTE (14-18 micra in diameter)

In the scale of maturation, the premyelocyte follows the myelohlast. The
granules begin their course of development and appear first in this cell as purple-
red dots, distinct in outline, few in number, and scattered through the cytoplasm.
Both the nucleus and the cytoplasm stain a shade less intensely than in the myelo-
blast. The premyelocyte is faintly peroxidase-positive, the intensity of the reac-
tion depending upon the number of granules present. Premyelocytes develop into
three different types of myelocytes which are classified according to the reaction
of their granules to the polychrome dyes, Le., eosinophilic, basophilic, and neutro-
philic, each of which becomes the precursor of its respective mature form, the
eosinophil, basophil and neutrophil.
S5
56 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

3. NEUTROPHILIC MYELOCYTE (12-18 micra in diameter)

In this cellrwhich is destined to become the segmented neutrophil, the neutro-
philic granular elements reach their highest point of development. Innumerable
wine-red or lavender-red granules cover the entire surface of the cell, almost ob-
literating the nucleus from view. As the granules increase in number, the cyto-
plasm becomes a lighter blue and fmally a pink-lavender color, and, at the same
time, the nucleus becomes more lightly staining, more irregular and indistinct in
outline. As the myelocyte advances toward the juvenile stage, the granules lose
the intense purple· red color and become more truly neutrophilic and smaller in
size. This older myelocyte is shown in Plate 5.

4. JUVENILE NEUTROPHIL (JUVENILE NEUTROCYTE) 10-18 micra in

diameter .
In this cell the nucleus becomes more definite in outline than that of the
myelocyte. It is slightly indented or frankly horseshoe shaped, and shows a
sharper nuclear membrane. The chromatin material is more dense than in the
myelocyte and the nucleoli have disappeared. The cytoplasm has generally lost
the blue color of the younger forms and stains neutrophilic, although the blue
color may persist to a later stage, when the sp.eed of maturation is more rapid
than normal. The granules are small, fine, and stain a lavender-pink color.

5. BAND OR STAB NEUTROPHIL (ra-IS micra in diameter)

The band differs from the juvenile mainly in the elongation of the nucleus
which may take the form of a uniformly curved band or of an irregular H, T, or S
.
shape. The nucleus stains more deeply as it ages .

6. SEGMENTED NEUTROPHIL (SEGMENTED NEUTROCYTE) (10-12 micra

in diameter)
The nucleus becomes lobed or segmented with a coarse, chromatin network
and stains a _bright violet color. The lobes vary in number from two to five or
more and often are connected by a single fine strand of chromatin. The cyto-
'plasmic granules are fine in textur:e, stain a delicate lavender-pink color, and
usually are evenly dispersed throughout the cytoplasm.

7. EOSINOPHfLIC MYELOCYTE (12 - I 8 micra in diameter)

This cell has an indistinct, round or oval, purple nucleus with a blue cyto-
plasm. It is characterized by large, coarse, almost square orange-red or, more
typically, cantaloupe-colored granules which are densely scattered over both the
cytoplasm and the nucleus. Sometimes these granules appear bronze or dirty-
yellow due to a persistence of basophilia. As they increase in number they appear
to be stuck together thus hiding the blue of the cytoplasm and obscuring tbe
nucleus. Occasionally, a .few dark blue granules are scattered through the cytq-
plasm.

8. JUVENILE EOSINOPHIL (ra-I8 micra in diameter)

Eosinophilic juveniles and bands are seen infrequently in the peripheral blood.
In the juvenile the nucleus is indented or horseshoe shaped and the cytoplasm
 lUOU.PIlOLOGY OF BLOOD CELLS 57
is pale blue. The granules are larger and more distinct in outline than in the
myelocyte.

9. SEGMENTED EOSINOPHIL (10-15 micra in diameter)

The nucleus of the eosinophil rarely divides into more than two segments.
The granules are large, round, often appear shiny, refractile, sharp in outline, and
are usually the color of ripe cantaloupe. The cytoplasm is a faint, delicate blue
and frequently fails to stain.

10. BASOPHILIC MYELOCYTE (10-18 micra in diameter)

Basophils are easily recognized by the presence of large, coarse, purple-black
granules, which are more irregular in shape and more variable in their staining
reaction than the eosinophilic granules. In the myelocyte the nucleus is indistinct
in outline and is usually dull-purple in color. The cytoplasm may be blue or dull
lavender. The granules arc usually so heavy and dark that it is impossible to dis-
tinguish the color of either_ the cytoplasm or the nucleus.

II. JUVENILE BASOPHIL (10-18 micra in diameter)

Due to the heavy granulation and the indistinctness of the nucleus it is diffi-
cult and of little practical importance to distinguish between basophilic myelo-
cytes, juveniles and bands. The only appreciable difference lies in the shape of
the nucleus. The juvenile form is shown merely to illustrate that these cells pass
through a maturation cycle similar to that of the neutrophils.

12., SEGMENTED BASOPHIL (10-12 micra in diameter)*

The nucleus of this cell segments but stains more lightly than that of the
eosinophil or neutrophil and appears buried beneath coarse, purple-black granules.

LYMPHOCYTES, MONOCYTES, AND ERYTHROCYTES

1. LYMPHOBLAST (15-20 micra in diameter). See Plate 3.

The lymphoid cells have their origin from a large mononuclear cell, the
lymphoblast, which is often indistinguishable from the myeloblast. These two
cells appear identical in size and staining properties. There are, however, a
few fine cytological distinctions. The nuclear chromatin of the lymphoblast
is coarser and less homogeneous than that of the myeloblast and is concentrated
at the edges into a perceptible nuclear membrane. The number of nucleoli
rarely exceeds three. A definite, clear perinuclear zone is characteristic of the
cytoplasm of Iymphoblasts and is rarely found in typical myeloblasts.

2. YOUNG LYMPHOCYTE (9-18 micra in diameter)

In this cell the nucleus is large in size, intense hlue-violet in color, round
or oval in shape, and there is present a dense nuclear membrane. The nuclear
chromatin is less dense than that of'the mature form. Surrounding the nucleus is
a wide zone of clear, sky-blue cytoplasm which contains a definite perinuclear
pallor and mayor may not contain a few, scattered, bright red granules. These
• The function of thO;! basophil is entirely unknown although, as Michels point!!o out, twenty.five different
hypotheses have been enume--ated for the functlOrt of this cell. Fot" an extretnely comprehensive discussion
of the basophil con:;uU,.. the chapter on m.ast cells by il-lichels in. Downt:y's Handbook of Hematology.
 PLATE II

MORPHOLOGY OF BLOOD CELLS

(GRANULOCYTES)

QJCQ)~
@)®®
@ 'l 8
r®1
Y
~
~ @;!~:
u ®~~
W
10 1
I. Myeloblast. 7. Eosinophilic myelocyte.
2. Premyelocyte. 8. Juvenile eosinophil.
3. Neutrophilic myelocyte. 9. Segmented eosinophil.
4. Juvenile neutrophil. 10. BasophiHc myelocyte.
5. Band neutrophil. H. Juvenile basophiJ.
6. Segmented neutrophil. 12. Segmented basophil.

CeIls drawn on large scale with particular view of showing individual cellular morphology of
the typical cell. For variations in morphology see other plates.
Plate IT.
Plate III.
 PLATE III

MORPHOLOGY OF BLOOD CELLS

(LYMPHOCYTES; MONOCYTES j ERYTHROCYTES)

t. Lymphoblast.
2. Large lympbocyte without azure granules.
3. Small lymphocyte.
4. Monoblast.
5. Immature, non-granular monocyte.
6. Mature, granular monocyte.
7. Plasma cell.
S. Primitive cell (stem ceil).
9. Megakaryocyte.
10. Megaloblast.
II. Normoblast.
1:2, ReticUlocyte.
60 THE DEVELOPMENT AND J\:IORPHOLOGY OF BLOOD CELLS

"azure" granules bear little resemblance to those of granulocytes and react nega-
tively to the peroxidase stain. They have been described by Naegeli as "chromi-
uial nuclear extrusions." They are not present in cells ready to divide and are
!arely found in lymphatic leukemia.

3. MATURE LYMPHOCYTE (7-15 micra in diameter)

As the lymphocyte matures the nucleus assumes a more marked affinity for
basic dyes. It is round or oval in shape, frequently indented, and contains
heavy masses and clumps of chromatin. The cytoplasm decreases in amount
until the mature form possesses only a narrow rim of sky-blue or deep hlue
cytoplasm or, in some instances, no cytoplasm is visible at all.

4. MONOBLAST (15-25 micra in diameter)

The monoblast, with its purple-red nucleus and deep blue cytoplasm, can-
'not be distinguished with certainty from the myeloblast by ordinary staining
methods, but it is .recognized presumptively by its association with other mono-
cytes more easily recognized. The monoblast in this plate was drawn from a
proved case of monocytic leukemia. This cell appeared larger and more irregular
in outline and the chromatin network more string-like than the ordinary myelo-
blast but these are uncertain criteria. Naegeli believes that the two cells are
identical but Sabin and her co-workers classify the monoblast as a separate cell
entity, basing their conclusion on its reaction to supravital staining. It has, as
other leukoblasts, a peroxidase negative cytoplasm.

5. INTERMEDIATE OR NON-GRANULAR MONOCYTE (12-20 micra in

diameter)
The young monocyte is, as a rule, larger than the large lymphocyte. It is
pastel in color reaction. The nucleus is a pale pink-lavender with a finely-woven
chromatin reticulum. It is indented, convoluted, eccentric in position and gives
an impression of depth and over-folding peculiar to this cell. The cytoplasm in
this young form is typically a cloudy-blue color. It may be smooth or irregular
in outline and appears to be no more than a sheer film of protoplasm.

6. MATURE MONOCYTE (12~20 micra in diameter)

·The mature cell is shown here equal in size to the younger form but it is
often smaller. The nudeus is more indented and convoluted and more nearly
approaches the horseshoe shaped nucleus of the monocyte described in the older
literature as the "transitional" cell. The light blue cytoplasm is literally dusted
with small lilac, azurophilic granules, finer in texture than those of the large
lymphocyte and pinker in color than those of the neutrophil. Protrusion of
the cytoplasm is of frequent occurrence. This cell is easily confused by the
!lmateur with the juvenile neutrophil and even veteran hematologists disagree
violently concerning its identification. Peroxidase stains do not settle the ques-
tion, since no one agrees upon the reaction of the monocyte to the stain. There
!lre some definite morphologic distinctions when the cells appear in their typical
forms. The monocyte is larger, the zone of cytoplasm wider and more pastel in
color, the granules are finer and more uniformly dispersed, the, pseudopodia are
 ~ORPHOLOGY of BLOOD CELLS 6,

entirely characteristic, the nucleus more convoluted, and the chromatin finer in
texture than the juvenile neutrophil. Under atypical conditions where cells vary
from the normal, supravital stains are required to separate the monocytic series
from other cell types.

7. PLASMA CELL (12-20 micra in diameter)

This is a large cell with an intensely violet colored nucleus which is typically
eccentric in position, round or oval in shape, and frequently appears to be almost
extruding from the cytoplasm. The chromatin material is coarse in texture and
has an arrangement which suggests the spokes of a wheel. The clear perinuclear
7.one is more pronounced in this cell than in any other leukocyte. The cytoplasm
is a brilliant blue and frequently contains vacuoles of all sizes (see plate 9).

8. PRIMITIVE CELL (20-30 micra in diameter)

This cell, with a pale lavender, loosely woven, somewhat coarse nucleus, a
blue nuclear cleft, and a pale halo of blue cytoplasm was drawn from a case of
acute leukemia of an unidentified type. Practically all cells in the blood smear
were of similar morphology. ~any of them contained fine granulation. There
is some evidence that these cells are more primitive than leukoblasts, perhaps
even younger than the so-called primitive free cell described by Sabin. On the
other hand they may be degenerate forms of leukoblasts. Some authorities con-
sider them cells of the myelogonirun and others (Piney) believe they are atypical
and degenerate premyelocytes.

9. MEGAKARYOCYTE (20-50 micra in diameter)

The precursor of blood platelets is a large irregular cell with a lavender
nucleus and pale blue cytoplasm which contains fine azure granules. This cell
was found in the peripheral blood of a patient with myeloid leukemia. It is
usually the largest cellular body seen in the marrow or the peripheral blood. See
Plate 13 for various types of megakaryocytes and their maturation ·cycle.

10. MEGALOJlLAST (10-20 micra in diameter or larger)

The megaloblast shown here was drawn from a case of pernicious anemia and
represents the type most frequently seen in peripheral blood. The nucleus is
similar in color and cllromatin arrangement to that of the myeloblast but is
easily distinguished from that cell by its smaller size. Another distinguishing
feature is the distinctness of the nuclear membrane. The true megaloblast never
shows radiating strands of chromatin typical of older erythroblasts. The cyto-
plasm is typically grey-blue in color, frequently irregular in outline, and gen-
erally pale near the nucleus. The larger type of megaloblast (see Plate I I) is
more difficult to distinguish from the myeloblast.

II. N ORMOJlLAST (7-9 micra in diameter)

This nucleated red blood cell is comparable in size to the normal circulating
erythrocyte. The nuclear chromatin stains a deep purple color and, in the young
form, is frequently arranged in a "cart-wheel" fashion. A. the cell matures the
6. THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

nucleus becomes more condensed, pyknotic, and almost black in color. The
cytoplasm of the normoblast is blue-pink in color in the early stages, but before
the cell loses its nucleus it stains as brightly pink as the mature erythrocyte.

12. RETICULOCYTE-(7-8 micra in diameter)

The reticulocyte is tbought to be an intermediate form between tbe nucleated
and non-nucleated erythrocytes. It is characterized by the presence of a fine
skein-like reticulum which cannot be demonstrated by_ ordinary staining methods.
These cells are best stained with brilliant cresyl blue for temporary observation
and counterstained with Wright's or Giemsa's stain for permanent preparations.

BIBLIOGRAPHY
MORPHOLOGY OF BLOOD CELLS
SER, ]. H., and WINTROBE, M. M,; j~Diseases of the Blood," Tice. Practice "f Medicine. 6,
1929 edition supplemented.
NAEGELI, 0.: Blutkrankheiten und Blutdiagnostik, ed. 51 Springer, Berlin, 1931.
SABIN, F. R.: Bone Marrow. Physiol. Rev., 8, 191, 19:18.
 CHAPTER 5
MYELOBLASTS AND MYELOCYTES
MYELOBLASTS

GENERAL D,SCUSSION

Myeloblasts w~re first described by Muller in r89I from a study of a case of

leukemia, although their significance as the stem cell for the granular series of
leukocytes was not understood until Naegeli, in '900, formulated the concept
that the myeloblast resides in the hone marrow and gives rise to granulocytes,
monocytes and erythrocytes. More recent investigators believe that a younger
cell than the myeloblast, the primitive free cell, is the resting cell of the bone
marrow which, upon stimulation, is quickly converted to the myeloblast. Further-
more, they do not adhere to the trivalency of the myeloblast, believing that its
sole function is the generation of the granular series of leukocytes.
The typical myeloblast occurring in the normal maturation cycle has been
presented in detail in the description of Plate 2. In brief summary, the chief
characteristics are: a round or oval nucleus with a finely woven nuclear network;
from three to five nucleoli without basi-chromatin clumping at the edges; no
definite nucleolar membrane; a sky-blue or ultramarine cytoplasm without a light
perinuclear halo. These characteristics offer some contrast to the lymphoblast"
with its coarse chromatin, fewer nucleoli, marked nuclear membrane, and dis-
tinctive perinu~lear pallor.
ATYPICAL TYPES

In Plate 4 are shown eighteen types of myelohlasts, the majority of which,

since they were selected from cases of myeloblastic leukemia, show atypical
variations from the normal. The first six cells, macromyeloblasts, are larger in
size than normal. They further show Auer bodies (cell 2), excessively brilliant
staining property of the cytoplasm (cells 3 and 5), protrusion of the cytoplasm
(cell 4), vacuolization (cell 5), and atypical division (cell 6). The next three
cells, normomyeloblasts, are approximately normal in size and, of these, cell
7 represents an entirely normal myeloblast. There is a scantiness and irregularity
of cytoplasm in cell 8 which is atypical. Cell 9 is a myeloblast with a fairly
typical nucleus but the neutrophilic cytoplasm has matured beyond the myeloblast
stage. This disorderly type of maturation is frequently seen in myeloblastic
leukemia. In some cases the nucleus attempts to mature first, by indenting and
lobulating, and the cytoplasm lags behind.
The last six cells are micromyeloblasts which resemble the intermediate
and small lymphocytes in size and staining properties and, for this reason,
often become diagnostic pitfalls. When they are present lin large numbers, they
63
 PLATE IV

MYELOBLASTS

oQQ
Q(j)
000
00010 II lZ

QQ8
I.
2.
G? Q 18
Macromyeloblast with round nucleus and sky-blue cytoplasm.
Macromyeloblast with oval nucleus and Auer bodies in the cytoplasm.
,3. Macromyeloblast with intense, blue cytoplasm. .
4. Macromyeloblast with irregular nucleus and protrusion of cytoplasm.
S. Macromyeloblast with vacuoles in a deeply staining cytoplasm.
6. Macromyeloblast in division.
7. Normomye]obIast.
S, Normomyeloblast with num~rous, irregular protrusions of cytQplasm.
9. Normomyeloblast in which the cytoplasm slams more acidophilic than normal but the nuc1Cli3
has not developed.
10. Normomyeloblast with a perinuclear area of azurophilic pro granulation.
ll. Normomycloblast with a large nucleus and only a remnant of cytoplasm.
12. NormolUyeloblast with atypical lobulation of nucleus.
I3, I4 and 15. Micromyeloblasts corresponding in size to intermediate lymphocytes.
16, 17 and 18. Mkromyeloblasts corresponding in size to small lymphocytes_ (Cell 17 has no
cytoplasm.)
Plate IV.
 Plate V .

, ..
 PLATE V

MYELOCYTES

QQQ
O ~rf7\
~~
,
.

()®11 IZ

C"n
'V0sfl
16 .
1. Premyelocyte with scanty granulation and centrally placed nucleus.
2. Large premyelocyte.
3. Premyelocyte with eccentric nudeus.
4, 5, 6, 7, 8 and 9. Early neutrophilic myelocytcs with blue cytoplasm and brilliantly stained
granule:;, (Myeiocyte' C of Sabin.)
10, II and 12. Late neutrophilic myelocytcs with more delicately stained grn.nules and cytoplasm.
(Myelocyte C of Sabin.)
13. Early eosinophilic myelocyte with dark, bronze granules and blue cytoplasm.
14 and IS. I~ate eosinophilic myelocytes with numerous brilliant granules.
16. Early basophilic myelocyte with blue cytoplasm and only a few granules.
17 and 18. Late basophilic myelocytes with num~rous granules and indistinct nudear outlines.
66 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

give the false impression of a chronic lymphatic leukemia. They differ mor-
phologically from lymphocytes chiefly in their finely stippled chromatin arrange-
ment, in the presence of definite nucleoli, and in the absence of a nuclear mem-
brane. They are identified definitely by the peroxidase-positive reaction of other
granular cells associated with them.
The peroxidase stain demonstrates the presence of an oxidizing ferment in
the cytoplasm of the granular series of cells with the exception of the myeloblast
which reacts negatively since it alone contains no granules. Other leukoblasts
likewise react negatively. This stain, however, is of considerable value in making-
a morphologic differentiation between the myeloblast and the lymphoblast since
early premyelocytes, which are almost invariably associated with myeloblasts,
may react oxidase-positive when the granulation is only barely perceptible and
easily overlooked with ordinary staining methods. As a general rule, there are
sufficient numbers of the more mature premyelocytes and myelocytes associated
with a questionable !llyeloblast to make its identification a certainty and to
make it unnecessary to rely upon the more uncertain morphologic distinctions.
OCCURRENCE AND D,STRIBUTION

Myeloblasts occur in large numbers in the spleen and liver during embryoni~
life and, for a long time after birth, constitute the greatest part of marrow cells
(NaegeJi). They are present in larger numbers in the bone marrow of children
than in adults. They do not occur normally in the peripheral blood except
occasionally in young children when the bone marrow is late in assuming its
post-natal hematopoietic function. They appear more frequently in the blood
of children in response to excessive stimulation of the bone marrow than under
similar conditions in the adult. Because of this it should be emphasized that
myeloblasts in the blood of children should be interpreted quite differently from
the finding of these cells in the blood of an adult. Myeloblastemia in children
oftentimes means a severe shift to the left due to an infectious process while the
same finding in an adult would suggest a leukemic process.
Myeloblasts appear in large numbers in the bone marrow and are seen
occasionally in the peripheral blood in severe and grave infectious processes,
post-hemorrhagic anemias, pernicious anemia, sickle cell anemia, von Jaksch's
anemia, and in all myeloid proliferations, especially myeloid leukemia.
A true myeloblastosis, in which the myeloblast is the predominant cell type
in both the bone marrow and peripheral blood, occurs only in the terminal, exacer-
bation stage of chronic myelosis and in acute myeloblastic leukemia. Frequently,
in the latter disease, tuere is a condition, described by Naegeli, of "hiatus leu-
caemicus" in which there appears in the reripheral blood only myeloblasts and
mature forms without any intermediate stages between the two. This is brought
about by the rather sudden inability of the leukemic bone marrow to produce
mature granulocytes and a consequent cessation of maturation at the myeloblast
stage. The mature cells seen in the peripheral blood have been produced days
before. Naegeli states that this "hiatus leucaemicus" is pathognomonic of the
leukemic state.
 A1YELOBLASTS AND ~YELOCYTES

MYELOCYTES
DESCRIPTION. (See Plate 5)
In the scale of maturation, premyelocytes and myelocytes represent inter-
mediate stages between non-granular and mature functioning· granulocytes. Cells
I, 2, and 3 closely resemble myeloblasts in their nuclear framework and cyto-
plasmic color but differ in the appearance of a few scattered, wine-red granules.
These granules do not conform to the color of either basophils, eosinophils, or
the more mature neutrophils. They appear similar to those of the large lympho-
cyte but are smaller and more irregular in size and are oxidase-positive. This
cell is capable of division and possesses the potentiality of developing in three
directions; a majority of them becoming neutrophilic myelocytes and the re-
mainder eosinophilic and basophilic myelocytes. The limiting of this potentiality
to the premyelocyte stage is necessarily hypothetical, since it is possible that
a certain number of myeloblasts or even younger cells are predestined for -the
production pf eosinophils and still otljers for neutrophils and basophils.
Cytoplasmic granulation reaches the maximum in myelocytes which are
divided into three cell types, neutrophilic, eosinophilic, or basophilic, depending
upon the reaction of their granules to the polychrome dyes. The myelocyte
may divide and reproduce itself or it may develop immediately into its mature
form, depending upon the need of the body for the production of new cells.
Mtcr the myelocytic stage, the cell is no longer capable of division.
Neutrophilic myelocytes (cells 3 through 12) vary in details of cytoplasmic
color, distinctness of nuclear outline, size of granules and nucleus, but have the
common characteristic of heavy, wine colored or reddish-lavender granulation
scattered densely over the cytoplasm and, as a rule, over the surface of the
nucleus. These granules are considered neutrophilic in reaction, since they are
neither basic-blue nor eosin-red in color. They are not the delicate, neutral
color of the mature neutrophil, but rather a brilliant combination of acid and,
basic dyes with a tendency toward the acid in well-stained preparations. This
results in the bright wine-red color which is especially pronounced in the
younger forms (cells 4 through 9). As the cell matures the granules stain more
pastel in shade and the cytoplasm gradually loses its blue color. The earlier
myelocytes, with brilliant blue cytoplasm and deeply stained granules (cells 4
through 9), are rarely seen in the peripheral blood except in myelosis. The
older and less intensely stained neutrophilic myelocytes (cells 10 through 12)
often appear in the blood stream during the so-called "shift to the left" in severe
infectious processes (Schilling). Thus, from a practical standpoint, it is impor-
tant to distinguish these various types of myelocytes, since the earlier types
would suggest a leukemic myelosis and the more mature types an infectious
process of considerable severity.
The earliest eosinophilic myelocyte (celJ '3) usually has a blue cytoplasm
with dull orange or bronze granules which ·are not so markedly eosinophilic as
in the later forms. Occasionally a cell occurs in which both eosinophilic and
basophilic granules appear. This phenomenon is supposed to indicate a dis-
 PLATE VI

THE DEVELOPMENT OF A MYELOCYTE

1. Young myelocyte with blue cytoplasm and briUiant granulation.

2 and 3. Later myelocytes.
4 and 5. Young juveniles with slight indentation of nutlei and a retention of blue in the cyto-
plasm.
6, 7, 8 and 9. Juveniles maturing by indentation of nUclei and development of a more delicate
type of granulation.
IO. Band (staff or stab form of Schilling).
II. Band showing narrowing and curling of nucleus.
12. Band just preceding segmentation.
:13. Segmenter with two. lobes. separated by a filament of chromatin.
14. Segmenter with three lobes separated by two filaments.
IS. Segmenter with four lobes separated by three filaments.
16. Segmenter with five lobes separated by four filaments (hypersegroented).
17. Segmenter with six lobes separated by five filaments (the senile neutrophil).
:l8. Segmenter with seven lobes separated by six fi.laments.
 Plate VI.

, .
,

~
,::'-:

..
 MYELOBLASTS AND MYELOOYTES 71
Cooke and Ponder have classified these cells according to the number of lobes.
In Arneth's classification cells I through 12 belong to class I, cell 13 to class
2, cell '4 to class 3, cell IS to class 4, cells 16, 17 and 18 to class S. Therefore, there
is little difference between these classifications except instead of counting filaments,
Arneth counts the number of lobes. Both classifications should be abandoned
in favor of the Schilling classification. For further details of the various methods
for making differential leukocyte counts, see Chapter 10.

BIBLIOGRAPHY
M YELOBLASTS
MULLER, H. F.: "Zur Lcukamie-Frage; zugleich ein Beitrag zur Kenntnis5 der Zellen und der
Zelltheilungen des Knochenmarks." Deutsch. Arch. I. klin. Med., 48, 47, 1891,
NAEGELI, 0.: Blutkrankheiten und Blutdiagnostik, ed. 5, J. Springer, Berlin, 1931.
SABIN', F.: uBone Marrow." Physiol. Rev., 8, 191, 1928,
SABm, F. R., AUSTRIAN, C. R., CUNNINGHAM, R. S., and DOAN, C. A.: j'Studies on the Maturation
of Myeloblasts into Myelocytes and on Amitotic Cell Division in the Peripheral Blood in
Subacute Myeloblastic LeukemiaY Jour, Exp. Med., 40, 845, 1924.

MYELOCYTES

ARNETH, J.: "Die neutrophilen weissen Blutkorpcrchen bci InfekHons-Krankheiten," G. Fisher,

Jena, 1904.
COOKE, W. E., and PONDER, E.: "The Polynuclear Count." Lippincott~ Philadelphia, 1927.
FARLEY, D. L., ST. CLAIR, H., and REISINGER, J. A.: liThe Normal Filament and Non-Filament
Polymorphonuclear Neutrophil Count; Its Practical Value as a Diagnostic Aid." Am. J. Med.
Sci., 180, 336, 1930,
GROAT, W. A., WYATT, T. C., ZIMMER, S. M., and FIEJ.D, R. E.: (IAcute Basophilic Leukemia."
Am. J. Med. Sci., 191, 457, 1936.
ISAACS, R: "Present Status of the Study and Treatment of Leucemia." J. Lab. and CUn. Med.,
17, 1006, 1932.
ISAACS, R., and DANIELIAN, A. C.: HMaintenance of Leukocyte Level and Changes During Irradia-
tion; A Study of the White Blood Corpuscles Appearing in the Saliva and Their Relation
to Those in the Blood." Am. J. Med. Sci., 174, 70, 1927.
KRACKE, R. R., and G.4.RVER, H. E.: UHypocytic Leukemia (Aleukemic Leukemia)." internat.
Clin., 14, 37, 1935.
PONS, C., and KRUMBHAAR, E. B.: "Studies in Blood Cen Morphology and Function. Extreme
Neutrophilic Leukocytosis with a Note on a Simplified Arneth CounU' Jour. Lab. and CUn.
Med., 10, 123, 1924.
ScHILLINC, V.: "The Blood Picture and Its Clinical Significances/' ed. 8. Translated by R. B. H.
Gradwohl. C. V. Mosby Co., St. Louis, 1929.
SCHILLING-TORGAU, V.: "Ein praktisch und zur Demonstration brauchbarcr DifferentiaUeukozyto-
meter mit Arnethscher Verschiebung des Blutbildes." Deutsch. Med. Wcltnschr., 37, IIS9, 19II.
 PLATE VII

LYMPHOCYTES

QQQ
()t)o
QQQ
000
n 10 lZ

QQQ
0
16
00 17 16,
I, 2, 3 and 6, Lymphoblasts drawn from a case of acute lymphatic leukemia.
4. Large lymphocyte with lobulated nucleus and vacuole in the cytoplasm drawn from a case
of infectious mononucleosis. (Lymphoblast?).
5 and 7. Large lymphocytes with azure granulation.
8, 9, 10, II and 12. Large lymphocytes.
13, 14, IS, 16, 17 and IS.,Intermediate and small lymphocytes,
Piate VII.
Plate VIII.
 PLATE VIII

MONOCYTES

I. Monoblast with one nucleolus and light blue cytoplasm.

2. Monoblast with two nUcleoli.
3. Monoblast with dark, basophilic cytoplasm and one vacuole.
4. Young monocyte with indented nucleus and no cytoplasmic granules.
5. Young monocyte with four vacuoles in the cytoplasm.
6, 7 and 8, Monocytes with slightly indented nuclei and azure granulation.
g. Monocyte with mature nucleus and no cytoplasmic granules.
10. Monocyte with a long averiolding nucleus, a cytoplasmic pseudopod and granular cytoplasm.
1 I. Monocyte with excessive granulation.
12. Monocyte with two pseudopodia and no granules,
13 and 16. Monocytes with blue non-granulated cytoplasm.
14 and 17. Monocyte5 with azure granulation near the hoi of the nucleus.
15. Monocyte with irregular cytoplasmic border.
18. Mature monocyte with hOf5e-shoe nucleus and small azure granules scattered over the surface
of the cytoplasm.
76 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

Other hematologists have been equally sincere in their belief that monocytes
are derived from lymphocytes. Conspicucius in this group are the members of
the monophyletic school, including Maximow, Pappenheim and others. Bloom
has been especially emphatic in denying the existence of a separate stem cell
for monocytes. He believes that monocytes are associated with lymphocytes by a
large number of transition forms.
In 1912, Schilling-Torgau first expressed the idea that the monocyte
should be separated on morphologic grounds from both lymphocytes and granulo-
cytes. Evidence was added to this theory in 1913 )Vhen Reschad and'Schilling"
Torgau reported the first case of monocytic leukemia and, therefore, the first
blood disease involving the monocytes as a separate strain of leukocytes. Since
that time, numerous cases have been reported in which supravital studies have
resulted in the accumulation of evidence in favor of the trialist"concept that
monocytes arise from connective tissue and have a separate cycle of maturation.
Mum confusion has arisen regarding the nomenclature of the cells of the
connective tissue and their relation to the cells of the peripheral blood. A large
group of investigators believe that monocytes arise from histiocytes (clasmato"
cytes). Doan and Wiseman have reviewed this question in detail and have cleared
up many controversial points.
As early as 189I, certain phagocytic cells of connective tissues were called
"clasmatocytes" (Ranvier). Metclrnikoff classified the phagocytes of the body
into two groups: first, microphages, a term for the circulating, neutrophilic phago"
cytes, and second, macrophages, a term for the phagocytic tissue cells, which
include the Kupffer cells of the liver and the large mononuclear phagocytes of
the tissues, splenic pulp, and lymph nodes. Marmand demonstrated that clas"
matocytes were derived from adventitial cells of blood vessels and during in"
flammatory processes these adventitial cells were converted into macrophages.
Maximow designated the phagocytes of inflammatory tissue as "polyblasts."
Ribbert found that the tissue phagocytes took up inert dyes, but fibroblasts and
muscle fibers failed to exhibit this phenomenon. Finally, Goldmann showed by
the use of pyrrhole blue reactions that clasmatocytes, adventitial cells and poly"
blasts were merely different names for the same cell. Later Aschoff and Kiyono
called this tissue cell a histiocyte and advocated the reticulo"endothelial concept
of phagocytosis. At that time, the most popular theory for the origin of the mono"
cyte was from the connective tissue macrophage (histiocyte, clasmatocyte, etc.)
and it was generally believed that tissue phagocytes and circulating monocytes
were merely stages of the developmental cycle of a single strain of cells. Dame"
shek concurs in this idea and points out that there are numerous transition forms
between histiocytes and monocytes. He believes that both cells are derived from
the same source, and that histiocytes become monocytes after mitotic division.
With the introduction of supravital technic in which living blood cells are
subjected to Janus green and neutral red dyes the relationship of circulating
monocytes to tissue macrophages has been seriously questioned. With this
st!11ninO'mptnnii "111'1'1pr'-'11<;:: ;n'UPc.:.tlO'!1trwc;). (~!1hin nn~n rlmnlnah!1m Wil;:.pm!1n)
h
a
 LYMPHOCYTES, rvloNOCYTES AND PLASMA CELLS 77
vacuoles clustered in the hof of the nucleus. This peculiar arrangement is not
found in clasmatocytes, lymphocytes or granulocytes. These supravital struc-
tures are not identical with the delicate azure granulation demonstrable with
polychrome dyes. These investigators have presented evidence to show that the
monocytes are present in both tissues and peripheral blood and can be separated
from clasmatocytes in their origin and cycle of development. The clasmatocyte
is a larger ceIl than the monocyte. It rarely appears in the peripheral blood and
is extremely phagocytic for large bodies such as red and white cells. With
supravital stains it shows pronounced, large, neutral red segregation vacuoles
ranging from yellow to deep maroon. The clasmatocyte lining the sinusoids of
the liver, spleen and bone marrow, has its origin from endothelium and has no
distinctive maturation cycle. The Kupffer cells of the liver and the splenic
macrophages are clasmatocytes.
The monocytes have their origin from the reticular cells of connective tissue
and show a maturation cycle as follows: (I) reticular cell; (2) primitive free
cell; (3) monoblast; (4) young monocytes; (5) mature monocytes. The cells
become motile and some remain in the tissues while others circulate in the periph-
eral blood. These cells differ in their cycle from other leukocytes since every
cell is capable of division, even the mature forms in the peripheral blood.
Thus, these investigators have divided the phagocytes of the so-called reticulo-
endothelial system in two types: (r) monocytes derived from the reticulum
and identical with the monocytes of circulating blood with an individual matura-
tion cycle similar to .lymphocytes and granulocytes; and (2) clasmatocytes
originating from endothelium, which remain in the tissues and do not circulate
in the peripheral blood under normal' conditions.
The phagocytes of the blood and tissues are shown as follows:
Tissue phagocytes Circulating phagocytes
Clasmatocytes Monocytes Monocytes Granulocytes
(Histiocytes)
THEORIES OF ORIGIN

From the foregoing discussion it is noted that there are five outstanding
theories for the origin of the monocyte: (r) it represents a "transitional" form
in the granular series; (2) it arises from the myeloblast; (3) it arises from the
lymphocyte; (4) it has its origin from the histiocyte (clasmatocyte); (5) it is
a separate strain of leukocytes with an individual stem cell, the monoblast, which
in turn arises from the reticular cell of connective tissue. The last concept is
summarized in the following diagram and shows the relation of the monocyte to
other circulating cells and to clasmatocytes (modified after Sabin et al.) :
Reticulum "'- Endothelium
Bone Marro/ Lym~hoid "'- Connective Tissue
/
Bone marrow
"'-
Generalized
Reticulum Reticulum Reticulum Endothelium Endothelium
I I I I I
Myeloblast Lymrhoblast Monoblast Megaloblast Clasmatocyte
I I I (Histiocyte)
Granulocytes Lymphocytes Monocytes Erythrocytes
 DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

FUNCTION AND OCCURRENCE

Monocytes are thought to be active in the phagocytosis of non-pyogenic

bacteria, as seen in syphilis, typhoid fever, and undulant fever, and also in the
ingestion of certain particulate matter (Doan and Wiseman). Sabin and her
associates have contributed to the knowledge of the function of monocytes fn
their extensive study of the role of these cells in tuberculosis. They have found
that the epithelioid cell of the tubercle is an altered type of monocyte, and that
monoblasts are' markedly increased around the tubercle bacilli prior to the
formation of the tubercle and also prior to the increase of mature monocytes
in the peripheral blood. A peripheral monocytosis in tuberculosis indicates the
active formation of tubercles and has resulted in the use of the so-called mono-
cyte-lymphocyte ratio. This is based on the belief that if monocytes are
abnormally increased in the peripheral blood in tuberculosis an active tubercu-
lous process is indicated, while predominance of lymphocytes indicates relative
quiescence. Anderson has studied the chemical nature of this response and has
shown that lipoids, especial1y tuberculo-phosphatides, are directly responsible for
monocytic proliferation. This has led to the suggestion that other lipoids may
possess a smiliar monocytoid stimulus (Doan and 'Wiseman), since there is fre-
quently a monocytic proliferation in conditions of disturbed lipoid metabolism.
Monoblasts and mature monocytes occur, respectively, as the predominant cell
types in acute monoblastic and chronic monocytic leukemia. Monocytosis is
found in chronic malaria but not in the acute stage of the disease. In the latter,
there is a granulocytic immaturity or a "shift to the left."
MORPHOLOGY (SEE PLATE 8)
The typical monoblast (cells I and 2) presents few cytological criteria for
positive differentiation from the myeloblast and lymphoblast. It closely re-
sembles the myeloblast in the finely-woven reticulum of the nucleus, but, like
the lymphoblast, contains only one or two nucleoli. The blue of the cytoplasm
is usually more basophilic than that of myeloblasts but less intense than the
cytoplasm of lymphoblasts. Under abnormal conditions, however, the color
varies from the delicate blue of cell I to the intense color of cell 3. As the mono-
blast matures the cytoplasm becomes a pale, milky blue and the nucleus becomes
indented (cell 4). Small, fine azure granules appear in the cytoplasm, usually
near the nucleus which continues to indent and appears to fold over upon itself
(cells '4 and '7). Cell 13 is a mature monocyte with a horse-shoe nucleus and
pale clue cytoplasm filled with azure granulation. Cell 10 shows a protrusion of
typical cytoplasmic pseudopodia. Under abnormal conditions these cells exhibit
many variations from the normal, such as excessive granulalion (cell II), irregu-
larity of cytoplasm (cells 12 and IS), vacuolization (cell 5), and maturation of
the nucleus without the appearance of cytoplasmic granulation (cell 9).
Doan and Sabin have shown that a majority of the monocytes of the circu-
lating blood are peroxidase positive, but a few of them are entirely negative. This
leads one to infer that monoblasts and young monocytes react negatively and
mature forms react positively to this stain. Similar reactions are obtained with
the indophenol blue synthesis.
 LYMPHOCYTES, MONOCYTES, AND PLASMA CELLS 79
The cells in Plate 8 illustrate the varied morphology of monocytes. There-
fore, it is illogical to present a drawing of only one cell and lahel this as a "char-
acteristic monocyte." In our opinion, a single characteristic monocyte, which
would represent the morphological characteristics of all of the cells of this group,
does not exist. The same statements may be applied to lymphocytes, granulo-
cytes and other types of cellular structures. It is easy to understand morphologic
variations in the cell type when one considers the varied factors that influence
maturation and cell division. A monocyte in its early stage of maturation may
show a nuclear development that proceeds much faster than that of cytoplasmic
granulation, or conversely, cytoplasmic granulation may occur at a more rapid
rate than nuclear development. It can be seen, therefore, that when variations of
this nature occur, a wide variety of morphologic pictures will be the result.
In the various cells of the series, the granules may be either numerous, scarce,
or entirely absent. The cytoplasmic color may vary from pale to intense blue.
The nucleus may be either round, lobulated, overfolding, horseshoe shaped, or
band shaped. A series of cells is shown in the accompanying plate in order to
illustrate these morphologic variations.

PLASMA CELLS
DESCRIPTION. (See Plate 9)
Plasma cells present a number of unique characteristics which distinguish
them from the other cells of the blood. The nuclei are round or oval in shape
and, as a rule, extremely eccentric in position (cells 6, 9, 12, '7 and IS), often
appearing partially extruded from tbe cytoplasm. The nuclear chromatin is ar-
ranged in dense masses and clumps which are separated and sharply outlined
(cells 9 and 10). Frequently, these masses are grouped in a radial arrangement
resulting in the typical "cart wheel" design of the nucleus (cell II). An oc-
casional nucleus 'has a large blue nucleolus (cell 3). There is a definite nuclear
membrane which is not always continuous but appears broken, Or rather limited
to the individual masses of chromatin (cell 9). The cytoplasm is usually in-
tensely blue in color and mottled in structure, but many cells have a lighter
sky-blue color similar to that of large lymphocytes (cell '3). Some authors
attribute this decrease of basophilia to a process of degeneration. It seems more
logical that it merely represents a maturation or aging of the cell. Vacuoles are·
frequently found in the cytoplasm (cells 7 and 8). The most distinguishing cyto-
plasmic characteristic is the occurrence of a distinct, colorless area around the
nucleus resulting in a sharp contrast between the deeply stained periphery and
the pale perinuclear zone (cell ,6). Osgood and Hunter state that acidophilic
granules may occur in an occasional cell under abnormal conditions. These
granules may be scattered over the cytoplasm, but usually they are clustered in
a girdle around the nucleus (cell 4). All plasma cells are peroxidase negative and
react similarly to lymphocytes in supravital preparations.
ORIGIN AND FUNCTION

In '931, Michels presented a comprebensive review of the plasma cell in

regard to its morphology, function and development. The high points of his
 PLATE IX

PLASMA CELI,s

I. Large plasma cell with oval nucleus, brilliant blue cyt,DpIasm and distinct pallor (peri-nuclear
pallor) around the nucleus.
2. TUrk cell with blue nucleolus and no peri-nuclear pallor. (Is a TUrk cell a plasma cellr)
3. Large plasma cell with round nucleus and blue nucleolus.
4. Plasma cell with peri-nuclear azurophilic granulation. (An extremely rare form,)
5. Large plasma cell with a bizarre nudeus.
6, Large plasma cell with nucleus apparently protruding from cytoplasm.
7. Plasma cell with large vacuoles.
8. Plasma cell with small vacuoles.
9 and 10. Plasma cells with sharp separation of nucle<Jr clumps.
II. Plasma cell with typical cart-wheel arrangement of nuclear chromatin.
12. Smaller plasma cen with extruding nucleus.
13. 14 and 15. Plasma cells corresponding in size and color of cytoplasm to 1arge lymphocytes.
16, 17 and 18. Plasma cells which in many respects reselllble megaloblasts.
Plate lX_
Plate X .

i'~ ....
~.'
 PLATE X

DEGENERATED AND FRAGMENTED LEUKOCYTES

1: and 2. Smudge forms with nucleoli from the blond of a patient with myeloblastic leukemia.
These cellular remnants are presumably degenerated forms of myeloblasts as indicated by the
presence of nucleoli.
3. Smudge form without nucleoli from myeloblastic leukemia.
4, 5 and 6. Cellular remains from a case of chronic myeloid leukemia (basket cells).
7. A lymphocyte with an atypical clover-leaf nucleus from oxalated b~ood.
8. A smudge form drawn from a case of acute lymphoblastic leukemia. The presence of a
nucleolus suggests that it is a degenerating lymphoblast.
9. A smudge form from a case of chronic lymphatk leukemia; probably a remnant of a more
mature lymphocyte since there is no nucleolus.
10, IIand 12. Disrupted monocytes from a case of monocytic lcuRcmia.
73. Ruptured neutrophll.
14. Ruptured eosinophil.
1$. Ruptured basophil.
82 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

summary illustrate the controversial issues which have evolved since the dis-
covery of this cell. Ramon y Cajal (r890) first descrihed its occurrence in
syphilitic condylomas. He considered the cell an embryonic type of normal con-
nective tissue and a derivative of lymphocytes. Unna, a year later, in a study
of patients with lupus coined the term "plasma cell" and claimed that it was a
pathological cell derived from connective tissue. Marschalk6 (,895), stressed
the spoke wheel arrangement of the nucleus and considered these cells normal
constituents of connective tissue arising from "emigrated hemic lymphocytes"
since he could find no transition forms between fibroblasts and plasma cells. He
believed, however, that plasma cells could be transformed into fibroblasts.
Since that time various investigators have shown these cells to be present
normally in interstitial tissue of organs and glands and have postulated numerous
theories concerning their origin. These theories are summarized by Michels
as follows: (r) a histiogenic origin from connective tissue cells (histiocytes,
clasmatocytes, tissue lymphocytes, fihroblasts, resting wandering cells, adventi-
tial cells, hemohistioblasts, etc.); (2) a hematogenic origin from emigrated
lymphocytes; (3) a mixed origin from emigrated lymphocytes (monocytes) or
preexistent tissue lymphocytes; (4) an origin from immature blood cells (myelo-
hlasts, erythroblasts, granulohlasts) through aberration or abortion. Michels
adheres to the third theory.
Michels' summary of the theories for the function of plasma cells is as follows:
(I) local absorption of chromatic material in areas where there is nuclear de-
struction; (2) transportation of nuclear material; (3) a transient, irritative,
physiological condition of lymphocytes at which times the cells function as
secretory corpuscles; (4) the elaboration of antitoxic substances; (5) phago-
cytosis.
Osgood and Hunter, in a study of plasma cell leukemia, state the hypothesis
that the plasma cell is a distinct entity and question the generally accepted
theory that these cells are altered or aborted stages of other cell types such as
lymphocytes, myeloblasts, and megaloblasts. They could not find any transition
forms between the plasma cell and any of the other cell types. They state that
plasma cells differ from lymphocytes in their large size ,and type of granulation,
and from lymphohlasts, in their amitotic type of division. Furthermore, plasma'
cells are easily distinguished from myeloblasts and fibroblasts and, although
some of the smaller forms closely resemble megaloblasts, they fail to develop
hemoglobin and never mature into normoblasts. The behavior of plasma cells
suggests that they comprise a separate strain of leukocytes; the plasma cell
is present normally in small numbers in the hone marrow and peripheraJ hlood;
it is subject to increased numbers in the blood in response to a stimulus as seen
in German measles; it migrates to tissues in various types of subacute and
chronic inflammatory processes; it is subject to benign and malignant forma-
tions and to leukemic and aleukemic manifestations.
In German measles, mUltiple myeloma, and plasma cell leukemia, Osgood
and Hunter have found large numbers of cells conforming to the description
of the Turk "irritation" forms; and they have observed many transitIon forms
between Turk cells and plasma cells. This suggests that Piney's lymphoblastic
 LYMPHOCYTES, MONOCYTES, AND PLASMA CELLS 83

plasma cells are actually TUrk forms, and that the TUrk cell may he the im-
mediate precursor of the plasma cell. Piney, Downey, and Naegeli are promi-
nent in the list of authorities who believe that plasma cells have their origin from
lymphocytes.
The cells illustrated in Plate 9 were drawn from Osgood and Hunter's case
of plasma cell leukemia. Cell 2 represents the young TUrk form with a more
homogeneous nucleus and a blue nucleolus. If one concurs with the idea that
the TUrk cell is the precursor of the plasma cell and accepts Doan and Wise-
man's theory that the plasma cell has its histiogenesis from a primitive cell, the
TUrk cell, therefore, should correspond in age to the myelohlast, lymphoblast, and
monoblast. Although the terms, "TUrk cell" and "plasma ceil," arc poor and
nondescriptive, it is hazardous to change their names until their origin is cer-
tain. If the TUrk cell is proved to be the progenitor of the plasma cell, it should
be called "plasmoblast'" and the plasma cell, "plasmocyte." The above theory
is shown in the following chart:
Reticular cell

'"
Primitive free cell

'"
Plasmablast (Turk cell)

'"
Plasmacyte (Plasma cell)
OCCURRENCE AND DISTRIBUTION

Plasma cells occur in small numbers in the blood of normal cold blooded
and warm blooded animals (Downey). These cells are found increased in the
blood of patients with German measles, in the infiltrations of syphilis, in multiple
myeloma, and in plasma cell leukemia (Osgood and Hunter). They are also in-
creased in the tissues in chronic inflammations, in lupus, in granulomata of the
skin, in epitheliomata and papillomata, in the nervous system in encephalitis, in
tubercles, and in plasma cell myelomata (Michels).

DEGENERATIVE LEUKOCYTES
(See Plate 10)

Smudge forms (cells I, 2, 3, 8 and 9) are said to represent the degeneration

or destruction of various mononuclear cell types. They appear to be nuclear
remains after loss of cytoplasm. It is possible that they indicate an increased
fragility of leukocytes. When nucleoli are present, they can 'be identified with
fair certainty as the remains of young immature leukoblasts. They are present
in normal blood only occasionally and in small numbers. The presence of smudge
forms in large numbers suggests either an excessive destructive process or an
increased fragility of leukocytes or both. These forms are found in large num-
bers in acute myeloblastic leukemia, acute lymphoblastic leukemia, and chronic
lymphatic leukemia, but only in small numbers in chronic myeloid leukemia.
A majority of smudge forms in the acute leukemias contain nucleoli. In chronic
lymphatic leukemia, the smudges arc often more numerous than the typical cell
84 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

types. For this reason smudge forms are often considered pathognomonic of
lymphatic leukemia. This is a misconception; they always occur in lymphatic
leukemia but they appear in any condition where there is excessive degeneration
or peripheral destruction of mononuclear forms.
It has been stated that smudge forms are degenerating lymphocytes and
that basket cells (cells 4, 5 and 6) are degenerating granulocytes. It is true
that the degenerating forms found in chronic myeloid leukemia have a more open
and loosely woven meshwork than those of lymphatic leukemia. Nevertheless,
it seems more probable that the smudge cell is an early stage and the basket cell
a later stage of the same process.
Crushing and rupturing of monocytes, neutrophils, eosinophils and basophils
(cells II, 12, 13, 14 and IS) occur in improperly made smears, especially when
too much pressure is applied to the drop of blood. These cellular remnants are
found in various abnormal states where there is excessive destruction of leuko-
cytes. In these cases their occurrence is probably the result of toxic agents or of
an increased fragility of the cellular elements. Eosinophils are frequently dis-
rupted in cases of eosinophilia and chronic myeloid leukemia.

BIBLIOGRAPHY
LYMPHOCYTES AND IVfoNOCYTES

ANDERSON, R. J.: "The Chemistry of the Lipoids of Tubercle Bacilli." Physiol. Rev., 12. 166,
1932.
ASCHOFF, L., and KIYONO, K.: "Zur Fragc der grossen Mononukldircn." Folia Haemat' 15, 383,
J

1913.
BLOOM, W.: "The Origin and Nature of the Monocyte," Folia Haemat., 37, I, 1928.
CUNNINGHAM, R. R.. SABIN, F. R., SUGIYAMA, F. R., and KUNDWALD, J. A.: "Role of the Mono-
cyte in Tuberculosis." Bull. JOh1lS Hopkins Hasp., 37, 231. 1925.
DAMESHEK, W.: "Acute Monocytic (Histiocytic) Leukemia." Arch. Int. }.led., 46, 7r8, 1930.
DoA...'T, C. A.: "Newer Aids to Diagnosis and Prognosis in Active Tuberculosis." Med. eli/I. N.
Amer., 14, 279, 1930.
DOAN, C. A., and SABIN, F. R: "Normal and Pathological }~ragmentation of Red Blood Cells.
The Correlation of the Peroxidase Reaction with Phagocytosis in Mononuclear Cel1s." Jour.
Exper. lIfed., 43, 839, 1926,
DOAN, C. A" and WISEMAN, B. K.: "The Monocyte, Monocytosis and Monocytic Leukosis: A
Clinical and Pathological Study." Ann. Int. Med., 8, 383, 1934.
GOLDMANN, E. E.: !'Die aussere und innere Sekretion des gesundcll und kranken Organismus im
Lichte der vitalen Farbung." Bcitr. z. klin. Chir" 64, 192, 1909.
MAXIMOW, A.: "Experimentelle Untersuchungen tiber die entztindliche NeubiIdung von Binde-
gewebe." Beitr. z. path. Anat. u. aUg. Path. Suppl" 5, I, I902.
MERKLEN, p" and WOLF, M,: HLe Monocyte. Cytologie et histiogenese du mononuc1eaire granu-
leux des tissus et du sang:" Ann. dJanat. path., 4, 621, I927.
METCHNIKOFF, E.: uLe~ons sur la Pathologie Comparee de l'Inflammation." G. Masson, Paris,
I892.
NAEGELI, 0.: n~!!Ikrankheiten und Blutdiagnostik, ed. 5, J. Springer, Berlin, 1931 j and Personal
Communication,
PINEY, A.: "Recent Advances in Hematology.H P. Blakiston's Son and Co., Inc., Philadelphia,
ed. 3, 193I.
RANVIER. L.: "De l'Origine des cellules du pus ct du role de ces elements dans les tissus en~
flammes." Compt. rend. Acad. d. sci. Par., 120, 922, r891.
 LYMPHOCYTES, MONOCYTES, AND PLASMA CELLS 85
REICH, C.: !fA case of monocytoid myeloblastic leukemia." N. Y. Slate Med. Jour., 32, 1193, 1932.
RESCHAD, H., and SCRILLlNG-ToRGAU, V.: Ueber eine neue Leukamie durch echte Uebergangsformen
(Splenozytenleuklimie) und ihre Bedeutung fUr die SelbsHindigkeit dieser Zellen." Munchen.
med. Wchnschr., 60, 1981, 1913.
RmBERT, H.: "Die Ausscheidung intravenous injizierten gelOsten Carmins an den Geweben." Ztschr.
f. AUg. Physioi., 4, 201, 1904.
SABIN, F. R.; "Origin of Blood Celis," Physiol. Rev., 2, 38, 1922.
SABIN, F. R., DOAN, C. A., and CUNNINGHAM, R. S.: "Discrimination 'of Two Types of Phagocytic
Cells in the Connective Tissue by the Supravital Technic." Contrb. to Embryol., 16, 125,
19 2 5.
SABIN, F. R., DOAN, C. A" and FORKNER, C. E.: "Studies on Tuberculosis." Jour. Exper. Med.
Supplemented, 3, 1, 1930.
SCIDLLING-TORGAU, V.: "Das Blutbild und seine klinische Verwertung." G. Fisher, Jena, 1912.

PLASMA CELLS
DOAN, C. A, and WISEMAN, B. K.: "The Monocyte, Monocytosis, and Monocytic Leukosis: A
Clinical and Pathological Study." Annals Int. Med., 8. 383, 1934.
DOWNEY, H.: "The Origin and Structure of the Plasma Cell 'of Normal Vertebrates, Especially of
the Cold Blooded Vertebrates, and the Eosinophils of the Lung of the Amblystoma.l> Folia
Haemat. Archiv., II, 275, 1911.
MICHELS, N. A.: "The Plasma Cell: A Critical Review of Its Morphogenesis, Function and Devel-
opmental Capacity under Nonnal and Abnormal Conditions." Arch. Path""':, 11, 775, 1931.
NAEGELI, 0.: Blutkrankheiten und Blutdiagnostik: ed. 5, J. Springer, Berlin, J93I.
OSGOOD, E. E., and HUNTER, W. C.: "Plasma Cell Leukemia," Folia Haemat' J Bd. 52. 369, 1934 .
. PINEY, A.: "Plasma CeH Leukaemia. Lymphadenosis Leucaemica PlasmaceUularis." Folia Haemat.
Arch' J 30, 173, 1924.
 CHAPTER 7
ERYTHROBLASTS, ERYTHROCYTES AND
THROMBOCYTES
ERYTHROBLASTS
ORIGIN AND DEVELOPMENT
In post-natal life erythrocytes have their origin from the endothelium lining
the capillary sinusoids of the bone marrow. A collapsed capillary bed, a slug-
gish flow of blood, and a low oxygen tension have been shown to provide the
favorable conditions for normal erythropoiesis (Sabin). In the healthy state
these conditions occur rhythmically and periodically in order to keep the rate
of production at sufficient speed to maintain the circulating erythrocytes at a
normal 1",,101. The stages in th" d"velopment of these cells have been studied
in the bone marrow during regeneration from an aplastic or hypoplastic state by
Doan and associates as follows: The endothelium of the blood vessels swells and
divides. When the equator of the division spindle is parallel to the wall of
the vessel, the outer cell becomes the endothelial wall and the inner daughter
cell becomes a megaloblast, the stem cell for this strain of blood cells. This cell
has been described in detail in Plate 3. It is a large cell with basophilic cyto-
plasma and a finely woven chromatin reticulum (Plate II, cells 5 and 6). The
cells are frequently larger than those shown on this plate, sometimes approaching
th~ size of the myeloblast.

DESCRIPTION. (See plate II)

In r880, Ehrlich distinguished two types of nucleated red blood cells, the
normoblast giving rise to the normocytes, and the megaloblast, seen only in the
embryo and in pernicious anemia giving rise to megalocytes. If megaloblasts are
stained while wet the nucleus shows from one to two nucleoli similar to those
of immature nuclei in the leukocytic series. Also the cytoplasm contains variable
amounts of hemoglobin. The average size of the megaloblast in the stained film
is about IS micra in diameter and the nucleus is about 12 micra in diameter.
The nuclear patterns of the megaloblast and normoblast are quite different. The
chromatin material in the normoblast is much coarser and usual! y appears in
clumps while in the megaloblast it is of a reticular type. Megaloblasts are seen
in normal bone marrow of the adult and of course in many anemic conditions.
Furthermore, intermediate stages between the megaloblast and normohlast can
be shown. Isaacs believes that the cell is a stage in the development of the
normoblast and that development into the latter cell is inhibited if the hemato-
poietic factor is deficient.
Many authorities do not concede the existence of the megaloblast in post-
natal life except in pernicious anemia. -They believe that the cells of the bone
87
 PLATE XI

NUCLEATED ERYTHROCYTES (ERYTHROBLASTS)

6)
15

I, 2, 3 and 4. Megaloblasts in division.

5 and 6. Early megaloblasts.
7, 8 and 9. Early macroblasts.
10 and II. Late macro blasts.
12. Erythroblast in process of karyorrhexis with basophilic stippling in cytoplasm.
13. Normoblast,
14. NormobJast in division.
15. Normoblast with nuclear extension or eccentric nucleus.
16, 17 and 18. Microblasts.
 Plate XL

• •
,>.~

•
.

• - •
•
 ERYTHROBLASTS, ERYTHROCYTES AND THROMBOCYTES 89
marrow called megaloblasts are merely early macroblasts. Cells 5 and 6 were
drawn from a case of pernicious anemia and we believe they correspond so
closely to similar cells which appear in small numbers in normal bone marrow
that it is impractical to separate them on morphological grounds.
There has been much confusion relative to the cells which represent inter-
mediate stages between the megaloblast with its basophilic cytoplasm and the
normoblast which contains hemoglobin and takes, the eosin dye. This inter-
mediate form is called erythroblast by Sabin and Haden and macro-normoblast
by Piney. The term "erythroblast" is somewhat confusing since all nucleated
red cells are erythroblasts whether they are mature or immature. The term
"macroblast," a contraction of the more cumbersome and more correct term,
macro-erythroblast, appears to be preferable. The intermediate macroblasts vary
in appearance and fall roughly into two classes, early and late forms. The early
macroblast (cells 7, 8 and 9) follows the megaloblast stage. The nucleus becomes
condensed and the chromatin freqnently assumes a spoke wheel arrangement.
The cytoplasm is basophilic but not so intense as that of the megaloblast. In
later macroblasts, hemoglobin begins to appear in the cytoplasm and the nucleus
becomes smaller and more pyknotic. These macroblasts differ from normoblasts
only in size and in the retention of a slight basophilia of the cytoplasm.
The next stage is the normoblast (cells 13 and IS) wbich is comparable in
size to the mature, circulating erythrocyte. It contains a dark, almost black,
pyknotic nucleus. Any nncleated red blood cell is capable of repeated divisions,
but normoblasts reproduce themselves at a more rapid rate than the more im-
mature forms. The smaller microblasts (cells 16, 17 and 18) are atypical forms
appearing only under abnormal conditions.
There are two critical stages in the life history of the erythrocyte, the megalo-
blast stage and normoblast stage. Castle's antianemic factor which is stored in
the liver seems responsible for the maturation from the megaloblast stage to the
normoblast stage. At this point hemoglobinization begins. Iron is the factor re-
sponsible for hemoglobin production and also influences division at the normoblast
stage and the growth of this cell to maturity.
The maturation of erythrocytes is shown in the following chart:

Endothelium
y
Megaloblast
y X hematopoietic factor
Early macroblast
y
Late macroblast
y
Normoblast
y
Reticulocyte Iron
y
Erythrocyte
 PLATE XII

ERYTHROCYTES

I. Hyperchromic macrocyte. J3. J4, 15 and 16. Reticulocytes.

2. Normochromic macrocyte.
3. Hypochromic macrocyte.
4. Polychromatophilic macrocyte.
5. Hyperchromic normocyte.
6. Normochromic normocyte.
7. Hypochromic normocyte.
8, Polychromatophilic normocyte.
9. Hyperchromic microcyte.
IO~ Normochromic microcyte.
II. Hypochromic micro~yte.
12, Polychromatophilic microcyte.
J7, 18, 19 and 20. Erythrocytes with basophilic
stippling.
21 and 22. Cabot ring bodies.
23 and 24. I-IoweH-J ally bodies.
25, 26 and 27. PoikiIocytcs.
28. Polychromatophilic poikilocyte.
29, ,30 and 31. Sickle cells (meniscocytes).
32. Polychromatophilic sickle cell.
33, 34 and 35. Ovalocytes.
36. Polychromatophilic ovalocyte.
 Plate XII.

,"

;'

J
{
 Plate XMl.

J
~' ..

-,,;

.. ..
Q' .-
• .\t
;;: ...
';c
,.
._
v:
'

"
~

'"
..c:J;
~

"
.It'
•..
\~
" ..
.;- ~:o\".~..".
_

H(>rf~n.$f!! GortF('r.
 PLATE Xln

THROMBOCYTES (PLATELETS)

. .:~~
~......:~.:.,
:'·;)5
I and :2:. Thrombohlasts with no cytoplasmic granu.lation (from bone marrow). Presumably
the precursor of the megakaryocyte. (Naegeli calIs these megakaryoblasts.)
3, 4, 5 and 6. Megakaryocytes with definite diffuse cytoplasmic granulation (from bone marrow).
j, g and 9. Later stages of megakaryocytes, showing cytoplasmic irregularity, pseudopodia, "and
beginning detachment (from peripheral blood in myeloid leukemia).
10. Late megakaryocyte or early giant thrombocyte with nuclear fragmentation (from peripheral
blood in chronic myeloid leukemia). '(
II, 12and 13. Clusters of large cellular thrombocyte~ (platelets),
14 and 15. Clusters of smaller thrombocytes (platelets).
92 THE DEVELOPMENT AND M01CPHOLOGY OF BLOOD CELLS

OCCURRENCE

Megaloblasts are present in small numbers in normal bone marrow but they
never appear in the normal blood except in a few conditions. Marrow smears
taken soon after birth show a preponderance of megaloblasts which may appear
in the blood of children in response to severe bone marrow stimuli. They occur
in pernicious anemia in relapse. In this condition the hematopoietic maturation
factor of Castle, which is produced in the stomach and stored in the liver, is
absent and maturation of erythrocytes stops at the megaloblast stage. The
bone marrow becomes crowded with megaloblasts, and some of these cells escape
into the peripheral blood. Megaloblasts are sometimes found in blood in leuke-
mias when proliferation and infiltration interfere with the cellular activity of the
bone marrow and the storage of the erythropoietic factor in the liver. Division
forms (cells 1, 2, 3 and 4) occur normally in tlle maturation cycle, but their
presence in the peripheral blood indicates a marked proliferation of the megalo-
blastic elements of the bone marrow and a maturation arrest af this leve!'
Macroblasts and normoblasts constitute about ninety per cent of bone mar-
row erythroid elements. They may appear in the peripheral blood when the rate
of blood loss or blood destruction considerably exceeds the rate of production of
new cells. Under ordinary conditions the bone marrow is capable of producing
new cells to compensate for reasonable loss so that in anemias due to hemorrhage
there is generally a reticulocytosis to show an increased bone marrow activitY7 but
only rarely a normoblastemia. Here a healthy bone marrow meets a need by the
speeding up of maturation, and by an increased production of new cells. When,
however, the rate of destruction exceeds the ability of the bone marrow to re-
produce, or the bone marrow. is incapable of normal maturation and division,
there is an <ilnemia and an erythroblastosis. This occurs frequently in hemolytic
jaundice, in sickle cell anemia, in von Jaksch's anemia, and in certain stages of
leukemia.

ERYTHROCYTES

DESCRIPTION. (See Plate 12)

The normal circulating erythrocyte (cell 6) of human beings is a non-

nucleated, bi-concave disk varying from 7 to 8 micra in diameter and from
2 to 3 micra in thickness. In unstained preparations it has a pale yellow color.
In the fixed, stalned smear it has an affinity for the acid dyes, hence the term
"erythrocyte," meaning red cel!.' The normal form has a central pallor as por-
trayed in cell 6.

CHEMICAL CONTENT

T1:te strncture of the living red bllJod cell is relatively simple since it does
~t contain a nucleus but apparently has a functional type of envelope contain-
ing a stroma charged with hemoglobin. The red cell is quite tough since it can
be pulled or stretched, twisted or wrinkled without permanent distortion. It
reacts to varying degrees of salt solution as does any anatomical membrane. The
 ERYTHROBLASTS, ERYTHROCYTES AND THROMBOCYTES 93

surface appears to be semi-fluid. The internal st,ucture is probably of a col-

loidal type.
The red cell consists of an organic matrix or stroma, water, and a surface
condensation of lipins which act as an envelope, or a cell membrane (Cooke).
Hemoglobin is the most important constituent, comprising about thirty per cent
of the main cellular bulk. The cell contains small quantities of phosphatides,
cholesterol, glucose, ure;]., creatinine, creatine, uric acid, glutathione, thionine,
adenine-ribros-nucleotide, the inorganic ions of potassium, chloride, magnesium
and phosphate, small quantities of copper, and iron which is essential for the
elaboration of hemoglobin (Musser and Wintrobe). The average red corpuscle
contains frDIll 28 to 30 n1icro-rnicrograms of hemoglobin. '
Davidson and Leitch have summarized the important factors known concern-
ing the chemistry and metabolism of the components of the normal erythrocyte.
The total solid constituents arc proteins, 4 per cent; hematin, 4.3 per cent; and
globin 89.3 per cent. The hemoglobin of the stroma is elaborated by the poly-
merization of the iron compounds of pyrrole with the amino acids of globin. A
deficiency of any of these substances may be the causative agent in anemia, b:ut
it has been shown that a deficiency of iron invariahly results in the hypo-
chromic, microcytic type of anemia. It has recently been discovered that an anti-
anemic factor, divorced entirely from iron constituents, is responsible for the
maturation of the megaloblast to the normoblast stage (Castle, et al.). A de-
ficiency of this substance results in the macrocytic, hyperchromic type of anemia.
The erythrocyte acts as a passive vehicle for the transportation of bemo-
globin which carries oxygen to the tissues and removes carbon dioxide. Haden
states that the life span of an erythrocyte is from two to six weeks, and that these
cells are flushed into the blood stream at the rate of two to six trillion daily, or
nearly two billion per minute. Since the number of red cells is kept at a fairly
constant level (discounting temporary fluctuations due to a change in plasma
volume) there is a balance between the rate of production and tlle rate of de-
struction. A disturbance in this balance results either in polycythemia or
anemia.
The mechanism of blood destruction is not thoroughly understood. Hemol-
ysis, fragmentation, and phagocytosis have been suggested as the most probable
methods of rem ..ving non-functional cells from the circulating blood. The first,
hemolysis, has been proved to occur only in pathological conditions. Rous 'has
presented evidence to show that, after fulfilling their function in the blood stream,
erythrocytes undergo fragmentation, and that the fragments are removed by
the phagocytes of the reticulo-endothelial system. During this process hemo-
globin is set free and is broken up into globin and hematin. Hematin loses
its iron and is changed into bilirubin and bile pigment. The spleen has been
incriminated as the destructive agent of fragile erythrocytes in hemolytic
jaundiCe.
FACTORS INFLUENCING NUMBER OF RED CELLS

The number of red cells varies in the different capillary beds of the body.
94 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

The variation, however, is only slight. It has been stated that the red cell count
of a normal person may vary 5 per cent during the course of a day. The cells
are delivered rhythmically with a range as much as a million cells per cubic
millimeter between the high and low counts. Apparently they are not affected by
intake of food. The number of cells increases with exercise, apparently because
of release of storcd cells into the general circulation, the cells probably being
stored in the splcnic capillaries. Probably fluctuation in fluid volume in the
vascular system accounts for most discrepancies in red cells since dehydration
causes marked increases in counts. The red cells apparently are no different in
the various races of people.
In normal people there is a definite increase of red cells within 30 minutes
after passage from low to high altitudes. The count increases from two to five
hundred thousand in 30 minutes and one million cells in 24 hours. It is seen,
therefore, in men during balloon ascensions or i~ aviators. It has been calcu-
lated that there is an increased count of five thousand cells per cubic millimeter
for every 1,000 feet elevation. These sudden changes have been ascribed to
rapid water ]OS5 from the lungs, changes in distribution in circulation, rapid
production of new cells, direct effect of air pressure decrease and increased ultra-
violet light in high altitudes. There is little evidence that red cells are influenced
by particular types of climate or temperature. Apparently the season of the year
influences the count very little.
Wintrobe concluded that the number of cells, amount of hemoglobin and
cell volume were correlated with increased weight in men and with surface area
in women. The injection of adrenalin into the body or the development of an
emotional state that would increase the secretion of adrenalin causes a slight tem-
porary increase in red cells which may be seen within just a few minutes after
injection and may last for several hours. This is thought to be caused by con-
traction of the spleen or release of stored cells from the liver. It is said that
massage may cause a local increase in the number of red cells.
ROULEAUX FORMATION OF RED CELLS

'Vhen suspended in various solutions~ including their own plasma or serum,

red cells may exhibit a tendency to stack upon each other like a stack of coins.
This is known as rouleaux formation. The cause of rouleaux formation of red
cells is not definitely known. Optimum formation takes place in saline concen-
trations of about seven tenths per cent. There appears to be no relation between
viscosity of a solution and tendency to rouleaux formation. Hypertonic solution
decreases it because of tl,e crenation of cells and hypotonic solutions because of
development of hemolysis. It occurs in both plasma and serum. Rouleaux is
accentuated in exudates but is not affected in transudates. Normal serum loses
the property of forming rouleaux after standing twelve hours. Freezing and
thawing do not influence it. The property is retained in dried serum when
brought up to its original volume. There seems to be no relation between specific
diseases and rouleaux forming properties. It has been described as a combina-
tion of surface tension of red cells and adhesiveness of cells influenced perhaps by
protein elements in the serum.
 ERYTIIROBLASTS, ERYTHROCYTES AND THROMBOCYTES 95

VARIATIONS IN SIZE, SHAPE AND COLOR

In tbe various anemic states the erythrocytes show marked variation in size,
shape and staining properties. These variabilities are called, respectively, aniso-
cytosis, poikilocytosis, and polychromatophilia. Erythrocytes may vary in size
frOlll 2 to 3 micra to 10 to IS micra in diameter. Macrocytes (cells I, 2,3 and
4) are cells larger than normal and may he deeply stained, normal in appear-
ance, or paler than normal. They occur in any condition where there is an ab-
normal or aborted type of erythrogenesis. The cell fails to mature properly and
loses its nucleus before the cytoplasm bas decreased to normal size. Macrocytes
occur chiefly in anemias dne to a deficiency, faulty absorption, or faulty storage
of the maturation factor for erythrocytes (Castle's anti-anemic factor). The
most striking examples are found in perniciou~ anemia, anemias of liver damage,
von Jaksch's anemia, anemias of the new born, and anemias associated with sprue,
carcinoma of the stomach, and Diphyltobo.thrium latum infestations.
In conditions due to dietary deficiency or to faulty absorption or storage of
iron as well as long continued blood loss, the cells become predominantly micro-
cytic, or smaller than normal. These cells are called microcytes (cells 9, lO, I I
and 12).
In the different types of anemias erythrocytes vary in their hemoglobin con-
tent and consequently in the intensity of their color reactions. Cells 2, 6 and 10
are Hnormochromic" since they contain a normal quantity of hemoglobin. Cells
with an excess of hemoglobin are "hyperchromic" (cells I, 5 and 9) ; those with a
reduced amount are "hypochromic" (cells 3, 7 and I I). The terms ((normo-
cythemia Uhypercythemia" and tlbypocythemia" refer respectively to a normal,
j "

an increased, and a decreased total number of cells. Osgood, Wintrobe, and

Haden have classified the anemias according to the number, size, and hemoglobin
content of the average erytbrocyte. Theoretically this would result in 27 dif-
ferent blood pictures. The following types of anemia are outstanding examples of
tllis method of classification:
Hypocythemic macrocytic hyperchromic anemia (pernicious anemia)
Hypocythemic microcytic hypochromic anemia (chronic blood loss)
Normocythemic microcytic bypochron1ic aneJuia (certain iron deficiencies)
Hypocythcmic normochromic normocytic anenlia (aplastic anemia)
The red cell picture is described as "polychromatophilic" when a considerable
number of cells stain basophilic. This term means a variation in staining prop-
erties and the darkly stained cells arc called polychromatocytes (cells 4,8, 12, 24,
28,32 and 36). These dark cells are considered identical with reticulocytes (Key).
When cells assume abnormal shapes (tailed cells, racquet cells, sickle cells, oval
cells) they are called poikilocytes (cells 25 through 36) and an increase of these
bizarre shapes is called "poikilocytosis." These variations are found in severe
anemias; tbe sickle cell is found in sickle cell anemia of the negro race and
ovalocytes occur only as a rare hereditary anomaly. .
96 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

RETICULOCYTES

Reticulocytes (cells 13, '4, 15 and 16) are intermediate stages between
nucleated and non-nucleated erythrocytes. How the nucleus is lost and replaced
by a mitochondrial reticulum is not fully understood. The processes of extrusion,'
fragmentation, and solution have been suggested. According to the studies of
Cooke, the nucleus is lost by a process of karyolysis or by pyknosis and karyolysis
with or without karyorrhexis. His work discounts the theory of nuclear extrusion
which, if true, must be the aunique examp1e of cellular hara~kari in biology."
In normal blood about I per cent of the red cells show a reticulum. The
reticulum can be stained with methylene blue, brilliant cresyl blue or neutral
red stain. The reticulum can not be seen in the unstained red cell and it is not
shown in dark field .ilIumination. The reticnlocyte is slightly larger than the
mature red cell. Reticulocytes are seen in increased numbers in any situation
where the production of mature red cells is greater than normal. If hematopoietic
substn.nces are provided the bone marrow that is deficient in this material, there
is a marked reticulocytosis caused apparently by rapid maturation of megalo-
blasts to the reticulocyte stage. Minot and Castle have stated that anoxemia
is the probable ul timate stimulating mechanism since exposure of animals to re~
duced oxygen tension is followed by reticulocytosis. However, in certain diseases
characterized by anoxemia reticulocytosis is not always present. Reticulocytes
apparently do not participate very much in rouleaux formation. They seem to
be more sticky than the mature cells since they adhere to foreign bodies. They
a.re more resistant to hypotonic salt solution and also more resistant to heat than
older cells. The reticulum substance probably is acid since it has an affinity for
basic dyes. The length of life of a reticulocyte is not known but Keith believes
it is about 120 hours.
After the reticulum disappears the next step in red cell development is the
appearance of a single highly refractive granule as described by Isaacs. He
believes this represents a normal stage of maturation of red cells. He states that
the granule moves about in the red cell with a dancing motion and it can be
squeezed intact from the cell. They may be seen occasionally free in the blood
plasm. The number of granules containing red blood cells varies during the day
and bears a reciprocal relationship to the number of reticulocytes, increasing
when the latter decrease, and the sum of the two cells is remarkably constant,
maintaining a level of 3 to 5' per cent throughout the entire day. A reticulo-
cytosis is the first sign of red cell regeneration and always precedes a rise in
the total erythrocyte count. For this reason, a reticulocyte count is considered
the most accurate index to the efficacy of liver extract therapy and is an excel-
lent index of blood regeneration. Reticulocytes are increased in the new born,
in pregnancy, in all anemias characterized by excessive bone marrow activity -
(anemia of hemolytic jaundice, anemia of leukemia, sickle cell anemia, anemia
following hemorrhage) and in the regenerative stages of pernicious anemia. They
are absent in aplastic anemia and in pernicious anemia during relapse.
BASOPHILIC STIPPLING

Cells containing coarse and fine punctate basophilia or basophilic stippling

 ERYTHRODLASTS, ERYTHROCYTES AND TI-IROMllOCYTES 97

(in cells 17 through 20) arc found in severe anemias and, to a marked degree, in
lead poisoning. The exact nature of this basophilic material is unknown. It is
not considered a part of the nonnal maturation cycle; it bears no relation to re-
ticulocytes, and it is probahly not related to nuclear fragmentation. Cabot ring
bodies (in cells 2I and 22) and Howell-Jolly bodies (in cells 23 and 24) are, prob-
ably, minute chromatin remnants. These bodies appear in red cells in severe
anemias, and indicate a pathological type of erythropoiesis.

THROMBOCYTES (BLOOD PLATELETS)

ORIGIN, MORPHOLOGY AND FUNCTION

Thrombocytes or platelets constitute the third class of formed elements of

the blood. Although they are not true cellular elements, they play an important
part in the normal physiology of the blood. The normal platelet is a small body,
about 2 micra in diameter with a blue background, upon which are super-
imposed clusters of acidophilic granules.
The enumeration of these pseudo-cellular constituents has become an impor-
tant laboratory procedure, especially in connection with the hemorrhagic diseases.
The normal number is from 250,000 to 500,000 per cu. mm. depending upon the
method of counting.
In a study of 6I individuals from a group of mill workers and their families
in which from 2 to IS platelet counts were made on each person, we found that
the children of this group had a larger numher of platelets than adults and that
men had a higher number than women.
A lowered number of platelets predisposes to hemorrhage and oozing of blood
from the mucous membranes and into the tissues. An increased number is
frequently responsible for intravascular thrombosis. The number of platelets be-
comes important in making a differential diagnosis between the various hemor-
rhagic diatheses, as illustrated in idiopathic thrombocytopenic purpura and hemo-
philia; these conditions are characterized clinically by profuse bleeding, but the
number of platelets is markedly lowered in the former and normal in hemophilia.
Numerous theories have been proposed for the origin of platelets; some au-
thorities believe that they are fragments of erythrocytes; others suggest that
they arise from leukocytes, from protein precipitates of blood plasma, from lymph
follicles, or from endothelial cells of the marrow. Wright has presented the most
plausible theory-that thrombocytes are detached portions of the cytoplasm of
bone marrow megakaryocytes. These giant cells, shown in 'Plate I3 (cells 3, 4,
5, 6, 7 and 8), are very large, measuring as much as 40 micra or more; the
nucleus is irregular, often lobulated, and the chromatin arranged like the weave of
a basket. The cytoplasm is usually a clear blue and contains numerous, irregular,
clumped, acidophilic, peroxidase-positive granules, which may hecome clustered
into a girdle around the nucleus or scattered over the cytoplasm. According to
N aegeJi these cells appear in the embryo simultaneously with the appearance of
thrombocytes. They are actively motile and presumably protrude their pseudo-
podia between the endothelial cells of bone marrow vessels. Portions of these
98 THE DEVELOPMEl'I'T AND MORPHOLOGY OF BLOOD CELLS

protrusions are pinched off and are swept into the peripheral circulation as non-
motile, pseudocellular platelets (cell IS). Larger pseudopodia (10, II and 12) and
late megakaryocytes (cells 7, 8 and 9) may escape into the peripheral blood
under abnormal conditions in a manner analogous to the appearance of other
immature hematopoietic elements during marked regenerative activity. This
occurs most frequently in chronic myeloid leukemia and during certain stages
of polycythemia.

TABLE IX (From Olef)

DISEASE TOTAL NO. OF PLATELETS DIFFERENTIAL PICTURE
Chronic blood loss ................ ... .. Normal or reduced Increase of small forms
(Group I)
Acute blood loss .... .................. . Initial drop followed by an Increase of small forms
elevation (Group I)
Polycythemia Normal, reduced or ele- Increase of small forms in
vated, usually elevated group I, or of large forms
in group 3
Polycythemia after phenylhydrazine Markedly increased .- , Increase of group I
therapy
Chronic lymphatic leukemia. . . . .. Reduced or normal
Aleukemic leukemia. Reduced
Chronic myeloid leukemh. . . Usually elevated Increase of groups I and 3
Malignancy . ..... . Frequently elevated but . Increase of group I or 3
may be normal or reduced
(rare)
Hodgkin's disease ........ . . Elevated . Normal (other investigators
, have found giant forms)
Essential thrombocytopenic purpura . . ... Markedly reduced Either increase of group I
or of giant forms
Jaundice ..... .................... ... ,. Reduced Increase of group I
Acute infections . .................... .. Reduced
Convalescence from acute infections ...... Elevated Increase of group I
Chronic infections. . . . . . . . . . . . . . . . . Increased or reduced Increase of group I
Tuberculosis. . . . . . . . . . . . . . . . . . . . . . Always elevated Increase of group I
Rheumatic fever. .. . . . . . . . . . . . . . . . . Elevated
Following operations. . . Decreased first 24 hours. Increase of group I
Elevated for two weeks
Anaphylactoid purpura. . Normal Normal
Hemophilia. . ... Normal Normal
Chronic nephritis. . . . Reduced Normal or increased num-
ber of group I
Hemachromatosis ........ . Reduced Increa;sc of giant platelets
SL'U!VY··········· . Normal Increase of group I

Cells I and 2 are reproductions of a cell type which is found in small num-
bers in the bone marrow and resembles the megakaryocyte in nuclear morphology.
This cell represents our conception of a more immature, rion-granular precursor
of the motile megakaryocyte, corresponding in age to other leukoblasts. We have
given it the name, thrombobla$l, and have assumed, by reason of its extravas·
cular position, that it originates from bone marrow reticulum. Naegeli refers to
this type of cell as a megakaryoblast.
The chief function of the thrombocytes is the liberation of a substance
essential to the retraction of the blood clot. Blood may clot promptly in the
absence of platelets but will fail to retract properly. A spongy, non-retractile
dot will not prevent hemorrhage from a cut surface and predisposes to a spon-
taneous oozing from the vascular system into the tissues and mucous membranes.
 ERYTHROBLASTS, ,ERYTHROCYTES AND THROMBOCYTES 99

OCCURRENCE

In a careful study of platelets in various pathological states,' a variation in

their size and cellularity is easily perceptible. This morphologic variability has
been studied extensively by Olef in an attempt to devise a workable differential
count and a correlation of various abnormal forms with certain diseases. He
has used a wet, stained preparation and has shown its superiority to the ordi-
nary dry-stained preparations. He has demonstrated that the size of plate-
lets is directly related to their functional activity since the large cellular platelet
is a "giant morphologically and a dwarf functionally" whereas the small form
is active functionally in regard to coagUlation and agglutination. The life span
of a thrombocyte is of short duration, probably only a few days.
Olef's normal differential platelet count is shown in the following chart.
His figures were obtained from counts on 45 normal adults.
Normal
Group Average Si"e Per-Gent
1.8 mina (one quarter of a red cell) ...... , ......... , ....... ' .............. ,',. 18.6
2.5 micra (one Ulird of a. red cdl) ... , .. , ........... , .. , ................... , .. , 63,3
3.6 micra or larger (one half of a red blood cell or larger) ..................... . 17·4
4 Irreb'1l1ar shaped platelets. . . . . . . . . . ............. . 0,7
Olef has continued his studies in a number of diseases. An outline of his
findings is shown in Table IX.
In summary of Olef's study, it is obvious that the form exhibiting tbe highest
relative and absolute elevation in regenerative, conditions is the small platelet.
He found, also, that an injection of epinephrine hydrochloride, causing an empty-
ing of blood reservoirs especially in the spleen, resulted in an increase of large
platelets. This has led him to the conclusion that the small functional platelet
is the young form and that the large, cellular type is the mature senile form.
From the evidence he offers, this conclusion is apparently justifiable, but it is
contrary to the order of maturation of the other elements of the blood where
the small mature cell is the functional cell and the larger, immature forms are
non-functional. The appearance of small platelets in ordinary regenerative proc-
esses is not necessarily indicative of immaturity, but could be interpreted as an
acceleration of maturation resnlting in the increased output of small func-
tional forms. Olef states, also, that intense regenerative activity results in the
appearance of increased numbers of large forms. This statement seems con-
tradictory to his conception of the senility of large non-functional forms when
we consider the sequence of events in other hyperplastic conditions. The intense'
stimulation of myeloid elements results in the appearance in the peripheral blood
of numerous large, immature, non-functional forms which "are destroyed in the
spleen. Epinephrine hydrochloride results in the emptying of vascular reservoirs
which include the bone marrow sinusoids, It is not illogical to believe that a sud-
den constriction might pinch off larger portions of megakaryocytes resulting in
the appearance peripherally of larger forms. We have seen giant forms more
frequently in chronic myeloid leukemia where there is an immaturity of cellular
elements. If the criterion of analogy is acceptable, it seems more probable
that large platelets are immature forms and the small normal forms are mature
and functional.
100 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

I BIBLIOGRAPHY
ERYTHROBLASTS

BLACKP'AN, K. D., BATY, J. M., and DIAMOND, L. K.: "The Anemias of Childhood." Oxford Jrfon.
on Diag. and Treat., 9, Oxford Univ. Press, New York, supplement to 193I.
CASTLE, W. B.: "Observations on the Etiologic RelationOihip of Achylia GastriG\. to Pernicious
Anemia. I. The effect of the administration to patients with pernicious anemia of the con·
tents of the normal human stomach recovered after ingestion of beef muscle." ~~me", Jour.
Med. Sci., 178, 748, 1929.
DOAN, C. A., CUNNINGHAM, R. S., and SABIN, F. R.: "Experimental Studies on the Origin and
Matur;tion of Avian and Mammalian Red Blood Cells." Contrib. Embryol., Carnegie Inst.,
Wash., Pub. No. 36z, 16, 1.63, 1925.
HADEN, R. L.: "Clinical Factors in the Production of Anemia and the Regeneration of Erythrocytes
and Hemoglobin." Mcd. CUn. N. Amer., 17, 887, 1934.
NAEGELI, 0.: Blutkrankheitelt WI-d Blutdiagnostik, ed. 5, J. Springer, Berlin, 1931.
PINEY, A.: "Recent Advances in Hematology," ed. 3, P. Blakiston's Son and Co.) Philadelphia,
1931.
ROBSCHEIT-RoBBlNS, F. S.: "Tbe Regeneration of Hemoglobin and Erythrocytes." Physiol. Rev.,
9, 666, 1929.
SABIN, F.: "Bone Marrow." Physiol. Rev., 8, 191, 1928.

ERYTHROCYTES
...
CASTLE, W. B.: "Observations on tbe" Etiologic Relationship of Achylia Gastrica to Pernicious
Anemia. I. The effect of the administration to patients with pernicious anemia of the con-
tents of the normal human stomach recovered after ingestion of beef muscle." Amer. Jour.
M ed. Sci., 178, 748, 1929. "
CASTLE, \V. D., and TOWNSEND, W. C.: "Observations on the Etiologic Relationship of Achylia
Gastrica to Pernicious Anemia. II. The effect of the administration to patients with per-
nicious anemia of beef muscle after incuQation with normal human gastric juice." Amer. Jour.
Mea. Sci., 178, 764. 1929.
CASTLE, W. B., TOWNSEND) W. C.., and HEATlI, C. W.: "Observations on the Etiologic Relation-
ship of Achylia Gastrica to Pernicious Anemia. III. The nature: of the reaction between
normal human gastric juice and beef muscle leading to clinical improvement and increased
blood formation similar to the effect of liver feeding." Amer. Jour. IJed. Sci., 180, 305,
1930.
CASTLE, W. B., HEATH, C. W., and STItAUSS, M. B.: "Observations on the Etiologic Relationship
of Achylia Gastrica to Pernicious Anemia. IV. A biologic assay of the gastric secretion of
patients with pernicious an~mia having free hydrochloric acid and that of patients without
anemia or with hypochromic anemia having no free hydrochloric add, and of the role of
intestinal impermeability to hematopoietic substances in pernicious anemia." Amer. JOllr. Med.
Sci., 182, 741, 193I.
COOKE, \V. E.: "The Structure of the Human Erythrocyte." Brit. Afea. Jour., 1, 433, 1930.
DAVIDSON, L. S. P., and LEITCH, I.: "The Nutritional Anemias of Man and Animals." Nutr.
Abstr. and Rev., 3, 901, 1934.
HADEN, R. L.: "Clinical Factors in the Production of Anemia and the Regeneration of Erythrocytes
and Hemoglobin." Med. CUn. N. Amer., 17.887.1934.
HADEN, R. L.: "Clinical Significance of Volume and Hemoglobin Content of Red Blood Cells."
Arch. Int. Mea., 49, 1032, 1932; and Ibid. "Classification and Differential Diagnosis of the
Anemias." Jour. Amer. Med. Assoc., 104, 706, 1935.
KEY, J. A.: "Studies on Erythrocytes, with Special Reference to Reticulum, Polychromatophilia,
and Mitochondria." Arch. Int . .i1fed., 28, SII, I1J2I.
MUSSER, J. H., and WINTROBE, M. M.: "Diseases of the Blood"; Tiee, F., Practice of Medicine, 6~
I'9Z9 edition supplemented.
 ERYTHROBLASTS, ERYTHROCYTES AND THROMBOCYTES 101

OSGOOD, E. E.; "Hemoglobin, Color Index, Saturation Index and Volume Ind-cx Standards:
Redeterminations based on the findings in 137 healthy young men." Arch. Int. Med., 37,
685, 1926,
Rous, P.: "Destruction of the Red Blood Corpuscles in Health and Disease." Physiol. Rev.,
3, 75, 1923.
WINTROBE, M. !vL: "Anemia: Classification and Treatment on the Basis of Differences in the
Average Volume and Hemoglobin Content of Red Corpuscles." Arch. Int. ]lled., 54, 256, :1934.

THROMEOCYTES

NAEGEU, 0.: Blutkrankheiten und Blutdiagnostik, ed. 5, J. Springer, Berlin, :1931.

OLEF, I,; "The Differential Platelet Count, Its Clinical Significance." Arch. Int. Med., 57, :1063,
:193 6 .
WRIGIIT, J. H.: "The Origin and Nature of the ~lood Plates," Boston Med. Surg. Jour" 154,
643, :1906.
 CHAPTER 8
SUPRAVITAL STAINING OF LEUKOCYTES
The method for staining the cells presented in Plate "4 is the combined Janus
green and neutral red technic recommended by Sabin; Doan, Cunningham and
Tompkins (see section on Technic). By this method blood cells may be studied
in the living motile state. Mitochondria are stained with Janus green and the
granules of leukocytes react to the neutral red. The nuclei of cells are not stained
but, with a little practice, the nuclear contours are perceptible. In addition this
method provides an opportunity to study the metabolic state of leukocytes by a
study of ueutral red vacuoles. We have shown only one cell with vacuoles (cell
10) since these bodies vary markedly _in size, number, and distribution at differ-
ent time intervals. With the exception of cell IOi the types of cells in this plate
represent their reaction at about twenty minutes after the preparations were made.
The myeloblast (cells I, 2 and 3) is a large, non-motile cell with an eccentric
nucleus. The cytoplasm is filled with small mitochondria. It resembles the primi-
tive free cell (not shown on this plate) which has a more centrally placed nucleus
and contains mitochondria that are larger in size and more diffuse in distribution.
In the myeloblast, the mitochondria are clumped and often concentrated at one
point of the cytoplasm, usually near the nucleus. These bodies may ·be rod-like
or granular in appearance.
The myelocyte A (cell 4) differs from the myeloblast in the appearance of a
small clump of neutral red granules in the region of the centrosphere. In mye[(}-
cyte B (cell 5) the neutral granules increase in number and become dispersed
through the cytoplasm. These two forms corresponds to the premyelocyte in
the classification of cells stained with polychrome dyes.
In myelacyte C (cell 6) the neutral red granules reach their highest peak of
development and fill the cytoplasm. The mitochondria are reduced to a small
group around the periphery. Eosinophilic and basophilic myelocytes are not
shown. Their granules react similarly to those of their respective mature forms.
In metamyelocytes (cells 7 and 8) which follow the myelocytic stage, there
appears a slight movement of granules without motility of the entire cell. The
nucleus indents in the juvenile (cell 7) and becomes band-like (ccII 8). In these
forms the mitochondria decrease in nmnber until only an occasional one remains.
The granules decreased in. size and are not so brilliantly stained. Eosinophilic and
basophilic juveniles are not shown.
The segmented neutrophil (cell 9) is actively motile and shows a streaming of
granules. These cells are characterized by numerous fine, yellow-pink granules
which sometimes appear as colorless droplets. There are ';0 large vacuoles until
the cell has stood for 30 or 40 minutes. At this time, brick red vacuoles appear
(see cell 10) and increase in number with a lapse of time. Vacuoles are found
103
 PLATE XIV

SUPRAVITAL STAINING OF LEUKOC

I J 2 and 3. Myeloblasts. II. Eosinophil.

4. Myelocyte, type A. I2. Basophil.
5. Myelocyte, type B. 13. Lymphoblast.
6, Myelocyte, type C. 14. Large lymphocyte.
7. E[l.rly metamyelocyte (juvenile), I5. Small lymphocyte.
8. Lllte metamyelocyte (band). 16: Monoblast.
9. Segmented neutrophil. 17. Young monocyte.
10. Segmented neutrophi1. r8. Mature monocyte.
 Plate XIV.

~'
,. ", ' p ••

..

....
._ ',~

.'
.... ,_ .,

...
_. ~, "

"

.II<>rl.e"~c Garver.
 SUPRAVITAL STAINING OF LEUKOCYTES 105

increased in the cells of patients with infectious diseases. Mitochondria are few
or entirely absent in the mature segmented neutrophil.
The granules of eosinophils (cell II) are large, uniform in size, golden yellow
in color, and, as a rule, rather uniformly distributed through the cytoplasm. At
times, they may be concentrated near the nucleus, which is usually two-lobed.
Eosinophils are motile~ and, upon standing, rarely develop more than one vacuole.
As time passes, the granules change from a golden color to a red-brown. Mito-
chondria occur only occasionally in these cells.
The granules of the basophil (cell 12) stain a brilliant, brick color. They are
s~mewhat variable in size, and are smaller than eosinophilic but larger than neu-
trophilic granules. The basophil is sluggishly motile and vacuoles and mito-
chondria rarely appear in its cytoplasm.
The lymphoblast (cell 13) is a large non-motile cell comparable in size to
the myeloblast. Mitochondria are arranged in a girdle around the nucleus or are
scattered over the cytoplasm. The former arrangement facilitates its distinction
from the myeloblast.
The lymphocyte (cell 14) has a wide zone of cytoplasm and a round or in-
dented nucleus. The mitochondria are frequently in the form of short, plump
rods and are characteristically clumped in one area of the cytoplasm, usually near
the nucleus. Neutral red bodies occur in small numbers. This cell is actively
motile and may be distinguished by its movement from the non-motile myelocyte
A, and from monocytes by the clumping of mitochondria and the scattered loca-
tion of the neutral red bodies.
The small lymphocyte (cell IS) differs from the larger type only in size. The
mitochondria usually show a greater tendency to clump at one side of the smaller
cell.
T1le monoblast (cell I6) resembles the myeloblast and lymphoblast in its re-
action to supravital stains but differs somewhat in the tendency of the mito·
chondria to cluster in the hof of the nucleus.
In the young monocyte (cell I 7) the nucleus is indented. A few neutral red
bodies cluster in a circle in the cytoplasm opposite the indented groove of the
nucleus and numerous mitochondria are scattered throughout the cytoplasm. This
type of monocyte and myelocyte A are practically identical in Plate I4. They are
distinguished easily in the actual preparation by their activity; premyelocytes are
non-motile and monocytes are actively motile, especially when the temperature
is slightly elevated in the hot box.
The mature monocyte (cell 18) differs little from the young form except
in the advanced development of the nucleus into a more elongated fo'rm and the
increase of neutral red bodies which are clustered characteristically in the bend
of the nucleus. The cluster frequently encircles a clear zone. Upon standing
vacuoles increase in size and number in both types of monocytes and eventually
become brick red, as in neutrophils.
Mature erythrocytes do not react to this stain. The reticular network of
reticulocytes stains red frequently but not consistently. The method is of con-
siderable value in studying the younger nucleated erythrocytes and establishing
their maturation cycle (Doan, Cunningham and Sabin).
I06 THE DEVELOPMENT AND MORPHOLOGY OF BLOOD CELLS

The supravital staining method has found a permanent place in hematological

studies since it permits the observation of cells in the living state and provides
a method for tracing the development and maturation of cells under physiologic
conditions. It has provided a new criterion for separating monocytes from
lymphocytes and granulocytes. It permits the study of the distrihution and de-
velopment of mitochondria, the occurrence of motility, and the metaholic activity
of the various types of leukocytes and erythrocytes. It has the disadvantage of
requiring an exact regulation of temperature, thus necessitating the use of a hot
box, and an immediate examination. It does not provide infallible criteria for
the exact separation of monoblasts, Iymphoblasts and myeloblasts as has been
expected by the critics of this method. It does not permit nuclear distinctions.
However, none of its adherents has advocated that' supravital studies supplant
fixed methods but rather that they be used as an adjunct to provide additional
information concerning the separation of cell types under certain given conditions.
The method is not practical for routine work and it does not add greatly to our
knowledge of the morphology of the normal, mature cells of the blood, but it is of
considerable assistance in establishing the predominant cell types in the atypical
leukemic states. It has contributed to our conception of the age of cells in the
bone marrow and other hematopoietic centers, especially in experimental animals.
It should be used in conjunction with other stains in studying the material from
bone marrow biopsy and all acute atypical leukemias.
After a thorough review of the value of supravital methods, Hall states that
as a routine laboratory procedure the dry smear method is decidedly superior to
the supravital technic, particularly from the standpoint of cell identification.
He states further that very little information of practical value can be gained
from utilization of the supravital method since the procedure is still in the ex-
perimental stage of development and has not been found to be sufficiently reliable
to justify its routine use.
Even though the supravital study of blood cells provides information as de-
scribed above, it seems that the method is not sufficiently established to employ
as a routine procedure in clinical work. At this time its use should be restricted
to various types of research problems in which it would appear to provide valu-
able supplementary information. Under no circumstances' should it supplant the
conventional staining methods.

BIBLIOGRAPHY
SUPRAVITAL STAINING OF LEUKOCYTES

CUNNINGHAM, R. S., SABIN, F. R, and DOAN, C. A.: "Development of Leukocytes, Lymphocytes,

and Monocytes from a Specific Stem-Cell in Adult Tissue," Contrb. Embryol., 16, 227, I925.
CUNNINGHAM, R. S., and TOMPKINS, E. H.: "The Supravital Staining of Normal Human Blood
Cells," Folia Haematol., 42, 257, 1930.
DOAN, C. A" CUNNINGHAM, R. S., and SABIN, F. R.: "Experimental Studies on the Origin and
Matura.tion of Avian and Mammalian Red Blood Cells." Contrb. Embryol., 16, 163, 1925.
HALL, B. E.: "Evaluation of the Supravital Staining Method." Chap. II. Downey's Handbook of
Hematology. Paul B. Hoeber, New York. 1938. (
 SUPRAVITAL STAINING OF LEUKOCYTES 10 7
SABIN, F. R., DOAN, C. A., and CUNNINGHAM, R. S.: "The Discrimination of Two Types of
Phagocytic Cells in Connective Tissue by the Supravital Technic." Contrb. EmbryoZ'J 16,
I2S, I9 25·
SABIN, F. R.: "Bone Marrow." Physiol. Rev., 8, IgI, 1928.
WILSON, C. P., and CuNNINGHAM, R. S.: HA Consideration of the Supravital Method of Studying
Blood in Cases of Mononuclear Cell Response." Folio Haematol., 38, 14, 1929.
 SECTION THREE
NORMAL BLOOD
NEUTROPHILIC LEUKOCYTOSIS
LYMPHOC YTOSIS, MONOCYTOSIS, EOSINO-
PHILIA, AND BASOPHILIA
THE LEUKOPENIC DISEASES
 SECTION THREE
LEUKOCYTOSIS AND LEUKOPENIA

CHAPTER 9
NORMAL BLOOD
The hematologic system consists of the circulating blood and all blood form-
ing tissues. In its collections of organs and tissues which function in correlation,
·it is comparable to other systems of the body but it is unique in the possessiou
of a fluid tissue, the circulating blood, which pervades the entire body. The sys-
tem is divided functionally into three organs or units; the erythron, the leukon,
and the thrombon, and although each division has an independent function, an
abnormality of one may throw the entire system into a state of unbalance. Each
unit is composed of both fixed and mobile tissue, the fixed tissue providing the
site of origin for the formed mobile elements which circulate in a state of suspen-
sion in the fluid plasma of the blood stream.

THE ERYTHRON

The erythron includes the circulating erythrocytes and fixed erythropoietic

tissue of the bone marrow sinusoids. The function of this organ is to produce
and maintain a number of circnlating erythrocytes adequate for the transporta-
tion of oxygen to the tissues of the body and the removal of carbon dioxide. The
hemoglobin content of the erythrocytes enables them to accomplish this gaseous
exchange. The mature circulating erythrocyte is a non-motile, non-nucleated,
bi-concave disc averaging from 7 to 8 micra in diameter. Its functional life
averages about seventy days.

THE LEUKON

The leukon in the adult consists -of three types of tissue: (,) myeloid tissue
of the bone marrow which produces the granular series of leukocytes; (2)
lymphoid tissue which generates the lymphoid series of leukocytes; and (3)
the scattered reticulo-endothelium and connective tissue which are responsible for
the production of monocytes and the fixed phagocytes of the body. The mature
functional leukocytes of the circulating -blood are motile, nucleated cell bodies
which play an important r6le in the defense of the body against noxious agents.
There are three types of cells, neutrophils, eosinophils, and basophils, depending
III
 PLATE XV

NORMAL BLOOD

® ll.

r. Neutrophils.
2. Lymphocyte.
Blood Findings (normal adult): Differential:

-flemoglobin . .16 gms. (Newcomer's method). Myelocytes .. 0%

R.B.C ..... . 5,100,000 per c,mm. Juveniles ... . 0%
W.B.C. 7,200 per c.rnm. Bands ...... . I%
Platelets 350,000 per c.rnm. Segmenters 63%
Total neutrophile. ..... 64%
Erythrocytesj normochromic and normocytic. Lymphocytes. 33%
Monocytes .. 2%
EosinophHs .... lo/q
Plate XY.
 NORMAL BLOOD "3
on the reaction of their cytoplasmic granules to polychrome dyes. All three types
contain oxidizing ferments in the cytoplasm which react positively to peroxidase
stains, but each type has an independent function and reacts to specific stimuli.
The neutrophils phagocytize certain types of bacteria, notably the pyogenic group.
Eosinophils are considered active agents in the removal of foreign proteins from
the tissues. They are found increased in certain parasitic and skin diseases as
well as allergic conditions but here their role is not understood. The function
of basophils is as yet undetermined. The mononuclear lymphocytes have no
oxidizing ferments in their cytoplasm. They are active in the process of walling
off areas of inflammation and are concerned in some manner in the defense of the
body against certain bacillary and virus diseases. Tbe monocytes are actively
motile, are called scavengers of the blood, and are known to phagocytize tubercle
bacilli, 'as well as finer particles of inert material, such as india ink, carmine, etc.

THE THROMBON

Wbether the thrombocytes and their precursors, tbe megakaryocytes of the

bone marrow, deserve the classification of an organ is open to question on histo-
logical grounds. Functionally these cells act as a separate unit of the hematologic
system. Circulating thrombocytes or platelets are thought to be pinched-off
pseudopodia of the giant megakaryocytes. These cytoplasmic fragments are
about one fourth the size of a red blood cell. They play some part in the forma,-
tion of a blood clot although in their absence the tissue juices may assume this
function. They are necessary elements, however, for the proper retraction of a
blood clot after it has formed, a process equally as essential to the control of
certain types of bleeding as coagulation itself.

BLOOD PLASMA AND SERUM

The blood plasma serves not only as a vehicle for the transportation of the
formed elements of blood but in addition carries the antibodies of the blood,
the elements essential for blood coagulation, and the numerous constituents neces-
sary for the maintenance and regulation of cellular metabolism. Blood serum,
a clear straw-colored liquid, is that part of the blood plasma which is left after
the blood coagulates and incorporates the formed constituents in a meshwork of
fibrin.

HEMOCONIA

In addition to the formed constituents, erythrocytes, leukocytes and plate-

lets, the blood plasma contains minute, dancing particles, called hemoconia or
blood dust, which are perceptible with high magnification in a wet unstained
preparation of blood. These particles do not stain and their nature is unknown.
Tbey appear similar in size to the granules in the cytoplasm of granular leuko-
cytes. Tbis has led to the supposition that they are extruded granules but no
114 LEUKOCYTOSIS AND LEUKOPENIA

conclusive proof has been given for this identity. We have noticed a decrease in
their number in aplastic anemia, a tremendous increase in chronic myeloid leu-
kemia and a pr'ogressive increase in samples of normal blood in physiological saline
which has been allowed to stand for a few days. The increase appeared to parallel
the disintegration of leukocytes.

BLOOD VOLUME

The total volume of blood is dependent upon body weight and surface area
and varies considerably with the method employed for its measurement. By
the dye method the volume is considered, roughly, to be about one twelfth of the
body weight or about 6000 cc. in the average adult. Lower values are found by
the carbon monoxide method.
DIAGRAMMATIC COMPOSITION OF '[HE BLOOD
(Webster and Koch)
Serum albumin
Serum Serum globulin
Gluc6se extractives, calcium
Plasma salts, sodimn and potassium
chlorides, carbonates, phos-
phates, etc. .

/Oxyhcmoglobin
~ Reel cells~Lecithin
C 11 lar---- Salts
el::ent~ White cells-fibrin ferment
~B100d platelets
Hemoconia

EXAMINATION OF BLOOD

For a thorough preliminary examination of the blood of a patient with a

sllspected blood dyscrasia the following procedures are recommended:
Routine blood count:
Hemoglobin estimation (Newcomer's or Sahli's Method).
Erythrocyte count.
Leukocyte count.
Differential count. (Schilling's nuclear index when immature cells are present.)
Color index.
Volume index. (Hematocrit method of Wintrobe.)
Reticulocyte count. '
Fragility test.
Coagulation time.
Clot retraction time.
 NORMAL BLOOD lIS
Platelet count.
Wassermann or Kahn.
Icterus index.
Peroxidase stain when the differential count indicates its need.
This appears to be a time-consuming list of procedures for a preliminary ex-
amination but for a careful hematologic study few of these tests can be omitted
unless the diagnosis is fairly certain before the blood is studied. Some of the
procedures may be unnecessary in certain clear cut diseases and others must be
added. For example, the fragility test and icterus index are unnecessary in a
frank case of chronic leukemia; the coagulation time may be omitted when there
is no history of hemorrhage; and the measurement of clot retraction time is un-
necessary when the platelet count is normal. Experience has shown that most
. of the procedures become necessary for the elimination of related blood diseases
before treatment can be instituted safely.
With sufficient practice every procedure in this list may be made on a single
sample of venous blood. The values obtained in the examination of venous blood
are just as reliable as those obtained from capillary or peripheral blood. Andre-
sen and Mugrage compared values of venous and peripheral blood in 120 subjects
and concluded that the findings were in close agreement and could be used
interchangeably. .
The following method has been found highly satisfactQry. (See Section 9
for detailed technic for each procedure.) An interval timer is set at the time of
venipuncture and 10 cc. of blood are drawn from the vein with a sterile dry
syringe or one previously rinsed with sterile, physiologic salt solution. The time
is noted on the interval clock and I cc. of blood is placed in an 8 mm. test tube
for measurement of the coagulation time by Lee ·and White's method. As
quickly as possible, 3 cc. of blood are placed in a sterile tube to be used later
for both the Wassermann and clot retraction time. Still working rapidly, 3 cc.
of blood are placed in a flask containing 0.05 cc. of a 20 per cent solution of
potassium oxalate and shaken a few times to prevent clotting. The procedure
up to this point should not require more than 20 to 30 seconds. Next, one drop
of blood is placed in each of the 12 tubes of a fragility set up and the rack shaken
t_o mix the blood and the hypotonic saline solutions. This should not require
more than 30 seconds. Next, three or four blood smears are made on glass slides
or cover glasses depending upon the method preferred. If the blood has not
clotted in the syringe by this time, 4 or 5 drops may be expelled upon a glass
slide and the erythrocyte and leukocyte pipets filled from this sample. The first
test tube, containing I cc. of blood, is inverted to note evidence of coagulation.
If the blood has clotted the coagulation time is within normal limits.
If the blood clots before the red and white cell pipets can be filled, the
counts may be done at leisure from the sample of oxalated blood which must be
shaken thoroughly immediately before the dilutions are made. Blood smears are
not made satisfactorily from oxalated blood since the leukocytes soon become dis-
torted and vacuolated and are difficult to stain and identify. In addition the
oxalated specimen is used for the hemoglobin determination, for the reticulo-
cyte count (cresyl blue method) and finally for the filling of the Wintrobe
hematocrit tube in which the volume of packed cells is determined. If desired,
Il6 LEUKOCYTOSIS AND LEUKOPENIA

the sedimentation time may be measured in this tube before it is centrifuged.

After centrifugation, the supernatant plasma' is used for the measurement of the
icterus index by comparison with a set of standard tubes recommended by Mur-
phy: If another measurement of serum bilirubin, such as the van den Bergh
test, is preferred, serum for this determination may be obtained from the second
test tube, containing 3 cc. of blood, after it has been allowed to stand for the
measurement of the clot retraction time. Serum from this specimen may also be
used for the Wassermann or Kahn test.
Finally, one of the blood smears is stained with Wright's stain. Schilling's
differential cell count and a platelet count is made upon microscopic examination.
When immature leukocytes are found which are difficult to classify, a peroxidase
stain may be made on one of the remaining blood smears. It is recognized that a
platelet count under these conditions is subject to error, but abnormal clumping
may be ruled out by a careful examination of the stained smear with the low
power lens of the microscope. The method provides merely a rough estimate of
the number of platelets.
The color index is calculated from the hemoglobin per cent divided by the
erythrocytes in per cent of normal. The volume index and other indices may be
calculated from figures obtained from these tests.

NORMAL HEMATOLOGIC STANDARDS

ADULTS

Hemoglobin (Newcomer's Method) :

Men: 14.5 to 16.9 Gm. per 100 cc. of blood.
Women: I2.5 to 15.0 Gm. per 100 ·cc. of blood.
Erythrocytes:
Men: 4.5 to 6.0 million per cu. mm. of blood.
_ Women: 4.0 to 5.5 million per cu. mm. of blood.
Leukocytes:
Men and women: 5,000 to 10,000 per cu. mm. of blood.
Differential Count:
Relative values Absolute MlulJS
(per cent) (per CIt. mm. oj blood)
Ncutrophils .. .......... , .. , .. 60-70 3,()()O--7,OOO
Basophils ... ........... , .... . 0·5-:- r o-roo
Eosinophils. '-3 50"-300
Lymphocytes . . 2()-4° I,Coo-4POO
Monocytes ....... . 2-6 to0-6oo

Color Index:- 1.0.

Volume of Packed Cells (Wintrobe hematocrit method) :
Men-46 cc. per 100 cc. of blood.
Women-42 cc. per 100 cc. of blood.
Average figure for both sexes-42-4 cc. per 100 cc. of blood.
Volume Index:-I.o.
Mean Corpuscular Hemoglobin: 27 to 32 micromicrograms.
Mean Corpuscular Hemoglobin Concentration: 33 to 38 per cent.
 NORMAL BLOOD II7

Mean Corpuscular Volume: 80 to 94 cubic micra.

Reticulocytes: 0.1 to 1.0 per cent of erythrocytes.
Platelets: 250,000 to 350,000 per cu. mm. of blood (Fonio's smear method).
514,000 (average figure) per cu. mm. of blood (Olef's method).
Coagulation Time:
2-6 minutes (slide method)
3-8 minutes (capillary tube method)
5-10 minutes (Lee and White's method)
Bleeding Time: 2-3 minutes.
Clot Retraction Time: Beginning retraction in I to 6 hours.
Resistance of Erythrocytes' to Hypotonic Salt Solutions;
Beginning hemolysis from 0.44 per cent to 0-42 per cent NaCl;
C~mplete hemolYsis from 0.34 to 0.32 per cent NaCl.
Icterus Index: 1-5.
Van den Bergh Reaction: Indirect reaction; normal serum contains 0.1 to 0·3
mg. bilirubin per 100 cc. of blood.
CHILDREN

Erythrocytes, leukocytes, platelets and hemoglobin values are high at birth.

The following normal values are given by Blackfan, et ai., for normal new born
infants;
Hemoglobin; 95-r40 per cent.
Erythrocytes; 5 to 8 million per cu. mm. of blood.
Leukocytes; 15,000 to 25,000 per cu. mm. of blood.
Platelets; 200,000 to 400,000 per cu. mm. of blood.
\Vithin the first three months there is a physiologic decrease in red cells and
hemoglobin (the latter frequently falling to a level as low as 65 to 75 per cent)
which is explained by Davidson and Leitch as the result of two factors; (I) a de-
crease in bone marrow activity due to the high oxygen tension of extra uterine life
and (2) a destruction of erythrocytes by hemolysis. The red cell and hemo-
globin values gradually rise until the normal level is reached at about the sixth
month.
The differential count likewise is altered during infancy. There is a neutro-
philia immediately after birth wbich gradually recedes to 25 to 50 per cent during
the first two weeks. Following this period there is a gradual increase to 40 to
60 per cent during the next five or six months, but a normal level is not attained
until abont the twelfth year. This relative decrease of neutrophils between the
second week and twelfth year is accompanied by a slight relative and absolute
lymphocytosis. Eosinophils, basophils, and monocytes occur soon after birth
in higher numbers than in adults, but normal levels are usually reached at the
end of two weeks. Immature red and white cells arc frequently encountered in
the blood of infants and children. Cellular immaturity in the blood of children
is not always considered of serious import but indicates an unstable, embryonic
hematopoietic system.
lI8 LEUKOCYTOSIS AND' LEUKOPENIA

The studies of Mugrage and Andresen on the blood of infants and children
of various ages, have been valuable.
TABLE X
AVERAGE VALUES OF HEMOGLOBIN, RED CELLS AND VOLUME OF 'PACKED CELLS AT
VARIOUS AGE LEVELS. TABLE IS COMPILED FROM THE VARIOUS TABLES AND CHARTS
OF MUGRAGE AND ANDRESEN.

Age of Hemoglobin Red Cells in Vol. Packed Cells in

Patienl in Grams MiUions % of Whole Blood
At birth {cord blood)................. :1'7.14 4.86 53.18
3days-2months............. 14.64 4.22 43.28
2- 4months ............. " II.I4 3.90 34.18
4-8months............... 12.29 4.23 37.31
8-I2 months.. ........ ..... II.SX 4.28 36.81
12-18months................. 11.73 4.25 36.28
18-24 months................. 12.66 4.31 38.28
2- 3 years................. 12·73 4.36 38.51
3- 4 years .. ,............... 13.18 4·44 39. 61
4- 5 years.... ..................... I3·43 4.43 40 . 10
5- 6years........ ........ ...... 13.27 4.40 39·93
6- 7 years ................. 13-34 4.41 39.87
7- 8 years ................. 13·33 4·37 39·77
8- 9 years ............... _. 13·64 4.44 40.90
9-10 years................ 13.87 4.49 41.23
Ie-II years. 13.99 4.54 41.70
II-I2 years................ 14.22 4.58 42.30
1:2-13 years.................... '14.49 4·66 43· IS
13-15 (male}......... ...... 14.90 4.81 43.;0
13-15 (female)............. 14.51 4.66 43·35
15-17 (male). ...... ......... 15.30 4.98 46.10
15-17 (female)... ..... ..... 14.79 4.69 44.20
17-19 (male)................. 16·57 5·05 49.55
17-19 (female)............... 14.75 4.65 44·33
19-2I (male)................. .16·45' 5·35 48.70
19-:21 (female) ........... __ ......... 14.54 4.63 43.16
Men ......................... __ 16.54 5.42 48.35
Women..... ............. 14·45 4.63 43.22

Colo~d~Of~~~~~d~e~=~:!~)~s ~~~~e~e~tPdi~i:~;t:~~. v~I~~~c~:~u~~i~e~~~~~~~~5 ~~R~n~~~

falls to adult level at three months (Andresen and Mugrage. Folia Hematologica, 61: 201, 1938).

It is possible that hemoglobin and red cell values may be slightly higher in
normal people living in high altitndes. Nelson and Stoker examined the red
cell and hemoglobin values of 350 healthy men, both urban and suburban resi-
dents, living in the midwestern portion of the United States at an altitude of
between 700 and ICOO feet above sea level. They found the average hemoglobin
was 15.03 grams per IOO cc. of blood, and the average red cells 5. II million cells
per cU.mm. There were no differences between city and country residents nor
between men working ont of doors ana within doors, and none between healthy
male negroes and whites. The hemoglobin determinations were carried out by
the Van Slyke oxygen capacity method in duplicate.
 NORMAL BLOOD 119

CHEMICAL CONSTITUENTS
Nonprotein Nitrogen: 25 to 30 mg. per 100 cc. of blood.
Urea: 12 to IS mg. per cent.
Uric acid: I to 3 mg. per cent.
Creatinine: I to 2 mg. per cent.
Sugar: 80 to 120 mg. per 100 cc. of blood.
Chlorides:
In plasma: 570 to 600 mg. per cent.
, In cells: 285 to 300 mg. per cent.
Calcium: 9 to 10 mg. per cerit in the plasma of adults; slightly higher (10 to 12
mg. per cent) in that of children.
Serum Protein, total: 6 to 8 Gm. per 100 cc. of blood.
Albumin: 4.5 to 5·5 Gm. per cent.
Globulin: 1.5 to 3.0 Gm. per cent.
CO2 Combining Power of the Plasma: 55 V;> 80 cc. of carbon dioxide per roo cc.
of plasma.
Cholesterol: 150-200 mg. per roo cc. of blood.

BIBLIOGRAPHY
NORMAL BLOOD
ANDRESE...."f, M. 1., and MUGRAGE, E. R.: "Venous and Peripheral Red Blood Cell Values." Am. Jour.
GUn. Path., 8, 4-6, 1938.
ANDRESEN, M. I., and MUGIUlGE, G. R.: "Red Blood Cell Values for Normal Men and Women."
Arch. Int. Med., 58, 1:36, 1936.
ANDRESEN, M. I., and MUGRAGE, G. R.: IIDiameter and Volume of Red Blood CeUs in Infants and
SmaU Children." Fol. Haemat., 61, 201, :£938.
BLACKFAN, K. D., BATY, J. M., and DIAMOND, L. K.: "The Anemias of Childhood." Oxford
Monographs on Diagnosis and Treatment, 9, Oxford Univ. Press, New York, supplemented
to :1931.
CASTI.E, W. R, and MINOT, G. R: 'I Pathological Physiology and Clinical Description of the
Anemias." Oxford University Press, New York, 1936 . .
DAVIDSON, L. S. P., and LXITCH, I.: "The Nutritional Anemias of Man and Animals." Nutr. Abst.
Rev., 3, 901, 1934.
MACItAY, H. M. M.: "Normal Hemoglobin Level During the First Year of Life." Arch. Dis. Child,
8, 221, 1933.
MUGRAGE, E. R.. and ANDRESEN, M. I.: "Values for red blood cells of average infants and children."
Amer. Jour. Dis. Child" 51, 775, 1936.
MUGRAGE, E. R., and ANDRESON, M. I.: "Red Blood Cell Values in Adolescence." Am. JOUY. Dis.
Cltild., 56., 99'1, 1935.
MuRPHY, VI. P.: HAn Easy Method of Estimating the Amount of Jaundice by Means of the Blood
Serum." New Eng. Jour. Med., 194, 297, 1926.
MUSSER, J. H., and WINTROBE, M. M.: "Diseases of the Blood." Frederick Tice. Practice of M edi~
cine, 6, 1921 edition, supplemented,
 PLATE XVI

NEUTROPHILIC LEUKOCYTOSIS

,
,

:3
~z
Rr\
~
4

1. Juvenile neutrophi1.
2. Band neutrophils. (toxic p;ranulation).
3. Segmented neutrophils (toxic granulation).
4. Segmented neutrophils (normal granulation).
5, Lymphocyte.
Blood Findings (patient with lobar pneumonia): Differential:

Hemoglobin .15 gms. (Newcomer's method). Myelocytes 1%

R,B,C, 4,850,000 pcr c.mm. Juveniles . 10%
W,B.C, 40,200 per c.mm. Bands ..... 31%
Platelets 240,000 per c.mm. Segmenters ..... 46%
Total neutrophils
Lymphocytes
Erythrocytes; normochromic and normocytic. Monocytes
Plate XVI.
 NORMAL BLOOD 113

on the reaction of their cytoplasmic granules to polychrome dyes. All three types
contain oxidizing ferments in the cytoplasm which react positively to peroxidase
stains, but each type has an independent function and reacts to specific stimuli.
The ueutrophils phagocytize certain types of bacteria, notably the pyogenic group.
Eosinophils are considered active agents in the removal of foreign proteins from
the tissues. They are found increased in certain parasitic and skin diseases as
well as allergic conditions but here their role is not understood. The function
of basophils is as yet undetermined. The mononuclear lymphocytes have no
oxidizing ferments in their cytoplasm. They are active in the process of walling
off areas of inflammation and are concerned in some manner in the defense of the
body against certain bacillary and virus diseases. The. monocytes are actively
motile, are called scavengers of the blood, and are known to phagocytize tubercle
bacilli, as well as finer particles of inert material, such as india ink, carmine, etc.

THE THROMBON

Whether the thrombocytes and their precursors, the megakaryocytes of the

bone marrow, deserve the classification of an organ is open to question on histo-
logical grounds. Functionally these cells act as a separate unit of the hematologic
system. Circulating thrombocytes or platelets are thought to be pinched-off
pseudopodia of the giant megakaryocytes. These cytoplasmic fragments are
about one fourth the size of a red blood cell. They play some part in the forma-
tion of a blood clot although in their absence the tissue juices may assume this
function. They are necessary elements, however, for the proper retraction of a
blood clot after it has formed, a process equally as essential to the control of
certain types of bleeding as coagulation itself.

BLOOD PLASMA AND SERUM

The blood plasma serves not only 8$ a vehicle for the transportation of the
formed elements of blood but in addition carries the antibodies of the blood,
the elements essential for blood coagulation, and the numerous constituents neces-
sary for the maintenance and regulation of cellular metabolism. Blood serum,
a clear straw-colored liquid, is that part of the blood plasma which is left after
the blood coagulates and incorporates the formed constituents in a meshwork of
fibrin. .

HEMOCONIA

In addition to the formed constituents, erythrocytes, leukocytes and plate-

lets, the blood plasma contains minute, dancing particles, called hemoconia or
blood dust, which are perceptible with high magnification in a wet unstained
preparation of blood. These particles do not stain and their nature is unknown.
They appear similar in size to the granules in the cytoplasm of granular leuko-
cytes. This has led to the supposition that they are extruded granules but no
1I4 LEUKOCYTOSIS AND LEUKOPENIA

conclusive proof has been given for this identity. We have noticed a decrease in
their number in aplastic anemia, a tremendous increase in chronic myeloid leu-
kemia and a progressive increase in samples of normal blood in physiological saline
which has been allowed to stand for a few days. The increase appeared to parallel
the disintegration of leukocytes.

BLOOD VOLUME

The total volume of blood is dependent upon body weight and surface area
and varies considerably with the method employed for its measurement. By
the dye method the volume is considered, roughly, to be about one twelfth of the
body weight or about 6000 cc. in the average adult. Lower values are found by
the carbon monoxide method.
DIAGRAMMATIC COMPOSITION OF THE BLOOD
(Webster and Koch)
Serum albumin
Serum Serum globulin
Glucose extractives, calcium
Plasma salts, sodium and potassium
chlorides, carbonates, phos-
phates, etc. .

_/Oxyhemoglobin
~Red cells~Leeithin
C llular------ Salts
el:ment~ White cells-fibrin ferment
~BJOOd platelets
Hemoconia

EXAMINATION OF BLOOD

For a thorough preliminary examination of the blood of a patient with a

suspected blood dyscrasia the following procedures are recommended:
Routine blood count:
Hemoglobin estimation (Newcomer's or Sahli's Method).
Erythrocyte count.
Leukocyte count.
Differential count. (Schilling's nuclear index when immature cells are present.)
Color index.
Volume index. (Hematocrit method of Wintrobe.)
Reticulocyte count. .
Fragility test.
Coagulation time.
Clot retraction time.
 NORMAL BLOOD II5
Platelet count.
Wassermann or Kahn.
Icterus index.
Peroxidase stain when the differential count indicates its need.
This appears to be a time-consuming list of procedures for a preliminary ex-
amination but for a careful hematologic study few of these tests can be omitted
unless the diagnosis is fairly certain before the blood is studied. Some of the
procedures may be unnecessary in certain clear cut diseases and others must be
added. For example, the fragility test and icterus index are unnecessary in a
frank case of chronic leukemia; the coagulation time may be omitted when there
is no history of hemorrhage; and the measurement of clot retraction time is un-
necessary when the platelet count is normal. Experience has shown that most
-of the procedures become necessary for the elimination of related blood diseases
before treatment can be instituted safely.
With sufficient practice every procedure in this list may be made on a single
sample of venous blood. The values obtained in the examination of venous blood
are just as reliable as those obtained from capillary or peripheral blood. Andre-
sen and Mugrage compared values of venous and peripheral blood in I20 suhjects
and concluded that the findings were in close agreement and could be used
interchangeably. -
The following method has been found highly satisfactory. (See Section 9
for detailed technic for each procedure.) An interval timer is set at the time of
venipuncture and 10 cc. of hlood are drawn from the vein with a sterile dry
syringe or one previously rinsed with sterile, physiologic salt solution. The time
is noted on the interval clock and I cc. of blood is placed in an 8 mm. test tube
for measurement of the coagulation time by Lee ·and White's method. As
quickly as possible, 3 cc. of blood are placed in a sterile tube to be used later
for both the Wassermann and clot retraction time. StilI working rapidly, 3 cc.
of blood are placed in a flask containing 0.05 cc. of a 20 per cent 'solution of
potassium oxalate and shaken a few times to prevent clotting. The procedure
up to this point should not require more than 20 to 30 seconds. Next, one drop
of blood is placed in each of the 12 tubes of a fragility set up and the rack shaken
to mix the blood and the hypotonic saline solutions. This should not require
more than 30 seconds. Next, three or four blood smears are made on glass slides
or cover glasses depending upon the method preferred. If the blood has not
clotted in the syringe by this time, 4 or 5 drops may be expelled upon a glass
slide and the erythrocyte and leukocyte pipets filled from this sample. The first
test tube, containing I cc. of hlood, is inverted to note evidence of coagulation.
If the blood has clotted the coagulation time is within normal limits.
If the blood clots before the red and white cell pipets can be filled, the
counts may be done at leisure from the sample of oxalated blood which must be
shaken thoroughly immediately before the dilutions are made. Blood smears are
not made satisfactorily from oxalated blood since the leukocytes soon become dis-
torted and vacuolated and are difficult to stain and identify. In addition the
oxalated specimen is used for the hemoglobin determination, for the reticulo-
cyte count (cresyl blue method) and finally for the filling of the Wintrobe
hematocrit tube in which the volume of packed cells is determined. If desired,
rr6 LEUKOCYTostS AND LEUKOPENIA

the sedimentation time may be measured in this tube before it is centrifuged

After centrifugation, the supernatant plasma' is used for the measurement of th,
icterus index by comparison with a set of standard tubes recommended by 1\1 ur·
phy~ If another measurement of serum bilirubin, such as the van den Berg}
test, is preferred, serum for this determination may be obtained from the secone
test tube, containing 3 cc. of blood, after it has been allowed to stand for th,
measurement of the clot retraction time. Serum from this specimen may also b,
used for the Wassermann or Kahn test.
Finally, one of the blood smears is stained with Wright's stain. SchilJing'~
differential cell count and a platelet count is made upon microscopic examination
When immature leukocytes are found which are difficult to classify, a peroxidas,
stain may be made on one of the remaining blood smears. It is recognized that ~
platelet count under these conditions is subject to error, but abnormal clumpin!
may be ruled out by a careful examination of the stained smear with the 10Vl
power lens of the microscope. The method provides merely a rough eslimate 01
the number of platelets.
The color index is calculated from the hemoglobin per cent divided by th,
erythrocytes in per cent of normal. The volume index and other indices may b,
calculated from figures obtained from these tests.

NORMAL HEMATOLOGIC STANDARDS

ADULTS

Hemoglobin (Newcomer's Method):

Men: 14.5 to 16.9 Gm. per roo cc. of blood.
Women: 12.5 to 15.0 Gm. per IOO cc. of blood.
Erythrocytes:
Men: 4.5 to 6.0 million per cu. mm. of blood.
_ Women: 4.0 to 5.5 million per cu. mm. of blood.
Leukocytes:
Men and women: 5,000 to IO,OOO per cu. mm. of blood.
Differential Count:
Relative 'lIaJlICS Absolu.te'MJlICS
(per cent) (per cu. mm. of blood)
Neuirophils . .......... . 60--70 3,000-7,000
Basophils ............ . 0.5-:-1 O-Ioo
Eosinophils, , ........ . I-3 SQ-300
I.ymphocytcs. 20-40 1 1000-4,000
Monocytes ......................... . 2-6 Ioo-6oo

Color Index:-r.o.
Volume of Packed Cells (Wintrobe hematocrit method):
Men -46 cc. per roo cc. of blood.
Women-42 ce. per IOO cc. of blood.
Average figure for both sexeS-42 -4 cc. per roo cc. of blood.
Volume Index:-r.o.
Mean Corpuscular Hemoglobin: 27 t(l 32 micromicrograms.
Mean Corpuscular Hemoglobin Concentration: 33 to 38 per cent.
 NORMAL BLOOD

Mean Corpuscular Volume: 80 to 94 cubic micra.

Reticulocytes: o.t to 1.0 per cent of erythrocytes.
Platelets: 250,000 to 350,000 per cu. mm. of blood (Fonio's smear method).
514,000 (average figure) per cu. mm. of blood (Olef's method).
Coagulation Time:
2-6 minutes (slide method)
3-8 minutes (capillary tube method)
5-10 minutes (Lee and White's method)
Bleeding Time: 2-3 minutes.
Clot Retraction Time: Beginning retraction in r to 6 hours.
Resistance of Erythrocytes· to Hypotonic Salt Solutions;
Beginning hemolysis from 0.44 per cent to 0.42 per cent NaCl;
C~mplete hemolysis from 0.34 to 0.32 per cent NaeL
Icterus Index: 1-5.
Van den Bergh Reaction: Indirect reaction; normal serum contains o. I to 0 .•,
mg. bilirubin per 100 cc. of blood.
CHILDREN

Erythrocytes, leukocytes, platelets and hemoglobin values are high at hirth.

The following normal values are given by Blackfan, et aI., for normal new born
infants:
Hemoglobin: 95-140 per cent.
Erythrocytes: 5 to 8 million per cu. mm. of hlood.
Leukocytes: 15,000 to 25,000 per cu. mm. of blood.
Platelets: 200,000 to 400,000 per cu. mm. of hlood.
Within the first three months there is a physiologic decrease in red cells and
hemoglobin (the latter frequently falling to a level as low as 65 to 75 per cent)
which is explained by Davidson and Leitch as the result of two factors: (I) a de-
cre""e in bone marrow activity due to the high oxygen tension of extra uterine life
and (2) a destruction of erythrocytes by hemolysis. The red cell and hemo-
globin values gradually rise until the normal level is reached at about the sixth
month.
The differential count likewise is altered during infancy. There is a neutro-
philia immediately after birth which gradually recedes to 25 to 50 per cent during
the first two weeks. Following this period there is a gradual increase to 40 to
60 per cent during the next five or six months, but a normal level is not attained
until about the twelfth year. This relative decrease of neutrophils between the
second week and twelfth year is accompanied by a slight relative and absolute
lymphocytosis. Eosinophils, basophils, and monocytes occur soon after birth
in higher numbers than in adults, but normal levels are usually reached at the
end of two weeks. Immature red and white cells are frequently encountered in
the blood of infants and children. Cellular immaturity in the blood of children
is not always considered of serious import but indicates an unstable, embryonic
hematopoietic system.
118 LEUKOCYTOSIS AND LEUKOPENIA

The studies of Mugrage and Andresen on the blood of infants and children
of various ages, have been valuable.
TABLE X
AVERAGE VALUES OF HEMOGLOBIN, RED CELLS AND VOLUME OF PACKED CELLS AT
VARIOUS AGE LEVELS. TABLE ISrCOMPILED FROM THE VARIOUS TABLES AND CHARTS
OF MUGRAGE AND ANDRESEN.

Age of Hemoglobin Red Cells in Vol. Packed Cells in

Patient in Grams Millions % of Whole Blood
At birth (cord blood).... ............. 17.14 4.86 53.18
3 days-2 months.................... 14.64 4.22 43.:28
2- 4months....................... 11.14 3.90 34.18
4-8months....................... 12.:29 4·23 37.37
8-12months....................... II.Sl 4.28 36.81
I2-18montbs....................... 11.73 4.25 36.28
[8-24 months....................... 12.66 4.31 38.28
2- 3years......................... 12.73 4.36 38.51:
3-4yeats......... 1:3.18 4.44 39.61:
4-5years ......................... 1:3.43 4·43 40.1:0
5- 6 years. 1:3.27 4·40 39·93
6- 7 years........................ 1:3·34 4·41: 39.87
7- 8 years............... 1:3·33 4·37 39·77
8- 9 years. ................. 1:3.64 4.44 40.90
g-1:oyears...... 1:3.87 4.49 41:.23
lQ-n years......................... 13.99 4.54 41:.70
II-I:l years...... 14.22 4-58 42.30
12-13 years......................... ·I4.49 4.66 43.15
13-15 (male)........................ I4.90 4.8I 43.)0
'3-'5 (female)............. 14.5' 4·66 43.35
15-17 (male)............... I5.30 4.98 46.10
15-17 (female)............. 14.79 4.69 44·20
17-19 (male)............... 16.57 5·05 49·55
'7-'9 (female)............. '4·75 4.65 44·33
19""""21: (male)............... 16·45 5·35 48.70
19""""21 (female) .... _........ 14.54 4.63 43.16
Men....................... ••...... 16.54 5.42 48.35
Women............................. 14.45 4.63 43·22
Hemoglobin determinations by oxygen capacity method. Subjects studied were residents of Denver,
Colorado (altitude SOOO feet). Average cell diameter and volume high during first three weeks and
falls to adult level at three months (Andresen and Mugrage. Folia Hematologica, 61: :W1, 1938).

It is possible that hemoglobin and red cell values may be slightly higher in
normal people living in high altitudes. Nelson and Stoker examined the red
cell and hemoglobin values of 350 healthy men, both urban and suburban resi-
dents, living in the midwestern portion of the United States at an altitude of
between 700 and IDOO feet above sea level. They found the average hemoglobin
was 15.03 grams per roo cc. of blood, and the average red cells 5.1 I million cells
per cU.mm. There were no differences between city and country residents nor
between men working out of doors and within doors, and none between healthy
male negroes and whites. The hemoglobin determinations were carried out by
the Van Slyke oxygen capacity method in duplicate.
 NORMAL BLOOD IIg

CHEMICAL CONSTITUENTS
Nonprotein Nitrogen: 25 to 30 mg. per 100 cc. of blood.
Urea: 12 to IS mg. per cent.
Uric acid: I to 3 mg. per cent.
Creatinine: I to 2 mg. per cent.
Sugar: 80 to 120 mg. per 100 cc. of blood.
Chlorides:
In plasma: 570 to 600 mg. per cent.
In cells: 285 to 300 mg. per cent.
Calcium: <) to 10 mg. per cent in the plasma of adults; slightly higher (,0 to 12
mg. per cent) in that of children.
Serum Protein, total: 6 to 8 Gm. per IDa cc. of blood.
Albumin: 4.5 to 5·5 Gm. per cent.
Globulin: 1.5 to 3.0 Gm. per cent.
CO 2 Combining Power of the Plasma: 55 to 80 cc'. of carbon dioxide per roo cc,
of plasma.
Cholesterol: '50-200 mg. per roo ce. of blood.

BIBLIOGRAPHY
NORMAL BLOOD
ANDRESEN, M. I., and MUGRAGE, E. R.: "Venous and Peripheral Red Blood Cell Values." Am. Jour.
Clin. Path., 8, 46, 1938.
ANDRESEN, M. 1., and MUGRAGE, G. R: "Red Blood Cell Values for Normal Men and Women."
Arch. Int. Med., 58. 136, 1936.
ANDRESEN, M. I., and MUGRAGE, G. R.: "Diameter and Volume of Red Blood Cells in Infants and
Small Children." Fol. Haemat., 61, 201, 1938.
BLACKFAN, I{. D., BATY, J. M., and DIAMOND, L. K.: ('The Anemias of Childhood." Oxford
Monographs on Diagnosis and Treatment, 9, Oxford Univ. Press, New York, supplemented
to 1931.
CASTLE, W. R., and MINOT, G. R.: "Pathological Physiology and Clinical Description of the
Anemias." Oxford University Press, New York, 1936._
DAVIDSON) L. S. P., and LEITCH, 1.: I'The Nutritional Anemias of Man and Animals." Nutr. Abst.
Rev., 3, 901, 1:934.
MACKAY, H. M. M.: "Normal Hemoglobin Level During the First Year of Life." Arch. Dis. Child,
8, 221, 1933.
MUGRAGE, E. R., and ANDRESEN, M. I.: "Values for red blood cells of average infants and children."
Amer. Jour. Dis. Child., 51, '775, 1936.
MUG RAGE, E. R., and ANDRESON, M. I.: uRed Blood Cell Values in Adolescence." Am. Jour. Dis.
Child., 56, 997, I93S.
MURPHY, W. P.: !IAn Easy Method of Estimating the Amount of Jaundice by Means of the Blood
Serum." New Eng. Jour. Med., 194, 297, 1926.
MUSSER, J. B., and WINTROBF., M. M.: "Diseases of the Blood." Frederick Tiee. Practice of Medi-
cine, 6, 1921 edition, supplemented.
120 LEUKOCYTOSIS AND LEUKOPENIA

NELSON, C. F., and STOKER, R.: uThe Hemoglobin Concentrations and Erythrocyte Counts of
Healthy Men." Fol. Haemat., 58, 333, 1937.
OSGOOD, E. E., and BAKER, R. L.: UErythrocyte, Hemoglobin, Cell Volume and Calor, Volume
and Saturation Index Standards for Normal Children of School Age." Amer. Jour. Dis. Child"
50, 343, 1935.
WINTROBE, M. M.: I1Ma:croscopic Examination of the Blood: Discussion of Its Value and Description
of the Use of a Single Instrument for the Determination of Sedimentation Rate) Volume of
Packed Cells, Leukocytes and Platelets, and of the rcterlls Index,» Am. Jour. M ed. Sci., 185,
58, '933·
 CHAPTER 10
NEUTROPHILIC LEUKOCYTOSIS (NEUTROCYTOSIS)
Leukocytosis may be defined as that condition in which the total number
of circulating leukocytes exceeds the normal. The increase in cells may be due
to an increase of anyone of the various types of leukocytes. The most com-
mon form is neutrophilic leukocytosis (neutrocytosis), or the increase may be
due to lymphocytes in which event it is a lymphocytosis, or it may be caused by
an increase in eosinophils resulting in eosinophilia or eosinophilic leukocytosis.

NORMAL NUMBER OF LEUKOCYTES

Since there are so many factors that play a part in regulating the number
leukocytes in the normal person the range of .normal for leukocytes is quite
ie. There is reason to believe that 5,000 leukocytes per cu. mm. may be the
•. ~rmal for one person while 10,000 would be normal for another. In various
text books tbe normal number is usually given at a certain figure but it is well
to remember that the normal leukocyte count of the same person may be quite
different on different days, and for that matter, at different times of the same day.
In healthy adults the counts vary between 5,000 to 10,000 cells per cu. mm., the
latter figure representing probably the upper limits of normal. We have found
the leukocytes to average about 8,000 per cu. mm. in healthy medical students
and have found similar figures in a group of seven hundred college women. The
counts of infants range between 10,000 to 15,000 cells per cu. mm. because of the
large number of lymphocytes.
It is advisable to think of leukocytes in whole numbers and only in this
way can a correct conception of cellular changes be obtained. It is possible for
the total number of lymphocytes to be increased and the total number of
leukocytes to be decreased simultaneously and it is possible for the neutrophils
to be decreased and the total number of leukocytes to be increased simultaneously.
Therefore, before one can properly evaluate the shifting cellular changes, the
whole numbers of circulating cells should be estimated.

PHYSIOLOGIC LEUKOCYTOSIS

Because of the importance of increased numbers of leukocytes, it is essential

that one be thoroughly familiar with the various factors that may produce
leukocytosis in the normal person.

BONE MARROW DELIVERY

It has been well established by Sabin and her co-workers that there occurs
daily a so-called "diurnal tide" of leukocytes with a corresponding intervening
121
 PLATE XVI

NEUTROPHILIC LEUI\:OCYTOSIS

.,

3
~Z.
.
.

1. Juvenile neutrophil.
2. Band neutrophils (toxic granulation).
3. Segmented neutrophils (toxic granulation).
4. Segmented neutrophils (normal granulation).
5. Lymphocyte.
Hood Findings (patient with lobar pneumonia): Differential:

Hemoglobin ... IS gms. (Newcomer's method), Myelocytes 1%

R.B.C . . 4)8$0,000 per c.rnm. Juveniles. . " 10%
W.B.C. 40,200 per c.m..._m. Bands .... 31%
Platelets 240,000 per c.rom. Segmenters .. . .... 46%
Total neutrophils 88%
Lymphocytes 9%
Erythrocytes; normochromic and normocytic. Monocy~es 3%
Plate XVI.
 NEUTROPHILIC LEUKOCYTOSIS (NEUTROCYTOSIS) 12 3

period of relative leukopenia. Thus, at two periods during the day there is a
physiologic leukocytosis occurring late in the morning and late in the evening
(10;00 to n;oo A.M. and n;oo to 12;00 P.M.). Also, there is a so-called
"rhythmic delivery" of leukocytes from the bone marrow into the peripheral'circu-
lation occurring about twice each hour. These changes influence the total number
of cells apparently and the cell changes are not restricted entirely to neutrophils
as would be observed in a pathologic neutrophilic leukocytosis.

EFFECT OF FOOD
eo vThere is considerable evidence to show that leukocytes are influenced by
the food intake. For many years it has been thought that there exists a so-called
"digestive leukocytosis." However, there is considerable question as to the exist-
ence of such a state in all persons. For that matter, it is more likely that there
exists a digestive leukopenia which occurs from a few minutes to two or three
hours after meals. This is based on the assumption that many leukocytes are
mobilized in the visceral circulation in connection with the process of food absorp-
tion, resulting in a transient peripheral leukopenia. Food does affect the number of
circulating leukocytes, especially in that type of individual who may be sensitive
to some particular food. Based upon this has been the work of Vaughn, in
showing that the ingestion of such foods results in a transient leukopenia and
based upon this he has advocated a diagnostic test for food sensitivity. N aegeJi
points out that in some patients the ingestion of carbohydrates may be followed
by leukopenia and in otber patients the ingestion of proteins or fats may have
the same effect, while still in others tbe ingestion of any of these foods may pro-
duce leukocytosis. Naegeli further points out that it is difficult to find eosinophils
during a hunger period in a normal person. All of this indicates that food inges-
tion does playa role in the number of circulating leukocytes, but the exact rela-
tionship at this time is quite obscure.

EFFECT OJ;' PREGNANCY

It has long been recognized that there exists a leukocytosis in pregnancy.
This seems to become more marked as pregnancy progresses toward term. The
full term pregnant woman has a leukocyte count that may vary from 10,000 to
20,000 cells per cu. mm., but in the first and second trimester this is not so evi-
dent. The leukocytosis of pregnancy has been attributed to many things, includ-
ing increased metabolic activity, the, presence of absorbed foreign material from
the fetus, the cellular damage that may be going on in the liver and kidneys,
although it should be pointed out that apparently the leukocytosis of the toxemic
states does not exceed that of the normal pregnant woman. We have carried out
blood studies on one hundred normal pregnant women and find that the leuko-
cyte count averages slightly more than 14,000 in the third trimester.

IN THE NEW-BORN
. The leukocytosis of the new-born has long been recognize.d. Some normal
mfants have as many as 20,000 cells per cu. mm. which gradually decrease
through the first few months of life, become rather stationary in number at the
124 LEUKOCYTOS1S AND LEUKOPJ£;NIA

age of about one year and ~~en gradually decrease to the normal of adults at
the age of puberty. There has been no explanation offered for this other than the
increased hematopoietic ac'c;vity of the bone marrow of the infant.

MUSCULAR ACTIVITY
It has been well demonstrated by Garrey and his associates that various
types of muscular activity will result in physiologic leukocytosis. Whether
or not this leukocytosis is due to increased adrenalin output or to muscular con-
tractions in which leukocytes may be mechanically forced into the peripheral
circulation is open to question. It has also been shown that a transient leuko-
cytosis is found during periods of fright, emotional upsets, anger, etc. In all
probability this is due to a stimulation of the sympathetic nervous system.
Naegeli speaks of "adrenaline leukocytosis" and it would appear that these condi-
tions are probably on the same basis. When this occurs, however, there is no
disturbance of the relative number of cells; the percentage of various cell types
remains relatively the same. Therefore, it would appear that this type of leuko-
cytosis has its basis in a so-called "redistribution phenomenon." It is generally
believed that only a small part of the capillary bed is in use at anyone time
and that a large number of leukocytes may be normally stored in the spleen and
other tissues as well as the capillary bed. However, the role of the spleen in
this process is questionable since Lucia and his associates were able to provoke
the same degree of leukocytosis after adrenalin injections in splenectomized pa-
tients as well as in normal persons. Under conditions of emotional upset, muS-
cular activity, stimulation of the sympathetic nervoUs system, overactivity of the
adrenals, etc., there occurs a dilatation of the vascular bed, possibly constriction
of the spleen and forcing of leukocytes into tbe peripheral circulation. The
regUlarity with which leukocytosis occurs following muscular activity has led to
an attempt to differentiate between hysterical spasms and true epilepsy on the
basis of the leukocyte count.

TEMPERATURE
It has been noted that body temperature has an influence on the number
of leukocytes. Rabbits can be placed in temperatures lower than normal and
this will result in a definite but transient leukocytosis. N aegeJi states that leuko-
cytosis is the rule after cold baths. It would appear that these factors cause the
peripheral dilatation of the vascular bed as mentioned before. On the other hand,
rabbits can be subjected to high temperatures and this will result in a lower
leukocyte count. This relationship may be of some practical importance in view
of the present trend to utilize artificial fever therapy in various diseases. Simp·
son has pointed out that there is a definite snstained increased number of leuko-
cytes in patients being treated with hyperpyrexia machines (Kettering hyper.
therm). The significance of high leukocyte counts nnder these conditions has
not yet been evaluated. It is not known what role they play in the protective
mechanism under such conditions. There is some evidence to indicate that
increased temperature per se will cause leukocytosis, but only if the marrow is
normal. High temperatures have little effect on an aplastic or damaged marrow
 NEUTROPHILIC LEUKOCYTOSIS (NEUTROCYTOSIS) 12 5

as evidenced by patients witb agranulocytosis having neutropenia associated

with fever.

ALTITUDE
It has been observed that the leukocyte count may be decreased when an
individual is first placed in a high altitude but that within two to three weeks
there results a fairly defmite and sustained leukocytosis rarely exceeding, however,
10,000 cells per cu. mm. We have observed patients living at sea level and going
to high altitudes who demonstrate this leukocytosis as well as a marked increase
in red blood cells. It should be emphasized that in practically all instances of
pbysiologic leukocytosis there is no relative increase of any particular cell type
and in this respect it can be distinguished from pathologic leukocytosis.

PATHOLOGIC LEUKOCYTOSIS (NEUTROPHILIC)

The neutrophil is the most actively amoeboid cell of the body., When studied
in the unstained state it undergoes constant change of shape because of the
projection of pseudopodia, very much like those of an amoeba. When attracted
to a body it makes very definite but slow progress toward it at an average speed
of 37 micra per minute, according to Henderson. The rate of motility is increased
with increased temperature and it is decreased if the reaction is altered very little
from the normal. If the acid reaction is stronger than pH 6.0, paralysis of the
cell may supervene.
The neutrophil consists of a nucleus in the center of the cell with cytoplasmic
material surrounding the nucleus in all directions and crowded with very fine re-
fractive granules. The chemical nature of these granules is entirely unknown.
Bunting quotes recent opinions on this question and states that they are highly
differentiated bodies of very complex composition and that they probably are
carriers of enzymes or enzyme-like substances.
Since neutrophils are actively amoeboid cells, their chief function is phago-
cytic in that they are constantly available to ward off invasion by pyogenic bac-
teria. Therefore, they constitute the first line of defense in many infectious dis-
eases. In addition to phagocytosis, they.release proteolytic ferments which break
down bacteria and cellular material into necrotic debris. It is likely that the
cytoplasmic granules are in some way concerned in this function. In nearly all
instances where the body is invaded by bacteria the neutrophils play some role
in defense, and consequently there are few infectious diseases that are not charac-
terized by increased numbers in the blood.

GENERAJ. DISCUSSION
In the pathologic types of neutrophilic leukocytosis there is not only an
increase in the total number of ceils, but also an absolute and relative increase in
the number of neutrophils. This type of leukocytosis is found chiefly in condi-
tions in which there is an inflammatory process produced by the pyogenic or pus
producing organisms. Infections with staphylococci, streptococci, pneumococci,
meningococci and !!ther organisms that produce pus, will invariably be accom-
126 LEUKOCYTOSIS AND LEUKOPENIA

panied by some degree of leukocytosis. Furthermore, the degree of leukocytosis

is influenced by the capacity of the organism to localize and there will be little
or no leukocytosis in any type of pyogenic infection in which the organism does
not localize. Staphylococci may prodnce multiple abscesses with leukocytosis
and streptococci may also produce similar abscesses with a coincident leuko-
cytosis but if the streptococci circulate in the blood stream and fail to localize a
marked leukocytosis would be most unusual. It appears, therefore, that destruc-
tion of tissue to some degree is essential for the production of leukocytosis and it
seems that the action of the organism, or of its toxin, is not the major factor in
bone marrow stimulation. This conception is further borne out by the leuko-
cytosis that can be produced by chemicals, such as the .injection of turpentine
with formation of a sterile abscess in the muscle.
Among the diseases that are characterized by a marked pathologic leukocy-
tosis are pneumonia, lobar and bronchial, but only if organisms are of the pyogenic
type, peritonitis, otitis media, mastoiditis, meningitis, multiple abscesses, fur-
uncles, solitary abscesses of considerable size, scarlet fever, diphtheria, erysipelas,
etc. We can not escape the belief that most often the degree of leukocytosis de-
pends on the amount and type of tissue involvement and that the major factor in
bone marrow stimulation is an unknown degradation product of tissue destruc-
tion. The effect of tissue changes on bone marrow stimulation is well illustrated
by the fact that varying degrees of myocardial infarction after coronary throm-
bosis are followed by leukocytosis in some instances as high as 40,000 to 50,000
cells per cu. mm., these being mainly young forms of neutrophilic leukocytes.
Pathologic leukocytosis may follow acute hemorrhage, particularly if the
loss of blood is considerable. It may reach quite a high figure under these condi-
tions. On the other hand, if the· bleeding is long continued and chronic, even
though the degree of anemia be the same as after acute hemorrhage, leukocytosis
will be very slight.
Leukocytosis is observed in all conditions in which there is acidosis. The
acidosis of diabetes is always accompanied by some degree of leukocytosis.
The injection of irritating chemicals is followed by leukocytosis as is so well
illustrated by the injection of five to six drops of turpentine into the muscles. This
produces a so-called "sterile abscess." We have observed a leUkocytosis of 25,000
following such an injection.
Pathologic leukocytosis is seen in malignancies, especially if far advanced.
It might be inferred that this is caused by secondary infections, but leukocytosis
often occurs in malignancies in which there is no secondary infection. Various
cachectic states are usually accompanied by a mild degree of leukocytosis. The
rise in white cells just preceding death has long been recognized and referred to as
agonal leukocytosis.
We have observed repeatedly the consistent leukocytosis that follows opera-
tive procedures even in non-infectious cases. This post-operative leukocytosis in
our opinion is due to tissue damage and trauma during the course of the opera-
tive procedure and there is little evidence that administration of anesthesia alone
will produce this. We have studied very carefully 20 non-infectious operative
cases for the hematopoietic changes that occur after operation and found that
the leukocyte count on the day afterward was double the original count in one-
 NEUTROPHILIC LEUKOCYTOSIS (NEUTROCYTOSIS) 12 7

half of the patients and the remainder showed some elevation of the count with
the peak of leukocytosis on the second post-operative day. Therefore, it should
be borne iIi mind that when the leukocytes are studied after operation this factor
of possible tissue trauma should be taken into consideration in evaluating the
findings.
The injection of many inert substances into laboratory animals produces vary-
ing degrees of leukocytosis. We have ·injected many rabbits with inert materials,
such as finely pulverized carbon and dyes (carmine), and found that a leuko-
cytosis developed after an initial period of leukopenia. The injection of dead
bacteria of practically any kind will cause a leukocytosis in rabbits. We have
also injected into rabbits finely divided colloidal preparations of gold, mercury
and sulphur and found that when these materials were injected intravenously and
intramuscularly there resulted a definite leukocytosis of mild degree. In an effort
to determine the leukocyte response following injections of sterile milk, 20 women
suffering with salpingitis were given daily intramuscular injections of sterile
milk (10 cc.). All of them responded with a leukocytosis reaching its peak from
the fourth to eighth hour after the injection. We have carried out similar experi-
ments with other types of non-specific proteins and a mild leukocytosis was the
usual result. If various tissues of the body are permitted to undergo varying
degrees of autolysis and the resulting products injected into animals, a leuko-
cytosis will usually result, occasionally to a marked degree (50,000 per cu. mm.).
Leukocytosis has also been produced in animals by various other methods.
In a study of the relationship of leukocytosis to leukemia Oliver and Katzman
injected animals periodically with sodium nucIeinate and sodium caseinate and
found extremely high leukocyte counts. They also noted that practically all of
the biliary salts would produce marked degrees of leukocytosis, sometimes re-
sembling leukemia. Glycocholic and taurocholic acids were especially capable
of producing this action. They stated "to our knowledge this is the first time
that leukemia-like blood changes as well as leukemia-like tissue changes have
been produced by acids found present in the normal bot:y metabolism."
In his effort to find an agent that is specific for leukocyte production, Net-
tIeship concluded that substances were released from the cytoplasm of the leuko-
cytes themselves that appeared to stimulate the bone marrow. He prepared
various tissue extracts to test their leukocyte producing properties and found
that only hemopoietic tissues gave positive results. He prepared an aqueous ex-
tract of the bone marrow, extracted this with cold ether to remove the fat, then
took the water fraction of this, treated it with heat or ethyl alcohol, with the
resulting precipitate producing marked leukocytosis in rabbits. Nordenson car-
ried out experiments on 43 patients by injecting them with sodium nucleinatc
and pentose nucleotide, and was able to produce a sustained leukocytosis with
these agents. He points out that the bone marrow in these cases must be previ-
ously undamaged.
Cuttle gave rooD mg. of ascorbic acidJo ten patients; six as controls and
four with leukemia. He did not notice any elevation of the leukocyte counts in
the normals and no decrease of cells in the leukemic patients. Dean and Solomon
injected hemolyzed autogenous dtrated blood intravenously into seven patients.
but leukocytosis was not marked or consistent.
 LEUKOCYTOSIS ·AND LEUKOPENIA

Because of the many factors that affect the total number of leukocytes and the
fact that the leukocytic changes are oftentimes so slight, it is necessary for careful
studies that leukocyte counts should be done if ·possible at the same time of day
and under the same conditions. Because of this there has arisen the term
"basal leukocyte count" which is analogous to the basal metabolic rate in the
estimation of oxygen consumption. A basal leukocyte count is one that is done
with the patient in a rested state, having taken no food immediately hefore-
hand. Thus, to determine the hasal leukocyte count the patient should report
to the laboratory without breakfast and should be given a rest period of one
hour before the count is made. Of course, other precautions are necessary, such
as the use of certified pipettes in the hands of the same worker, if repeated daily
counts are done. As stated before, leukocytosis can occur because of a general
redistribution of those cells that are already present in the peripheral circula-
tion. The second method by which leukocytosis comes about is by direct stimula-
tion of the bone marrow and this is the type produced in the pathologic group.
Naegeli states that the bone marrow response is the direct indicator of the severity
of the infection and the resistance of the individual. It seems reasonable that the
bone marow output is directly stimulated by a number of agents. Bone marrow
output may increase as a result of decreased oxygen tension. It seems reasonable
to assume that bone marrow maturation and delivery of leukocytes are accelerated
by the action of bacterial toxins, the products of tissue degradation, small doses
of radiation, and chemical agents. Probably the most marked stimulation results
from the unknown factor that is usually referred to as the maturation factor.
This may be one of the nucleic acid derivatives (adenine sulphate; guanine hydro-
chloride; pentnucleotide). In any event, the factor that is responsible for in-
creased delivery of neutrophils from the bone marrow has not yet been discovered
and it affords, naturally, one of the most intricate problems in the field of
hematology.
The bone marrow should be regarded as a widespread and far flung organ
whose function is to manufacture and deliver neutrophilic leukocytes, as well
as red cells and platelets. Apparently it does this in the normal individual in such
an efficient way as to maintain the leukocyte count at its normal figure. Thus, it
is easy to assume the existence of a hypothetic substance which regulates the
speed of maturation of granulocytes and, furthermore, perhaps another substance
that regulates the mechanism of their delivery. It is not difficult to conceive of
this substance as being manufactured in some unknown tissue in the body, stored
in another, and liberated as needed, similar to the rme of insulin in the metabolism
of sugar.
Under normal conditions the bone marrow is supposed to deliver to the
peripheral circulation only those cells that are matured and functional. There-
fore, the normal neutrophils consist largely of fully segmented types in which
the nuclear lohes are definitely divided with only a small per cent (4 per cent)
of young neutrophils in which the lobes are not divided. Therefore, if an examina-
tion of fue blood shows immature forms to be increased in number, whether or
not the total leukocyte count is increased, this should be accepted as evidence that
the bone marrow is subjected to undue stimulation and it should be einphasized
here that no other interpretation can be placed on it. As we have pointed out
 NEUTROPHILIC LEUKOCYTOSIS (NEUTROCYTOSIS) 129

before, there are many factors that are capable of stimulating the bone marrow
to increased activity. Therefore, the presence of immature granulocytes does not
necessarily mean the presence of an infectious process but it merely means that
the bone marrow is being subjected to an undue and excessive stimulating agent.
It is quite difficult_ to evaluate the significance of the total blood cell count.
A high cell count no doubt means that tbe bone marrow is capable of an effective
resistance. A low cell count on the other hand does not mean that the bone
marrow is incapable of resisting. It probably emphasizes the point brought out
by Watson and Sarjeant that a low leukocyte and neutrophil count may result
from the emigration of large numbers of leukocytes to the involved infected area,
and the larger the area the greater the number of cells involved.

DIFFERENTIAL COUNT

As stated before, the normal blood contains from 65 to 70 per cent of adult
segmented neutrophils. In many instances of infection the total number of
leukocytes is not increased but the percentage of neutrophils is increased. If a
patient has a high leukocyte count and a high percentage of neutrophils, this can
-be accepted as evidence of increased bone marrow output. If a patient has a
normal leukocyte count and a high percentage of neutrophils, this also can be in-
terpreted as being the result of the excessive bone marrow stimulation, perhaps
with poor resistance, or a poor capacity of the bone marrow to respond. But if
a patient presents a normal leukocyte count and a normal percentage of neutro-
phils, it is still possible that the bone marrow is SUbjected to undue stimulation
and this can be ascertained only by a careful study of the circulating cells. There-
TABLE XI
THE RELATIONSHIP OF THE VARIOUS METHODS OF NEUTROPHILIC CLASSIFICATION

Cooke Filament
Pons~ and Non-fil-
Schilling Arneth Krumbhaar Ponder ament Type oj Cell
Myelobl...' .......... .. Class I . I lobe Non-fil. Myeloblast
Premyelocyte . ........ . Class I I lobe Non-fiI. Premyelocyte
Myelocyte ............ . Class I I lobe Non-fil. Myelocyte
Juvenile .............. . Class I Metamyelocyte I lobe Non-fil. Metamyelocyte
Band (Stab) .......... . Class I Non-segmented I lobe Non-fil. Young neutrophil
Segmenter ............ . Class II Segmented 2 lobes Fil Filamented
(Two lobes)
Segmenter. . . . . . . . . . . .. Class III Segmented 3 lobes Fil. Three lobes
Segmenter. . . . . . . . . . . .. Class IV Segmented 4 lobes Fil. Four lobes
Segmenter. . . . . . . . . . . .. Class V Segmented 5 lobes Fil. Five lobes
Segmenter. . . . . . . . . . . .. Class V Segmented 5 lobes Fil. More than five
lobes (Macro-
polycyte)
fore, as a general rule regardless of the total number of leukocytes, or the total
percentage of neutrophils, the most accurate criterion for the evaluation of exces-
sive bone marrow stimulation is the presence of immature circulating leukocytes.
This fundamental concept has led to many attempts on the part of various work-
ers to classify the immature cells, to evaluate their significance and to formulate
an expression which would indicate the degree of bone marrow activity. Thus
have resulted such cellular classifications as the Schilling index, the Arneth count,
the Cooke and Ponder classification, the Pons-Krumbhaar classification; and the
130 LEUKOCYTOSIS AND LEUKOPENIA

filament non-filament grouping of leukocytes_ The differences and similarities of

these various classificationsr are shown in Table XI.

SCHILLING CLASSIFICATION
From a close study of this table it is seen at once that all of these methods
have the same purpose in view. Thus, Arneth c.lassified all granulocytes into
five classes with subgroups depending upon the number of lobes in the nucleus.
Cooke and Ponder's classification is exactly the same although not quite so com-
plex. The filament non-filament count is similar except the number of fila-,
ments is counted rather than the lobes. Schilling recognized the fact that
these classifications are deficient in some respects. They do not take into
account the degree of immaturity of the cell beyond the juvenile or metamyelo-
cyte stage. Thus, the Arneth count, the Cooke and Ponder classification and the
filament non-filament count are of some value, but they classify only those cells
that have reached the adult stage. On the otlier hand, the Schilling classification
designates all types of cells from the myelocyte down and all of the segmented
cells are simply referred to as segmenters. There would seem to he little reason
for classifying neutrophils according to the number of lobes in the nucleus since
we believe that when the neutrophil is delivered from the hone marrow it is at the
segmentation stage and the subsequent division of lobes occurs in the peripheral
blood and within itself is of little importance. Therefore, we prefer to use the
Schilling classification which, in our opinion, affords the most valuable indica-
tion as to the degree of immaturity of the neutrophils and the degree of bone mar-
row stimulation. The Schilling classification of neutrophils should be included
in every routine differential count. The well trained laboratory technician can
classify these cells with little difficulty and the method offers considerably more
information than can be obtained from any of the other methods of classification.
In reporting differential cell counts we use the following classification:
MYe}OCYtes
Total Neutrophlls { ~ands (Stabs)
. uveniles
Lymphocytes Segmenters
Monocytes .
Eosinophils
Basophils

During the course of the examination if the leukocytes present an unusual

degree of nuclear segmentation, a second count is made in which the numher of
nuclear lobes is recorded. If the immature cells are markedly increased, this is
reflected in the differential count. In general, the degree of immaturity is an
indicator to the severity of bone marrow stimulation but it does not necessarily
follow that the degree of immaturity is equivalent to the severity of a certain
infectious process. When the degree of cellular immaturity is marked, this is
commonly referred to as a shift to the left, or a shift toward immaturity.
It is important that the findings in the Schilling index he carefully inter-
preted. As is the case with so many recently advanced laboratory procedures,
there is a tendency for one group of workers to over-emphasize the importance' of
the procedure and for another group to decry its value. It is unwise to utilize
these findings as an aid to prognosis. It is unwise to accept cellular immaturity
as positive proof of an existence of an infection because, as pointed out above,
many things can stimulate the bone marrow and infection is only one of these.
 NEUTROPHILIC LEUKOCYTOSIS (NEUTROCYTOSIS) 13 1

This can be well illustrated by some instances of food poisoning, where no bac-
terial infection is present and yet these patients may show a high leukocyte count
with a marked shift toward immaturity. Immaturity of cells, therefore, must be
interpreted in its proper light; .that is, it is simply an indication that i'he bone
marrow is subjected to excessive stimulation. Some bone marrows respond with a
high leukocyte count and a large number of' mature neutrophils; others respond
with a high leukocyte count and large numbers of very young neutrophils; while
still another may respond with a normal leukocyte count and a normal percentage
of extremely immature neutrophils. From this, it might be inferred that the
degree of resistance could he based upon such findings but this is inadvisable
because it should be borne in mind that the bone marrow response, as evaluated
by the Schilling index, is a result of a combination of factors among which are:
the general resistance of the individual; the capacity of the marrow to respond;
the type of stimulation, and the severity of the stimulating agent.

DEGENERATIVE INDEX
The first blood count was recorded in 1853, and in 1879 Ehrlich classified the
three types of neutrophilic leukocytes. The procedure was further improved by
the invention of the blood diluting pipette in 1867 and counting chamber in 1877.
In '904, Arneth made the first attempt to classify the leukocytes according to
age. Then came the classification of Schilling in 19II, followed by those of
Cooke and Ponder, Pons and Krumbhaar, and finally the filament and non-filament
count suggested by Farley and his associates. As pointed out before, all of these
developments in leukocyte studies have centered around three basic findings:
first, the total number of circulating neutrophils; secondly, the numbers of each
type, and thirdly, the immaturity of the cells. There has been proposed a further
study of leukocytes which may add additional evidence in diagnosis and this is a
study of the so-called basophilic or toxic granulation. It has been noted in certain
severe infectious statcs that the cytoplasmic granules in many of the leukocytes
are not stained in the normal way, but they appear more basophilic than normal.
This type of leukocyte is illustrated in cells 2-3, Plate 16. Based upon the
number of toxic leukocytes there has been proposed the so-called "degenerative
index" suggested by Kugel and Rosenthal. The degenerative index is computed
by dividing the total number of neutrophils into the total number that show toxic
changes. An example of this computation is shown in Table XII.

TABLE XII
METHOD FOR COMPUTING TlJE DEGENERATIVE INDEX
13asophilie
Total Normal Granulation
Neutrophlls ............ 75% 20% 55%
Eosinophils. . . . . . . . . . . . 0
Basophils. . . . . . . . . . . . .. 0
Lymphocytes. . . . . . . . .. 15%
Monocytes. . . . . . . . . . . .. 10%
Number of neutrophils showing
basophilic granulation 55
Degenerative index = Total number of neutrophils ... 15 = 73
Percentage of neutrophils showing basophilic granulation om 73
13 2 LEUKOCYTOSIS AND LEUKOPENIA

The degenerative index, therefore, represents the percentage of neutrophils

that show basophilic granulation. In general, it is said that the presence of toxic
cytoplasmic granulation indicates, not only the presence of a snspected toxemia
of infection, but also the severity of it. We have found that the state of toxic
granulation has afforded us very little information as an aid in diagnosis but like
other neutrophilic changes, it must be carefully correlated with the clinical fea-
tures of the patient.

BIBLIOGRAPHY

ARNETH, J.: "Die Neutrophilen Weissen Blutkorpcrchen bci Infections. Krankhcitl:!D." G. Fisher.
Jena, 1904.
COOKE, W. E., and PONDER, E.: "The Polynuclear Count." The J. B. Lippincott Company, Phila-
delphia,1927. r
CUTTLE, T. D.: "Observations on the Relation of Leukocytosis to Ascorbic Acid Requirements."
Quart. J. Med., 31, 575, 1938.
DEAN, S. R., and SOLOMON, H. C.: "Intravenous Autohemotherapy with Hemolyzed Blood: Tech-
nique and Leucopoietic Response," Jour. Lab. Clin. Med., 23, 775. 1938.
EHRLICH, p,; "Ueber die Specifischen Granulationen des Elutes." Arch. J. Physiol., 571, 1879.
FARLEV, D. L., ST. CLAIR, H.,.and REISINGER, J. A.: "Normal Filament and Non-filament Poly~
morphonuclear Neutrophil Count. Its practical value as a diagnostic aid." Amer. Jour. Med.
Sci., 180, 336, '934.
FITz-HuGH, T., JR.: "The Age of the Leukocyte in Relation to Infection." Jour. Lab. and CUn.
Med., 17, 975, 1932.
GARREY, W. E" and BRYAN, W. R.: "Variations in White Blood Cell Counts." Physiol. Reviews,
15, 597, 1935.
HADEN, R. L.: If Qualitative Changes in Neutrophilic Leukocytes." Amer. Jour. GUn. Path., 5,
354, 1935. .
KUGEL, M. A., and ROSENTHAL, N.: 'lPathologic Changes Occurring in Polymorphonuclear Leuko-
cytes During Progress of Infection." Amer. Jour. Med. Sci., 183, 657. 1:932.
LUCIA, S. P., LEONARD, M. E., and FALCONER, E. H.: "The Effect of the Subcutaneous Injection of
Adrenalin on the Leukocyte Count of Splenectomized Patients and of Patients with Certain
Diseases of the Hematopoietic and Lymphatic Systems." Amer. Jour. Med. Sci.} 194, 35, 1937.
NAEGELI, 0.: Blutkrankheiten und Blutdiagnostik, ed. 5. J. Springer, Berlin, 1:923.
NETTLEsnIP, A.: "Leukogenic Bone Marrow and Leukocyte Extracts." Amer. Jour. Clin. Path.,
10, 265, 1940. Ibid. "Leukocytosis Associated with Acute Inflammation." 8. 398, 1938.
NORGENSON, N. G.: "Experimental Leukocytosis in Man." Quart. J. Med., 32, 3U,' 1:939·
OLIVER, S., and KATZMAN, D.: "On the Relationship between Leukocytosis and Leukemia." Folia
Haem .• 59, 289, 1938.
PONS, C., and KRUMBHAAR, E. B.: "Studies in Blood Cell Morphology and Function. Extreme
Neutrophilic Leukocytosis with Note on Simplified Arneth Count.'1 Jour. Lab. and GUn. Med'l
10, 123, 1924.
SABIN, F. R., CuNNINGHAM, R. C., DOAN, C. A., and KrNnwALL, J. A.: "The Normal Rhythm
of the White Blood Cells." Bull. Johns Hopkins Hosp., 37, 141 1925.
SIMPSON', \V.: Personal Communication. 1935. (On effect of hyperpyrexia on the leukocyte count.)
VAUGHN, W. T.: liThe Leukopenic Index. Food Allergens." Preliminary report. Jour. Allergy,
5, 601, 1934; and Jour. Lab. and Glin. Med., 21. 1218, 1936.
WATSON, C. H., and SARJEANT, T. R.: "Significance of a Low Leukocyte Count in Ac'U:te Pyogenic
Infections." Canadian Med. Asso. Jour., 39, 460, 1938.
 CHAPTER 11
LYMPHOCYTOSIS, MONOCYTOSIS, EOSINOPHILIA AND
BASOPHILIA
LYMPHOCYTOSIS

The normal number of circulating lymphocytes in the adult is between 2,000

and 4,000 per cu. mm., while in children it"is between 4,000 and 6,000. About 75
per cent of these are small lymphocytes and the remainder large lymphocytes,
which are supposed to be the more immature types. (See Plate 7 for detailed
description of lymphocytes.) We see little reason to differentiate between the
large and small lymphocytes and see no reason for separating them in the routice
differential count. Oftentimes this is difficult to do with any degree of cer-
tainty since only the large type with typical azure granules can be definitely
identified. In certain instances, however, the differentiation is of some impor-
tance; in particular, those of extreme lymphoid hyperplasia where the diagnosis
of a leukemic state is in question. As a rule, it may he assumed that the more
immature the lymphocytic picture, the more marked is the lymphoid hyperplasia.
In this respect the lymphoid immaturity is similar to the granulocytic immaturity
of the bone marrow, except it does not have as widespread diagnostic application
as the latter.
Lymphocytosis may be either relative or absolute. If the total number of
lymphocytes is normal and the number of granulocytes below normal, there then
exists a relative lymphocytosis. It is obvious that the term is a misnomer though
accurate. It carries the implication that the lymphocytic picture is altered where
as a matter of fact there actually exists a nentropenia with the lymphocytic pic-
ture normal. This illustrates the desirability of dealing with whole numbers in-
stead of percentages of cell types.
Any disease characterized by lymphoid hyperplasia is usually accompanied
by a lymphocytosis of varying degree. The lymphoid hyperplasia secondary to
infectious processes, in which the glandular enlargement may be local and re-
stricted to one area, may be accompanied by increased lymphocytes in the blood,
and if lymphadenopathy is 'generalized, lymphocytosis is practically certain to
be present. Thus, such diseases as glandular tuberculosis, secondary syphilis,
Hodgkin's disease, lymphosarcoma and infectious mononucleosis will present vary-
ing degrees of lymphocytosis.
Various infectious states of the oral cavity, if accompanied by cervical
lymphadenopathy, will result in increased lymphocytes in the blood. For ex-,
ample, Vincent's angina, acute and chronic tonsillitis, pharyngitis, etc., may give a
lymphocytic increase.
Many diseases are stated to be characterized by lymphocytosis. Among these
are typ~oid fever, typhus fever, undulant fever, malaria in its chronic stage, small-
pox, chickenpox, measles, especially whooping cough, tuberculosis in the healing
133
 PLATE :;eVIl

LYMPHOCYTOSIS

tr"\
~\J
<;' V 1l

'0
09 I
1

1. Lymphocytes.
2. Lymphocyte with azure granules.
3. Neutrophil.

mood Findings (child with whooping cough): Differential:

Hemoglobin . .16.2 gms, (Newcomer's method)· MyeIocytes ...... 0'%'
R.B.C. 5,35°,000 per c.mIl]. Juveniles ........ 4%
W.B,C. 39,000 per c,rom· Bands ...... 6%
Platelets 310,000 per (:.mjll· Segmenters ...... 100/0
Total neutrophils . . . " 20%
Erythrocytes; normochromic and normocytic. Lymphocytes ...... . .. 80%
Plate XV IT.
Plate XVIII.
 PLATE XVIII

MONOCYTOSIS

~
~
n

I. Monocytes with granular cytoplasm.

2. Monocyte without granulation.
3. Neutrophil.
4. Lymphocyte.

Blood Findings (patient with active pulmonary tuber- Differential:

culosis);
Hemoglobin 1:4.5 gms. (Newcomer's method). Myelocytes 00/'0
R,B.C. . 4,500,000 per c.mm. Juveniles ... 2%
W.B,C. 10,200 per c.mm. Bands 8%
Segmenters .......... 56 %
Platelets .... . .............. , 292,000 per c.mm. Total neutrophUs . 66%
Erythrocytes; normochromic and normocytic. Lymphocytes ............... 16%
Monocytes .. , ........ " • 18%
 LEtfKOCYTOSIS AND LEUKOPENIA

stage and syphilis in the stage of lymphadenopathy. In some of these, however,

particularly the acute exanthemata, the lymphocytosis is more apparent than real,
because a leukopenia may be present at the expense of the neutrophils, with
the lymphocytic picture unaffected. .
• Lymphocytosis is not found as a response to the pyogenic bacteria, since the
lymphocyte is not a phagocytic cell, but its function is more mechanical than
the phagocytic cell, in that its chief function is to isolate bacteria and foreign
material by a walling off process. Therefore, in most bacillary diseases, especially
those of chronicity, the response is lymphatic.
Lymphocytosis i, said by Naegeli to follow various vaccinations (smallpox,
typhoid). The most extreme degree of lymphocytosis is seen in whooping cough,
and is present with sufficient consistency to make its finding of considerable diag-
nostic importance. In the presence of a characteristic cough and other clinical
findings, an extreme lymphocytosis establishes the diagnosis in this disease. How-
ever, not every case of whooping cough is accompanied by lymphocytosis.
The roseola infantum or exanthem subitum of children is accompanied by
a marked lymphocytic predominance. Though the lymphocytes do not rise to an
extremely high level, there is also a neutropenia that accentuates it all the more;
nevertheless, there is a t{ue lymphocytic increase.
No doubt there. exists a type of person characterized by generalized lymphoid
overgrowth, as seen in the so-called status lymphaticus, and who is said to have
the "lymphoid diathesis." This type of person often shows a true lymphocytosis.
Exophthalmic goiter is oftentimes accompanied by the lymphatic reaction,
whereas the simple colloid goiter is not. This seems to be true only in those
patients that are undergoing the so-called involution changes, in which the thyroid
gland has undergone a period of aativity but is retrogressing, manifested 'by the
appearance of fIbrosis, degenerative changes, and lymphoid tissue in the gland.
Some of these show germinal centers of lymphoid tissue and this type of case is
likely to have a lymphocytosis. In hyperthyroidism of the active type, the
lymphocytosis may precede the involution changes. The frequency with which
lymphoid changes occur in this disease has led to the belief that the administra-
tion of iodine may be responsible for the lymphocytosis. However, this is un-
likely, since the administration of iodine in the normal subject produces' no such
lymphocytic predominance in the blood.
Lymphocytosis is said to occur in Addison's disease of the adrenal cortex,
though this has not been verified. We have not observed this in the few cases that
we have seen.
The disease that has appeared in recent years known as infectious mononu-
cleosis, or benign lymphadenosis, presents a marked lymphocytosis at its height,
with a wide variety of immature types that may give a hematologic picture
similar to a leukemic state. We have seen cells in this disease that, in out
opinion, are unmistakable lymphoblasts. (See chapter on infectious mono-
nucleosis. Plate 47.)
Any disease, even though it is pyogenic in the beginning, that requires a long
period for the healing process, may present a lymphocytosis. Naegeli refers to
this as postinfectious lymphocytosis.
LV:MPIIOCYTOSIS, lVIONOCYTOSlS, EOSINOPHILIA AND BASOPHILIA I37

On the clinical service of Dr. M. Hines Roberts, in the division of pediatrics

in Emory University, we have studied the blood of mauy children with profound
hypochromic anemia because of malnutrition, and we are convinced that a lympho-
cytic predominance is a part of this picture. The leukocyte count is usually high
(IS,OOO) and the total number of lymphocytes from 10,000 to 12,000. Therefore,
there is also an actual neutropenia. Furthermore, in the healing or recovery phase
of various leukopenic diseases of unknown cause we have noted the consistent
rise of lympbocytes to such an extent that we wonder if the lymphocyte does not
attempt sometimes to fill the place of the absent neutrophil.
The extent of the lymphocytosis in tuberculosis is of some practical impor-
tance. Since a lymphocytic predominance is usually associated with the healing
process, not only in tuberculosis, but in all chronic infectious states, the assump-
tion has become firmly established that a lymphocytic predominance in this dis-
ease indicates a stage of healing and quiescence, while a monocytic predominance
indicates activity of the process. This occurs so consistently that the use of the
monocytic-lymphocytic ratio to evaluate the progress of a patient with tubercu-
losis has become widely adopted. This would appear to be based on logical
premise since the first line of defense against the invasion of the tubercle bacillus
is a zone of monocytes, this followed by the more leisurely walling off process of
the lymphocytic cells.
Lymphocytosis in extreme degree is seen in the lymphatic leukemic states.
The number of small lymphocytes in chronic lymphatic leukemia may reach one
million per cu. mm. Also the lymphocyte count reaches high levels in the acute
types of lymphadenosis. In these instances, the diagnosis is readily made because
of the high lymphocytic counts and the presence of immature blast cells. In those
cases in which the total count is only slightly above normal or even below normal,
it is oftentimes a difficult problem to distinguish between a benign lymphocytosis
and a true leukemic state and the differentiation rests upon the identity of the cir-
culating lymphocytic cells. (See plates on lymphocytes, lymphoblasts, and
lymphadenosis. )

MONOCYTOSIS

In the normal adult the monocytes constitute from 5 to 10 per cent of the
circulating white cells, or from Sao to 1,000 per cu. mm. If it is true that these
cells arise from the reticula-endothelial system, then monocytosis may be regarded
as an expression of overactivity of that particular tissue. On the other hand,
if they arise from the myeloid tissue, as Naegeli contends, then these cells should
be increased in all types of infectious processes which are ordinarily attended
with increases in neutrophilic leukocytes. There is some evidence that this is true.
Thus, the monocytosis that follows bone marrow stimulation in pigeons after
injection of tubercle bacilli, the monocytosis after certain anginal conditions of
the soft tissues of the mouth, the infectious character of infectious mononucleosis
:vith an incre:",e in monocytes, the early response of monocytes and neutrophils
l~ th~ formatIon of the tubercle, and the monocytoid phase of cellular regenera-
tIOn 10 recovery from agranulocytosis, as pointed out by Rosenthal, ;upport this
 LEUKOCYTOSIS AliD LEUKOPENIA

belief. Therefore there is some basis for Naegeli's expression that mono·cytosis
is closely parallel with myeloid activity. It would appear more likely that mono-
cytes are capable of being produced and delivered from the bone marrow because
this tissue, like many others, includes a considerable amount of reticula-endothe-
lium. All of this does not mitigate the concept that, for the most part, these
cells are formed in the widely distributed reticula-endothelium and they may
arise from any of these tissues, which are found in the lnarrow, liver sinusoids,
splenic channels and lymph nodes. Therefore, because monocytes are found in
considerable numbers in the marrow under certain conditions is no reason why
one should assume that they are produced there exclusively. We can see no
reason for this bitter controversy for the origin of monocytes when one person
contends they arise from reticulo-endothelium and another contends that they
arise from the bone marrow tissue. They may arise in either place, and one
needs only to consider the distribution of tissue to understand this.
In malaria it is usually stated that there exists a mo'nocytosis, with which
we agree, but only in the chronic stages of the disease. We have never been
able to demonstrate a true monocytosis in malaria in the acute, newly developed
case; this type of the disease usually has a neutrophilic leukocytosis of mild
degree with a moderate shift to immaturity, including numerous metamyelocytes
which mt\y be mistaken for monocytes. It is oftentimes impossible to distinguish
between a heavily granulated monocyte and a metamyelocyte. Supravital studies
may eventually do this but in our opinion the criteria of cell identification in
supravital studies may fall into the same errors as the criteria in stained prepara-
tions. We have placed monocytic cells in a microscopic field and invited some
of the best hematologists in America to identify them, and the results are as
variable as if a group of sophomore medical students had attempted the identifi-
cation. This merely shows the disagreement concerning the morphology of mono-
cytes. In view of these differences of opinion concerning the appearance of these
cells on the part of experts and in view of the fact that these cells probably can
be formed in a number of tissues, it seems like quibbling over minutiae to spend
time in controversy on this point. We are likely to become lost in a maze of
cytologic details.
Monocytosis exists in the peripheral blood in those conditions that can be
designated clinically as subacute. It is seen in the various stages of most of
the lymphogranulomata, in chronic malaria, von Jaksch's anemia, syphilis (stage
of invasion), tuberculosis during the active and progressive phase, typhoid fever
in the early stages, early Hodgkin's disease almost invariably, and late Hodgkin's
disease usually, chronic stages of amebic dysentery, infectious mononucleosis,
and after removal of the spleen.
It should be emphasized that any of these diseases may not be accompanied
by monocytosis at any time during their course, and that most of them show
monocytosis only during certain stages of the disease. This depends on the um!er-
lying pathology. Thus, in typhoid fever there is neutrophilic leukocytosis in the
stage of breaking down and ulceration <)f Peyer's patches, but monocytosis only
in the early invasion of the area, and later in the healing process. Also in tubercu-
losis there is neutrophilic leukocytosis in the active stage of invasion and early
LYMPHOCYTOSIS, MONOCYTOSIS, EOSINOPHILIA AND BASOPIIILIA 13t;

formation of the tubercle, this followed by monocytosis, and this followed in turn
by lymphocytosis. Therefore, it is not essentially correct to state that a certain dis-
ease is accompanied by a certain cellular output in the blood. This depends entirely
upon the pathologic process that is going on in that particular person at that time.
We have observed that a monocytosis follows splenectomy after varying
periods of time. This tends to confirm the impression that a large part of the
reticulo-endothelium is removed at splenectomy and compensation is necessary
in the form of monocytic output from the marrow, liver sinusoids, and the lympli
,nodes. Sometimes this is so marked that there is a generalized lymphadenopathy
during the process. We have studied carefully one patient who had a splenectomy
eight years ago, and who maintains consistently a monocytosis with a slight
generalized lymphadenopathy. Also, we have seen others who develop monocy-
tosis only a few months after splenectomy.
In our experience most patients with infectious .mononucleosis show at one
time, usually in the late stages, a definite monocytosis with the predominant cell
the adult monocyte. However, we have seen others in whom the picture is essen-
tially one of lymphoid overgrowth. It is a paradoxical disease in so far as the
blood picture is concerned. Then there are cases in which the predominance is
lymphocytic, this later developing into monocytosis. This inconsistency, no
doubt, has resulted in the confusion in terminology, in which some call it in-
fectious mononucleosis and others benign lymphadenosis.
Monocytes reach high levels in many cases of monocytic leukemia. Some
of these eventually become typical myeloid types. (See section on monocytic
leukemia.) Again we would emphasize that monocytosis cannot be labeled as a
characteristic of any certain disease;. that it occurs in many diseases at various
phases; that in general it is a phagocytic response for the walling off and removal
of large organisms and foreign material; that the various hematologists disagree
as to what a monocyte is, what it looks like, and where it comes from; and
finally, that a thorough knowledge of underlying pathologic processes is necessary
in any disease to understand or explain why monocytosis occurs at certain times.
EOSINOPHILIA (EOSINOPHILIC LEUKOCYTOSIS)
The normal number of circulating eosinophils varies from I to ;, per cent of
the total white cells, or 100 to 200 cells per cu. mm. The eosinophil differs from
the neutrophil mainly in containing a type of cytoplasmic granulation that is much
larger, much more shiny, more refractile and more acidophilic in character so
that the eosin stained granule is a bright orange red. Eosinophilic granulation
is peroxidase positive and many eosinophils show a tendency toward basophilic
changes as evidenced by the blue tinge to the orange colored granules in some
of the cells.
The granules in eosinophils do not show the characteristics of fat. They are
not digested by tryptose or by autolytic enzymes. They are soluble in concen-
trated alkalis and adds and in warm acetic ;l.cid. They contain I I per cent of
iron, according to Barker. Weidenreich states that the granules are composed
of hemoglobin or products of hemoglobin breakdown and that these are taken up
by the cells and deposited in the cytoplasm as eosinophilic granules.
 ,PLATE XIX

EOSINOPHILIA

1. EosinophiIs (showing color variation in cytoplasmic granulation).

:2, Neutrophil.

Blood Findings (patient with trichiniasis): Differential:

Hemoglobin ....... 12.6 gms. (Newcomer'~s method). Myelocytes .. 0%
RB.C. .. . . " 4,340,000 per c.rum. Juveniles ... 3%
W.B.C, ......•.... 1I,8oo per c.mm. Bands ... , . . , ... 5%
Pla.telets .. 320,000 per C.mm. Segrncnters . .. .34%
Total neutrophils
Lymphocytes
Eosinophils ..
Erythrocytes i slightly hypochromic and normocytic.
LYMPHOCYTOSIS, MONOCYTOSIS, EOSINOPHILIA AND BASOPH1LIA 141

Eosinophils are sometimes increased in normal individuals. These instances

are said to be constitutional or hereditary in nature. We have studied several
people who maintain an eosinophilia of 10 to 25 per cent and who demonstrate
no other abnormalities whatever. Therefore, the mere presence of excessive
eosinophils does not necessarily indicate' a disease process.
Eosinophils are at times increased in the myeloid leukemic states as com-
pared to the number of neutrophilic cells. Scarlet fever in its early stages is most
often accompanied by a definite eosinophilia of 5 to 10 per cent. After the dis-
ease has progressed this disappears and a neutrophilic leukocytosis becomes evi-
dent. Then again in the' healing phase, or late stage of scarlet fever eosinophilia
again becomes a prominent part of the picture. This has led to the belief that
the initial phase of scarlet fever may be an allergic type of reaction.
Eosinophils are quite consistently increased in infestation with various intes-
tinal parasites; thus, infestation with hookworms, various tapeworms including
the fish tapeworm, the round worms, the pin worms, and others cause varying
degrees of eosinophilia. Apparently, this is not due to the mechanical features
of the infestation since it has been shown that the injection of an extract from
the round worm (Ascaris) will produce eosinophilia. It seems probable, there-
fore, that these parasites produce an unknown type of agent capable of stimulating
these cells by action on the bone marrow. Eosinophilia may be present in
amebiasis and in filariasis. Probably the most marked increases are those that
accompany trichiniasis after the cysts have formed in the muscles.
Practically all varieties of the so-called allergic diseases are accompanied by
eosinophilia. It is consistently found in all types of asthma, hay fever, angio-
neurotic edema and various other manifestations of the allergic, or hypersensitive
state. It occurs so consistently in these conditions that its mere presence is
regarded as an important diagnostic sign. The nasal and oral secretions of
allergic patients will show more eosinophils than the normal. The fluid of blebs
and wheals in the allergic phenomena eventually become filled with numbers of
eosinophils. It is stated that an eosinophilia in most of these instances precedes
the appearance of the clinical symptoms. Eosinophilia is said to be a consistent
finding in a number of skin diseases, these including psoriasis, pemphigus, pruritis
and the types of eczema that are allergic in nature. The presence of eosinophilia
is so remarkably consistent in the allergic states that this finding in various skin
conditions would indicate that the disease in question, in all probability, is on
an allergic basis.
The marked eosinophilia that follows the bite of the black widow spider is
probably also an allergic phenomenon. In this particular instance the presence
of eosinophils becomes an important diagnostic sign since the bite of this insect,
is followed by marked abdominal pain and muscular rigidity which often simu-
lates an acute pyogenic process in the peritoneal cavity. Therefore, a marked
eosinophilia leads one to suspect the true nature of the process and prevent an
~nnecessary operative procedure. We have seen this happen in at least three
Instances.
An increase of eosinophils is thought to indicate the healing process after
acute infections. Appendicitis is oftentimes labeled subacute because of eosino-
 LEUKOCYTOSIS AND LEUKOPENIA

philic infiltrations into the appendiceal submucosa. The same is true of other
acute infectious diseases that are subsiding. It has been suggested that eosino-
philia is a frequent finding in arthritis. Naegeli, however, studied carefully one
hundred cases of arthritis and found no evidence of eosinophilia in this disease.
Eosinophilia is marked in cases of periarteritis nodosa. This disease is
thought to be caused by a filterable virus and is characterized by necrosis of. the
arterial media with surrounding fibrinous exudation. Finally there is a cellular
infiltration and granulation tissue in and about the arterial walls. A high eosino-
philia is one of the most characteristic diagnostic signs. In occasional instances
the count' may go as high as 50,000 cells per cU.mm. Eosinophilia may also
occur in certain cases of chronic benzene poisoning, according to the observations
of Hunter at the Massachusetts General Hospital.
Bracken reported that eosinophils are seldom if ever seen in the early stages
of pneumonia and that their appearance in the blood in the later stages of the
disease is a sign of good prognostic import, indicating an early clinical improve-
ment. Furthermore, eosinophils did not appear in the blood at any time in those
patients who subsequently came to a fatal termination.
Allin and Meyer noted various degrees of eosinophilia in over one third of
a large number of patients under treatment for pernicious anemia with various
preparations of liver extract. In some instances it persisted after cessation of
liver therapy. It has been stated that the oral.administration of liver is likely to
be followed by eosinophilia.
It should be borne in mind that the percentage and total number of eosino-
phils vary widely in the normal person and that the presence of eosinophilia does
not necessarily mean that the patient suffers from an allergic state or from para-
sitism, but when it is presen't in connection with other clinical findings indicating
this, it becomes evidence of a confirmatory nature. I have observed a consid-
erable number of patients with moderate degrees of eosinophilia, .and an occa-
sional one with an extremely high per cent of these cells, in whom it was not
possible, after careful study, to determine the cause. Some will show a per-
sistence of the state for years while in others it appears to be only temporary.
Apparently ill such instances the eosinophilia is of little significance, and is com-
patible with good health.
INCREASED BASOPHILS

Basophils are seldom if ever increased in the blood beyond their normal
number of about 50 per cubic millimeter, and such a condition as basophilic
leukocytosis is unknown. Even in lead poisoning where marked regenerative
changes occur in the form of punctate erythrocytic basophilia, there is no increase
in these cells. Any increased number should excite a suspicion of a possible
leukemic disorder.
The function of the basophil is unknown, although at least 25 different hy-
potheses have been suggested. Doan and Rinehart have suggested that the
basophil cytoplasmic granules are responsi61e for some type of secretory function, .
possibly the production of heparin. Eagle also points out that the basophils are
probably the primary source of heparin, since the tissues that contain the largest
LYMPHOCYTOSIS, MONOCYTOSIS, EOSINOPHILIA AND BASOPHILIA 143

number of these cells yield the largest amount of that substance. It should
be borne in mind that the basophil granules are water soluble. Therefore the
cells are seldom recognized in tissue sections since the fixation is usually in an
aqueous solution of formalin. For a comprehensive review of basophils consult
Michels in Downey's Handbook of Hematology.

BIBLIOGRAPHY

ALLIN, R. N., and MEYER, O. 0.: '~The Development of Eosinophilia Following Liver Therapy."
Jour. Lab. and CUn. Med., 26, 457, 1940.
BRACKEN, M. M.: "The Prognostic Significance of EOsinophils in the Blood in Pneumonia."
Amer. Jour. Med. Sci., 198, 386, 1939.
HUNTER, F. T.: "Chronic Exposure to Benzene (Benzol): Clinical Effects." Jour.lnd#st. Hyg. and
Toxicol, 21, 331, 1939.
KIRK, R. C.: "The Clinical Significance of Eosinophilia." Jour. Lab. and CUn. Med., 23, 1I37, 1938.
LE:ElOWICH, J., and HUNT, H. D.: uThe Diagnostic Significance of Eosinophilia in Periarteritis
Nodosa.1I Am. Joqr. Clin. Path., 10, 642, 1940.
MICHELS, N. A.: "The Mast Cells." Chap. IV. Downey's Handbook of Hematology. Paul B.
Hoeber. New York. 1938.
RmGOEN, A. R.: "Eosinophile Leukocytes and Eosinophilia." Chap. III. Downey's Ha.ndbook
of Hematology. Paul B. Haeber. New York. 1938.
TUltLEY, L. A., and DOUGlIER.TY, T. F.: HThe Relation of Lymphocytes to the Activity of Myco·
bacterium Tuberculosis." Jour. Lab. and Clin. Med.• 25, 828, 1940.
 CHAPTER 12
THE LEUKOPENIC DISEASES
Leukopenia may be defined as that condition in which the number of circu-
lating leukocytes is below the normal, without reference to the type of leukocyte
that is involved. Thus, leukopenia may exist at the expense of the ueutro-
phils, in which case it would be a neutropenia, or the lymphocytes may be de-
creased, resulting in a lymphopenia or lymphocytopenia. In some instances,
however, all of the white cells may be decreased to an equal degree, this being
a true leukopenia. Of the various leukopenic states, that characterized mainly by
a diminution in the neutrophils is the most important, not only because of its
relative frequency but also because of its seriousness.
Only since 1922, when the disease agranulocytosis was first reported by
Schultz, has the true significance of the leukopenic state been fully apprecia..ted.
Before that time a white cell decrease was regarded as a valuable diagnostic sign
in some diseases, but it was not recognized that a decrease in the number of pro-
tecting leukocytes would soon be followed by wholesale bacterial invasion. In
recent years a considerable amount of study has been given to this subject, mainly
because of the appearance of agranulocytosis.
Lawrence (1941) emphasizes the fact that physicians generally do not give
sufficient consideration to the various mechanisms by which the state of leuko-
penia may develop. He believes that leukopenia may be brought about through
five different mechanisms as follows:
(I) Diminished production of white cells by
a. Simple inhibition
b. Maturation arrest
c. Aplasia of marrow
d. Infiltration of marrow
(2) Increased elimination of white cells from the vascular· system into infected
areas such as a large abscess.
(3) Increased destruction of white cells in peripheral blood, although no good
examples of this apparently are available.
(4) Redistribution of cells in the vascular system as in the injection of foreign
protein.
(5) Redistribution of white cells through the entire body as seen in aleukemic
leukemia.

THE BONE MARROW IN LEUKOPENIA

The neutrophils are pr~duced in the bone marroW and released 'in sufficient
numbers to maintain the normal number in the peripheral blood. In this process,
there are two distinct factors that appear to playa rOle in their production and
144
 THE LEUKOPENIC DISEASES I45
delivery. First, the maturation factor is that hypothetical and undemonstrable
substance that regulates the orderly maturation of the myeloblast into the fully
segmented neutrophil, and secondly, there is apparently a delivery factor that
regulates the process of the release of these mature cells into the blood stream.
(See section on origin and development of the granulocyte.) If the maturation
factor is inhibited or diminished, there will result a decreased production of
neutrophils, and likewise if there is interference" with their delivery, a decreased
number will also be the result. Furthermore, leukocytes may be produced and
delivered in adequate numbers, but there is reason to believe that toxic agents
may destroy them in the peripheral blood, although this has not been fully
demonstrated.
A study of the bone marrow in the leukopenic states indicates that it fails
to produce a sufficient number of cells, rather than their increased destruc-
tion in the peripheral blood. If the marrow is subjected to depressing in-
fluences, there may result either an arrest of the cell development at immature
levels, notably at the myeloblastic level, or there may be an actual decrease in
the number of immature cells producing the mature types, even though there is no
arrest of the normal maturation. From a study of bone marrow in cases of
agranulocytosis, we are convinced that both types of pathology may be present.
Fitz-Hugh and Krumbhaar first called attention to the bone marrow characterized
by arrest of cell development at the myeloblastic level, this resulting in a so-called
hyperplasia of the cells at that level; thus has arisen the concept that .bone mar-
row in leukopenic patients may be "hyperplastic" (see Plate 5I). We have studied
the marrow from some cases of agranUlocytosis and have found no maturation
arrest but rather a quantitative decrease in the number of cell producing elements
and hematopoietic centers with the cellular structures largely replaced by fat.
Also we have studied the marrow of one patient that was crowded with myeloblasts
and, therefore, "hyperplastic" at a time when the peripheral leukocyte count was
only 400 cells per cu. mm. and at autopsy five days later the marrow was "aplas-
tic" (Plate 51), the myeloblasts having disappeared with only scanty stroma
and fat remaining. Furthermore we have observed a definitely aplastic marrow
from sternal puncture and a month later at autopsy found most of the long and
flat bones crowded with red, granular, cellular, myeloblastic cell producing mar-
row, reflected in the peripheral blood with a high leukocyte count before death.
Hence, we see little reason for the controversy as to whether the marrow of
agranulocytosis is "aplastic" or "hyperplastic," because either may exist in dif-
ferent patients or in the same patient at different times, and for that matter may
exist in the same patient simultaneously in different bones.

FACTORS DEPRESSING GRANULOPOIESIS

INFECTIONS
It has long been known that certain infectious diseases are accompanied by
leukopenia. These leukopenic states are usually of mild degree, with the leuko-
cyte count seldom less than 3000 cells per ell. mm, Also the neutrophils are
usually present in their normal proportions though in some instances they may
 LEUKOCYTOSIS AND LEUKOPENIA

be involved to a greater extent than the other cells. We have never observed a
leukopenia secondary to an infectious state in which the neutrophils entirely
disappeared, and if these cells are present in considerable numbers (50 per cent
of 3000 cells), we look upon this as evidence that the process is one following
and resulting from infection. .
Leukopenia has long been observed in many of the infectious states, such as
measles, German measles, mumps, influenza, malaria, undulant fever, typhus
fever, early typhoid fever, and during overwhelming pyogenic processes, especially
during the early stages. In all of these, there is usually a neutropenia compared
to the diminution in other cell types. We have seen patients with typhus fever
exhibit a leukopenia of less than 1000 cells per cu. mm. with only 10 per cent
neutrophils.
A leukopenia may also occur in the disease known as histoplasmosis of
Darling. This syndrome is characterized by intermittent pyrexia, leukopenia,
secondary anemia, enlargement of the spleen and liver, with focal necrosis of the
lungs, liver, spleen and lymph nodes. The organisms are usually found in blood
cultures antemortem or at autopsy in the spleen. A rather marked neutropeuia
is a characteristic of the disease. (See p. 553.)
Leukopenia, as well as severe anemia and thrombocytopeuia, has been re-
ported in tuberculous splenomegaly. I have observed a severe leukopenia of over
one year's duration in a patient who was subsequently shown to have diffuse
miliary tuberculosis, which involved the bone marrow.
The mechanism by which these leukopenic states are produced is not under-
stood. It is usually attributed to the action of bacterial toxins on the bone mar-
row, and it should also be considered that the injection of dead bacteria in the
lower animals results in'transient leukopenia of several hours duration.

CHEMICALS
Of tbis class of agents that are capable of depressing bone marrow function,
Benzene (CeH.) is outstanding. The daily injection of small doses of benzene
into rabbits is invariably fo1l9wed by marked leukopenia within a few days.
Most often the cell depression also involves the red cells and platelets as well.
If given in very small doses, however (0 cc. daily), this chemical seems to exert
a selective affinity for the granulopoietic tissue so tbat the animal may die with
a clinical picture that is indistinguisbable from agranUlocytosis, even to the devel-
opment of secondary infections in the oral cavity. If benzene is inhaled there
is little action on the hematopoietic tissues in most people for the reason that it is
chemically changed or detoxified before it reaches the blood forming tissues. If it
is taken by mouth by human beings or administered orally to rabbits a marked
leukopenic state is the result. It has not been many years since it was used to
reduce the white cell count in the leukemic states. It seems unlikely that it
reaches the bone marrow as such but rather is converted into another hemato-
poietic depressant hy oxidation. We have. presented some evidence to indicate
that the resulting agent may be quinone.
There are many chemicals containing. the benzene ring as their nucleus.
Some of these are known to produce leukopenia in human beings and in lower
 THE LEUKOPENIC 'DISEASES 147
animals. Aniline, when injected into mice, produces marked leukopenia in some
of them, does not affect others, while in others it produces marked degrees of
leukocytosis. Amidopyrine (Kracke), arsphenamine (McCarthy and Wilson),
dinitrophenol (Bohn), and the many combinations and variants of these have
produced agranulocytosis and other leukopenic states in the human. Lin and
Isaacs have reported the production of severe grades of leukopenia in Swiss mice
that have been injected with potassium dicarboxybenzanthracene.
There is evidence that other non-benzene chemical agents may depress the
bone marrow function, these including the gold salts, used in the treatment of
tuberculosis (Dameshek), gasoline fumes (Hamilton), arsenic (Lawson, et al.),
and probably many others.
In a consideration of substances depressing the granulopoietic activity of
the marrow it should be remembered that no substance is likely to have a total
selective affinity for only one embryonic cell type in the marrow but it is more
likely that an agent capable of depressing the leukocytes and producing leuko-
penia will likewise affect the red cells and platelets as well. Thus, if a single
agent is shown to be capable of depressing the marrow in one of its cell types,
this same agent should be regarded with suspicion with respect to all other cell
types of the marrow. Indeed, it would be most remarkable if a depressant agent
confined its action to only one marrow cell type and one may expect various
degrees of leukopenia, erythropenia, and thrombopenia when the marrow is sub-
jected to a noxious agent. This leads to speculation as the relationship between
agranulocytosis, aplastic anemia, so-called hypoplastic anemia, bone marrow
failure, and the aleukemic types of leukemia. It is possible that many of these
are variants of the same depressing influences on bone"marrow.

RADIATION
That internal radiation with alpha particles can affect the marrow output
in a number of ways has been shown by Martland. In radiation osteitis there
is likely to develop a leukopenic state of considerable severity with more or less
involvement of the other cellular elements. The occurrence of simple leukopenia
is rare. More often the red cells and platelets are involved as well, resulting in a
clinical picture in which a profound anemia or a hemorrhagic syndrome may be
outstanding features. In the final stages the marrow tends to become completely
acellular with fibrous tissue replacement. This type of marrow depression is
seen in industry where workers are using radioactive substances in their work,
as was seen in the famous instance of fatal aplastic anemia developing in a group
of young women who pointed brush tips between their lips when painting clock
djals with a luminous radioactive substance (Gamma rays). Bone marrow
depression has been reported in miners handling these substances, in people
drinking radioactive water, and in the earlier days in the therapeutic use of
radium before adequate protective measures were established.
Roentgen rays are capable of depressing bone marrow function, this being
dependent upon the size of the doses, length of exposure, and no doubt also on
the susceptibility of the individual. Roentgenologists are often concerned over
the possibility that they may suffer bone marrow damage from constant exposure
 LEUKOCYTOSIS AND LEUKOPENIA

to X-ray, but this seems quite unlikely under the present protection afforded by
modern equipment. However, exposure to large doses of radiation produces de-
generated leukocytes in the peripheral blood, and a marked bone marrow depres-
sion with a hematologic picture of aplastic anemia. Although it· is true that
radiation may be dangerous to an occasional person who is exposed to it over a
long period of time, there is little or no danger associated with ordinary thera-
peutic' radiation. Kornblum and associates have studied this question and came
to the conclusion that therapeutic radiation affects the blood but little, and if so,
it does not affect the circulating cells at all, but may produce a mild depressing
effect on the hematopoietic system. The effects seem to be primarily on the
lymphocytes, then the neutrophils, monocytes, eosinophils, and basophils, in the
order named. They conclude that both therapeutic roentgen and radium therapy
may caUse a physiological hypofunction of the hematopoietic organs rather than
a dysfunction because of cellular damage, and that it is of such minor degree as
to be of no clinical significance and may he entirely disregarded.
A recently introduced diagnostic test involving the use of thorium dioxide
has centered attention on the effect of this substance on bone marrow. Tripoli
states that he observed no ill effects, but other observers (Pohle and Ritchie) have
noted aplastic anemia after small doses in rabbits.
There have been no systematic studies made on patients subjected to exces-
sive action of sunlight, except perhaps in the normal groups. We have been im-
pressed by a number of changes seen in the blood of patients on their way to and
from Florida, where excessive exposure to sunshine seems to be the chief object
of the journey. We have observed several patients with leukopenic syndromes
who apparently improve after reaching a higher altitude and no longer subject
themselves to excessive radiation from sunlight, and who again become more leuko·
penic, more anemic, and more thrombocytopenic after being exposed to sunlight
for a considerable period. We cannot escape the conclusion that in some people
excessive sunlight depresses the bone marrow to a variable extent. The question
of altitude and deficient oxygen because of rarefied atmosphere also has to be
considered. However, it may be well to regard sunlight as a therapeutic aid
in the same class as other therapeutic agents; that is, as a two-edged sword that
is capable of doing actual damage if used to excess.

DIET
,There is some evidence that an inadequate diet may result in development
of the leukopenic state, presumably by depression of granulopoiesis. Langston
and Day have reported the deve!opment of marked leukopenia, anemia, and
thrombopenia in monkeys after prolonged feeding with a diet deficient in Vitamin
G. Also Miller and Rhoads have produced similar leukopenias with ulcerative
stomatitis in dogs fed on a deficiency diet that causes black tongue. These intima-
tions are sufficiently well substantiated so that the diet of the neutropenic patient
should be carefully considered. Also, in a study of possible depressing marrow
factors in all leukopenic patients, their dietary habits should be closely investi-
gated with particular reference to deficiencies.
 THE LEUKOPENIC D,SEASES '49

DRUGS
In 1931 we pointed out that certain drugs may cause agranulocytosis and
since that time this concept has been amply confirmed. Among the drugs that
have been incriminated are amidopyrine, dinitrophenol, sulfanilamide, sulfapyri-
dine, sulfathiazole (not neo-prontosil), and neoarsphenamine, and the many com-
binations and modifications of these, especially those of amidopyrine. No less
than .00 cases of agranulocytosis have been reported as having followed the ad-
ministration of this drug alone. It has been shown clinically that the administra-
tion of amidopyrine to a susceptible person results in a marked depression of
the leukocyte count (Madison and Squier; Sturgis; and others). Also its admin-
istration to rabbits and other animals, although not producing marked depression
of the leukocyte count, often results in severe bone marrow damage, evidenced
by loss of granulopoietic centers, fibrous tissue replacement and increased marrow
fat (Climenko). In general, all efforts to produce agranulocytosis by administra-
tion of drugs to animals have been unsuccessful, including the feeding of amido-
pyrine to a wide variety of animals. An excellent summary of this is given by
Butt, Hoffman, and Soli who produced agranulocytosis in two dogs that had re-
ceived large doses of amidopyrine over periods of many months.
Unfortunately, it is difficult to restrict the use of this drug, since it has been
combined with so many other preparations by the different manufacturers, and the
resulting products given such a wide variety of uninformative names, that it is
almost impossible to determine whether or not many preparations contain the
drug. Especially is this true of the "patented" preparations (Kracke and Par-
ker). For a list of some of tbe more widely used proprietary preparations see
Table XIII.
Agranulocytosis may develop after a single dose of amidopyrine, or after its
long continued use. This brings up the question of its mode of action, whicb
would appear to be allergic in type in a person with a hypersensitive marrow,
especially in those instances where the leukocytes decrease after a single dose.
We admit that the designation of any process as being "allergic" is highly un-
satisfactory and non-informing.
We have attempted to show that the depressing action of amidopyrine re-
sults from atypical oxidation of the drug, presumably in the gastrointestinal tract,
to a product more toxic, such as quinone or hydroquinone, but there seems to be
present another factor of susceptibility in the "sensitive" person, especially in
view of the disease developing after a single dose. Some believe (Herz) that its
depressant action is dependent upon the attached pyrazalon group rather than the
central benzene ring. This assumption is based largely on the fact that phenyl-
hydrazine, a pyrazalon derivative, is destructive of red blood cells but the analogy
does not necessarily follow. Furthermore, the depressing action of arsphenamine
and dinitrophenol which contain no pyrazalon group would mitigate such a
conception.
Physicians should prescribe these drugs with caution and use them sparingly,
and always, if possible, under conditions whereby the blood picture may be
checked at frequent intervals. We have recently observed a fatal case of agranu-
ISO LEUKOCYTOSIS AND LEUKOPENIA

TABLE XIII
A PARTIAL LIST OF AMERICAN' PROPRIETARY PREPARATIONS THAT DID OR Do
CONTAIN AMIDOPYRINE"

NAME OF DRUG MANUFACTURER

Allonal. ....................... Hoffmann-La Roche, Inc.
Alphebin ....................... Gane & Ingram, Inc.
Amarbital. ..................... A. W. Kretschmar, Inc.
Amidol ........................ Flint, Eaton & Co.
Amido-Neonal Abbot Laboratories.
Amidonine .............. ...... ,Pitman-Moore Co.
Amidophen .................... Eli Lilly & Co.
Amidos ........................ National Drug Co.
Amidotal Compound ............ William H. Rorer, Inc.
Amifeine ....................... McNeil Laboratories, Inc.
Aminal ........................ Smith-Dorsey Co.
Am-Phen-Al .................... G. S. Stoddard & Co., Inc.
Ampydin ...................... National Aniline & Chemical Co., Inc.
Amy tal Compound ........... ' Eli Lilly & Co.
Amnalgia . : .................... William S. Merrell Co.
Antabs ........... ~ ............ William S. Merrell Co.
Baramid ............ : .......... McNeil Laboratories, Inc.
Barb-Amid ..................... Sutliff & Case Co., Inc.
Benzedo Compound ............. Abbott Lahoratories.
Cibalgine ....................... Ciba Co., Inc.
Cinchopyrine ................... Abbott Laboratories.
Compral ....................... Winthrop Chemica! Co., Inc.
Cronal. ........................ Cronal Co., Inc.
Dymen ........................ Coland Laboratories.
Dysco ......................... Abbott Laboratories.
Eu Med ........................ Oralee Co.
Gardan ............ ' ............ H. A. Metz Co.
Gynalgos ....................... John Wyeth & Brother, Inc.
I-Iexin . .............. , ...... , . . Hexin, Inc.
Ipral-Amidopyrine ........ : ..... E. R. Squibb & Sons.
Kalns ........................ Johnson & Johnson.
Lumodrin ...................... Winthrop Chemical Co., Inc.
Midol ......................... General Drug Co.
Mylin. . . . . . . . ............. Mifflin Chemical Corp.
Neonal Compound .. ' ........... Abbott Laboratories.
Neurodyne .............•....... Chicago Pharmacal Co.
Nod ........................... Reader Drug Co.
Optalidon ...................... Sandoz Chemical Works.
Peralga . . ................... Schering & Giatz, Inc.
Phenamidal .................... Upjohn Co.
Phen-Amido! ................... Carroll Dunham Smitlr Pharmacal Co.
Phenopyrine .................... Cole Chemical Co., Inc.
Pyramidon ..................... H. A. Metz Laboratories, Inc.
Pyraminal ...................... I!. A. Metz Laboratories, Inc.
Seeqit ......................... Laboratories JAQ, Inc.
Y east-Vit ...................... Yeast-Vit (U. S. A.), Inc .
• List supplied by Council on Pharmacy and Chemistry, American Medical Association (May
1935). This list does not include many of the large group of lipatent medicines" or secret formula
remedies.
 THE LEUKOPENIC DISEASES

locytosis in a negro boy after one dose of neoarsphenamine. This, of course,· is

exceptional but illustrates its toxic action on the bone marrow. Aplastic anemia
has been observed following a single dose of this drug. Many instances have
come to our attention in which it llppears that Atabrine, used in the treatment of
malaria, has caused marked leukocyte depressions, and even quinine has been
suspected in some cases.
The leukocyte depressing activity of dinitrophenol is clearly indicated on
the basis of a considerable .number of cases following its use. For this and other
reasons its use should be abandoned.
, Although the development of agranulocytosis reached its peak in 1934, pre-
sumably from the indiscriminate use of amidopyrine, since that time there has
been a gradual decrease in the incidence of the disease, until within recent years
the advent of the sulfonamide group of drugs is responsible for another group of
cases of serious leukopenic depression. This group of drugs, including sulfanila-
mide, sulfapyridine, and sulfathiazole, have the benzene ring as their central
structure and are therefore potentially dangerous with respect to depression of
bone marrow. There have now been reported over roo cases of agranulocytosis
from the administration of this group of drugs. Fortunately it is· a rare compli-
cation. Although complete agranUlocytosis may not always develop, a consider-
able number of patients may show varying degrees of leukopenia. Britton and
Hawkins studied So patients that had been treated with sulfanilamide and r6
of these developed a mild leukopenia during this time. Long and Bliss in a study
of 408 patients treated with sulfanilamide reported two cases of agranulocytosis.
The exact mechanism of drug action in these cases is not known. The bone
marrow is identical with that seen in cases of amidopyrine depression, that is,
there is an arrest of cellular maturation at the myeloblastic level. The degree of
leukopenia may be equal to that in the amidopyrine cases, but most often is not.
Agranulocytosis follows the administration of these drugs only after they
have been given over a considerable period of time. Leukopenia does not Occur
in the first few days of medication, and it has been stated that the drug must be
given for ten days in fairly large doses before there is danger. In this respect the
mechanism of action seems to be different from that of amidopyrine, since serious
depressions may occur after only a single dose of this drug.
The red cells and platelets are usually not affected. There seems to be no
relationship of depression of white cells to development of acute hemolytic anemia.
After the disease develops it can not be distinguished in any way from agranu-
locytosis from other causes. Jones and Miller reported a patient who recovered
from agranulocytosis caused by amidopyrine and who later was confronted witb
the necessity of taking sulfanilamide, and he developed a depression of leukocytes
on both occasions. On the other hand, Long and Bliss treated a patient with subse-
quent doses of sulfanilamide and noted no change in the white cells. They
referred to a patient who had had previous attacks of the disease and later had
to take the drug again but the second treatment did not cause another attack of
agranulocytosis.
When leukopenia develops in the course of treatment with the sulfonamide
group of drugs, it is essential that medication be stopped if it is possible to do
 LEUKOCYTOSIS AND LEUKOPENIA

SO. However, in severe infections such as those caused by hemolytic streptococci

in the blood stream or various types of meningitis, it is often not feasible to do
'this but to continue medication and simply take a chance on the leukopenic
disorder.
Another effect of this group of drugs is the production of an unusual degree
of neutrophilic leukocytosis in an occasional patient. The white cells may rise
to 40, 50, or 60 thousand per cu.mm. Apparently the patient shows no ill effects
from the production of such a leukocytosis, but it may be confusing, particularly
in the patient who at that time is showing clinical improvement.
In an occasional instance the leukocytosis associated with administration of
sulfanilamiae may reach an astounding figure. Spence and Roberts have re-
ported an instance of 140,000 cells per cU.mm.

HORMONES
There is some evidence to indicate that certain hormonal disorders may be
accompanied by leukopenia and the inference is drawn that bone marrow activity
may be regulated by the action of one of these. The hormone most involved is
that of the adrenal cortex. Adrenalectomized cats often have marked leukopenia
presumably from a cortical adrenal deficiency (Britten and Corey). We have ob-
served several patients who presented a syndrome of deficiency of cortical extract
and have noted the marked leukopenia that is present. It consists of extreme
weakness and fatigue, marked hypotension, varying degrees of pigmentation and
a severe leukopenia. In two of these we have been able to demonstrate the
marked adrenal cortical destruction at autopsy. Also we have studied carefully
one patient with a severe aplastic anemia and a leukocyte count that remained
below 1000 cells for months, who finally died with generalized miliary tuberculosis
involving the bone marrow, with total destructioll of the adrenal cortex.
Although it has been stated that the leukocyte count is affected by the activity
of the thyroid gland, we have been unable to confirm any significant changes in
patients with hyperthyroidism or myxedema.

MISCELLANEOUS
There are many instances of severe bone marrow depression for which an
etiological agent cannot be discovered. Thus, there is the so-called marrow hypo-
plasia of the aged which is characterized by weakness, easy fatigue, anemia, de-
creased white cells, red cells, and platelets. This may be an example of physio-
logic failure of marrow activity in the occasional person, just as various parts
of the vascular system finally undergo degenerative changes.
In aplastic anemia is seen the most severe bone marrow failure, which may
occur at any age, in which the marrow loses its capacity to produce cells of any
kind, or in very small numbers. Its etiology is entirely unknown, the course
progressively downward, and the outcome invariably fatal.
Bone marrow depression with gramilocytopenia is also a finding common to
certain other blood dyscrasias, especially pernicious anemia in relapse and
aleukemic leukemia.
Lawrence reports the development of spontaneous agranUlocytosis in a cat
 THE LEUKOPENIC DISEASES 153
from which the liver was removed, a suspension made, and the supernatant fluid
used for the injection of five additional cats, this resulting in the disease in two
of the five animals. This process was continued until 13 transmissions had been
effected. He has concluded that there is a transmissible disease characterized by
neutropenia and that the causative agent is a filterable virus.

MALIGNANT NEUTROPENIA

(Agranulocytosis) (Agranulocytic Angina) (Acute Granulopenia) (Angina

Agranulocytica) (Mucosistis Necroticans Agranulocytica) (Idiopathic
Neutropenia), Etc.

HISTORY
Although this disease has occurred infrequently for many years, as shown
by the four cases reported by Brown in 1902, modern interest dates from 1922
when the disease was fully described by Schultz in Germany. In 1924 the first
American case was reported by Lovett. Since that time it has apparently increased
in frequency until 1500 deaths were reported in the United States alone from '93'
until '934. Since 1934, there has apparently been a decline in the number of
cases, as shown by the records of the United States Bureau of Vital Statistics.
This appears to be because of the caution employed by physicians in the use of
amidopyrine and its combinations.

ETIOLOGY
As stated before in the discussion of factors depressing the bone marrow, it
seems likely that most cases of this disease follow the administration of small
or large doses of amidopyrine and apparently the drug is harmless unless ingested
by a susceptible person. Plum carefully investigated the 88 patients in his series
in Denmark and stated that the only feature common to all of them was a history
of symptoms calling for the use of analgesics and that 52 out of 56 patients were
discovered to have been treated with amidopyrine before the onset of the disease,
and in only one case was it possible definitely to exclude this drug. Plum con-
cludes that in Denmark amidopyrine is by far the most frequent cause of the
disease, and that since the importation of amidopyrine in Denmark has been
prohibited hy government decree, the disease agranulocytosis has practically dis-
appeared.
No doubt some cases of agranulocytosis develop from other causes, some of
which include the administration of organic arsenical compounds, sulfonamide
drugs, dinitrophenol, exposure to benzene, excessive radiation, dietary deficiencies,
etc. There is little evidence for the assumption of a bacterial etiology. The world
wide distribution of the disease corresponds closely to the use of such drugs as
mentioned above. In overwhelming infectious states the bacterial toxins may
depress granulopoiesis of the bone marrow to the point of a mild leukopenia hut
not completely to agranulocytosis. Reznikoff has stressed the importance of
chronic fatigue as a possible causative agent in the disease. However, it is more
154 LEUKOCYTOSIS AND LEUKOPENIA

likely that the neutropenia, possibly previuusly unrecognized, is the cause of the
fatigue.

INCIDENCE
Malignant neutropenia occurs at all times of the year with no seasonal pre-
ponderance. It is chiefly a disease of middle age, from the fourth to fifth decades,
although no age is exempt. It affects the female sex in a ratio of about two to
one. It is peculiarly a disease of white people, since there is only an occasional
record of its occurrence in a negro, and whenever this is found, there is usually a
history of administration of neoarsphenamine. It is prevalent in people of the
better economic class, as shown by its rarity in charity hospitals. Most cases are
reported from Germany and the United States, with a lesser number from France
and Italy and it is comparatively rare in England. It has also been reported
from other parts of the world, including thtl Orient.
Many observers have noted that the disease occurs among people of the
"medical group," including physicians, thdr families, nurses, dentists, hospital
employees, etc. It apparently is eight times more frequent in this than in any
other group (Kracke and Parker). We have shown that this is because this class
of people are prone to use many of the ll"wer pain relieving remedies,..most of
which contain amidopyrine. In the cases ill Denmark reported by Plum abouf 10
per cent occurred in persons who were 'in scme way connected with medicine.

DESCRIPTION
The disease may occur suddenly from full health or the onset may be in-
sidious, oftentimes as a complication of sOllle preexisting illness. The first signs
may be fatigue, weakness, and lassitude and collapse in an occasional patient.
The temperature is usually elevated and the pulse rate in proportion. The
throat is red and injected and there mayor may not be diffuse ulcerations in
the pharynx. In some patients there is widespread destruction of the oral tissues
with secondary invasion by various bacteria. There may Qe ulcerations about
the vagina or anus or any other areas normally inhabited by bacteria. The ulcers
are characterized by a lack of yellow color which is ordinarily due to the accu-
mulation of pus cells, since there are no neutrophils available to form such an
exudate. Some of the lesions may appear blackened or dark colored as if gan-
grenous. Many patients show no ulcerations of any kind and the outstanding
feature may be a severe edema of rhe soft tissues of the neck. Otherwise the
physical examination is usually negative. Late in the disease a bronchopneu-
monia or blood stream infection may' De present.
Murphy states the following clinical description of the disease: "The typical
attack of acute granulocytopenia or agranulocytosis is generally preceded or ac-
companied by evidence of an infection, perhaps most often focal in type but occa-,
sionally generalized. The onset is usually abrupt with severe headache, high
fever, rapid pulse, chill, and frequently generalized aching, sore throat and
spongy sore gums. The onset may simulate an attack of so-called 'grippe' al:
though the degree of prostration is generally far greater than the physical
signs of the illness warrant. Following this abrupt onset there may occur,
 THE LEUKOPENIC DISEASES ISS
if treatment and consequently improvement is delayed, a gangrenous or
necrosing lesion involving the throat and tonsillar region, the buccal or other
mucOUS surfaces as the vagina, cervix, and regions or even the mucosa of the
intestinal tract. With the latter involvement diarrhoea may occur. The ulcera-
tions or necroses are not to be considered as the primary infection but rather as
secondary to the granulocytopenia. During the course of the illness there may
occur a rash, herpes labialis, nausea and vomiting, dysphagia and occasionally
epigastric pain and tenderness. Bronchopneumonia has been present frequently
~n the terminal stages and this may be accompanied by jaundice."

HEMATOLOGIC FINDlNGS
The outstanding finding and the chief diagnostic criterion is the marked
decrease or total absence of the neutrophils in the peripheral blood. These cells
are first to disappear followed by the monocytes, and finally the lymphocytes are
diminisbed to a low level. For example, if the total leukocyte count is only 600
cells per cu. mm. and all of these are lymphocytes, it is obvious that approxi-
mately 2000 lymphocytes per cu. mm. are absent. Strictly, then, the disease
is characterized by a diminution in all of the white cells. This seems difficult
to reconcile with the concept that agranulocytosis is a disease of the bone mar-
row function, unless most of the lymphocytes also are produced in the marrow.
There is usually little or no anemia, and the platelets are normal, if Schultz's
original description of cell changes is taken as the diagnostic criterion, but it seems
unlikely that a noxious agent would depress one cellular type and fail to de-
press another. The probable reason the agranulocytic patient is not anemic is
that the average patient does not live long enough for anemia to develop.
On theoretical grounds, a patient should show no evidence of clinical anemia for
some days, even though red cell production is stopped completely. Therefore,
we see no justification for the exclusion of agranulocytosis from the diagnostic
possibilities because the patient is anemic or because the red cells and hemoglobin
are low. For that matter, is there any reason why an anemic patient cannot
develop agranulocytosis? Indeed, most patients are suffering from pre-existing
diseases of various kinds before they develop agranulocytosis. If not, they prob-
ably would not be taking amidopyrine and other drugs.
There are some who state that the patient with agranulocytosis does not
show platelet deficiencies and hemorrhages. We find various hemorrhagic states
in many patients, both clinically and at autopsy, and we do not rule out the dis-
ease because they are hemorrhagic. To our mind it would be quite remarkable
if they did not show a hemorrhagic tendency, especially those that survive for
some time and whose duration of illness is prolonged. Attention has been cen-
tered on the leukocytic decrease because this finding is predominant and the
various complications, including the ulcerations and infectious states, probably
develop because of the leukocytic decrease and the loss of protection afforded by
these cells. The appearance of monocytes in the blood has been interpreted by
Reznikoff and also by Rosenthal as indicating a resumption of cellular output, and
a good prognosis.
 LEUKOCYTOSIS AND LEUKOPENIA

PATHOLOGY
The underlying pathology is apparently-in the bone marrow, and may be one
of two types. Either the marrow becomes quantitatively aplastic and does not
produce a sufficient number of white cells, even though the maturation process is
orderly, or there may be an arrest of maturation at the myeloblastic level and
overcrowding of the marrow with myeloblasts. The laUer concept is stated by
Custer to be the rule, based on his thorough studies of marrow from eleven
patients. We have examined the marrow from about thirty patients and find both
types of pathology (see Plate 51).
The histologic picture in the areas of infections and ulcerations is char-
acterized by an absence of neutrophils and therefore, an absence of true pus_ The
cellular infiltrations are' largely lymphocytic, and from this arises tIie question
of whether or not all of the circulating lymphocytes are used to ward off infec-
tion and therefore the lymphocytes are decreased in the blood.

COURSE
The acute, severe development of the disease usually terminates fatally within
a few days. The more chronic, insidious type may be complicated by various in-
fectious processes and may finally recover or terminate fatally. The temperature
remains high, the patient weak, fatigued, oftentimes irrational, delirious, and per-
haps comatose. The course depends upon the type and extent of the complicating
infectious states. The patient may be quickly overwhelmed with infection or may
undergo a stormy convalescence over a period of weeks. The clinical course,
therefore, is variable to an extreme degree, and is characterized by its inconstancy.

TREATMENT
No person has treated a sufficient number of cases of agranulocytosis by one
method alone to make dogmatic statements about the value of any specific
therapeutic agent. During the last ten years a number of substances have been
proposed that are claimed to reduce the mortality rate. These include radiation
to long bones, repeated transfusions, nucleic acid derivatives, liver extract therapy,
and many others. '
Radiation to long bones has been used, presumably with the assumption that
the marrow of flat bones is incapable of granulocytic production, and therefore
it has been directed to the inactive marrow of the long bones, in an effort to stimu-
late it to granulopoiesis. We have used this in many cases, employing the so-
called "stimulating" doses, but we have failed to observe any benefit from its use.
The use of radiation is based on nmch speculation and few facts, when it is as"
sumed that a certain dosage wiII stimulate bone marrow to increased production,
especially when so little is known about the state or pathology of the marrow.
There is much evidence to indicate that any dose of radiation is destructive
to all cells, and not stimulating to the process of maturation.
The use of nucleic acid derivatives by intramuscular injection has been widely
advocated. Among the first of these used were adenine sulphate and guanine
hydrochloride. These have been supplanted largely by the use of pentnucleotide,
 THE LEUKOPENIC DISEASES 157
which is a proprietary preparation, is more readily available and more highly
advertised, and therefore more wiilely used. Jackson recommends that it be used
in large doses (more than 40 cc. daily), and attributes failures to small doses.
However, in many patients violent reactions occur, and apparently it is not well
tolerated in a certain group. Furthermore, as with any other therapy, the patient
may die before the drug has time to produce beneficial results, and this, no doubt,
accounts for some failures. We have observed little benefit from its use, although
our experience with it is relatively limited.
, Liver extract has been advocated as an efficient agent. We have used inten-
sive liver therapy (intramuscular injections of the equivalent of 100 grams of
fresh liver twice daily) in a few cases and it is our opinion that it has decided
value and will produce increased delivery of granulocytes in 24 to 48 hours. Here
again, if the patient is overwhelmed with infection, or in extremis, this or no
other agent is of value. We prefer to treat these patients with liver extract rather
than with pentnucleotide.
Transfusions are useful as supportive measures, although it is not likely that
they are of value in stimulating the depressed marrow function or even for cellular
replacement, with the exception of the red cells. We feel that many patients
are kept alive with transfusions until a specific therapy has had time to produce
stimulation of cellular output.
The production of a septic state for granulocytic stimulation is not necessary
since the patient too often provides this. On the other hand, occasional patients
seem to improve after the development of sepsis (ulcers, abscesses). We have
used the injection of turpentine intramuscularly in an effort to stimulate marr9W
production by the formation of a sterile abscess, but it is not recommended.
To summarize, the treatment of agranulocytosis should include:
I. Daily intensive intramuscular liver therapy, and liver by mouth if possible.
2. Repeated daily transfusions of 200 cc. to 300 cc. of blood.
3. A high caloric diet, good nursing, adequate cardiac and respiratory stimulation,
supportive and genoml measures, as indicated.
4. The withdrawal of all pain relieving drugs that contain the benzene ring, and
also all of the so-called barbiturates. Sedation may be accomplished by the
use of narcotics such as morphine, codeine, dilaudid, etc.
In their 1940 review of the literature of blood diseases, Isaacs and his asso-
ciates conclude that the most effective management of the patient with agranulo-
cytic angina is as follows: I. Simultaneous administration of pentnucleotide in-
tramuscularly in doses of 40 ce. daily and of yellow bone marrow extract orally
in daily doses of 300 to 500 grams. 2. Treatment of the lesions of the mouth and
ti)roat with bland saline irrigations, and avoidance of surgical intervention in
these areas during the acute process. 3. Prohibition of all drugs such as amido-
pyrine, sulfanilamide, and allied substances that are known to cause granulocyto-
penia. 4. Use of blood transfusions in the occasional patient, who has had anemia
prior to development of agranulocytosis or who shows anemia during the course
of the disease.
Jackson and Tighe summarized the results of treatment in 390 cases of
158 LEUKOCYTOSIS AND LEUKOPENIA

agranulocytic angina based on a survey of the literature and their personal ex-
perience. They present the effectiveness of various types of treatment as follows:
With no specific therapy, the mortality rate is 70-80 per cent; with transfusions
74 per cent; with x-rays 67 per cent; with liver extract 62 per cent; with pent-
nucleotide 35 per cent; with adenine sulfate 20 per cent; with leukocytic cream 17
per cent; and with yellow hone marrow extract 10 per cent. However, because
of the fact that the number of patients treated by each method is quite small,
and tbe fact that patients were extremely few on whom only one method is used,
it is difficult to present accurate figures as to comparative mortality rates.

PROGNOSIS
Apparently more patients recover now than five years ago, presumably be-
cause of the liberal use of liver extract,-and pentnucleotide, and because benzene
drugs are seldom used during the illness. The, outlook for the patient depends
upon the condition present when the diagnosis is first made, and the outlook is
bad for those overwhelmed with infections. Therefore, early diagnosis is impor-
tant. It is estimated that approximately fifty per cent recover, while the mortality
rate in earlier years was from eighty to ninety per cent. The patients who have
recovered may lead an active life without fear of recurrent attacks if the cause
of the first attack is ascertained and they carefully refrain from ingestion or con-
tact with the causative agent. There are some who seem to go into a stage of
chronic neutropenia and exhibit symptoms of this syndrome at irregular intervals.
Such patients live in constant fear of a second attack, and may develop a "leu-
kopenic anxiety neurosis." However, we have under observation eight people who
have recovered from acute typical agranulocytosis, and find that they can lead
an active, normal life. .

CHRONIC NEUTROPENIA

Chronic neutropenia may be defined as that condition characterized by a pro-

longed and ·constant decrease in the number of circulating neutrophils below the
normal level. This brings up the question of what is the normal. Undoubtedly,
this varies in different people. In our publications on this question (Roberts mid
Kracke) we have assumed that if the number of rteutrophils is below 4000 per cu.
mm., that person is considered leukopenic.
Chronic neutropenia may exist in normal people who show no evidence what-
ever of disease. Occasionally the leukocyte count may fall to a low level (3000-
3500 cells per cu. mm.) and the individual show no symptoms. This has led us
to believe that a few people have very low leukocyte counts as their normal figure,
just as we also believe that a red cell count of 3,50°,°00 to 4,000,000 is quite nor-
mal for certain people, particularly those of sedentary occupations. There is no
more reason for instituting intensive therapy for· the correction of a symptomless
chronic neutropenia than the giving of repeated transfusions for the correction
of a symptomless mild anemia; yet some of our clinical colleagues seem addicted
to this practice. ,
Chronic neutropenia may be characterized by certain symptoms. These are
 THE LEUKOPENIC DI~EASES 159

mainly weakness, excessive fatigue, a tendency to easy tiring, and a predisposi-

tion to intercurrent infections. We have studied the records of 8000 patients in
the private practice of Dr. Stewart Roberts and find this syndrome closely cor-
related with a chronic neutropenia. In this type of person the use of a single
dose of liver extract intramuscularly oftentimes elevates the leukocyte count and
produces a marked improvement in the sense of well being and alleviation of
the symptoms mentioned above. In others it seems to produce no effect what-
ever. However, it is worthy of a trial.
There also exists a type of chronic neutropenia in which the leukocyte count
remains at a dangerously low level (1500 to 2000 cells per cu. mm.). These
patients usually complain of an indefinite train of symptoms. Many of them
eventually turn out to be early cases of aplastic s.nemia or finally eventuate into
some form of aleukemic leukemia. Others seeII) to go on for year after year,
with marked fluctuations in the leukocyte count. They are susceptible to infec-
tious processes, particularly of the upper respiratorY tract. They seem to improve
during the period of active infection when the leukocyte count is relatively ele-
vated. Careful search should be made in these patients for possible noxious agents
that may be responsible, sucn as drugs, cherpica'lo, 'lTf'"dutqaate U'(,A, etc. '.lIle
best type of treatment seems to be the use of liver extract by mouth and by injec-
tion, liberal doses of iron, a high caloric diet, adequate rest, freedom from worry,
and other general measures as indicated.

BLOOD DYSCRASIAS CHARACTERIZED BY NEUTROPENIA

APLASTIC ANEMIA
Aplastic anemia is a disease that is characterized hematologically by severe
erythropenia, thrombopenia, and neutropenia. The bone marrow is aplastic and
fails to produce and deliver these three types of cells. Neutropenia, therefore,
is an important and serious aspect of aplastic anemia, because the patient is
always threatened with loss of resistance and tissue invasion with various types
of bacteria. The neutropenia of this disease way reach extremely low levels,
sometimes to only 200 to 300 cells per cu. mm. It does not respond to treatment.
The leukocytes usually remain low in spite of various measures employed to stimu-
late them. (For details of treatment see section on Aplastic Anemia.) The usual
leukocyte range is from IDOO to 3000 cells per cu. mm., the decrease being at the
expense of the neutrophils.

ALlj:UKEMIC (HypOCYTIc) LEUKEMIA

In this disease is seen a profound decrease in the number of neutrophils which
remain low for long periods of time, although in the acute, more fulminant types
of stem cell leukemias, the leukopenia may change to a marked leukocytosis with
the cells composed of the types predominant in that particular type of leukemia.
We have studied a patient who had only 500 cells per cu. mm. on one day, with
the count reaching 60,000 per cu. mm. 48 hour;; later, and this repeated many
times before death. In cases of aleukemic leukemia in which the cell count is
e"tremely low, if the count rises to a normal level or above, this is diagnostic
160 LEUKOCYTOSIS AND LEUKOPENIA

evidence that the disease is of leukemic nature as contrasted to aplastic anemia,

in which the cell count seldom if ever rises to normal figures, even though the
course of the disease be considerably prolonged.

PERNICIOUS ANEMIA
The leukopenia of pernicious anemia has long been recognized. It occurs
only during periods of relapse. It seems that the deficiency in red cell maturation
finally involves the granulocytic series as well. This gives rise to the speculation
as to the presence of a granulocyte stimulating factor being produced in the
stomach and stored in the liver. When these patients receive adequate treatment
the neutropenia is speedily corrected and the count rises to normal long before
the red cell picture reaches the normal level.
Certain other diseases are accompanied by leukopenia at various times dur-
ing their course. This is notably true in Banti's disease, and might be explained
on the hasis of advanced destruction and cirrhosis of the liver and therefore im-
paired function in the storage of the hematopoietic substance. The lymphogranu-
lomata, metastatic multiple myelomata, chloroma, bone marrow tuberculosis,
Hodgkin's disease, glandular tuberculosis, lymphosarcoma, bone marrow car-
cinomatosis, etc., may at times exhibit a neutropenia.

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TRIPOLI, C. ]., :;tnd VON lIAAM, E.: "The Effects of Toxic and Non-Toxic Doses of Thorium
Dioxide in Various Animals." Proc. Soc. Exp. BioI. and V!d., 29, 1053, 1932.
 SECTION FOUR
FACTORS INFLUENCING ERYTHROPOIESIS
CLASSIFICA TION OF THE ANEMIAS
HEMOGLOBIN AND ITS DERIVATIVES;
THE PORPHYRINS
THE HYPOCHROMIC ANEMIAS
-
IDIOPATHIC HYPOCHROMIC ANEMIA
THE HEMOLYTIC ANEMIAS
HEMOLYTIC JAUNDICE
HEMOGLOBINURIA .
HYPOCHROMIC ANEMIA OF LEAD POISONING
PERNICIOUS ANEMIA
:PISEASES CHARACTERIZED BY MACROCYTIC
ANEMIA
APLASTIC ANEMIA
SICKLE CELL ANEMIA
ANEMIAS OF CHILDHOOD
 SECTION FOUR
THE ANEMIAS

CHAPTER 13
FACTORS INFLUENCING ERYTHROPOIESIS
AGE

Red cell and hemoglobin values are high at birth. Between the second week
and the third month of life, there is a physiological decrease of hemoglobin wbich
gradually rises to normal at about the sixth month. After this period, age appears
to have little influence on these values. It is recognized, however, that certain
anemias are known to occur more frequently at certain age levels. For example,
pernicious anemia occurs rarely in children, but has a high incidence in middle
and old age.
The effect of old age per se on the blood level is still uncertain and the fre-
quency of anemias is due, probably, to causes other than age itself. From Wil-
liamson's figures, computed from the hemoglobin determinations in I9I9 normal
individuals, cellular values in senility are as 'high as those of young people. Lasch
and Triger have shown, in a study of ISO patients over 60 years of age, that cer-
tain types of anemia occur frequently in old people and they have observed 64
cases of pernicious anemia in this group. They have found that other hyper-
chromic, macrocytic anemias and aplasia of the bone marrow occur not infre.
quently in the aged and that these conditions respond poorly to treatment.

SEX

Until the age of puberty there is little difference in the blood values in males
and females. After thiG period, the values are higher in men than in women. The
lower values of women have been attributed to menstruation. In a study of 3 12
college women over a period of two years, we have been unable to find any cor-
relation between the menstrual cycle and erythrocyte and hemoglobin values.
Davidson and Leitch have found the incidence of nutritional anemias low in chil-
dren and male adults and high in adult females, particularly at the childbearing
age. They attribute this to increased iron requirement at certain stages of a
woman's life.
166 THE ANEMIAS

INDIVIDUAL REQUIREMENTS

The limits for normal values for red cells and hemoglobin are wide and elastic
and depend considerably upon individual requirements. Individuals engaged in
strenuous manual labor appear to require a larger number of red cells and more
hemoglobin than those of sedentary habits. Broun has shown that exercise plays
an important r6le in the maintenance of an active hematopoietic system and that
strenuous activity exerts a stimulating effect upon the bone marrow.

BLOOD LOSS

The effect of blood loss upon the hematopoietic system is dependent upon the
amount lost and the speed at which the loss occurs. Acute loss in which as much
as one-third of the total blood volume is depleted quickly and to a marked degree
results in shock and usually death. Rapid infusion of fluids and transfusion of
blood, in this instance, becomes a life saving procedure. When the loss is not
rapid enongh to bring about a depletion of blood volume sufficient to cause im-
mediate circulatory embarrassment, the recovery is spontaneous. The bone mar-
row responds by rapid regeneration within 24 to 48 hours after acute hemorrhage.
This is evidenced in the peripheral blood by an increase of reticulocytes, leuko-
cytes, and platelets. Hemoglobin regeneration is slower than that of erythrocytes
so that the color index is low during recovery. The volume index is frequently
above one during the height of .the reticulocyte response.
Chronic blood loss produces a hypochromic type of anemia in which the
severity is dependent upon the amount of blood loss and the chronicity of the
hemorrhage. The marked regenerative blood picture following acute hemorrhag'
is absent in the chronic type. For further fUildamental details of the mechanisrr
and results of blood loss, the reader is referred to the chapter on the hypochromi(
anemias.

BLOOD DESTRUCTION

When red blood cells are destroyed, hemoglobin pigments are released in
great quantities in the blood plasma, as evidenced macroscopically by the yello"
color of blood serum in the hemoiytic anemias. Iron liberated during hemolysi,
is stored and used again (Haden). Hemolytic anemia, therefore, is not char·
acterized by iron deficiency and does not respond to iron therapy.
The bone marrow responds to excessive eryfurolysis by increased productior
which is reflected in the blood by reticulocytosis, normoblastemia, and other reo
generative changes. This compensatory activity of bone marrow may becomE
extensive and may result in the so-called "bone marrow crisis," in which the blood
picture is characterized by marked erythroblastosis and leukocytosis.
Accelerated blood destruction may occur as a result of many factors. Con·
stitutional anomalies of erythrocytes such as sphericity and sickling render
them excessively susceptible to hemolysis. These peculiarities are found, reo
 FACTORS INFL UEN CING ERYTHROPOIESIS

spectively, in chronic hemolytic jaundice and sickle cell anemia. How these
cellular abnormalities cause an increased destruction is not understood. Haden
suggests that the abnormal spherocytes, characteristic of hemolytic jaundice,
are incapable of absorbing as much fluid as the normal cell without hecoming
hemolyzed, and that there is a definite relationship between the point of initial
hemolysis and the size, shape, and thickness of red cells. Others believe that
an exaggerated destructive mechanism is the causative factor and the cellular
anomaly is only a secondary sequel.

ROLE OF THE SPLEEN

T.he incrimination of the spleen as a destructive agent in chronic hemolytic

anemia and in certain types of thrombopenic purpura has gained general ac-
ceptance due to the effectiveness of splenectomy in a majority of both 'conditions
(Doan, Curtis and Wiseman). It is not Imown, however, just what splenectomy
accomplishes in these conditions despite the clinical success of the operation.
Wiseman has discussed two theories for the pathogenesis of congenital hemolytic
icterus: first, the inherited fragility of red cells has its origin in a defect of the
bone marrow; and second, the spleen has become hyperactive in its hemolytic
activity and the bon~ marrow, after a long continued effort to supply the loss,
produces cells of inferior quality. He believes that the disappearance of exces-
sively fragile cells and the restoration of normal hematopoiesis following splenec-
tomy favors the acceptance of the second theory.
Doan and associates have observed that the spleen acts both as a depressant
and a stimulant in the establishment of a hematopoietic equilibrium; that it may
exert a "selective effect on one or more of the formed elements of the blood:';
and that the rationale for the removal of atrophic spleens in certain anemias is
based on the phagocytic activity of the spleen for blood cells. They have recom-
mended the removal of the spleen in acute hemoclastic crises as well as in chronic
hemolytic anemia and idiopathic purpura hemorrhagica. They have performed
sixteen successful splenectomies in the following diseases: hemolytic jaundice,
thrombopenic purpura, Banti's syndrome, myeloid and lymphatic leukemia, poly-
cythemia vera, and hypoplastic anemia. : They state that liver extract is con-
tra-indicated in the treatment of congenital hemolytic jaundice since they regard
the pathogenesis to be in the spleen and not in the bone marrow. It has been
their experience that "an exacerbation of the hemolytic diathesis frequently fol-
lows the administration of liver." Doan, Wiseman and Erf have re-emphasized
recently that the spleen is the causatiye agent in hemolytic jaundice. They
propose the idea that the faulty mechanism lies in the inherited overactivity of
the reticulo-endothelial system.
, The question has not been settled, but it is diffiqIlt for the adherents of
splenic pathology to explain why splenectomy, despite its clinical success in hemo-
lytic jaundice, does not always correct the erythrocyte abnormality. It is equally
as difficult for the sponsors of erythrocytic pathology to explain why the pecu-
liarity of the red cells frequently exists without anemia.
Rich has reported the occurrence of a splenic lesion in sickle cell anemia
168 THE ANEMIAS

which he believes is the causative agent in the production of this anemia and
Diggs has described a pathological change in tbe spleen occurring persistently in
the disease, This anemia, however, is not benefited by splenectomy.
In addition to these intrinsic factors inherent in the red cells themselves, in
the spleen, 6r in b,one marrow, there are numerous extrinsic factors, such as in-
fections, chemical poisons and parasites which exert a destructive effect on
erythrocytes and produce a severe, hemolytic anemia.

IRON

Within the last fifteen years, great advances have been made in the know,l-
edge of the relationship of dietary factors' to blood formation. Unquestionably,
iron is an important factor in normal hemoglobin production. When the diet
is deficient in iron the red cells soon become deficient in hemoglobin (hypo-
chromic) and smaller in size (microcytic) than normal.
From extensive clinical and experimental research, Whipple and Robscheit-
Robbins and their collaborators have evaluated the hemoglobin producing power
of numerous foods and tissues by feeding these substances to dogs rendered
markedly anemic by bleeding, They have found that kidney, liver, and apricots
are rich in material effective for hemoglobin regeneration and have confirmed
the satisfactory therapeutic effect of inorganic iron on hypochromic anemia.
TABLE XIV
HEMO~LOBlN POTENCY OF DIET FACTORS AND OF IRON: AVERAGE VALUES
(Excerpt from Whipple's chart, Nobel Lecture)
Total Net
Hemoglobin
Average
OutpuJ per
Did Faclor Daily Intake aWks.Gm.
Pig liver, 300 Gm.... .................... . 93
Liver extract, 55 Gm..................... . 56
(equivalent to 300 Gm.)
Pig Kidney, 300 Gm...................... . 69
Beef heart, 300 Gm...................... . 49
tio~cw:),~~e~~.~ .~~:::::::::::::~::::: 4'
53
Iron, 400 mg ... ......................... . 94
Salt mixture-Fe, 6 mg.......•............. 9
Salmon bread, 400 Gm ................ .... .
Whether Iron is used directly to build up the hemoglobin molecule or .whether
it is utilized as a stimulant for growth and metabolism of red cells is as yet an
unsolved question, The recent trend favors the conception that iron therapy
replaces a deficiency in hemoglobin·building materials despite the necessity for
massive doses in obtaining therapeutic results (Ottenberg), Iron by mouth must
be administered far in excess of the amount utilized because of the poor absorp-
tion of this metal in the intestinal tract, but Heath, Strauss and Castle have
demonstrated that iron injected intramuscularly into anemic patients is used
almost quantitatively in the production of hemoglobin. In some cases, iron
administration seems to stimulate the bone marrow in a manner similar to the
action of liver in pernicious anemia. This is evidenced by an initial reticulocyte
 FACTORS INFLUENCING ERYTHROP01ESIS 16 9
/

rise followed by a gradual increase of hemoglobin and red cells. Hemoglobin is

produced more quickly than red cells, thus raising the color index to normal. Con-
versely, liver therapy in pernicious anemia causes an increase first in red blood
cells with a consequenflowering of the color index to normal.
Davidson and Leitch have summarized the important factors in the chemistry
and metabolism of the components of normal erythrocytes. The total solids of
red blood cells are proteins, 4 per cent; hematin, 4.3 per cent; and globin, 89.3
per cent. Hemoglobin exists in the organic matrix or stroma of red cells in a
highly concentrated solution and is elaborated by the polymerization of four
hemochromogen molecules (iron compounds of pyrrole) with the amino acids of
globin. The materials necessary for erythrocyte formation, therefore, are the
organic constituents of stroma, pyrrole, iron, and the amino acids of globin. It
follows that a deficiency in the diet of any of these substances may become the
causative agept in anemia.
Heath states that in the average healthy man there are 2.7' Gm. of iron cir-
culating in the blood as hemoglobin iron, a little over 0.3 Gm. as functioning tissue
iron"and '-3 Gm. stored for emergency iron, giving a total of only 4.3 Gm. in the
entire body. The optimum intake is only IS mg. per day. Iron can enter the
body very easily but it can not be excreted in very large quantities. Diarrhea
reduces the iron that may be taken in by mouth and achlorbydria interferes with
its absorption. Hemorrhage always causes loss of iron. Under normal condi-
tions blood has five times the iron concentration of any other tissue. Heath
further points out tbat there is constant iron loss from menstruation; about 25
mg. of iron are required to produce a rise of 1 per cent in hemoglobin. If 5 mg.
are absorbed daily for five days the hemoglobin will rise I per cent and in 50
days 10 per cent. I
More information has accumulated concerning the relation of iron to hemo-
globin formation than to any of the other constituents. Food iron is largely
ferric iron and to be absorbed it must be rendered "soluble, ionizable, and ultra-
filterable," and its absorption is dependent to'a great extent upon its solubility
in the acids of the stomach (Davidson and Leitch). The extraction of iron from
food compounds, or the utilization of ferric iron administered therapeutically,
may be inadequate in patients with achlorhydria. In this event, it may become
necessary to administer the more soluble ferrous salts. Bethell and associates
have shown that, in iron deficiency anemias, patients with achlorhydria require
larger doses of iron for an adequate response than patients with normal gastric
secretion.
Robscheit-Robhins states that the site of ahsorption of iron is the duodenum
and upper small intestine, but the entire intestinal tract is capable of exercising
this function to a limited degree. The absorbed material is carried by the blood
and lymph to the various sites of storage, localized chiefly in liver and spleen.
She suggests that the liver, spleen, and bone marrow exercise the function of
iron metabolism. Whipple and Robscheit-Robbins have demonstrated that the
amount of iron stored in the liver varies considerably in the different blood dis-
 THE ANEMIAS

orders; it is high in aplastic anemia and pernicious anemia, normal in leukemia,

and low in anemia due to blood loss.
The experiments of Gottlieb on splenectomized rats lend support to the
idea that iron is stored in the spleen and that the reticulo-endothelial cells of
this organ play an important role in iron metabolism. He concludes that the
inevitable development of an anemia following the removal of the spleen of
normal rats, despite the administration of large doses of iron, is the result of
the removal of cells vitally concerned with iron metabolism.
The prevalence' of iron deficiency anemia in adults and children and the
rapid cure of the anemia by iron therapy has led to a detailed study by many
investigators of the underlying causes and the laboratory diagnosis of this type
of anemia. In general, these anemias may be caused by the following factors:
(r) dietary iron deficiency, (2) deficiency of other substances necessary for
building the hemoglobin molecule, (3) faulty iron metabolism, (4) inadequate
iron absorption or storage, (5) increased demands for iron which exceed the rate
of production, and (6) depletion of iron reserves by blood loss. Anyone or a
combination of these conditions may lead to the production of a blood picture
characterized by hypochromia and microcytosis.
Heath and associates emphasize the fact that a poverty of hemoglobin is
not the only manifestation of an iron deficiency. With an inadequate iron
diet, the body first relinquishes iron from the hemoglobin molecule in red cells;
and, as the demand becomes more imperative, iron is taken from the tissues.
Dystrophy of the nails in idiopathic hypochromic anemia exemplifies an inade-
quate iron supply at the nail roots. Atrophy of the skin and atrophic changes of
the tongue are probably other examples of iron poverty in the tissues.
Another interesting point in relat'ion to iron deficiencies is the recent con-
tribution of Cruz and of Rhoads and associates, that the anemia following hook-
worm infestation is an iron-deficiency anemia. They were able to prevent
anemia in cases highly infested with parasites by iron administration later sub-
stituted by an iron-rich diet. The effects of the worm or its toxins on the body
were of little importance in comparison to the interference with the absorption
of iron by the intestinal mucosa.

COPPER
Numerous investigators have presented evidence that copper is involved in
blood formation as a catalytic agent in tbe metabolism of iron. Support is
given to this thesis by the experiments by Sachs, Levine and Fabian in deter-
mining the copper and iron content in human blood. The average copper content
in normal men was found to be higher (0.r36 mg. per cent) than in normal women
(0,13r mg. per cent). The average iron content for men was SO mg. per cent and
for women 4S mg. per cent. These figures indicate a greater susceptibility to
anemia in women because of the lower iron and lower copper content. The
authors believe that individuals susceptible to anemia require larger amounts of
copper to stimulate hematopoiesis.
 FACTORS INFLUENCING ERYTHROPOIESIS

TABLE XV

IRON AND COPPER IN HUMAN BLOOD

Mg. Iron Mg. CoPP"

per IOO CC. per IDO ce.
One hundred normal males...................... 50,or 0.r3 6

~~;~n~;r:~m~l~hlrdr~it:::::: ~::: ~ ~ ~::::::::::: !~:~~

0.13 1
0.171
Six newborn infants .. ........ ,....... .. ........ 51.79 0. 083
~Twenty-three adolescent boys. .......... .... .... 47,91 0.1545
Twenty-nine adolescent girls. . . . . . . . . . . . . . . . . . . . 45.17 0.13 87
Ten menstruating women (first day) ...... , . . . . . . 44.58 0.135 1
Ten menstruating women (last day). . .. . . . . . . . . . . 43· 4S 0.14 1 5
Fifteen newborn infants (Cesa.rean). . . . . . . . . . . . . . 53.60 0. 103 0
Fifteen Cesarean mothers............. ......... 40.1I 0.2120
Eleven newborn infants........ .. ...... .. ....... 54.34 0.0980
Eleven mothers {normal delivery).... ... .. ... . ... 41 ·37
All figures from the publications of Sachs and. associates, Creighton University'"
School of Medicine, Omaha, Nebraska.

In analyzing the blood of patients with various anemias, they found an

inverse relationship between the copper and iron content of whole blood with
an increase in copper accompanying an iron deficiency. They believe that a
true copper deficiency does not exist in adults, and, for this reason, copper therapy
is an unnecessary adjunct to iron therapy in anemia except in rare cases in
infants.
Hart, Steenbock, Waddell and Elvehjem, in their work on the nutritional
anemias of rats, have found that these animals are rendered markedly anemic
by a milk diet, and that the resulting anemia simulates rather closely the nutri-
tional anemias of young infants. They have presented evidence that the anemia
in rats is not corrected by iron therapy unless augmented with small amounts
of copper. Other workers are equally certain that copper is an unnecessary
adjunct when adequate doses of iron are administered. In evaluating the thera-
peutic effect of copper, Parsons concludes that this metal is a necessary supple-
ment to iron only rarely in the anemia of infants, when there is a deficiency of
both iron and copper in the liver.
Elvehjem and Sherman base their .conclusions concerning the merit of cop-
per therapy in anemia on the concept that iron is assimilated and stored in the
liver but cannot be utilized in hemoglobin production without the presence of
copper as a catalyst.
Mackay has treated one group of patients having nutritional anemia with
4}/'-9 Gm. of ferric ammonium citrate daily and the second group with the same
dosage plus the daily administration of 1/20 gr. copper sulphate. A study of her
results does not indicate that copper is of any benefit in this type of anemia. She
concluded that "although copper deficiency may occur in isolated cases of nutri-
tional anemia, it plays no part in the great majority of cases seen in London and
that this probably holds good for the other parts of the world."
 THE ANEMIAS

PROTEIN

Drabkin and Miller have reported the correction of milk anemia in rats
by the addition of pure amino-acids to the milk which contained a quantity
of iron insufficient in itself to produce the result. The amino acids that seemed
most effective for hemoglobin regeneration. were arginine, glutamic acid and
their salts. Iron supplemented with these acids brought about a complete re-
covery from a severe grade of milk anemia. As an explanation of these results,
they propose the hypothesis that these organic substances are sources of pyrrole
radicals; that both iron and pyrrole are essential materials in the building of
hemoglobin; and that an excess of either one or the other may facilitate their
union to form hemoglobin.
Elvehjem and associates concur in the idea that certain organic groups such
as those derived from amino acids are essential building stones for the hemoglobin
molecnle, but they are not in agreement with Drabldn and Miller that amino
acids are effective in the cure of milk anemia. They assume that the favorable
, results with these acids were due to the presence of traces of copper in com·
bination with the amino acids. After carefully repeating the experiments of
Drabkin and Miller, they came to the conclusion that the combination of large
amounts of pure glutamic acid with whole milk and iron will not correct milk
anemia in rats, It seems evident that although proteins ·are necessary for a well
balanced diet and are linked in some manner with hemoglobin metabolism, their
relation to nutritional anemias has not been proved.
Unsuccessful efforts have been made to link the extrinsic hematopoietic fac-
tor of Castle with the proteins of beef muscle and yeast. This extrinsic factor
is fou;'d in high concentration in these materials but cannot be identified with
any single nueleo-protein (Strauss and Castle). The treatment of pernicious
anemia with defatted, desiccated hog stomach by Sturgis and Isaacs suggested
the possibility that some enzyme may act on the protein of hog stomach tissue to
produce a hematopoietic factor or that the stomach wall contains an active anti·
anemic principle which elicits a response in pernicious anemia equal to that of
liver extract. The relationship of protein derivatives has not been proved, but
the probability of the elaboration of an llcntianemic factor by the stomach has
become a certainty.

GASTRIC FACTORS

Although it has been known for years that gastric abnormalities are found
coincident with certain anemias, it was not until after the discovery of liver·
therapy for pernicious anemia, and the classification of this anemia as a deficiency
disease, that attention was centered seriously on the etiologic relation of the
stomach to the anemic states.
In their work on dietary factors in anemia, Whipple and his co-workers.
were the first to observe that the feeding of liver and kidney was effective in the
treatment of certain tY(les of anemia, but it was not proved until I926 by the
 FACTORS INFLUENCING ERYTHROPOIESIS 173
classical experiments of Minot and Murphy that the administration of laxge
quantities of liver to patients with pernicious anemia effected a remarkable im-
provement, both clinically and hematologically. They found tbat the oral ad-
ministration of liver elicited a response by reason of a bone, marrow reaction
reflected in the peripberal blood by an immediate marked reticulocytosis and
a milder normoblastemia, followed by a steady and gradual rise of red cells
to a normal level. Cohn and associates succeeded later in preparing concen-
trated liver extracts for parenteral administration. During the next few years
many investigations were made to determine the exact nature of the liver sub-
stance which still remains unknown. It is thought to be a small nitrogenous
compound and is not limited to the liver but may be found in stomach tissue,
brain, placenta and kidney (Minot, Nobel lecture). This work has made it
possible to correlate the interrelation of liver therapy, anemia, and gastric ab-
normalities.
In I928, Castle and Locke called attention to the etiologic relationship
of achylia gastrica to pernicious anemia, and in the next year Sharp, ,and Sturgis
and Isaacs, demonstrated the presence of an antianemic factor in defatted, des-
iccated hog stomach. Daily feeding of IS to 30 Gm. of this material produced
a reticulocyte response comparable to that induced by liver therapy. During
the same year the work of Castle and his co-workers proved that the effective
hematopoietic stimulus is elaborated in the normal stomach by the action of an
unknown secretory product upon some substance in the diet. They were able
to produce a remarkable clinical and hematological response in patients with per-
nicious anemia by feeding eight untreated cases daily for ten days with normal
hnman gastric juice recovered by stomach tube after the ingestion of beef muscle.
The response was as spectacular as that of the liver extract control group.
Three patients were fed beef muscle alone with no improvement.
These results led to further study of the nature of this SUbstance by Castle
and Townsend. Three patients were fed with normal gastric juice alone, four
patients with normal gastric juice at night and beef in the morning; and ten
patients with a mixture of incubated gastric juice and beef muscle. Response
was found only in the tllird group and this led to the conclusion that some inter-
action of normal gastric juice and beef muscle results in a product effective in
pernicious anemia .
. - Subsequent work by Castle and associates has demonstrated that the inter-
action of a specific intrinsic factor in normal gastric juice with an extrinsic
factor in the diet resnlts in the elaboration of a hematopoietic factor which stimu-
lates the production of red cells. This factor is called the "X" hematopoietic or
the "antianemic" factor and is probably absorbed in the intestine and stored in
the liver and kidneys. The actual site of interaction of the gastric and food
factors is not known. The stomach contents of pernicious anemia subjects are de-
?cient in the specific intrinsic factor and such victims are incapable of elaborat-
mg the "X" hematopoietic factor in sufficient quantity to maintain an adequate
number of erythrocytes. -
Castle and his co-workers have shown that some patients with pernicious
174 THE ANEMIAS

anemia are not totally deficient in the intrinsic factor. Small residual amounts
which may remain in the' early stages of the disease are probably responsible for
the "spontaneous" remissions which frequently occur. Goldhamer has suggested
that in pernicious anemia the factor is deficient in quantity rather than quality,
and that the degree of anemia is dependent upon the amount of the intrinsic
factor produced.
To summarize the results presented above, the following concepts are of
fundamental importance in hematopoiesis. An anti-anemic, or "X" hematopoietic
factor, is produced by the union of an "intrinsic" gastric factor with an "ex-
trinsic" dietary factor. This hematopoietic factor, if properly absorbed, is stored
in the liver and, to a lesser degree, in the kidneys, brain and placenta. Whether
these tissues exert a further metabolic action on the fac(or is problematical.
The factor, if present in sufficient quantity, governs orderly erythropoiesis in
the bone marrow. A deficiency of the factor or an interruption of its formation,
absorption, or storage results in a cessation of maturation and division of
erythrocytes at the megaloblast stage and consequently an abnormal develop-
ment and an inadequate number of red cells. The circulating blood in this
deficiency is characterized, therefore, by a deficiency of reticulocytes and a
macrocytic, hyperchromic anemia. The replacement of the factor in the form
of liver extract or stomach preparations supplies the bone marrow with the
deficient substance and brings about the return to a normal, orderly produc-
tion which is evidenced, first, by reticulocytosis and, finally, by the produc-
tion of erythrocytes, normal in size and function. Pernicious anemia is a
classic example of a constitutional inability to produce the "intrinsic" factor in
the stomach and, therefore, fall~ into the category of the deficiency states.
There is a paucity of knowledge concerning the exact nature of these fac-
tors. According to Castle, the intrinsic factor is heat-labile; it can be separated
from hydrochloric acid, pepsin, rennin and lipase of the gastric juice; and
it is not present in the saliva or the duodenal contents. Fouts, Helmer, and Zer-
fas have subjected gastric juice to ultrafiltration in an attempt to isolate the
intrinsic factor. They confirmed !:astle's observations that this factor can be
separated from the enzymes of the stomach, pepsin and rennin, by casein precipi-
tation and demonstrated that vacuum distillation is necessary before the intrinsic
factor will pass through the ultrafilter. Numerous investigators bave attempted
to isolate the "intrinsic" factor from the stomach and the "X" factor from liver
extract. While considerable progress has been made, especially in the purifica-
tion of liver extract, as yet neither factor has been isolated.
Greenspon has presented a different interpretation of the nature of the
anti-pernicious anemia principle in the stomach. In a series of experiments he
found evidence that gastric juice alone, and therefore, the intrinsic factor, was
effective in pernicious anemia when peptic activity was prevented. He suggested
that the extrinsic factor in beef muscle is unnecessary ·for the formation of the
anti·anemic factor, and that this food merely depepsinizes gastric juice. Later
investigators have not confirmed these results and the opinion prevails, at the
present time, that Castle's original hypothesis is valid (West, Ungley and Mof-
fett, Hanes, et al.).
 FACTORS INFLUENCING ERYTHROPOIESIS 175

The extrinsic factor has not been identified. It is present in marmite (auto-
lyzed yeast), beef muscle, rice polishings, wheat embryos, eggs, liver, and certain
liver extracts, but neither a nucleo-protein nor vitamin B2 in yeast has been
identified with the extrinsic factor.
West has given an excellent summary of the knowledge relative to the
actual nature of the antianemic principle in liver. It is soluble in water and
in slightly acid 95 per cent alcohol and is not soluble in ether. It contains car-
bon, hydrogen, oxygen and nitrogen. It is precipitated with phosphotungstic
acid and with Reineke's salt. Its potency is destroyed with the salts of gold,
silver, platinum, and mercury, and with normal sodium hydroxide at room tem-
perature or by hoiling with mineral acids. Its potency is not lessened clinically
by heating to 100 C. at pH 5.0.
The observations of Jones and his collaborators on the changes in the gastric
mucosa in pernicious anemia add to the accumulation of evidence that the dis-
ease is a deficiency state. Gastric atrophy occurred more frequently during re-
lapse, although not present in every case of pernicious anemia studied. Evidence
of chronic gastritis was always demonstrable and appeared to precede the anemia.
Adequate therapy resulted in the disappearance of atrophic change, and of the
inflammatory process, but the ability to produce hydrochloric acid was not
resumed.
Miller and Rhoads have produced an anemia in swine, closely similar to
pernicious anemia in man, by feeding a diet modified after that which produces
black tongue in dogs. The condition paralleled pernicious anemia in gastro-
intestinal change with loss of the intrinsic factor in addition to the characteristic
macrocytic blood picture.
Another interesting c,?ntribution is the demonstration by Wakerlin ,of an
anti-anemic principle in normal human urine which upon injection into pigeons
elicited a reticulocyte response similar to that following liver injection. An ex-
tract prepared from urine and given intramuscularly to a patient with pernicious
anemia brought about an improvement in the blood picture superior to the re-
sponse of the same patient with liver extract in a previous study.
In view of these hypotheses relative to the incrimination of the stomach
in pernicious anemia, total gastrectomy resulting in immediate achylia, theoreti-
cally should produce a macrocytic anemia. Some investigators have found the
hematopoietic system undisturbed following gastrectomy (Hurst). Ivy has
demonstrated that an anemia results in some instances in gastrectomized dogs.
It is worthy of note that five dogs whose blood picture remained normal after
the operation developed a marked anemia during pregnancy, being unable, pre-
snmably, to respond to the extra demands of the pregnant state.
In 1929, Finney and Rienhoff reviewed the reported cases of total gas-
trectomy. Of the nine patients who lived for a year after the operation, two
developed a pernicious anemia-like blood picture. Since that time an occasional
case has developed a similar anemia following gastric operations. A number of
diseases which interfere with gastric function frequently are accompanied in
the late stages by a macrocytic" anemia which, in some cases, responds to liver
17 6 THE ANEMIAS

therapy. On the other hand, "a number of gastrectomized patieuts show little
alteration in the blood picture. This fact was considered contradictory to the
incrimination of a stomach defect as the sole etiologic agent in pernicious anemia
until the experiments of Meulengracht (I934, I935) offered an interesting and
logical explanation. He demonstrated that desiccated materia] from the pyloric
region of the stomach and duodenum of pigs was rich in the hematopoietic
principle effective in pernicious "anemia and that the cardiac and fundic regions of
the stomach were relatively inactive. He has been successful, therefore, in
localizing the site of formation of Castle's "intrinsic" factor. This suggests that
the glands of both the pylorus and the upper duodenum are non-functional in
pernicious anemia, but in gastric resection the glands of the duodenum, in some
cases, are capable of producing sufficient quantity of the intrinsic factor to pre-
vent the development of a macrocytic anemia.
Schenken and his associates prepared an extract from the liver of a patient
who had died from scirrhous carcinoma of the pyloric part of the stomach. It
failed to produce reticulocytosis when administered to a patient with typical
pernicious anemia. Another extract was prepared from the liver of a patient who
had died with gastric. carcinoma which involved the entire stomach except the
pyloric portion. The administration of this did produce reticulocytosis. This
would indicate that hematopoietic material is obtained from the pyloric portion:
In a comprehensive report, Castle summarizes the three possible conditions
from which pernicious anemia and related macrocytic anemias may result: (I)
lack of the specific intrinsic factor of the stomach; (2) lack of the extrinsic factor
in the diet; (3) failure of absorption or utilization of the products of the inter-
action of the intrinsic and extrinsic factors.
Singer has devised an animal experiment for determining the' presence or
absence of tbe hematopoietic factor in gastric juice. Gastric fluid, aspirated after
histamine stimulation, was injected into two white mice subcutaneously or intra-
muscularly. One mouse was given 2 to 3 cc. and the second 5 to 8 cc. of filtered
and neutralized (NaHCOa) gastric juice. Reticulocyte counts were made on
the blood of the mice on two successive days before the injection, and daily for
five days after injection. Using the gastric contents of 4I patients with per-
nicious anemia, he found no rise in reticulocytes (negative reaction) in the blood
of the mice, but found a definite rise (positive reaction) in the same experiment
using gastric juice of forty patients with various other types of anemia.
There is considerable evidence that, in addition to its relation to pernicious
anemia, the stomach plays an important rOle in other deficiency anemias, notably
the hypochromic anemia due t6 iron deficiency. Although inadequate intake of
iron is the most prominent causative factor, hypochromic anemias are found
in some individuals whose diet shows no deficiency in iron. Davidson and Leitch
state that achlorhydria is known to affect the utilization and absorption of iron;
food passes through an achlorhydric stomach at a more rapid rate than normal,
thus partially inhibiting the liberation of iron; furthermore, absorption of iron
in the jejunum is facilitated when the reaction is acid rather than alkaline, as is
the case when the stomach contents are deficient in hydrochloric acid.
 FACTORS INFLUENCING ERYTHROPOIESIS '77
In adult women there is a high incidence of simple hypochromic, microcytic
anemia, invariably associated with achlorhydria which may precede the anemia
and may persist after the blood deficiency has been corrected by iron therapy.
This leads to the assumption that this type of anemia is an absorption anomaly
resulting from gastric dysfunction. On the other hand, there are numerous
patients with achlorhydria who show no evidence of anemia.
It seems probable that jf achlorhydria predisposes to faulty iron absorption;
an anemia results more frequently in women who, according to Davidson and
Leitch; are subject to increased iron demands at certain age levels. Evidence for
this is found in the incidence of simple achlorhydric anemia which occurs in the
ratio of about three to one in women.
In addition to the production of an intrinsic portion of the hematopoietic
factor and the regulation of iron absorption, the stomach may influence the
hematopoietic system by hyperactivity. Morris and associates have suggested
that an excessive secretion of addisin (their term for the hematopoietic factor of
gastric juice) is responsible for polycythemia iu which there is an overproduction
of erythrocytes. They advocated gastric drainage as a therapeutic measure in
this condition. Recently Briggs and Oerting have had excellent results in treat-
ing polycythemic patients by gastric lavage.

LIVER STORAGE

Since the discovery of a hematopoietic principle in liver tissue and a similar

or identical substance elaborated by the stomach, both effective in the treat-
ment of pernicious anemia, it follows that this antianemic factor must be pro-
dnced in the stomach and stored in the liver.
Confirmation of this conception is found in the conclusions of Goldhamer,
Isaacs and Sturgis that the active maturation stimulant for erythrocytes is pres-
ent in the liver at least two months before birth; that it may be entirely absent
from the liver of inadequately treated cases of pernicious anemia and present
in those who have received proper therapy; that it may be absent in conditions
other than pernicious anemia where there is liver damage of sufficient extent to
prevent adequate storage; and that there may be certain types of liver damage
which inhibit the delivery 9f the active principle which is stored in normal
amounts.
Davidson states that in extensive liver disease, the damage to the liver in-
hibits its function as a storage organ for the hematopoietic factor, thereby allowing
the occurrence of a megaloblastic blood picture. There is considerable evidence
..hat liver diseases are frequently complicated by a moderate degree of macrocytic
,nemia, but there is a diversity of opinion concerning the response of this anemia
to the administration of liver. It is possible that the active principle may nndergo
some change in the liver before it will act on the bone marrow, and a damaged
liver may be incapable of performing this function. This may account, in part,
ior the many failures of liver therapy to correct the anemic state in instances of
liver disease.
 Wintrobe and Shumacker have reported 43 patients with various hepatic
disorders and in all there appeared a mild macn)cytic anemia characterized by
spontaneous remissions. In contrast to the bloo,! picture of pernicious anemia,
there was little variation in size and shape of red corpuscles. Ten of the anemic
patients had no free hydrochloric acid in the stomilch contents. From their clini-
cal observations, they suggest that the hematopoietic prin~iple produced by the
interaction of the intrinsic and extrinsic factors is stored in the liver, that it may
undergo some further elaboration there, and in some cases of liver damage, its
utilization is partially or wholly inhibited. Thi'Y state that such disturbance
may lead to an anemia either as a result of impaired storage or "by a combina-
tion of partial gastric disturbance and incompletE liver damage."
Recent studies corroborate these findings a~ shown by one case of Gold-
hamer and four cases of Van Duyn of hepatic cirrhosis, with a well developed
macrocytic anemia and normal gastric acidity. van Duyn states that undoubt-
edly the hematopoietic principle formed in the stomach passes through the portal
vein to the liver where it is stored. Minot and Ca.tle quote Schwartz as asserting
that a macrocytic anemia develotls only when an unknown function of the liver
is impaired and that this anemia responds to liver therapy, but these authors
state that in such anemias, "liver extract is usually entirely without effect."
That there are certain diseases of early infancy in which impaired liver
function and macrocytic anemia are closely associated is suggested by Van
Duyn; in congenital syphilis, anemia infantum, pseudoleukemia, and erythrohlas-
tosis foe talis, a study of liver tissue indicates that this organ has not relinquished
its fetal erythropoietic function. His belief that the macrocytic anemia ac-
companying these conditions is caused by the failure of the liver to assume adult
function seems well founded.
Wright observed 12 cases and studied 41 case records of portal cirrhosis.
The average erythrocyte count was 4,000,000 and the leukocyte count, 5,840 per
cu. mm. The color index was above r.o in 90 per cent of those cases with a red
cell count below 5,000,000 per cu. mm. Gastric contents were studied in eight
patients and in six instances there was achlorl1ydria. He suggests the term
"pernicoid, anemia" for the macrocytic anemias following liver damage and
lists a group of conditions other than pernicious anemia in which a macrocytic
anemia might occur. He states that "these seemingly unrelated conditions have
one thing in common that all is not well in Glisson's capsule." His list includes
chronic hepatomegaly, metastatic carcinoma of the liver, acute catarrhal jaundice,
chronic passive congestion of the liver, erythroblastosis foetalis, congenital syphilis,
arseniureted hydrogen gas poisoning, infections of the gallbladder, Weil's disease
(spirochaetosis), and thrombosis of the portal vein.
In a study of 94 patients with portal cirrhosis, Heath found that macro-
cytic anemia occurred more frequently in the more severe forms and the later
stages of the disease. His analysis of animal eJ'periments in which liver dam-
age and macrocytic anemia were produced by chloroform and phosphorus poison-
ing is in. agreement with the clinical association of the two conditions.
In a comprehensive review of 132 cases of various hepatic disorders, Wintrobe
 FACTORS INFLUENCING ERYTHROPOIESIS '79
offered support of the hypothesis that the macrocytic anemia of liver diseases
arises from an inadequate storage of the hematopoietic principle: (I) there was no
evidence that the anemia was caused by any factor other than the hepatic dis-
ease; (2) the anemia, except in instances complicated by infection or blood
loss, was either macrocytic or normocytic, was morphologically similar if not
identical with a mild type of pernicious anemia, was subject to spontaneous re-
'missions, and responded in some instances to liver therapy; (3) macrocytic
anemia occurred more frequently in cases of cirrhosis where the lesions were
extensive and was not found in acute necrosis where liver damage was only
slight; (4) there was some evidence that the patients were able to form the
hematopoietic factor normally and therefore able to consume various amounts
of the factor which may explain the moderate degree of anemia; (5) foci of
blood formation were found in the spleen of patients with macrocytic anemia, thus
affording another similarity to pernicious anemia.
Higgins and Stasney have produced an experimental cirrhosis of the liver
in rats by daily exposure for 45 minute periods to the fumes of carbon tetra-
chloride over a period of 12 weeks. Typical atrophic cirrhosis of the liver
developed, accompanied by a severe macrocytic anemia which was directly re-
lated to the degree of cirrhosis. They believed that the anemia was caused by a
marked destruction of erythrocytes.
Castle and Minot believe that it is illogical to attribute the macrocytic anemia
associated with liver damage entirely to the interference in the storage of the
anti-anemic factor in the liver. They state that it is difficult to explain the de-
ranged hematopoiesis unless the kidney as well as liver is damaged, since the
kidney is known to share in the sto~age of the hematopoietic factor. They point
out that the usual explanation of the failure of many cases to respond to liver
extract rests upon an unproved assumption that the liver has a metabolic as
well as a storage function. In addition, they state that macrocytic anemias are
associated with conditions unrelated to pernicious anemia and that a deficiency
of liver extract is not the only cause of macrocytosis. In the opinion of these
authors, the etiology of the anemia of cirrhosis of the liver is obscure.
The question raised by Minot and Castle indicates the need for further study
of the problem. Kidney storage of the hematopoietic factor, however, rather than
damaging the hypothesis might explain the reason for the mild degree of anemia
in liver damage as compared to the severity of pernicious anemia and, in addi-
tion, the reason why a number of cases of liver damage are unaccompanied 'by
anemia of any type.
It should be emphasized that macrocytosis occurs in disorders other than
pernicious anemia and diseases of the liver. Any condition which interferes with
the production, absorption, and storage of the hematopoietic principle, whether
it is a deficiency in the diet, faulty absorption, or a diseased liver, will usually
be accompanied by a macrocytic type of anemia. An excellent example of a
dietary deficiency resulting in macrocytic anemia is found in the tropical anemia
reported by Wills. She was able to effect a complete remission of this malady
by the administration of marmite (autolyzed yeast) which is a rich source of
180 THE ANEMIAS

the extrinsic factor of Castle. Furthermore, any condition which is charac-

terized by bone marrow dysfunction, such as prolonged hyperplasia, will eventu-
ally produce a macrocytosis. A macrocytic anemia is seen frequently in tbe
leukemic states, and in these cases with a normal or low leukocyte count the
erroneous diagnosis of pernicious anemia is often made.
There is evidence that the liver acts as a store house for iron as well as
the "anti-anemic" principle. It is not illogical, therefore, that liver damage
may interfere with iron metabolism as well as storage of the bematopoietic factor
and lead to a macrocy\ic, hypochromic anemia. This is seen in some cases of
erythroblastosis of childhood where the liver has not assumed adult function
and in the leukemic processes where there is widespread leukemic infiltration
of the liver. In both instances there is bone marrow dysfunction, but the effect
of liver damage on the storage of the anti-anemic faclor and iron should be
considered ..

YEAST AND VITAMIN B2

The discovery by Castle and his co-workers tbat marmite (autolyzed yeast)
is rich in the extrinsic hematopoietic factor has stimulated efforts to determine
what fraction of yeast is effective in the treatment of the various types of macro-
cytic anemias. Wills has studied the effect of marmite in the alleviation of
tropical macrocytic anemia which is a deficiency anemia with normal gastric
contents but similar hematologically to pernicious anemia. She found that IS
to 30 Gm. of marmite administered daily produced a complete remission of this
anemia. Marmite is one of the richest sources of vitamin B 2 • The possibility
that this vitamin was the extrinsic factor of Castle led Wills and Chick to in-
vestigate the effect of various vitamins on tropical macrocytic anemia. Vitamins
A, C, B" B2 and B4 were administered without curative effect and whole yeast
did not possess the therapeutic value of marmite. Vitamin-free marmite was
found to be equally as effective as marmite containing its original vitamin con-
tent. This led to the conclusion that although tropical macrocytic anemia is due
to a dietary deficiency of the extrinsic hematopoietic factor, this factor is not
identical with vitamin B 2 • Other investigators, including Castle, have corrobo-
rated this work by finding vitamin B2 ineffective as the extrinsic factor in the
treatment of pernicious anemia. Gildea and associates have reported that dogs
kept on a diet deficient in vitamin B developed demonstrable histologic lesions
and definite signs of a disturbance of the central nervous system.
The therapentic effect in pernicious anemia of fresh and autolyzed yeast and
of wheat germs has been studied in 18 patients by Ungley and James. Ten of
these patients exhibited a mild hematopoietic response which was explained
on the prohability that certain cases of pernicious anemia retain the ability
to secrete small amounts of the intrinsic factor and when the extrinsic fac-
tor is fed in large quantities, as in yeast, some response occurs. Davidson
offers the explanation that yeast may contain a small quantity of the intrinsic
factor as well as the extrinsic factor. He condemns, however, the substitution
 FACTORS INFL UEN CING ERY1'IIROPOIESIS

of yeast for liver extract or desiccated hog's storJ1ach in the treatment of per-
nicious anemia.

VITAMIN .C

It has been demonstrated that a deficiency of vitamin C in the diet results

in an anemia in both adults and cbildren with I'n accompapying hemorrhagic
diathesis. The anemia may be macrocytic, norm(lcytic or hypochromic in type.
Tlte more severe cases are generally of the macroCytic variety unless bleeding is
sufficient to result in depletion of hemoglobin by blood loss. Mettier, Minot and
Townsend have found liver and iron ineffective in the treatment of typical cases
but obtained good therapeutic results with foods rich in vitamin C, namely,
orange juice and raw liver pulp. Vaughan has obtained a good response by the
administration of crystalline vitamin C. Dunlop and Scarborough have found
that pure ascorbic acid administered daily relieved the anemia and stopped
hemorrhage and ecchymoses. Mettier and Chew have produced the anemia
experimentally by maintaining guinea pigs on a vitamin C free diet. The ad-
ministration of orange juice caused a reticulocyte response fo1lowed 'by a cure
of the anemia.

THYROID SECRETION

There are numerous references to the suppo;;ition that the secretion of the
thyroid gland is related in some manner to hemlltopoiesis since a deficiency of
thyroid secretion frequently produces an anemia. Kunde, Green and Burns have
produced an experimental hypothyroidism in rabbits which was accompanied hy
a macrocytic anemia, normal gastric findings, and a hyperplastic bone marrow
in no way resembling the marrow picture of pernicious anemia. Thyroid therapy
produced a reticulocytosis and relief of the anemia. Experimental hyperthy-
roidism resulted in an initial polycythemia followed by a hypochromic anemia.
Stenstam reported eight cases of pernicious anemia associated with hyper-
thyroidism. He reviewed the literature on this subject and after a discussion
of the various possibilities accounting for it he concluded that the association of
the two diseases was merely coincidental.
According to Lerman and Means, in myxedettla of human beings there occurs
a hypochromic anemia and more rarely a macrocytic anemia as well as a high
incidence of gastric anacidity which probably bears some relation to the de-
velopment of the anemia. Thyroid therapy cures the anemia, but the cure is
hastened in the hypochromic variety by iron and in the macrocytic type by liver
extract.

PITUITARY SECRE'fION

Dobbs and his collaborators have shown th~t intravenous or intramuscular

injections of an extract of the pituitary gland caused a marked macrocytic anemia,
18. THE ANEMIAS

a hyperplasia of the bone marrow, a reticulocytosis, hemorrhage into the stom-

ach, and an increased production of bile. The experiments suggested to the
authors that the pituitary body has some control on hematopoiesis by its influ-
ence on the stomach and reticulo-endothelial system.
Meyer and his associates removed the hypophysis from a group of rats and
found that there was invariably an anemia and a subnormal reticulocyte count.
In these animals the administration of liver was not followed by a reticulocytosis.
However, the administration of antuitrin growth hormone produced a variable
degree of reticulocytosis with increased red cells and hemoglobin. From these
experiments it is intimated that the hypophysis may play some role in hema-
topoiesis.

PREGNANCY

That pregnancy is indirectly a predisposing factor to anemia has been

emphasized by Wills, Leigh, Moore, First and Goldstein, Roberts, Whitby, Lyon,
and many others. The relation of this type of anemia to the pregnant state is
illustrated by a quotation from Bland and associates': "The most interesting
fact noted was the remarkable and rapid recovery, in a large per cent of the
women, within a few days after delivery."
The recognition that a great many anemias are deficiency conditions has
led to a clearer understanding of the etiology of the anemia of pregnancy.
Strauss and Castle stress a deficiency hypothesis and emphasize two causes in this
type of anemia; first, a dietary deficiency; and secondly, an ineffectual absorp-
tion of food by a disturbed alimentary tract. Of their 20 cases, 9 were found
with adequate diet and normal gastric findings and not one of these developed
anemia. These authors continued the' study of the gastric contents of 24
additional patients through the entire period of pregnancy and found a re-
duction in hydrochloric acid between the fourth and fifth to the seventh and
eighth months with a definite rise in the last two months. They concluded
that 75 per cent of these women failed to secrete normal amounts of hydrochloric
acid during that period of pregnancy, and that hemoglobin deficiency was defi-
nitely related to this decline in acidity.
Strauss has reported a series of 40 patients which illustrate the varioUs
types of anemias prevalent during pregnancy. His classification includes (1)
physiologic anemia due to the effect of hydremia, (2) a hypochromic and micro-
cytic type, and (3) a macrocytic anemia. Thirty patients were grouped under
the classification of hypochromic anemia. Of these, 17 had post-histamine
gastric anacidity, while 10 were deficient in hydrochloric acid and 2 had nor-
mal gastric findings. All but one of the patients capable of secreting hydrochloric
acid gave a history of an iron deficient diet and all responded to iron admin-
istration. The others revealed a history of poor diet or showed gastric defects
which no doubt int.erfered with iron absorption.
In the remaining 10 patients, a characteristic macrocytic anemia was demon-
',rated, accompanied by achlorhydria in five and hypochlorhydria in four. All re-
 FACTORS INFLUENCHiIG ERYTHROPOIESIS

sponded to liver therapy, although the liver was supplemented by iron in a few
instances. Nine of these patients gave a history of dietary deficiency of protein
animal food. In regard to the etiology of the anemias in this last group, a
majority can he traced to a lack of the intrinsic factor in the gastric juice and
the others to a lack of the extrinsic factor in the diet.
In his summary, Strauss includes the following points which are of consider-
able fundamental importance in regard to the etiology and treatment of anemias
of pregnancy: (I) the physiologic anemia is due to hydremia; (2) hypochromic
anemias are due to a dietary deficiency, to a deficiency caused by gastric an-
acidity, or to the extra demands of the fetus, and all are ameliorated by large
doses of iron; (3) macrocytic anemias are caused by a lack of the intrinsic
faclor in the stomach or of the extrinsic factor in the diet and may be corrected
with liver or by liver supplemented with iron; (4) these anemias may be pre-
vented by dietary prophylaxis.
Mackay has studied the hypochromic anemias of pregnancy and has found
the hemoglobin level of 368 pregnant women and mothers to be 13.7 per cent
lower than the average figure for healthy women. Whether dietary deficiency
of iron or achlorhydria with consequent deficient absorption of iron is respon-
sible for the hypochromic anemia of these patients is a debatable question but,
regardless of cause, she recommends routine iron therapy during the pregnant
state.
In regard to this question, Davies and Shelley have presented,· from a study
of 50 normal pregnant women and 2 I with anemia, the following conclusions:
(I) Normal women show no decrease in red cells and hemoglobin during preg-
nancy and in these women gastric secretion is normal; (2) women showing a
permanently deficient gastric secretion are prone to develop a hypochromic
anemia during or after pregnancy, which is easily corrected by iron therapy;
(3) multiple pregnancies, menorrhagia and a dietary iron deficiency result in
:l picture identical with lCachlorhydric" anemia.
In a later study of 200 women during the last four months of pregnancy,
Corrigan and Strauss have found that of 100 women treated with 0.5 Gm. ferrous
sulphate daily, none had less than 70 per cent hemoglobin post partum and of
the 100 patients receiving no iron, 24 had less than 70 per cent hemoglobin.
They conclude that hypochromic anemia may be prevented, to a great exient, by
the administration of iron during the latter months of gestation.
Macrocytic anemia occurs with less frequency in pregnancy than the hypo-
chromic type. When present, it is conditioned by a deficiency of the extrinsic
factor in the diet or the intrinsic factor in the stomach. As a rule, patients are
able to produce the hematopoietic factor normally after the pregnancy terminates.
The blood picture in this anemia closely resembles pernicious anemia both clini-
cally and morphologically. Heilbrun has reported a thorough bone marrow
study in a patient with a macrocytic anemia of pregnancy, which he believes is
due to a dietary deficiency of the extrinsic factor since gastric secretion" was
normal. The marrow closely resembled that seen in pernicious anemia and in
 THE ANEMIAS

sprue during relapse, and the ceIiular constituents became normal with proper
therapy.

OXYGEN TENSION

In a discussion of the factors governing hemoglobin and erythrocyte pro-

duction Robscheit-Robbins states that it has become wen established by the
Pike's Peak and Peru expeditions that red celIs and hemoglobin values rise
immediately upon ascension to a high altitude. The initial increase is due prob-
ably to changes in plasma volume and this polycythemia may persist. The
theory has been. advanced that oxygen deficiency causes a contraction of the
spleen and a consequent emergence of blood cells from this storehouse to the
peripheral blood." Sabin states that low oxygen tension is one of the factors
favoring erythrogenesis and Davidson considers insufficient aeration in utero
as the cause of high hemoglobin and erythrocyte values at birth.
Sabin has reviewed the numerous animal experiments that have been carried
out to clarify this subject. When mice were placed for 33 days in a chamber
with an atmospheric pressure corresponding to an altitude of 30,000 feet, a de-
cided increase in erythrocyte and hemoglobin values occurred. A hyperplasia
.of the bone marrow was found in rabbits SUbjected to low atmospheric pressure.
Jordan and Speidel suggest that the accelerated rate of erythropoiesis at high
altitudes is due to a change in carbon dioxide concentration.
Tyler and Baldwin have demonstrated that oxygen tension influences red
cells and hemoglobin values in rats under experimental conditions. Sixty-six
adult and II young rats were exposed to air pressures ranging from 650 to 360
mm. of mercury for periods of from 2 to '4 days. Twenty-one of the surviving
"adult'rats showed an initial polycythemia followed by a severe grade of anemia.
Thirteen adult rats developed an anemia following exposure without an inter-
mediate polycythemia. Seven young rats showed an increase of red cells after
exposure. They consider this anemia in adult rats similar to the anemia fol-
lowing polycythemias in human beings which has been reported by many
investigators. "

TEMPERATURE

Huggins, Blocksom and Noonan have determined the temperature of red

active marrow in the bones of the body-trunk and the proximal portions of the
limbs and of the inactive marrow in the distal extremities of various laboratory
animals. They found, during muscular rest, a temperature gradient in the bone
marrow of the rabbit, rat, and pigeon. The bone marro\,\, of the cranium, sternum,
ahd the more centrally located bones of the extremities maintained a constant
temperature approximating that of the peritoneal cavity. The temperature of
the bone marrow in the distal extremities was subject to the same cooling process
as other soft tissues in the extremities and w;'s found to have a temperature of
from 4 to 8 degrel"," f;. less than central marrow.
 FACTORS INFLUENCING ERYTHROPOIESIS 1 85

In later experiments on rats these authors fixed the distal end of the tail
of these animals into the peritoneal cavity through an abdominal incision. The
bone marrow of the tail inside the cavity, which is normally inactive yellow mar-
row, became actively hyperplastic under this changed environment. The bone
marrow of the tail remaining outside consisted of inactive yellow marrow. Pre-
sumably, the temperature of the peritoneal cavity stimulated bone marrow ac-
tivif,y in the transplanted portion of the tail.

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FACTORS INFLUENCING ERYTHROPOIESIS

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CASTLE, W. B., and MINOT, G. R.: HPathological and clinic:al description of the anemias." Oxford
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COHN, E. J., MINOT, G. R' J ALLES, G. A., and SALTER, W. T.: liThe nature of the materialln
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r86 THE ANEMIAS

CORRIGAN, J. C., and STRAUSS, M. B.: "The Prevention of hypochromic anemia ill pregnancy."
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CRUZ, 0.: 'IPatogenia da anemia na anciIostomose. Portadores de parasitos.-ReIat;io entre
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DAVIDSON, L. S. P.: "Autolyzed yeast products in treatment of anaemia." British Mea. Jour., 2,
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DIGGS, L. W.: IISiderofibrosis of spleen in sickle cell anemia." Jour. Amer. Med. Assoc., 104,
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DOAN, C. A., WISEMAN. B. K., and ERF, L. A.: I'Studies in hemolytic jaundice." Ohio State
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DonBS, E. C., HILLS, G. M., NOBLE, R. L., and WILLIAMS, P. C.: HThc posterior lobe of the
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DMBKIN, D. L., and 1\fILLER, H. K.: "Hemoglobin production. II. The relief of anemia due to
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juice in pernicious anemia." Jnur. Amer. Aled. Assoc., 106, 2058, 1936.
 FACTORS INFLUENCING ERYTHROPOIESIS

HAkT, E. B.) STEENBOCX, H., W'ADDELL, I., and ELVEKJEM, C. A.: ulron in nutrition. VU. Cop-
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Quart. Jour. Med., 1, 157, 1932.
IVY, A. C., MORGAN, J. 'E., and FARR.ELL, J. I.: HEffects of total gastrectomy: Experimental achylia
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Gynec. Obst., 53. 6lI, 1931.
JAn'E, R. H.: "Severe anemia of aplastic type associated with sclerosis of thyroid gland." Arch. Int.
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JORDAN, H. E., and SPEIDEL, C. C.: "The behavior of the leukocytes during coincident regeneration
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JOSEPHS, H.: "Mechanism of anaemia in infancy." Bull. Johns Hopkins Hosp., 51, 185, 1932.
KUNDE, M. M., GREEN, M. F., and BURNS, G.: "Blood changes in experimental hypo- 3.nd hyper-
thyroidism (rabbit)," Amer. Jour. Physiol.~ 99, 469, 1931-1932.
LAs~, F., and TRIGER, K.: Uber das normale und pathologische rote Blutbild im boberen
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"Diseases of the blood and blood forming organs." Year Book oj Gen. Med., 364, 1934. Year
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LEIGH, H.: "The anemias of the pre-natal state." Texas State Jour. Med., 27, 234, 1931.
LERMAN, J., and MEANS l J. H.: "Treatment of the anemia of myxoedema." Endocrinology, 16,
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LYON, JR., E. C.: IIAnemia in late pregnancy; preliminary report." Jour. Amer. Med. Asso·c.,
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of food rich in vitamin C on blood formation." Jour. Arne:r. Med. Assoc., 95, 1089, 1930.
METTIER, S. R., and Cmw, W. B.: "Anemia of scurvy: Effect of vitarnine C diet on blood forrna4
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188 THE ANEMIAS

MEULENGRACHT, E.: "The presence of an anti-anemic factor in preparations of dried stomach sub~
stance from the cardia, fundus and pylorus respectively/' Acta. Med. Scandinav., 82, 352,
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and blood forming organs." Year Book Gen. Med., .356. Year Book Publishers, Chicago,
IU~ _
MEYER, O. 0., STEWART, G. E., THEWLIS, E. W., and RUSCH, H. P.: uThe hypophysis and hema-
topoiesis." Fal. Haem., 57, 99, 1937. .
MILLER, D. K., and Ra:oAPs, C. P.: "The experimental production of Joss of hematoietic ele-
ments of the gastric secretion and of the liver in swine with achlorhydria and anemia." Jour.
CUn. Investigation, 14, 153, 1935.
MLNOT, C. R.: liThe development of liver therapy in pernicious anemia." Lancet, 1, 361, 1935.
MINOT, C. R., and CASTLE, W. B.: I'Diseases of the blood and blood forming organs." Year
Book of Gen. Med., I934. 1935, I936. _
MINOT, C. R., and MURPHY, W. P.: "Treatment of pernicious anemia by special diet." Jour.
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1934·
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 FACTORS INFLUENCING ERYTHROl?OIESIS

SCTIENKEN, J. R., STASNEY, J., and HALL, \V. K.: uThe antianemic principle in the human liver in
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Proc. Soc. E.:Pe-r. Bioi. and Med., 32, 1607, 1935.
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Jour. Amer. Med. Assoc., 105, 432, 1935.
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WHIPPLE, G. H., and ROBSCHEIT-RoBBtNS, F. S.: t~Hemoglobin prodUcing factors in human
liver. I. Normal, infection and intoxication." Jour. Exp. Med., 57', 637, 1933.
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with standard diets." Amer. Jour. Physiol'J 53, 236, 1920. '
W~PU:, G. H., and ROBSCHEIT-RoBDINS, F. S.: "Hemoglobin producing factors in the human
liver. III. Anemias---primary aplastic and secondary-leukemias." Jour. Exper. Mea., 57,
671, 1933.
WmTBY, L. E. H.: uAnemias of pregnancy." Jour. Obst. and Gynaec., Brit. Emp., 39, 267, 1932.
WILLIAMSON, C. S.: "Influence of age and sex on hemoglobin." Arch. Int. ~fed'J 18. 50S, 1916.
WILLS, Lucy: HTropical macrocytic anemia." Lancet, 1, II'}2, 1934,
WILLS, Lucy: "Treatment of 'Pernicious anemia of pregnancy' and 'Tropical anemia' with spe-
cial reference to yeast e»tract as a curative agent." Brit. Med. Jour., 1. 1059, 193 1 •
WISEMAN', B. K.: Discussion of paper of Doan. Curtis, Wisetnan. Jour. Amer. Med. Assoc.
104, 145, 1935.
 THE ANEMIAS
WINTROBE, M. M., and 81IUMACKElt, JR., H. S.: t~Occ:uJfel'l.ce of macrocytic anemia in association
with disorder of the liver t9gether with a consideration of the relation of this anemia to
pernicious anemia." Bull. Johns Hopkins Hosp.~ 52, 38:7, 1933.
WINTROBE, M. M.: "Relation of direase of the liver to anemiaj type of anemia, response to
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WRIGHT, D.O.: "Macrocytic anemia and hepatic cirrhosis." Amer. Jour. Med. Sci., 189, lIS, 1935.
 CHAPTER 14
CLASSIFICATION OF THE ANEMIAS
In building a classification of the anemias, recent advances in the field of
hG_matology must be taken into account. Many false concepts have been per-
petuated by regarding the anemic state as a condition primarily of the peripheral
blood. An anemia should be considered a sign of an alteration in bone marrow
activity. The development of the concept that the peripheral blood and hema-
topoietic centers act as a unit, the "erythron," has been traced by Haden. All
morphologic changes from the normal in the circulating blood are merely indi-
cators of the efficiency of that unit. A shift to immaturity in the blood cells may
indicate, at one time, a healthy active marrow with a temporarily lowered
threshold reacting to meet an emergency. At another time, cellular immaturity
may reflect a severe process of bone marrow dysfunction in which maturation is
unable to proceed beyond an embryonal level. For this reason, an attempt to
correlate morphology and etiology in building a classification is difficult. A
classification which over-emphasizes one and neglects the other is obviously
inadequate.
In the light of modern advances concerning the origin and mech"nism of
the anemias formerly designated as "idiopathic," the old classification into
"primary" and "secondary" types has been discarded. Osgood, Wintrobe, and
Raden have pointed out the tendency of the various anemias to exhibit certain
fundamental morphologic characteristics and have evolved an excellent descrip-
tive classification based on the size and hemoglobin content of red cells.
This type of classification, confined strictly to number, cell size and hemo-
globin content, is represented by the following descriptive tenns: '
NUMBER HEMOGLOBIN CONTENT SIZE
Normal ........... Normocythemic l\formochromic Normocytic
Decreased ......... HypocYthemic Hypochromic Microcytic
Increased ......... Hypercythemic Hyperchromic Macrocytic
Obviously, by estimating the number, size, and hemoglobin content of the
erythrocytes, an anemia may be described accurately by the 'lse of these terms.
There are 27 mathematical combinations but fewer types of anemia, since the
size and hemoglobin content is frequently correlated. In general, the majority
of anemias are hypocythemic; hyperchromic anemias are always macrocytic;
and hypochromic anemias are nsnally microcytic or normocytic. Anemias in
which the erythrocytes are normal in number (normocythemia) are usually
hypochromic in type. Occasionally, in children and in adults with polycythemia a
hemoglobin deficiency (hypochromia) is found with an elevated erythrocyte count
(hypercythemia) .
There are, in addition, certain types of anemia in which the cells exhibit
I9I
 THE ANEMIAS

distinctive morphologic characteristics, such as the sickling in sickle cell anemia

and the sphericity of the cells of hemolytic jaundice.
Both Ottenberg and Haden have compiled comprehensive- classifications
based on pathogenesis in light of recent advances in etiology of pernicious anemb
and other deficiency anemias. Castle and Minot have compiled a ciassificatinn
based chiefly on etiology. They have grouped the anemias into two large
classes: first, the anemias due to blond loss and destruction, characterized by
hyperactivity of the bone marrow; and second, anemias due to decreased blood
production with hypoactivity of the bone marrow.
In the following classification an attempt is made to correlate clinical and
labor.atory findings, to group the anemias in suth a' manner as to indicate the
type of therapy known to be effective, to place the majority of macrocytic anemias
with pernicious anemia in the rank of deficiency Slates, and to emphasize etiology
in the light of Castle's investigations. Until Out knowledge of etiology is com-
plete any classification is necessarily tentative.

TREATMENT
r. AnemIas d'ue to oraod foss.
A. Extra vascular loss.
I. Acute hcmOTrhage (bone marrow hyperplastic), .................. Transfusion.
2. Chronic loss (bone marrow hypoplastic; blood pictUft:: hypochromic) , . Iron.
B. Intravascular destruction-hemolysis (hyperplasia of the bone marrow).
I. Acute hemolytic anemia of Lederer.
2. Chronic famIlial hemolytic jaundice.... .. ........ . ......... . . Splenectomy.
3. Anemia of infectious diseases (examples, syphilis, P!),roxysmal hc~ Transfusion, arsphena-
moglobinuria, malaria) mine, quinine.
4. Anemia due to the destructive action of chemicals (lead, nitro-
compounds, sulfonamide dmgs, etc.).
5. Sickle cell anemia.
II. Deficiency Anemias (hypoplasia of the bone murrow).
A. Iron deficiency alleLuias (usually hypochromic and luicrocytic) ........ Iron and iron supple-
mented ?lith copper,
yeast, vitamins or
thyroxin in certain
rare anemias of
childhood.
I. NuLritional anemias.
(a) Hypochromic nutritional anemia of adults.
(b) Hypochromic nutritional anemia of children.
(c) Hypochromic nutritional anemia or pregnancy.
2. Anemias duc to faulty absorption of iron.
(a) Anemia of coeliac diseases (chronic diarrheal
ulcerative coliti:s, etc.).
(b) Simple achlorhydric anemia (idiopathic microcytic
hypochromic anemia).
(c) Anemia. associated with intestinal parasitism (htlokworm, etc.).
3. Allemias clue to defective storagc of iron in the livel'.
(a) Anemia of prematurity.
(6) Anemia of twins.
(c) Anemia of offspring of anemic mothers.
B. Deficiency of the anti-anemic factor (usually characterized by a

I. 7:>~~~Z~~ ~~Th~c~i~~;i~~5i~r~~~?;ic factor........ , ... . . ..... Liver. extract.

(a) Pernicious anemia.
(b) Certain types of ;;.nemi.as of pregnancy.
(c) lvlacrocytic :lI~cllli::ts as a result of stomach injury
(carcinoma; gastric resection, etc.).
2. Deficiency of the '·extrinsic" gast.ric factor. . . . . . . . ............ Marmite, liver extrn.ct.
(a) Tropical macrocytic anemia of pregnancy.
(b) Certain types of macrocytic anemia of pregnancy.
(c) Macrocytic anemia of sprue and pellagra.
 CLASSIFICATION OF THE ANEMIAS 193
,3.Deficiency due to faulty absorption of hematopoietic factors.
(a) Macrocytic anemia of pregnancy......... . . Liver and iron.
(b) Macrocytic anemia of intestinal parasites
(DiPhylobothrium tatum).
(c) Macrocytic anemia associated with sprue.
(d) Macrocytic anemia Df pellagra.
(e) Macrocytic anemia of coeliac disease (idiopathic steatorrhoea).
4. Anemias due to deficient storage of "x factor" in the liver.. . . . . . Mayor may not re-
spond to liver.
(a) Macrocytic anemias of liver damage (cirrhosis, pregnancy with
toxemia, phosphorus and chloroform poisoning).
m. A.nemias of Bone marrow damage and dysfunction ...................... No therapy.
A. Due to toxic action of bcn7..o1, arsphenamine, gold compounds,
infections, nitrogen retentioll 1 etc.
B. Due to injury by X-ray and radium.
C. Due to disturbance of function by leukemias, malignancies and
osteosclerosis.
D. ~~i?~~~~~i~?;S!~~~~~1 of the bone marrow.
2. Congenital and erythroblastic anemias of childhood (von Jaksch's
anemia, Cooley'S anemia, etc.).
3. Banti's disease, "Splenic Anemia.". .. _ ... Splenectomy.

BIBLIOGRAPHY
CLASSIFICATION OF ANEMIAS

CASTLE, W. R, and M:(NOT, G. R.: "Pathological Physiology and Clinical Description of the
Anemias." Oxford University Pre5.9 1 New York, 1936. Reprinted from Oxford Loose-Leal
},Iedidne. _
HADEN, R. L.: "Clinical Significance of Volume and Hemoglobin Content of Red Blood Ceils."
Arch. Int. Med_, 49, 1032, 1932.
HADEN, R. L.: I'Classiftcation and Differential Diagnosis of the Anemias." Jour. Amer. Med,
Assoc .• 104, 706, 1935.
HADEN, R. L.: "Clinical Factors in the Production of Anemia and Regeneration of Erythrocytes
and Hemoglobin." A-fed. elin., N. Amer., 17. 887, 1934-
OSGOOD, E E.: "Hemoglobin, Color Index, Sailuation Index and Volume Index standards: Re~
determinations Based on the Findings in 137 Healthy Young Men." Arch. Int. Med., 87,
685, ]9 2 6.
OJ.TENllERG, R.: "Rec1assiftcation of the Anemias.'1 Jo-ur. Amer. Med. Assoc., 100, 1303, 1933.
v..~INTROBEJ M. M.: 'IAne-mia: Classification and Treatment on the Basis of Differences in the
Average Volume and Hemoglobin Content of Red Corpuscles/' Arch. Int. Med., 54, 256,
1934·
 CHAPTER 15
HEMOGLOBIN AND ITS DERIVATIVES
AND
THE PORPHYRIN COMPOUNDS
HEMOGLOBIN

Hemoglobin is the coloring matter of the erythrocytes and is held in the

loose stroma of the red cells in snfficient qnantity to give each cell a pale amber
color. When the celIs are present in large numbers, as in blood, the color is red.
The chief function of the red cells is to store this pigmel)t and to transport it in
-the circulation. To this end the normal red ceIl is a non-nucleated biconcave disc
and is so designed that it has a large surface area, permitting free exchange of
gases (oxygen and carbon dioxide). Hemoglobin nrst appears in the young red
ceIl in the bone marrow, even during its normoblastic stage. About 10 Gm. of
hemoglobin passes through the lungs per second. The efficiency of hemoglobin,
as an oxygen carrier can be appreciated when it is considered that 100 cc. of water
at body temperature at an oxygen pressure of 100 mm. of mercury stores only
0.33 cc. of the gas whereas the same amount of blood under the same conditions
°
will take up 2 cc. or 60 times as inuch. In other words, if water filled the vascular
system instead of blood, it would take 350 liters to do the same job.
Hemoglobin is a conjugated protein consisting of an iron-containing pigment
that is combined with a protein of the histone class called globin. The iron con-
taining pigment is known as heme. The pigment heme constitutes about 4 per
cent and the protein globin about 96 per cent of the hemoglobin molecule.
Hemoglobin has a molecular weight of approximately 68,000, and of this the atoms
of iron account for only 224. Therefore the percentage of iron in hemoglobin is
0.336. Human blood contains about IS Gm. of hemoglobin per 100 cc. and since
iron is only 0.336 per cent of this, the quantity of the metal in roo cc. is about
50 mg. and that in the total blood of the average adult is only about 3 Gm. The
total amount of hemoglobin in the average adult is about one kilogram, Or about
14 grams per kilogram of body weight.

STRUCTURE OF THE HEMOGLODIN MOLECULE

As stated before hemoglobin is an iron-containing pigment, combined with

globin. The central structure of the iron-containing pigment is a porphyrin.
Porphyrins are pigments which either alone or in combination are fonnd through-
out the entire plant and animal life; for example, a porphyrin is the pigment
basis of chlorophyl. When porphyrins are conjugated with other substances, they
form the various bases of blood and tissue pigments in different animals; for
example, a pigment found in the feathers of certain South African birds is a
porphyrin combined with copper. Such substances are called metallo-porphyrins.
When proto-porphyrin is combined with iron this forms the metallo-porphyrin
of the blo~d pigment.
194
 HEMOGLOBIN AND ITS DERIVATIVES 195

Heme is not peculiar to hemoglobin but is found in other substances; after

it is united with the protein globin, it is then known as a hemochromogen. If
globin is the protein with which heme is combined, the resulting hemochromogen
forms the basis of the blood pigment of all vertebrate life. Therefore hemoglobin
is an iron plus a porphyrin plus a globin compound. Hemoglobin itself is not
quite so widely distributed as heme, for it is confined only to the animal kingdom.
It is found only in the blood of all vertebrates and a few of the invertebrates.
In the muscles of mammals it is known as muscle hemoglobin or myoglobin.
The construction of 'the hemoglobin molecule may be summarized in the
following table:
Union of four pyrrol nuclei equals a porphyrin compound.
Porphyrin plus various metals equals metallo-porphyrin.
Protoporphyrin plus iron equals heme.
Heme plus a nitrogenous substance equals hemoclxromogen.
Heme if joined to iron in the ferrous state plus globin equals hemoglobin.
Oxidized heme with iron in the ferric state plus hemoglobin equals methemo-
globin.
From the above equations it can be seen, therefore, that the porphyrin com-
pounds play an extremely important role in the formation of the hemoglobin
molecule. When hemoglobin undergoes various chemical alterations, a number
of bodies may be derived from it, and each of these possesses a specific absoq;)tion.
spectrum, wbich aids in its identification. These are as follows: oxyhemoglobin,
methemoglobin, carbon monoxide hemoglobin, sulfhemoglobin, hemochromogen.
hematin, acid hematin and alkali hematin.
DERIVATIVES

OXYHEMOGLOBIN
The iron content of hemoglobin is responsible for its combination with oxygen.
These two elements (iron and oxygen) unite according to the Law of Definite
Proportions, two atoms of oxygen uniting with each atom of iron. The resul ting
combination of oxygen and hemoglobin is a very unstable one and is known as
oxyhemoglobin. The term, reduced hemoglobin, means just simply hemoglobin
or that it has given up its oxygen which has not been replaced by any other gas.
The oxygen capacity of a gram of hemoglobin is 1.34 cc. Thus the capacity of
roo cc. of normal human blood'is about 20 cc. of oxygen, and the total amount of
blood in the average adult will take about 1200 cc. which ordinarjly is used in five
minutes with the body at complete rest.
Hemoglobin under normal conditions exists in the ,blood under two forms;
these being reduced hemoglobin (Hhb) and oxyhemoglobin (Hhb02 ). Oxy-
hemoglobin can be classed as one of the reepiratory pigments, and there are various
forms of oxyhemoglobin as shown by the fact that different oxyhemoglobin
crystals can be obtained from the blood of each species of animal due to species
specificity of globin. The oxyhemoglobin acts as a slightly stronger acid tban
does reduced hemoglobin and therefore in its formation some carbon dioxide is .
set free from sodium bicarbonate.
 THE ANEMIAS

METHEMOGLOBIN
Methemoglobin is the true oxide of hemoglobin, since oxyhemoglobin is an
oxygenated compound, and methemoglobin is an oxidized compound. According
to Remington and Hemmings, in order for methemoglobin to develop, there must
be present an aromatic amino group unsubstituted or potentially free and capable
ot undergoing oxidation with the formation of a hydroxyl amide derivative or a
reversibly oxidizing system. A chemical such as aniline may form many times
its equivalent of methemoglobin. Aniline undergoes oxidation in the animal
body with the formation of para-aminophenol, which passes readily and reversibly
to para-imino-quinone. This can oxidize one equivalent of hemoglobin to met-
hemoglobin, become reoxidized in the tissues and enter into the reaction with
hemoglobin again. In this way it acts as a catalyst converting more and more
of the blood pigment to methemoglobin until an equilibrium is established. Simi-
lar to aniline, such preparations as acetanilid, phenacetine, sulfanilamide, sulfa-
pyridine, drugs of the nitrite group, nitro and amino compounds, nitrobenzene,
dinitrobenzene, etc. are capable of producing this reaction.
Meulengracht and Lundsteen studied I I cases of chronic acetanilid poisoning.
They described the blood as bluish red like mulberry juice and the color did not
change on shaking. Methemoglobin was present only in minor quantities and no
snlfhemoglobin was found. The color was proved to be a derivative of para-
midophenol. This penetrates the red cell where it becomes oxidized, bound to
hemoglobin, and then transformed into methemoglobin. They al.so found anemia
in nine of the eleven cases with a hyperplastic bone marrow indicating excessive
hemolysis of cells.
According to Smith, when 40 per cent of the oxyhemoglobin is transformed
to methemoglobin there wiil be clinical signs of poisoning in the form of cyanosis,
and spectroscopic bands will be seen. Furthermore, if 75 per cent is transformed,
this will result in death. Discoloration of the skin becomes evident when the
methemoglobin amounts to as much as 3 Gm. per 100 cc. of blood.
Bensley has reported two cases of familial idiopathic methemoglobinemia
and stated that only five cases have ever been reported previously. He considers
it the manifestation of some inborn error of metabolism. In summary, methemo-
globin is a compound of oxidized heme, with native globin.

CYANOSIS
The problem of cyanosis has become more important in recent years since the
widespread administration of the sulfonamide group of drugs, particularly sulfa-
nilamide and sulfapyridine. In some instances cyanosis may develop after a
small dose and in other patients much larger doses are required. The mechanism
of this cyanosis has been a subject of controversy. Colebrook and Kenny first
called attention to it and assumed that it was caused by the development of large
amounts of sulfhemoglobin in the blood, and issued a caution against the use
of sulfur compounds at the time the drug was given.
Practically all observers agree that methemoglobin is formed to a variable
extent, hut, as Marshall points out, it is doubtful if it is formed in sufficient
 HEMOGLOBIN AND ITS DERIVATIVES 197
quantities to account entirely for the cyanosis. On the other hand Hartman and
his associates maintain that methemoglobin is entirely responsible for it. How-
ever, Marshall has noted almost 100 per cent of functional hemoglobin present in
markedly cyanotic patients and in some of these he was not able to find methemo-
globin so, therefore, he looked elsewhere for an explanation. He later suggested
that the peculiar chocolate-brown color of the blood was probably caused by
the formation of a dye capable of actually staining the red blood cells. In this
connection, Ottenberg and Fox found that exposure of sulfanilamide to ultra-
violet radiation in the presence of oxygen, causes the immediate oxidation of the
drug to a violet color, and then when added to hemoglobin produced immediately
a marked conversion to methemoglobin. They have suggested that· one of the
colored products of oxidation might be responsible for the cyanosis, either by
reason of its own color or through the production of methemoglobin or, more
likely, by both. They point out that the addition of sulfanilamide to hemoglobin
in vitro produces no change, unless the sulfanilamide has previously undergone
this oxidation. In any event, the occurrence of marked degrees of cyanosis in
patients receiving drugs of the sulfonamide group seems to have little significance
and should give no cause for alarm. Wendell has stated that the chief abnormal
pigment in the blood of patients showing cyanosis from sulfanilamide appears to
be methemoglobin and he recommends the intravenous injection of small amounts
of methylene blue which causes rapid disappearance from the blood of methemo·
globin, which is quickly replaced by an equivalent amount of hemoglobin. Hart-
man and his associates also recommend the intravenous injection of methylene
blue in doses of 1-2 mg. per kilogram of body weight, repeated every four hours
until the cyanosis has become depleted.

SULFHEMOGLOBIN
This hemoglobin derivative is produced by the action of hydrogen sulphide
and oxygen on hemoglobin in vitro. It is closely related to methemoglobin but
has different bands in the absorption spectrum. It has been reported along with
methemoglobin in the blood. of patients who have shown cyanosis. Discombe
maintains that there was no doubt that in the cases he reported sulfhemoglobin
and not methemoglobin was present. It seems likely that before this condition
can develop there must be absorption of hydrogen sulphide from the intestinal
tract. The action of the coal-tar drugs in producing sulfhemoglobinemia is prob-
ably due to hydrogen peroxide, which is formed from the auto-oxidation of para-
aminophenol which is known to be produced in the tissues in cases of acetanilid
poisoning. The treatment for sulfhemoglobinemia is the same as that for meth-
emoglobinemia, which consists of immediate withdrawal of the agent producing
the condition and the possible use of methylene blue intravenously.

CARBON MONOXIDE HEMOGLOBIN

Carbon monoxide has a marked affinity for hemoglobin. At only a few
millimeters of partial pressure, carbon monoxide has the same combining capacity
as oxygen at a partial pressure of several hundred millimeters. Because of this,
the recovery of people who are asphyxiated with this gas js slow. The effects of
198 THE ANEMIAS

carbon monoxide asphyxia are probably those of ailOxemia. When as much as

40 per cent of the hemoglobin is comhined with tbis gas there will result usually
a rather deep coma, marked central nervous system symptoms, and often-times
death may ensue. Blood may become free of gas but the patient may subsequently
sink into another coma and die, perhaps as a result of central nervoUS system
damage. The blood can be recognized grossly sometimes hy its hright cherry red
color, which is even brighter than normal arterial oxygenated blood. This is
reflected in the skin of the patient, this heing described occasionally as a cherry
red skin color. The spectroscopic hands of carhon monoxide hemoglohin are
characteristic. Frequent sources of poisoning include artificial illuminating gas,
exhaust of motor vehicles, exposure to the gas in industries, etc. Treatment con-
sists of removal from the source of poisoning and inhalation of oxygen mixed
with :C02 to stimulate the respiratory center.

THE PORPHYRINS

The description of hematoporphyrin in 1871 begins the history of the

porphyrins in relation to hematology. Hematoporphyrin, the iron-free pigment
formed by the action of powerful reagents on hemoglobin or hematin, was the
first porphyrin to be ohtained in crystalline form. The slight differences in the
absorption spectra of the various porphyrins were not noted by early investigators.
Therefore the term hematoporphyrin has been used to designate any porphyrin
found in the urine, feces, meconium, and bile. Now that specific porphyrins have
been identified, the term has lost its usefulness.
The essential step in the preparation of the porphyrins is the splitting off
of iron from heme, the protein-free part of hemoglobin. At the same time the
side chains of the pyrrol nuclei are modified so that type III porphyrin can be
prepared. It has been shown that there are four artificial isometric porphyrins,
namely, etioporphyrin!; I, II, III and IV, which serve as reference substances for
all porphyrins, whether they be natural or synthetic. Porphyrins corresponding
to etioporphyrin III, induding hemoglohin and chlorophyl, and to etioporphyrin I
have been found in nature; porphyrins corresponding to etioporphyrins II and IV
have only been obtained artificially. The common framework of the porphyrins,
is built up of four pyrrole rings linked together by four carbon atoms in the form
of methane groups.

PROTOPORPHYRIN
Protoporphyrin in the form of its ferrous complex is the pigment fraction
of hemoglobin and corresponds in configuration to etioporphyrin III. , Most if
not all of the protoporphyrin in the erythrocytes is present in the reticulocytes.
This has been demonstrated by Watson and Clark in clinical material and by
experiments in which they gave rabbits phenylhydrazine. From these rabbits
blood samples were obtained in which all of the cells were reticulocytes, and the
largest amounts of protoporphyrin were found in these samples. The function of
the protoporphyrin of the reticulocytes is unknown but two possibilities have been
suggested by Watson, (1) that it is to be used in the construction of further
 HEMOGLOBIN AND ITS DERIVATIVES 199

h'emoglobin after the reticulocytes have entered the circulation, and (2) that it
serves as a respiratory enzyme in the reticulocytes which, in cont.rast to mature
red blood cells, have a measurable oxygen consumption.
Increased amounts of protoporphyrin have heen found in the feces of hemo-
lytic jaundice patients and according to Watson, this may be reticulocyte proto-
porphyrin which is liberated as tbe reticulocytes mature and which gains acce"
to the feces through the bile, in which protoporphyrin is demonstrable.

COPROPORPHYRIN I
Coproporphyrin, which bears its name because of its greater concentration
in the feces, is excreted normally in both urine and feces and in increased amounts
during heightened erythropoiesis.
Coproporphyrin may be either type I or type III. On the other, hand, all of
the known respiratory pigments are type III derivatives (hemoglobin, myoglobin,
cytochrome C and catalase) . Type I porphyrins cannot be derived by degradation
of these type III compounds. T\ms, there is a simultaneous production of type I
and type III porphyrins, a phenomenon designated as the "dualism of the porphy-
rins." Studies thus far have not confirmed the belief that the liver converts
protoporphyrin III to coproporphyrin I. Dobriner has presented evidence which
indicates that normally there is a constant ratio between the rates of production
of the two types of porphyrins.
The amounts of coproporphyrin I excreted normally are measured in frac-
tions of milligrams with much larger amounts in cases of pernicious anemia,
hemolytic jaundice, and in idiopathic porphyria. Dobriner has shown that in the
majority of diseases characterized by increased ,excretion of porphyrin in the
urine the porphyrin excreted is coproporphyrin I. These diseases include: catar-
rhal jaundice, obstructive jaundice, hemolytic jaundice, atrophic cirrhosis of the
liver, Hodgkin's disease, lymphosarcoma of the liver, and lobar pneumonia. These
pathological conditions are often accompanied by liver insufficiency which dimin-
ishes excretion of the porphyrin through the bile. Nevertheless, large increases
in urinary porphyrin appear to be due more to increased production than to
faulty excretion. Studies made on cases of pernicious anemia, hemolytic jaundice,
and refractory anemia indicate that there is some relationship between the forma·
tion of coproporphyrin and the rate of erythropoiesis in the marrow.

COPROPORPHYRIN III
In cases of acute porphyria, studied by Dobriner, and in lead poisoning studied
by Dobriner and by Watson, the excessive porphyrin formation was shown to be
usually of the type III. Coproporphyrin III has also been isolated from normal
urine and in increased amonnts after administration of arsenic, mercury, and
sulfanilamide.
In a series of cases undergoing sulfanilamide treatment, Remington and
Hemming have demonstrated porphyrinnria due to the pigments coproporphyrin
III and small quantities of coproporphyrin I. It was concluded that the drug
probably exerts a toxic action on both the liver and the bone marrow. This
porphyrinuric activity is shared by most drugs of the sulfonamide series and also
20C. THE ANEMIAS

by a number of simple aromatic amines tested, particularly aniline and its deriva-
tives. The porphyrinuric activity was found to run parallel with general toxicity.
In general, all substances capable of producing porphyrinuria are capable
of producing methemoglobinemia. Therefore the chemical grouping necessary
for increasing porphyrin excretion is the same as that given above for methemo-
globin formation. The question arises as to whether the excreted coproporphyrin
III is derived from the methemoglobin of the blood stream. Although it is prob-
able that porphyrins do not arise from the normal breakdown of hemoglobin, it
is also probable that an alternative route exists in certain pathological conditions
whereby porphyrins are derived from an increased breakdown of blood pigment.
It is suggested that methemoglobin, when once formed, is degraded, at least in
part, by a mechanism whicb leads ultimately not to bile pigment but to porphyrin.
UROPORPHYRIN I
The formation of uroporphyrin was thought in the past to be limited to
idiopathic porphyria which may be regarded as an "inborn error of metabolism"
but more recently it has been demonstrated by Dobriner in cases of sulfonal
poisoning. Uroporphyrin and coproporphyrin, as well as the others, are similar
hy spectroscopic examination and with the heavy metals form complexes having
spectroscopic properties similar to those of hemin.
In those rare instances in which porphyrins are present in abundance in the
urine and feces from birth, the dark red urine is the warning sign. Garrod has
pointed out that congenital porphyria may be a mendelian recessive cbaracter.
The subjects of congenital porphyria have a peculiar sensitivity to light, probably
due to unusual amounts of porphyrin in the tissues. The subjects develop,
especially in summer, a form of skin eruption upon the exposed parts of the body
which recurs, year after year.
DEUTEROPORPHYRIN
Following bleeding in the gastro-intestinal tract, deuteroporphyrin is present
in the feces. It is very similar in absorption to coproporphyrin and the fact that
copro-, deutero-, and protoporphyrins may be present in the same feces makes
careful fractionation necessary before the type or amount of porphyrin present
may be determined" (Watson).
HEME AND HEMOCHROMOGEN
The porphyrins seem able to combine with iron to form a series of hemes
which can be crystallized as hemins (the hydrochloride of heme), usually known
as Teichman's crystals. Porphyrin is relatively inert chemically and can be
rendered more active by adding iron.
The name hematin has always been used for a number of hemoglobin deriva-
tives in which globin was supposed to be separated from heme. When an excess
of acid or alkali is added to hemoglobin, substances obtained are known as "acid
hematin" and "alkaline hematin" respectively and a neutralized form known as
"neutral hematin."
The porphyrin substances active in cellular respiration are not simply porphy-
rins but are hemochromogens. The respiratory catalysts are also hemochromogens
 HEMOGLOBIN AND ITS DERIVATIVES 201

which are compounds formed when heme reacts with a nitrogenous substance.
Many nitrogen compounds, including cyanIde, ammonia, amines, pyridine, and
nicotine have this property. It was formerly assumed that hemochromogen
represented the reduced form of the iron-containing pigment group of hemoglobin
and that hematin and hemochromogen corresponded to oxyhemoglobin and reo
duced hemoglobin minus their protein moieties. It has now been demonstrated
by Anson and Mirsky that the globin of the original hemoglobin is still present
in the case of hemochromogen.
While molecular oxygen is unable to convert hemoglobin into methemoglobin,
it will convert reduced heme into its true oxide. Therefore, it is apparent that
neither heme nor hemochromogen is suited for the carriage of oxygen in the
blood. Nature has obtained a substance which combines with oxygen loosely and
from which the latter can be liberated by first combining heme with globin, so
forming a hemochromogen which is hemoglobin. Hemoglobin is estimated to
contain four heme units with a combined molecular weight of at least 68,000.
The biological fitness of hemoglobin is the result of this combination of heme
with globin.

BIBLIOGRAPHY

.I.ANSON) M. L., and MIRSKY, A. E.: (IHemoglobin, the heme pigments and cellular respiration.1!
Physiol. Rev., 10, 506, .1930.
2. BENSI.EY, E. H., RI:mA, L. J., and MILLS, E. S.: "Familial idiopathic methaemoglobinaemia."
Quart. Jour. Med. J 31, 325, .1938.
3. BEST, C. H., and TAYLOR, N. B.: "The physiological basis of medical practice." The Williams
and Wilkins Co. 2d Ed. 194Q.
4. COLEBROOK, L., and KENNEY, M.: "Treatment of human puerperal infections and of experi-
mental infections in mice with protosil." Lancet, 1, 1279, .1936.
5. DlSCOMDE, G.: "Sulphaemoglobina.emia following sulfanilamide treatment." Lancet, 1, 626,
1937·
6. DODRINER, K.: 'I Porphyrin excretion in the feces in normal and pathological conditions." Jour.
Biol. Chem., 120, lIS, 193'1.
'1. DODRINER, K.: "Urinary Porphyrins in Disease.'l Jour. Bioi. Chem., 113, I, 1936.
8. GARROD, A. E.: "The inborn factors in disease." The Clarendon Press. Oxford. 1931; p. 1I6.
9. HARTMAN, A. F., PERLEY, A. M., and BARNETT, H. L.: "A study of some of the physiological
effects of sulfanilamide, methemoglobin formation and its control." Jour. CUn. Invest., 17,
699, 193 8 .
:roo MARSHALL, E. K., JR., and W AI.ZL, E. M.: HO n the cyanosis from sulfanilamide." Bull. Johns
Hopkins HosP., 61, 140, 1937.
II. MEULENGRACIIT, K, and LUNDSTEEN, E.: "Die cyanase und anlimie bei chronischer acetanilid
vergiftung." Folia Hematol., 63, 89, 1939.
12. Ot"l'ENBERG, R., and Fox1 C. L., JR.: "Explanation for the cyanosis o~ sulfanilamide therapy."
Proc. Soc. Exp. Bioi. and Med., 38t 479, 1938.
13. REJ!..IINGTON, C., and HEMMmG, A. W.: "Porphyrinuric action of drugs related to sulfanilamide.
Comparison with reported toxicity, therapeutic efficiency and causation of methaemoglobi~
naemia. Definition of the structure responsible for porphyrinuric action." Biochem. Jour.,
33, 960, 1939.
14. WATSON, C. ].; "Porphyrins and diseases of the blood." Symposium on Blood. Univ. of Wis.
Press. 1939 j p. 14.
IS. WENDEL, W. B.: liThe control of methemoglobinemia with methylene blue." Jour, Clin. Invest.,
18, 179, 1939.
 CHAPTER 16
HYPOCHROMIC ANEMIA
GENERAL CONSIDERATIONS

Hypochromic anemia is the hematologic term applied to that large group

of anemic states in which the red cells have as their chief characteristic a sub-
normal amount of hemoglobin. As pointed out in the previous chapter, this
term has its. origin purely on morphologic grounds and hemoglobin content and
is strictly a hematologic classification. It does not take into account, of course,
the etiologic factors producing the various hypochromic anemias. Strictly, it
refers to a state of the blood in which the loss of hemoglobin has exceeded the
loss of red blood cells. The color index, therefore, is below one and each cell
does not carry its normal quota ·of hemoglobin.
The hypochromic anemias arise from many causes. They are usually second-
ary to some other disease process, and therefore, the condition is usually a symp-
tom of some other disease, the exact nature of which should always be ascertained
if possible. Hypochromic anemias have often been called "secondary anemias,"
and the two terms are still used synonymously by many people.
It is, without a doubt, the most common form of anemia encountered. It
is our impression that 90 to 95 per cent of all the anemic states are of the hypo·
chromic type. This results, of course, from the multiplicity of causes and this
is particularly true if one takes into account the large number of mild types
of the condition that are seen so commonly as a result of inadequate diet. It is
important to bear in mind that a patient may present a profound degree of hypo-
chromic anemia with the number of red cells at a normal level. Thus, red cell
counts of 4,500,000 to 5,000,000 per cu. mm. are frequently seen with a hemo-
globin content of 40 or 50 per cent. It is obvious that such a condition represents
an actual degree of anemia that may be expressed at 40 or 50 per cent, that is, at
the hemoglobin level. Then again, hypochromic anemia may present a red cell
count of only 2,500,000 per cu. mm. and the hemoglobin content at 40 per cent.
In the latter case, of course, the degree of anemia is equal to the former in so far
as oxygen carrying capacity is concerned. Therefore, it should be emphasized
that irrespective of the number of red cells, the hemoglobin percentage is the
most accurate and reliable criterion as to the degree of anemia. The number
of red- -cells is of less importance in estimating the degree of anemia in a given
patient. On the other hand, the number of red cells is of considerable impor-
tance in determining the type of anemia present.
In the different types of hypochromic anemia the red cells may be of various
sizes, but as a general rule they are either normal in size, or they are smaller than
normal. It is seldom that an anemia is encountered in which there is marked
hypochromia with the average red cell diameter larger than normal. This does
occur, however, in rare instances; for example, there may exist for " time a
202
 HYPOCHROMIC ANEMIA 203

hypochromic state in pernicious anemia being treated intensively with liver

extract when the average red cell diameter may be larger than normal and the
hemoglobin content is unable to keep pace with the rapid cellular regeneration.
Also hypochromia is not unusual in the macrocytic anemias of sprue and preg-
nancy. In the microcytic anemias in which the average cell diameter is smaller
than normal, hypochromia is the rule. It is doubtful if there exists an anemia
characterized by small cells, over-filled with hemoglobin. In such anemias, there-
fore, there are two factors responsible for the general anemic state of the
patient; one of these is hypochromia per se in which each cell carries less than its
normal amount of hemoglobin and the second one is the fact that the average
cell is smaller than normal. Furthermore, it is important to bear in mind that
hypochromic anemia may exist in certain phases of any blood dyscrasia at' certain
times, this being when hemoglobin production lags behind red cell production.
This may exist at any time when intense bone marrow regeneration is going on
and because this may occur it should be remembered that the diagnosis of per-
nicious anemia oftentimes is not dependent on the hemoglobin content of the red
cells. Especially must this be borne in mind when in recent years the use of liver
has become so wide spread and patients may present themselves for their first
examination during a time when there is rapid red cell production, due to previous
administration of liver.

SYMPTOMS OF ANEMIA

Symptoms resulting from anemia vary widely in their manifestations. In

general, they include varying degrees of pallor, fatigue, headache, general weak-
ness, tendency toward faintness, perhaps disturbances of sight such as black
spots before the eyes, especially upon exertion, palpitation of the heart, and
usually an increased pulse rate. If the anemia is extreme there is likely to be
dyspnea, and shallow breathing. The edema which is occasionally observed is
probably due to impaired circulation because of insufficient oxygenation of heart
muscle. It should be borne in mind that the degree of anemia may be quite
marked without showing symptoms and also quite marked witliout the patient
showing any unusual pallor. We have observed patients with rosy cheeks whose
red cell counts were only 2,000,000 per cu. mm. and hemoglobin 40 per cent.
Apparently these are instances of unusual peripheral vascular dilatation and
yet the patient was suffering with symptoms of marked anemia. What is still
more important we have observed many patients who presented a marked pallor
and yet whose red cells and hemoglobin were quite normal. Therefore, one
should be hesitant in estimating the degree of anemia according to the pallor of
the skin. Examination of the conjunctivae is far more important. Symptoms
produced by anemia will also depend upon the amount of activity of the patient.
An individual who is constantly at bed rest may exhibit no symptoms of a severe
anemia, yet if he should indulge in moderate muscular activity the symptoms will
become very pronounced.
It is useless to set forth any low figure to which red cells and hemoglobin
may fall and still be compatible with life.. Thls varies so widely in different peo-
204 THE ANEMIAS

pIe that it is of little val,ue. We have observed an untreated patie", with per-
nicious anemia in relapse whose red cells numbered 500,000 per cu. mm. and hemo-
globin 10 to IS per cent. This patient was perfectly comfortable while at bed
rest but would become unconscious when raised to a sitting position.

ANEMIA OF ACUTE HEMORRHAGE

The anemia that results from the sudden loss of large quantities of blood,
whether it be outside or within the body, is dependent upon the rate and .volume
of the blood loss. The clinical symptoms of this condition are well known and
consist essentially of varying degrees of restlessness, rapid shallow breathing,
a cold clammy skin, a rapid feeble pulse with low blood pressure, and prostration.
A blood examination taken at this time will usually show very little deviation from
the normal of red cells and hemoglobin. If the blood is examined at the time of
hemorrhage, or immediately following, before there has occurred a restitution of
fluid loss, it should show no changes in its composition of red cells and hemoglobin.
However, within just a few minutes the blood platelets will be increased in the
circulation and, therefore, the coagUlation time is decreased. Also, within one
or two hours there is an ontpouring of neutrophilic leukocytes including many of
the immature varieties with a shift to the left of cell types. If the patient is
adequately treated with various fluids this will restore the blood volume within
one or two hours and if blood is examined at that time this will be the first
evidence of an anemia. Blood findings under such conditions may have im-
portant practical significance in those instances where it is desirable to differ-
entiate between surgical shock and hemorrhage. In the former there is no altera-
tion of cellular content, and in the latter there is also no alteration, if the blood
is taken during or immediately after the suspected hemorrhage, but the anemia
becomes evident only during and after restitution of blood volume with fluids.
At this time the red cells have become diluted in tbe blood stream and will
be found to be low in number. The hemoglobin, however, will be correspondingly
low. In 24 to 48 bours will appear the first signs of hypochromic anemia be-
cause by that time the red cell production will have been speeded up, reticulocytes
will appear in the blood stream but the hemoglobin per cent will not be apprecia-
bly increased. Also the leukocytes may reach a high level of 20,000 to 25,000
per cu. mm. The reticulocytes will be markedly increased and will remain so
until approximately a normal red cell picture is reached.
The changes described here occur only in the normal individual who has a
normal bone marrow capacity. It should be borne in mind, that if the hemorrhage
occurs in an individual already weakened by infection, etc., that the bone mar-
row capacity to respond may be considerably impaired.
An excellent example of the bematologic picture following sudden blood loss
may he seen in those individuals who donate unusually large quantities of blood
for transfusions. In conducting a students bureau for blood transfusions over a
number of years, we have observed certain changes occurring in the hematologic
values of blood donors. It can be stated with a fair degree of assurance that the
young healthy adult who gives 500 cc. of blood at periodic intervals, not oftener
 HYPOCHROMIC ANEMIA 20 5
than one a month, will show no effects from continuing this over a long period of
time (namely, two years). Such individuals will show a slight rise in reticulo-
cyles immediately after giving blood. In a few days this has subsided to normal
and apparently the cellular values are normal again at that time. We believe
it is unwise for an individual to give blood transfusions oftener than intervals of
30 days. We have observed a few students who were giving too many trans-
fusions at various hospitals throughout the city and have observed moderate
degrees of hypochromic anemia that existed over a period of months. We found
one such student with a red cell count of 2,500,000 and hemoglobin of 40 per
cent following six transfusions within a period of two weeks.

ANEMIA OF CHRONIC BLOOD LOSS

This type of anemia involves many more factors than those considered in
acute blood loss. The reason for this is the fact that seldom does the patient
present marked blood loss over a long period unlE;s, there are varying degrees
and types of underlying pathology producing this. Therefore, such a patient
usually presents a combination blood picture brought about not only by the
loss of hlood, but oftentimes hy actual deficient hematopoiesis which in many
instances is due to inhibition of hone marrow activity by sepsis, parasites, mal-
nutrition, fever, etc.
The outstanding feature of such an anemia is not only the decreased number
of red cells, but the great decrease of hemoglobin content and, consequently, a
low color index with a true hypochromia. Examination of blood films will usually
show a moderate microcytosis and since there are usually septic processes de-
pressing the marrow, reticulocytes are low and the red cells show varying degrees
of central achromia. Furthermore, there may be considerable variation in size
and shape of red cells with poikilocytes being numerous in extreme chronic Cases.
The symptoms of such a condition, of course, are those of any severe anemia.
Attention should be directed toward discovering the underlying cause of the
loss of blood. It should he pointed out that in this instance, as with other
types of chronic hypochromic anemia, the use of intensive correction therapy
may obscure the basic underlying pathology just as a dose of morphine may
obscure the symptoms of acute appendicitis.

ANEMIA OF ACUTE INFECTIONS

Varying degrees of anemia are seen in practically all types of acute in-
fections, particularly if the infection is widespread or systemic in nature. This
type of anemia is apparently brought about by various modes of action. The
infecting organisms, particularly if in considerable numher in the blood stream,
may exert a destructive effect on red corpuscles with which it comes in contact.
Furthermore, its toxins may be capable of a similar effect. Thus, in pr010nged
blood stream infections with the streptococcus hemolyticus a marked degree of
hypochromic anemia may be produced. Also the organism or other products
may exert an inhibitory effect on bone marrow production. Then in those types
206 THE ANEMIAS

of parasitic invasion of red cells, as seen in malaria, the cells are destroyed me-
chanically by disruption. In the more chronic types of infection, such as those
produced by the Streptococcus viridans, there may be a marked increase in the
number of phagocytic white cells, monocytes, and these cells may engulf or
devour many of the red corpuscles.
Of all the factors conoucive to the production of anemia in acute infec-
tions, probably inhibition of bone marrow activity is the one of more importance
than any other. The hematologic picture that accompanies the acute infections
varies quite widely and the anemia is not hypochromic unless the process is
sufficiently prolonged. It is not unusual to observe the red cell count markedly
decreased, perhaps to z ,000,000 cells per cu. mm. and hemoglobin decreased
correspondingly, or perhaps even lower, resulting in a moderate hypochromic
anemia usually accompanied by neutrophilic leukocytosis. Therefore, in the use
of transfusions as therapeutic agents in acute infections, it is quite possible that
two purposes are served: first, the new blood may exert a destructive influence
on the invading agent; and secondly, it will serve to correct the existing anemia.
There is no reason to believe, however, that it accomplishes the latter through
bone marrow stimulation; more likely it corrects the anemia with replacement
of new functional red cells.

ANEMIA OF CHRONIC INFECTIONS

It has long been recognized that profound anemia may accompany pro-
longed infections. It is probable that it is produced by increased destruction
or increased utilization of red cells in conjunction with inhibition of hone marrow
production. Also the patient with a prolonged infection usually has other
factors such as impaired appetite with inadequate food intake and consequently
the anemias resulting may be partly due to nutritional deficiency. This type
of anemia is seen in many diseases, such as typhoid fever, malaria, tuberculosis,
chronic syphilis, nephritis, prolonged febrile diseases, such as Brill's disease, un-
dulant fever, prolonged influenza, subacute endocarditis, ulcerative colitis, pyelitis,
and many others.
A study of the red cells in these conditions usually shows them to be normal
in size, in some instances slightly microcytic, practically always with a slight or
profound hypochromia. Therefore, the volume index is normal or lower than
one. The color index is low. The blood plasma is usually pale and not icteric.
Treatment includes measures usually employed for any type of hypochromic
anemia (see section on treatment) with emphasis always on the removal of the
cause.

ANEMIA OF MALIGNANCY

It has long been recognized that various maligna~t processes are almost
invariably accompanied by hypochromic anemia. This occurs so consistently
that the presence of a moderately severe hypochromic anemia in an individual of
 HYPOCHROMIC ANEMIA 20 7

cancer age, combined with loss of weight, often leads the clinician to suspect a
malignant process. It appears that any type of malignancy in almost any tissue
is capable of producing anemia, even though the process is not grossly extensive
and there is no evidence of bone marrow involvement. Therefore, it has long
been thought by some that the malignant process itself either elaborates a product
capable of bone marrow inhibition, or in its growth utilizes a product necessary
for red cell production.
Although the exact mechanism of the anemia of malignancy ,is unsettled, there
does seem to occur degrees of anemia that are entirely out of proportion to the
extent of the malignant growth. The anemia usually seen in such states is defi-
nitely hypochromic. The red cells are reduced in number, the hemoglobin reduced
to a greater extent and the color index below one. The red cells in the stained
smear may be microcytic or they may be normal in size. They may exhibit
varying degrees of anisocytosis and poikilocytosis. Seldom is macrocytosis ob-
served in the anemia of malignancy except in cases of carcinoma of the stomach
and in other malignancies in which there is metastasis to the liver.
It seems probable that in carcinoma of the stomach the intrinsic factor may
become decreased in amount resulting in a macrocytic type of anemia and in
cases of liver damage, usually due to metastatic carcinoma in that organ, the
resulting anemias may also be of the macrocytic type. Furthermore, we have
observed some instances of macrocytic anemia with the volume index above one in
cases of a far advanced carcinoma of the lower intestinal tract. As a general
rule, however, it may be stated that the anemia of malignancy is of the bypo-
chromic type, severe in character and probably produced by interference in the
construction of the hemoglobin molecule which may be brought about by the
liberation of some unknown substance from the growing tumor. Mogensen studied
the blood in 75 cases of gastric cancer and found that the average hemoglobin was
72 per cent; however, 31 of these patients had no anemia whatever. Therefore,
failure of a patient to have anemia does not militate against the diagnosis of
cancer of the stomach.
Benign tumors are rarely accompanied by hypochromic anemia unless they
are of sufficient size to produce other conditions conducive toward the anemic
state. For example, far advanced uterine fibroids are usually accompanied by
anemia but this is brought about probably by the excessive uterine bleeding that
accompanies the condition. A patient may present a lipoma or fibroma of tre-
mendous si~e and yet exhibit no degree of anemia whatever. On the other hand,
a squamous cell carcinoma of the uterine cervix, or of the skin, that may be
only one· twentieth of the size of the benign tumor may produce a profound de-
gree of anemia.

ANEMIA OF PARASITIC INFESTATION

Almost any type of infestation with intestinal parasites is accompanied by

hYpochromic anemia. Apparently there is only one parasite, the fish tapeworm,
D,phyllobothrium tatum, that produces the macrocytic hyperchromic type. Birke-
208 THE ANEMIAS

land, in an excellent review on this subject, states that the anemia is indistinguish-
able from pernicious anemia, clinically, hematologically, and pathologically. He
points out further that achloryhydria is present in 84 per cent of the cases; that
the anemia is usually cured after the expulsion of the worm, but he also points
out that the presence of the worm is not the only factor in the production of the
anemia. Therefore, he believes that the fish tapeworm infestation is only the
precipitating factor producing the anemia. Furthermore, the fish tapeworm may
directly Dr indirectly enlarge the intestinal wall or the toxins may be absorbed
and act directly on the blood forming organs. It would appear from Birkeland's
review, that the fish tapeworm is the precipitating factor producing a pernicious
anemia-like blood picture in the person who is already susceptible to the disease.
This is supported by the fact that only one out of 5,000 people with the infesta-
tion develop the characteristic anemia.
Any type of infestation with intestinal parasites is likely to be accompanied
by a severe degree of aneinia; of these, hookworm infestation is one of the most
widespread, particularly in the subtropical and temperate regions.
Apparently most of the symptoms of the patient wtih hookworm are due to
the anemia. The pallor, the marked weakness, the excessive fatigue, the loss
of appetite, the gastro-intestinal symptoms, including abdominal cramps, are
apparently due in a large part to the anemic state. In a consideration of the
cause of hookworm anemia, hemorrhage appears to play very little part. It
has been thought by some that the worms actually suck blood from the intes-
tinal mucosa. However, it appears more likely that the hookworm elaborates
a substance that depresses bone marrow function in its capacity to form the hemo-
globin molecule. There may be an actual interference with absorption of iron
from the intestinal tract. If the 'latter concept is true, then the anemia of hook-
worm disease should be classified as one of the nutritional anemias. We have
observed many able bodied, healthy young men who carry light infestations of
hookworm and who showed absolutely no evidence of anemia. Therefore, in onr
experience, the presence of considerable numbers of hookworm in the intestinal
tract is not necessarily accompanied by anemia. On the other hand, we have
also observed numerous patients who presented the most profound degree of
hypochromic anemia but in whom the number of hookworms apparOO1tly was
very few. Therefore, there appears to be little relationship between the number
of hookworms present and the degree of anemia.
The anemia of hookworm infestation is usually moderately severe with a low
red cell count with the hemoglobin quite low in proportion resulting in a color
index of 0.5 or even less. The total leukocyte count is normal but there is a
moderate to marked eosinophilia. The red cells are usually of nowal size
and shape and they present invariably a central pallor. In severe cases they ap-
pear as mere rings. Also the anemia of hookworm disease may be characterized
by normal red cell count and an extremely low hemoglobin content. Thenlifore,
it seems that there is no inhibition of bone marrow production but a marked in-
terference with the elaboration of the hemoglobin molecules. This, in our opinion,
is brought about by an undefmable substance elaborated by the worm itself. This
 HYPOCHROMIC ANEMIA

blood picture is usually speedily corrected after the patient has received proper
treatment for the disease which consists in the eradication of the parasite. Infes-
tation with other intestinal parasites including the various round worms and fiat
worms present a similar blood picture.

ANEMIA OF MALARIA

In long-standing cases of malaria, anemia is a predominant part of the pic-

ture. Although the patient may present the fehrile reactions characteristic of
the disease, there may also exist a marked pallor and other signs and symptoms
referable to the long standing anemia. Examination of the blood reveals usually
only a moderately reduced erythrocyte count with markedly reduced hemoglobin
content. The volume index is below one and the red cells show a central pallor,
the extent of which is dependent upon the degree of hemoglobin reduction. In
cases of so-called "chronic malaria," there are signs of basophilic degeneration in
many of the red cells. These changes include varying degrees of polychromato-
philia, extensive basophilic stippling, so much so that one may suspect lead poison-
ing in some instances, and a moderate degree of anisocytosis and poikilocytosis.
The leukocyte count is usually normal or decreased, an actual leukopenia
existing at the expense of the granulocytic cells. Therefore, there is a relative
but not an absolute lymphocytosis. It seems that the anemia of malaria is caused
by several factors. First, there is constant invasion and disruption of the
circulating red cells; also, there probably exists some degree of bone marrow in-
hibition because of the tendency of malaria parasites to invade the younger
cells, the reticulocytes. In addition, malaria calls forth large phagocytic cells
from the endothelial system apparently for the purpose of engulfing and destroy-
ing parasites and in the course of this process some of the red cells are subject
to similar destruction. Furthermore, the spleen is enlarged. Its phagocytic
activity is increased and red cells are phagocytosed more readily by the fixed
endothelium. If it is possible to eradicate the parasites, then the anemia will
clear up of its own accord within the course of time.
Acute and quickly developing malaria, however, does not present any ap-
preciable degree of hypochromic anemia. \Ve have studied many cases of estivo-
autumnal malaria that were characterized by relatively sudden onset, and by
extremely widespread parasitic invasion of the red cells, and in these cases
there seldom existed any considerable anemia. Also, it should he pO,inted out
that in this type of malaria the leukopenic state is not present but there is more
often a leUkocytosis caused by an increased number of granulocytes, with a
moderate cellular shift to immaturity.

ANEMIAS OF PREGNANCY

Anemias of pregnancy may be divided into two general types: First, and
by far the most common type, is that known as the hypochromic, whereas the
macrocytic type is relatively rare. In the hypochromic type, of course, the
210 THE ANEMIAS

designation refers to hemoglobin content of th~ red cells, whereas in the macro-
cytic type the name owes its origin to the size of the red cells. The designa-
tion between the two, paradoxical as it may seem, is quite satisfactory because
the macrocytic type is seldom hypochromic.
There are many factors that contribute to the anemic state in the preg-
nant woman. First, and probably the most important, is the sudden and pro-
gressively increasing demand on the part of the fetus for iron and other red cell
building material which may produce a hypochromic anemia of severe degree.
Secondly, the period of gestation may be accompanied by various gastrointestinal
disturbances, nausea, vomiting, etc., so that the pregnant woman is not able
to take in, retain, and utilize a normal amount of food. Thirdly, the state of
pregnancy may be accompanied by various manifestations of toxemia; these in·
c1uding chiefly, fever, damage to the kidneys and to the liver, this resulting in
some instances in impaired liver function so that the liver is unable to properly
store, metabolize and dispense the hematopoietic substance. Furthermore, it
should be borne in mind that the red cell and hemoglobin content of the average
woman in many areas borders on an anemic state so that the addition of pregnancy
merely accentuates an already existing anemia. Dieckmann and Wegner estab-
lished the fact that there is usually a 25 per cent increase of plasma volume in the
blood in the last trimester of pregnancy, with a corresponding degree of relative
red cell reduction. This of course, may largely account for the marked accentua-
tion of the anemic state in the last trimester. Bethell has confirmed this finding
and stresses protein deficiency as a common anemia producing factor. Bethell
points out that the incidence of anemia in the pregnant state is quite high
and in his series of 158 clinic cases there were 54 per cent who were anemic.
Most often this was the hypochromic or iron deficiency type probably caused
by inadequate diet. He considers that if the hemoglobin falls below IO Gm. per
100 cc. of blood this indicates an actual lack of iron, and that if the red· cell
count is below 3,500,000 cells per cU.mm. associated with macrocytosis, this sug-
gests protein deficiency. Of course, both types may be present in the same patient.
The studies of Strauss have done much to clarify the classification of this
group of anemias. He states that the chief symptoms occur usually about the
middle of pregnancy and are initiated by an insidious development of lack of
well being and excessive fatigue. In the more severe cases there are considerable
pallor, varying degrees of edema, sometimes considerable dyspnea and occasion-
ally prostration. Some patients show atrophy of the papillae of the tongue,
In an examination of 30 consecutive patients with hypochromic anemia 'that
was severe, he found achlorhydria in 17, hypochlorhydria in 10 and only two
showed a normal acidity of the gastric contents. These could be classed, in
view of the gastric anacidity, as marked cases of idiopathic hypochromic anemia
which probably existed to some degree before pregnancy but which were precipi-
tated by the extra demands of the pregnant state. Then there is a class of patients
during pregnancy that present a severe grade of hypochromic anemia with no dis-
turbance of gastric acidity and who do not present the atrophic changes of the
tongue. These anemias are due probably to a simple iron deficiency and can be
classified as such.
 HYPOCHROMIC ANEMIA 211

Finally, there is the anemic state of pregnancy which has been given the
name of "pernicious anemia of pregnancy," but better known as the macrocytic
anemia of pregnancy. It is not identical with true pernicious anemia since the
condition usually disappears after delivery. This condition is said to occur in
about one in five thousand pregnancies. The etiology is relatively obscure but
numerous theories have been proposed to account for it. It is thought by some
to be a reaction of a damaged bone marrow to the added demands of pregnancy.
Others have attributed it to action of an unknown toxin from the placenta which
mayor may not be identical with the same toxin that may cause nephritis and
eclampsia. This anemia, because of its similarity to true pernicious anemia, may
be regarded as a temporary pernicious anemia produced by pregnancy but which
disappears upon its completion. It usually occurs more often in multiparae and
the patient usually is in mid-pregnancy before the symptoms become pronounced.
These consist of unusual degrees of weakness, dyspnea and palpitation. The appe-
tite becomes poor and some of the patients present a sore mouth and tongue, with
diarrhea. The skin color may be the lemon yellow of pernicious anemia. Various
circulatory complications such as hemic murmur, edema, and rapid pulse may
be present. The blood picture shows a marked reduction in red cells, usually
below 2,000,000, with reduction of hemoglobin but the color index is usually above
one. The stained smear shows the characteristic picture of pernicious anemia in
relapse including macrocytosis, and much variation in size and shape of cells with
many bizarre forms. The leukocytes are reduced in number at the expense of
the neutrophils. Gastric anacidity is the rule although it should be pointed out
that this type of anemia can be seen rarely in a pregnant woman who shows free
hydrochloric acid in the stomach. It seems, therefore, that there are four definite
types of anemia of pregnancy: First, hypochromic anemia with achlorhydria;
secondly, hypochromic anemia with normal gastric findings; thirdly, a pernicious
macrocytic anemia with achlorhydria; and fourth, a pernicious macrocytic anemia
with normal gastric findings. Then in addition to these there occur, no doubt,
various types of anemia as a result of the different states of toxemia in the preg-
nant woman.
As in other anemias, the patient with hypochromic anemia is best treated
with large doses of iron and if achlorhydria is present, with hydrochloric acid.
The macrocytic anemias are best treated with liver_ extract supplemented with
iron and hydrochloric acid. Some require transfusions. (For details of treat-
ment of anemias see section on treatment.)
We have made repeated examinations of the blood and gastric findings of
100 consecutive pregnant negro women. We found that 2S per cent of these
showed achlorhydria and not more than 50 per cent had a normal total gastric
acidity. Practically all of these women were anemic with less than 65 per cent
hemoglobin (Sahli). As pregnancy progressed the anemia became more accentu-
ated. There seemed to exist some correlation between gastric anacidity and the
macrocytic type of anemia. In five cases in which macrocytes were numerous,
there was no free hydrochloric acid. Also in all cases in which there was gastric
212 THE ANEMIAS

anacidity the macrocytic ~ype of anemia predominated over the hypochromic

type.

ANEMIA OF HYPOTHYROIDISM

It has long been noted that hypothyroidism, or myxedema, may he accom·

panied by a severe form of aneInia. The marked pallor seen in that condition may
be accounted for in part by the anemia but not entirely. Furthermore, every
case of hypothyroidism is not necessarily accompanied by anemia but it occurs
with sufficient frequency to suggest a close relationship. The anemia produced
may be of the macrocytic type with a color index of more than one, especially
in thyroidectomized animals, and it responds to injections of thyroxin. It has
been thought that many cases of myxedema are accompanied by a gastric an-·
acidity, but in our experience this is a rare finding.
The anemia of hypothyroidism may also be of the hypochromic type which
occurs more frequently ilian the macrocytic type. Inasmuch as both types of
anemia are seen in this disease, it would appear that the active principle of the
thyroid gland is concerned in hematopoiesis and thyroid substance is usually
employed in treatment. It is also advisable to supplement this therapy with con-
siderable doses of liver and iron. Furthermore, the hypochromic type can be cor-
rected by the institution of treatment with iron and liver without the administra-
tion of thyroid extract. Bomford made careful studies on ten cases of myxedema
and found three different types of anemia. One was a simple hyperchromic
type, the second a hypochromic form, and the third the addisonian hyperchromic
type. He states that the first of .these responds to thyroid administration, the
hypochroInic type responds to iron, and the third responds to both liver and
iliyroid. He concluded that thyroxin is not necessary for ilie normal maturation
of red cells. For relation of thyroid gland to hematopoiesis, see p. 18 I.

ANEMIA OF INADEQUATE IRON INTAKE

The fact that practically all hypochromic anemias respond in variable de-
grees to <the administration of large doses of iron indicates th~t many of the
hypochromic anemic states are due entirely or in part to this deficiency. Iron is
the single most valuable component necessary in the hemoglobin molecule. An
iron deficiency anemia may develop because of an inadequate iron intake, or
because of a failure in its absorption or utilization.
Moderate degrees of hypochromic anemia seem very common in people who
otherwise appear to be in good health. This seems to be brought about in
some people by voluntary diet restriction, in others by various peculiarities in
diet and in those who are ill because of an inability to take a sufficient amount of
iron in their restricted diets. Many of the anemias, therefore, that have been dis-
cussed in the preceding chapters are caused in part at least, by an inadequate
iron intake. A hypochromic anemia often develops in infants and children on
this basis. Apparently the gastric findings bear little or no relation to the failure
 HYPOCHROMIC ANEMIA 213

to utilize iron once it has been absorbed. The anemias of iron deficiency can be
easily corrected by the institution of treatment in which large quantities of in-
organic iron are given by mouth.
There have been numerous reports in recent years concerning a type of
anemia that occurs predominantly in children. This anemia, frequently called
"goaes milk anemia/ J or simply "milk anemia" or "alimentary anemia" is prob-
ably an iron deficiency type and is caused in infants and small children by being
pJaced on a ·diet of milk almost exclusively. This results in an inadequate food
intake of other types and since milk contains insufficient iron for the maintenance
of hemoglobin production, tbe anemia slowly and progressively develops. It can
be relieved by correction of the diet. The occurrence of this type of anemia in
recent years refutes the time worn phrase that milk is the perfect food and that
one could subsist entirely upon it.
Hypochromic anemia of malnutrition, caused by lack of iron, in our experi-
ence, seems to be a frequent finding in many people who are provided with a
diet that is adequate in other respects. In a hematologic study of 3 I 2 young ap-
parently healthy Southern college women, the average red cell count was 4,085,000
per cu. mm. but the average hemoglobin value was only 70 per cent (Dare).
This mild hypochromia could develop because of voluntary diet restriction and
lack of exercise.
We have also studied the blood of over 100 cotton mill workers, and found
an average of 4,220,000 red cells in adults and 50 per cent hemoglobin, with the
children showing essentially the same findings. Careful study of diet of this
group showed it to be woefully inadequate, and the severe hypochromia was
speedily corrected by a dietary supplement of 30 grains of ferric ammonium citrate
daily.

ANEMIA OF VITAM;rN 0 DEFICIENCY

It is well known that a definite clinical syndrome of vitamin C deficiency in

both infants and adults results from the inadequate intake of vitamin C. The
disease usually develops in those infants whose dietary intake has been inadequate
and who have been fed on various proprietary foods. It is now recognized that
the scurvy of infants known as Barlow's disease is the same disease as the scurvy
of adults. The condition is characterized by a generalized malnutrition, by
marked pallor with bleeding gUl)1s and various other types of hemorrhages over
the body.
The anemia that accompanies this deficiency is nearly always of the hypo-
chromic type. It may be due in part to an associated iron deficiency or as a result
of profuse hemorrhages of considerable degree, but certainly to some extent
to the actual vitamin deficiency itself.. The hemorrhages are probably due to a
vascular weakness (see section on purpuric diseases), since the platelets are not
altered in number and the coagulation and bleeding times are normal.
Every case of vitamin '0 deficiency is not necessarily accompanied by anemia,
but most of them do present varying degrees of the hypochromic type. There are
very few changes in the leukocyte or platelet pieture.
214 THE ANEMIAS

It has been shown by Mettier and his associates that these patients can be
treated with adequate doses of iron and liver without vitamin C in the diet
with no effect on blood formation. When vitamin C is added to the diet, an
immediate reticulocyte response follows. Therefore, this suggests that in many
other types of anemia which do not respond promptly to treatment with liver
and iron the addition of suitable dosage of vitamin C (cevitamic acid) might
be indicated.
There also exists a vitamin (: deficiency of mild degree sometimes called "sub-
clinical scurvy." This condition is characterized by a chronicity of symptoms,
prolonged periods of iII health, gradually increasing pallor, slow and insidiously
developing hypochromic anemia, and finally, an out-cropping of hemorrhages.
We have studied patients presenting this picture who were referred to us because
of the hemorrhagic syndrome. They invariably show a moderate hypochromic
anemia which is corrected with the administration of vitamin C in the form of
cevitamic acid. In such instances it is not necessary to supplement this treat-
ment with iron and, of course, this illustrates the desirability of correcting the
basic pathology of an anemic state rather than treating the anemia.

ANEMIA OF GASTRO-INTESTINAL DISEASE

Varying degrees of hypochromic anemia may occur as a result of disorders of

the stomach or intestines whether or not they are accompanied by blood loss.
Furthermore, it is not necessary that there exist organic pathology in order to
produce anemia because functional disorder such as hypermotility of the intestinal
tract may within itself prevent the absorption of an adequate amount of iron
and other agents necessary for building the hemoglobin molecule. Varying de-
grees of hypochromic anemia have been reported in such diverse conditions as
esophageal stenosis, complete or partial gastro-intestinal resection, gastro-enter-
ostomy, various intestinal stenoses and fistulae, diarrhea and dysenteries of all
kinds.
Especially is anemia likely to develop if there is co-existing hemorrhage or
if the diet is inadequate which so often is the case. It has also been noted in
certain cases of sprue and pellagra even though the usual picture in these two
diseases is of the macrocytic, hyperchromic type.
We have been impressed with the marked degree of hypochrOlp.ic anemia
in various types of so-called "colitis," including chronic ulcerative colitis of
unknown etiology, amebic dysentery and the so-called "mucous colitis" so fre-
quently observed in nervous women. All of these conditions characterized by
numerous stools daily which are watery and contain blood, mucus and pus; will,
in Our opinion produce the most profound degrees of hypochromic anemia. There
is no reason to believe that the infection itself or the loss of blood from the
colon· is adequate to account for the anemic state entirely. Therefore, it might
be possible that a diseased colon would have its function for absorption con-
siderably impaired so that an inadequate amount of iron and other products
would be absorbed in that area.
 HYPOCHROMIC ANEMIA 21 5

Recently, we have been impressed with the severe degree of anemia seen
in cases of amebiasis in which the infection was limited apparently to the colon
and the liver was not involved. Severe infections of the colon in amebiasis are
accompanied by profound hypochromic anemia with the color index quite low
so that the red cells oftentimes appear as mere rings. In these, of course,
treatment should be directed toward the removal of the offending organism in
addition to large doses of iron for the correction of the anemia. Oftentimes the
red cell count is very low and in two instances we have seen a remarkable
regeneration of red celts manifested by reticulocytosis following the institution
of proper therapy for amebic dysentery. Particularly has this been true after
the injection of emetin hydrochloride.

ANEMIA OF CHLOROSIS

This disease in the older literature occupied many pages of the average clin-
ical textbook but it is seldom seen in modern times; this is thought to be because
of better nutrition in modern diets. So-far as can be determined, the older cases
of chlorosis with extreme degrees of hypochromic anemia and green pallor in
young women were brought on by defective diets, and achlorhydria, coupled with
the chronic blood loss of excessive menstruation. The disease is characterized
by the symptoms of profound anemia with marked weakness, dyspnea, irregu-
larity of the menses, various vasomotor disturbances and a peculiar green color
of the skin from which it derives its name.
The importance of excessive menstruation in young women in producing
anemia has probably been overemphasized, unless the bleeding is profuse. In
studies of 300 young college women, we can find no relation between hematologic
values and amount of menstrual flow. A group of 16 women who used an average
of 22 pads daily for six to eight days, were found to have hematologic values
equal to the larger group with normal menstrual flow.
The blood picture in chlorosis is one of an extremely marked hypochromic
anemia with a very low color index. The red cells are definitely microcytic and a
lenkopenia of granulocytic cells often occurs. It is quite possible that the old
chlorosis may be identical with the disease seen in older women today known as
"idiopathic microcytic hypochromic anemia." It responds to the administration
of large doses of iron just as other types of hypochromic anemia.

TREATMENT OF HYPOCHROMIC (SECONDARY) ANEMIAS

There are no diseases of the blood in which such brilliant results can be ob-
tained as in the various types of hypochromic. anemia, if the proper treatp1en!
is used. Therefore, all varieties of anemia should be thoroughly studied and
properly classified, since it is generally true that only the hypochromic types re-
spond to the administration of iron and the hyperchromic macrocytic types (0
the administration of the deficient hematopoietic principle in the form of liver
extract. It follows then that the hypochromic types are not corrected by liver
 PLATE XX

MICROCYTIC HYPOCHROMIC ANEMIA

(SECONDARY ANEMIA)

q
t~--~.'~.~
.. . . :- .

~ ,:

I. Segmented neutrophil.
2. Band neutrophil.
3. I.ymphocyte. ,
4. Erythrocyte with basophilic stippling and polychromasia.
5. Microcyte.
6. Poikilocyte.
7. Normocyte.
Blood Findings (patient with chrQnic post-hemorrhagic Differential:
anemia):
Hemoglobin .. 5 gms. (Newcomer's method). Myelocytes 0%
R.B.C. . . . . . . . • . . . . . . 3,000,000 per c.mm. Juveniles 1%
W.B.C ... 9,800 per c.mm. Bands 6%
Platelets 500,000 per c.mm. Segmenters 6r%
Total neutrophils ......... 68%
Color Index . 0·5 Lymphocytes z80/0
Volume Index 0.6 Eosinophil::; . 1%
Reticulocytes, 8.0% Basophils 1%
Monocyte::; .............. 2%
Erythrocytes; hypochromic and microcytic with anisocytosis, poikilocytosis, polychromato-
philia, and basophilic stippling.
Plate XX.
 HYPOCHROMIC ANEMIA 21 7

extract, yet many patients in whom the type of anemia has not been carefully
determined have been given useless and expensive liver preparations while their
anemia could be corrected with the relatively inexpensive iron therapy.
In outlining the treatment for any case of hypochromic anemia it is quite
important that its cause should be determined and eliminated if possible. Thus,
the anemia of hookworm infestation, of chronic infection, or prolonged colitis,
may be partially corrected by the administration of large doses of iron, but the
improvement is only temporary if the cause is not discovered and eradicated.
Therefore, the first consideration always should be to ascertain why the patient
has the anemia. After all, anemia is only one finding common to many different
diseases.
As pointed out before, most patients with hypochromic anemia have a normal
or nearly normal number of circulating red celis, and the deficiency is mainly
one of hemoglobin. There is little indication for the use of any preparation
designed to stimulate the bone marrow to increased production of cells, but rather
the problem is to provide the necessary building materials for the hemoglobin
content of cells.

USE OF TRANSFUSIONS
It is seldom that transfusions are indicated in the correction of hypochromic
anemia, and without doubt, it is much overdone. Especially is this true in in-
stances of long standing chronic anemia. Even though the hemoglobin may be
only 25 per cent and the red cells quite low, this within itself is not an indication
for the transfusion of blood, provided the patient shows no emergency symptoms
referable to the anemic state. In some instances of anemia following, the loss of
large quantities of blood, the procedure is indicated, but mainly to restore a
depleted blood volume. There are times when a patient does not seem to respond
to the administration of what appears to be an adequate amount of iron, and in
such cases a transfusion of 500 cc. of blood seems to provide some type of stimulus
that initiates a satisfactory response to therapy. To use it routinely, however,
in severe hypochromic anemia, is as Castle and Minot state, "an expensive and
clumsy method of securing a therapeutic result."
It should be remembered that patients with low hemoglobin values offer
additional risk from reactions after transfusions. Also, the addition of new red
blood cells, even though it is followed by immediate signs of improvement, is no
different from providing a palliative agent for the use of pain, since it does not
correct the basic pathology and may serve to obscure the most valuable diag.
nostic findings. On many occasions we have observed profoundly anemic patients
given a so-called emergency transfusion of 500 to 1000 cc. of blood and then a re-
quest made for hematological studies to determine the type of anemia. This is
inexcusable, since the hematologic findings may become so altered that it may
be impossible to carry out exact diagnostic studies.
If the anemia is associated with various infectious states, transfusions may
serve to produce benefit for the infection by the addition of immune bodies in the
blood plasma of the donor. Many times it is necessary to give one or more
218 THE ANEMIAS

transfusions in preparing a patient for a necessary surgical procedure, in which

instances it is justifiable.
The administration of fluids should be carried out very cautiously in severe
anemias. Gibson and his associates at Harvard University have pointed out that
intravenous administration of fluids, even in cases of severe dehydration may
dilute the red cells to the danger point. They point out further that the hematocrit
level of red cells is a better criterion of erythrocyte volume deficit than either
red cell counts or hemoglobin determinations. It should be pointed out too that
blood plasma will effect the same results. Blood plasma should never be used
in cases of severe anemia, regardless of the typ~. It merely dilutes the red cells
in the vascular system, so that the anemia is accentuated.
The injection of sodium chloride causes a decrease in the hemoglobin con-
tent by further dilution of the blood plasma. This has been shown by Sheftel,
who found that in normal people, if from 5 to IS Gm. 6f sodium chloride in 500
cc. of water was given one hour after a light breakfast, there was a fall of
hemoglobin averaging over I I per cent within a few hours. Furthermore, the
unexplained variations that occur from day to day in patients who are under
treatment for anemia can sometimes be explained on this basis, that is, the
amount of ingested salt from three to six hours before the determination of the
hemoglohin. Sheftel points out that this is caused by diffusion of water from the
tissues into the blood and of salt from the blood into the tissues, causing an
increased blood volume temporarily.

USE OF IRON
The administration of suitable preparations and adequate amounts' of iron
is the single most valuable method of correcting all types of hypochromic anemia.
This has been recognized for'more than a hundred years, but only comparatively
recently has the full importance and value of iron therapy been fully appreciated,
especially since the reports of lVIeulengracht who stressed the necessity for very
large doses.
Iron as a necessary factor in building the hemoglobin molecule has been
discussed in detail in a previous section (factors influencing erythropoiesis). In -
summary, an ordinary well balanced diet contains sufficient iron for the average
demands (I mg. daily in adults, but several times as much for females in the
reproductive period, and for growing children) in the form of soluble iron salts,
iron hydroxide combined with protein, and hemin derivatives from ingested
meats. Not all of this is available for absorption since it is necessary that it be
broken down into soluble iron salts (Elvehjem). When the iron reaches the
stomach it is acted upon by the hydrochloric acid and pepsin and goes into solu-
tion. Then passing into the duodenum it is further acted upon by intestinal
ferments and is reduced to the bivalent ferrous state after which it is capable of
being absorbed.
Absorption ·takes place mainly in the duodenum and to some extent in the
pyloric portion of the stomach. Apparently it is facilitated by an acid medium.
Therefore, in gastric achlorhydria the addition of dilute hydrochloric acid is
 HYPOCHROMIC ANEMIA 21 9

important. After absorption it is eitber carried directly to the bone marrow

where it is taken up by the erythroblast and utilized in building the hemoglobin
molecule, or it is stored, perhaps acted upon further by catalytic agents, and then
released for utilization by the erythroblastic tissues. Thus it is obvious that hemo-
globin deficiency may be brought about by: first, a deficient iron intake; secondly,
failure of the iron to go in solution due to gastric anacidity; thirdly, failure of
absorption in the duodenal area; and fourth, interference with its conversion into
hemoglobin.
Because of the possible factors influencing its utilization, it has become neces- .
sary to give large amounts in treatment. The black stools of the patient receiving
only small amounts are a rough indication that it is not being absorbed in its
entirety. Much of it may be absorbed and then stored but not used in building
hemoglobin, and as Witts has stated, it can be regarded as "a frozen credit which
the patient is unable to liquefy."
That unknown factors play a part in the metabolism of iron is indicated by
the occasional instance of a patient receiving a large daily dose which should
be quite adequate (50 grains ferric ammonium citrate) with no effect on the
anemia while if the dose is doubled, immediate increases in hemoglobin values
become evident. Also Bethell and his associates have pointed out that a daily
dosage of five grains of reduced iron may be without benefit when given in three
doses but if given in ten doses may beCOme highly effective, this indicating an
inability to absorb a sufficient amount three times daily and furtber indicating
a maximum absorptive capacity.
It seems, therefore, in order to obtain maximum response from the admin-
istration of iron, it should be given in suitable amounts, using preferably an
absorbable preparation, into a stomach that contains hydrochloric acid, in some
instances in small divided doses. Witts states that "the therapeutic activity of
preparations of iron by mouth is directly proportional to their solubility and to
the ease with which they yield free ions of ferrous iron."
After iron is absorbed it is possible that other factors are necessary for its
conversion into a structure suitable for hemoglObin usage. In this connection it
has been stated that copper is a catalytic agent,' based on the fact that animals
on a copper and iron free diet will not respond to iron therapy alone but require
additional copper tlierapy.to produce increased hemoglobin values (Joseph, Elveh-
jem). Although this may be true in the experimental animal deprived of copper,
it does not seem to be true in man, presumably because there is present at all
times in man an adequate supply of copper, and the amount taken in food,
though quite small, is adequate for maintenance purposes. There is general
agreement that copper is not necessary in the therapeusis of hypochromic anemia.
McGhee, however, carried out careful hemoglobin studies on 140 cotton mill
workers in one village, giving them a daily quart of milk in which was a copper-
cohalt-manganese alloy in the form of a metal strip and noted an average bemo-
globin gain of 14 per cent in the entire group with 98.5 per cent showing some
degree of hemoglobin increase. However, he apparently failed to take into
account the hemoglobin regenerative effect of a quart of milk daily added to
220 THE ANEMIAS

the poor diet of a mill worker. Barer and Fowler have concluded that the admin-
istration of copper is unnetessary as an adjunct to iron therapy in any type' of
anemia. Tyson and his associates carried out experiments on 300 young white
rats studying the effect of various iron preparations on blood and also on the
blood of expectant mothers (human). They concluded that copper did not en-
hance the value of iron either in the animals or in the patients.
Hutchison in England carried out very careful metabolism studies on nine
infants and shows that the administration of copper enhances the conversion of
iron into hemoglobin, and that when iron is given in doses so small as to not raise
hemoglobin appreciably, if copper is subsequently given, the metal is then mobi-
lized and converted into hemoglobin. He concludes that it is probable that copper
in some form is necessary for normal erythropoiesis.
To summarize the question of the use of copper in the treatment. of hypo-
chromic anemia, it would appear to be of value in certain resistant cases of
idiopathic hypochromic anemia in adults, also perhaps in accelerating recovery
in nutritional anemia in children, and in securing optimum hemoglobin regenera-
tion when it has been lowered in the body by the excessive demands of growth,
pregnancy, and certain infectious diseases. Heath, in his discussion of iron
deliciency at the University of Wisconsin symposium on blood diseases, con-
cluded that copper is apparently a necessary substance in hemoglobin formation
as shown in small animals, and that other metals such as cobalt and manganese
probably have a similar influence, but that the value of administration of such
substances is doubtful.
The possible role of calcium in potentiating the action of iron has been re-
cently brought out by Orten and his associates who noted the supplementary
value of calcium in conjunction with iron in a series of experimentally produced
anemic rats. This would imply that even though a patient may receive adequate
doses of iron, the addition of calcium may result in its activation, absorption and
utilization. Kato fed a group of rabbits and dogs with iron and cobaltous sulphate
with a control group receiving iron alone. In the iron and cobalt group he was
able to produce polycythemia and a much higher iron content in the blood than
in the control group. He concluded that cobalt exerts an accelerating effect in
the synthesis of hemoglobin and the formation of erythrocytes. In these animals
the spleen and marrow contained very little deposition of iron, suggesting that
cobalt enables the iron to be more completely utilized in hematopoiesis than when
iron is given alone. In all of his animals he used IS grains of ferric ammonium
citrate and 10 grains of cobaltous sulphate as the daily dose.
As stated before, in order for iron to be efficacious, it should be ionizable and
absorbable. Witts believes that the ferrous salts are much superior to the fer-
ric salts for this reason and states that the dosage of the ferrous salts can be
much less to produce the same effect. There is general agreement that organic
iron preparations have little value and therapy should consist only of the inor-
ganic salts. Heubner found that ferric iron is not absorbed in dogs, while the
ferrous salts are readily absorbed. However, Heath and his associates have shown
that the injection of the ferric salt is effective. Therefore, it seems that either
 HYPOCHROMIC ANEMIA 221

preparation may be effective for hemoglobin production after it has gained en-
trance to the tissues, and that the ferrous salts have advantages only so far as
absorption is concerned.
The following table, modified from Witts, shows .the various commonly used
preparations of iron, with the daily dose, the iron content, and the average per
cent utilized in hemoglobin regeneration.

TABLE XVI
Daily dose Iron (;ontent
Prepa:ration (grams) (milligrams) Utilizatwn
Reduced iron (metallic). . . . . . . . . . . . . . . . . 2-6 1500-5000 I%
~!~
"Ferrous chloride ........................ 0.25--0.5 1"00- 200
Ferrous sulphate. . ..................... 0.6 ISo
Ferrous lactate ................... , . . . . . 1.5 300 8%
Ferrous carbonate {Blaud's mass)......... 4.0 400 7%
Ferric citrate........................... 2.0 400 6%

!:~~=i:~~~~~~:.::::::::::::::: '~~~
3%
3H .%
3%
According to Strauss, the following official U.S.P. preparations will give
maximum hemoglobin regeneration if used in the doses indicated.

Preparation Daily Dosage

Reduced iron (metallic) ....................... 3 grams (45 grains)
Ferrous carbonate mass (Blaud's) ............. " 4 grams (60 grains)
Ferrous carbonate pills (Blaud's) ........ ....... 4 grams (60 grains)
Iron and ammonium citrate .................... 6 grams (90 grains)
Ferrous sulphate ............................. I gram (J 5 grains)

Strauss points out that the first three in the above list are relatively insoluble
and the last two freely soluble, and that in patients who exhibit achlorhydria
the more soluble products will give better results.
It is necessary to give iron in divided doses, preferably with or just after
meals, since its admixture with food will give a wide distribution over consid-
erable absorptive surface and the dilution produced is sufficient to obviate un-
pleasant symptoms of gastro-intestinal irritation which usually are variable
degrees of nausea, abdominal cramplike pains, and diarrhea. Since some patients
are unable to take ferric ammonium citrate in suitable dosage the ferrous salts
should be used since the requisite quantities are much less.
It is our practice to use ferrous sulphate in daily doses varying from
15 to 45 grains, divided in three doses after meals. This product is usually pre-
pared in capsules of 3 grains each. Therefore, in the average case of hypo-
chromic anemia, three capsules after meals is usually adequate treatment.
If the administration of the above dosage results in nausea, abdominal pain
and diarrhea, then other ferrous preparations, such as the carbonate, lactate,
chloride and even reduced iron, can be tried. All iron preparations can be given
in capsules or in coated tablets, but there seems little advantage in using the
so-called enteric coated tablets. No coating should be used that would prevent
the iron being acted upon by the gastric contents, since, as pointed out before, this
222 THE ANEMIAS

action is probably necessary for solution to take place. Some patients can not
tolerate large doses of any of the iron preparations even ferrous sulphate. For
this group of patients Reznikoff and Goebel have recommended the use of ferrous
gluconate. They state that it results in a satisfactory reticulocyte response, a
high utilization of iron and a satisfactory increase in hemoglobin. They recom-
mend the oral administration of this compound in such patients.

TABLE XVII

VARIOUS TYPES OF WIDELY USED IRON PREPARATIONS

A1Jail- Avail- Avail-
able aM. able
Iron Liver Capper
Daily per per per Retail
TtaJemark Manufacturer [ron Compound Dose Diem Diem Diem Pric(J
Bepron John Wyeth Ferrous-ferric 3 tbsp. 40 5 9 2 .8 $3.75 per
Bro. saccharate pint
Cofron Elixir Abbott Labs. Ferric ammo- 3 tbsp. 99 60.0 0·4 :2.00 per
nium citrate .12 fl. oz.
Cupriferrum E. R. Squibb & Ferric ammo- 9 caps. 7 65 4·5 :1.'25 pet

Feosol
Son
Smith, Kline &
nium citrate
Ferrous sulphate 4 tabs. 160
'00
r.oo per
French Labs. 'co
Ferric Ammo- Lederle Labs. Ferric ammo- 9 caps. 765 1.25 per
mum Citrate mum citrate 100
Ferro-Catalytic Chas. E. Frosst Ferrous car- S caps. 550 4. 0 J.75 per
Co. bonate 50
Gluco-Ferrum* Van Pelt & Ferrous gIu~ 2 tsp. 36 1.00 per
(elixir) Brown conate 8 oz.
Hebulon* E. R. Squibb & Ferrous sulphate 6 ca.ps. 240 Q6.0 3.25 per
Son '100
Hematinic Plast- Bovinine Co. Ferrous sulphate 3 plas. .75 per
ules (plain) 50
Hematinic Plast- Dovinine Co. Ferrous sulphate 6 plas. zoo 21.0 1.50 per
ules (with liver) So
Iron and Ammo- Sharp and Ferric ammo- 2 caps. '70 .75 per
nium Citrate) Dohme nium citrate 100
(green)
r(~~;:k~rlate Parke, Davis
Co.
Ferric cacodylate lee.
(inj.)
1.5 I.75 per
30 'Ce.
61 per
F;h~~pb~~~-
Iron and Copper Eli Lilly Co. 5 tabs. 4. 0 1.00
100
Ironyl E. R. Squibb & Ferrous , cc. 8 6.50 per
Son adenylate 10 ce.
Jeculin'" Upjohn Ferric ammo- 9Ca.~. 102 92 . S ::1.49 per
nium citrate 84
Lel::tron no. S5 Eli Lilly Co. Ferric ammo- 12 caps. 408 5·5 2.75 per
nium citrate (Liver and 84
Stomach
Cone.)
Lirimin* Sharp & Dohrne Ferrous sulphate 2 caps. 120 .48 6.00 per
100
Naferon Parke1 Davis Fernc sodium 6 caps. 375 1.65 per
Co. citrate roo
'" .. denotes presence of vitamins.
Note: Ferrous sulphate is one of the best and inexpensive.
ik:r~j~S~~ 1~d1~:ti~~fo~ok~~i!j~Odroe:~.
There is no indication for use of liver and iron mixtures.

As in all other medication, the dose requirements vary in different patients.

One patient may require only IS grains of ferrous salt daily to produce maximum
 HYPOCHROMIC ANEMIA 223

effect, while another may require 50 grains daily. In general, the more severe
the anemia, the more iron is required. It is desirable to establish the requirement
of each patient and this can be done only by the trial and error method, that is,
giving an arbitrary initial dosage and following this with frequent hemoglobin
determinations to ascertain the response. The achlorhydric patient requires a
maintenance dose of iron even after complete restoration of blood values. Thus,
in achlorhydric microcytic anemia a patient may require 50 grains of ferrous salt
daily to restore the normal blood _values, and 10 grains daily for an indefinite
period thereafter. In such patients we usually advise one capsule (3 grains)
with meals. Iron capsules can be kept on the table and become just as permanent
a fixture as a salt shaker, and the patient suffers no inconvenience in continuing
the therapy. According to Heath, administration of iron in acid buffer mixtures
will cause greater blood production than when given in alkaline solutions, but the
administration of acid with iron is not to be recommended. Also, if iron is mixed
with a large amount of mucin; its effect is very definitely inhibited. Other factors
inhibiting utilization of iron include the absence of thyroid secretions or vitamin
C, and the presence of various diseases such as infections and nephritis. Even
though there is plenty of iron available a hypochromic anemia may develop and
in such instances iron therapy will not alleviate the anemia.
In patients who seem unable to swallow capsules the iron can be prepared
in elixirs and syrups. This is particularly true of children. Wben it is used in
liquid form the ferrous salts are desirable because of their solubility and the fact
that much smaller amounts are necessary for maximum effect.
There is general agreement that parenteral tberapy with iron is unnecessary
except in the rare instances where it cannot be taken by mouth. According to
Heath the intramuscular dose should not exceed 30 mg. per day. However,
Strauss states that from 100 to 200 mg. of ferric ammonium citrate may be in-
jected daily. There is always danger of untoward reactions, manifested by severe
local pain at the site of injection, general redness and flushing, palpitation, pre-
cordial pain and oppression, nausea, tachycardia and vomiting. The advantage
of iron injections is reasonable assurance that it is all utilized.

SUPPLEMENTARY MEASURES TO IRON THERAPY

Since the advent of liver extract some writers have advised that injections
of liver he given to supp!<iment the iron. In an occasional instance this may be
necessary but should not be done in all cases, for the main reason that it is not
needed and serves therefore as a useless and unnecessary 'expense to the patient.
Murphy advises the intramuscular injection of 3 cc. of liver extract (prepared
from roo grams of liver) at weekly intervals "to hasten the improvement of the
blood and especially the patient's general physical condition." Such supple-
mentary treatment may be indicated in that patient whose red cell count is low
on the assumption that there is a coexistent deficiency of the hematopoietic
principle, but in those in whom the red cells are normal or nearly normal, there
is little indication for its use. A majority of patients will show satisfactory
response on adequate dosage of iron without liver. Whipple has shown that the
224 THE ANEMIAS

fraction of liver precipitated by 70 per cent alcohol (tbe so-called secondary

anemia fraction) in combination with iron is more effective than either prepara-
tion alone, when used in the experimental anemia of dogs. (The anti-pernicious
anemia fraction is soluble in 70 per cent alcohol.) Also in an occasional patient
tbere may occur a point at which the hemoglobin risc ceases after a previous
satisfactory and sustained regeneration, and weekly injections of liver extract may
provide the necessary stimulus to furtber regeneration. Barker and Miller
treated II patients witb hypochromic microcytic anemia with iron for 10 days,
and then gave the Whipple liver fraCtion. This was followed by a second
reticulocytosis, and they imply tbat tbe so-called secondary anemia liver fraction
is of some value in the hypochromic anemias. However, Barer and Fowler have
concluded that the Whipple fraction of liver is of no service in the hypochromic
anemias.
In certain types of anemia the supplementary nse of vitamin C (cevitamic
acid) is indicated, especially if there exists a dietary deficiency that would involve
. a deficiency of this vitamin. In the hypochromic anemia accompanying the
scorbutic syndromes it is quite essential. An adequate amount can be obtained
in from six to twelve ounces of orange juice daily or by the oral administration
of 100 mg. of cevitamic acid daily.
In tbe anemia of myxedema thyroid extract should be used in quantity suf-
ficient to alleviate all symptoms which include the anemia. Also iron should
be given and if the anemia is macrocytic in character liver extract is indicated.
It is advisable to regulate the diet in order to insure an adeqnate intake of
vitamins and other types of iron in small amounts that may playa role in hemo-
glohin production. However, there is no evidence to indicate tbat the iron of the
diet is important if the patient" is receiving adequate therapy. In general the
diet should be liberal in animal proteins, fruits and green vegetables. After the
anemia is corrected the diet is quite important, particularly if the anemia devel-
oped as a result of inadequate iron intake, such as that seen in alimentary milk
anemia or so-called goat's milk anemia in children.
The following foods are stated by Bridges to be of most value in hemoglobin
regeneration:

Apples Calf liver Pig kidney

Apricots Chicken gizzard Prunes

Beef kidney Chicken liver Raisins

Beef spleen Fish liver Raspberries

Bone-marrow Lamb liver

Brains Peaches
 HYPOCHROMIC ANEMIA 225

The following Jist of foods is given by Bridges as containing the largest

amounts of iron in the order given:
Blood Fruit salad, canned Hazel nuts
Beef juice Dry beef Almonds
Parsley Dandelion greens Chestnuts
Lima beans, fresh Watercress Beef, lean
Apple butter, canned Walnuts, hlack Puffed wheat
Bran Calf liver Swiss chard
Beef liver Dates, dried Veal kidney
Pistachio nuts Prunes, dried Brazil nuts
Molasses Pineapple, canned Figs, dried
Egg-yolk Beef heart Peaches, canned
Kidney beans, canned Currants, dry Oysters
Raisins Shredded Wheat

RESPONSE TO ADEQUATE TREATMENT

The response to treatment depends upon the cause of the anemia, the pres-
ence of complicating diseases such as infections, the adequacy of treatment, and
is therefore quite variable. In general, the blood values can be fully restored
to normal in all anemias of the hypochromic type. The rate of hemoglobin in-
crease is the single most reliable criterion to judge its efficacy. In the severe types,
it may begin with increases as much as 2 per cent daily, but seldom over that,
and if the patient shows a consistent gain of I per cent daily, the response can
be considered maximum or satisfactory. As the hemoglobin slowly rises toward
the normal level, the daily rate of increase becomes less. There is an actual
marked reticulocytosis in the early stages which usually ranges about 5 per cent
and is not as marked as that seen in pernicious anemia during regeneration. In
patients with normal red cell counts at the beginning, the reticulocyte rise may
not be evident since there is no need for increased numbers of red cells. Witts
has stressed as a criterion of response the "double reticulocyte rise," the initial
one within three or four days after institution of therapy and the second one
after 20 to 3D days. He states that the administration of ferrous salts in particular
results in tbe second rise. An occasional normoblast may be seen during active
regeneration. In the more chronic types of anemia, the usual signs of basophilic
degeneration, including stippling, the variation in size and shape of cells, and
the central pallor of the stained cell, all gradually disappear., Finally blood values
are restored to normal.
There is also remarkable improvement in the well being of the patient. The
signs and symptoms referable to the anemia disappear; the glossitis and Safe
tongue are healed as well as labial fissures; intestinal symptoms, as diarrhea,
cramps, etc., are alleviated. There is a return of appetite. The achlorhydria in
some patients is not affected, and in these instances maintenance therapy is neces-
sary. In others there is return of normal gastric findings. This bears out
Apperly's statement that achlorhydria in some patients develops as a result of the
anemia, rather than as a qmse. Grooving and brittleness of the nails are cor-
226 THE ANEMIAS

rected since there is presumably an adequate supply of iron at the. nail bed.
In severe cases certain secondary hemorrhagic states as purpura and uterine
bleeding are corrected.
All patients do not respond even with adequate treatment. Those with
carcinoma or acute and chronic infections, intoxications, and continued fever
apparently have sufficient inhibition of marrow activity to nullify the effect of
treatment. In these instances, the disease causing the anemia is still present and
the anemia is only a· part of the disordered mechanism. This is also true of
pernicious anemia since Zerfas and Smithburn have shown that the same factors
prevent satisfactory response· in that disease.

BIBLIOGRAPHY
HYPOCHROMIC ANEMIA

ASHBY, W.: "Determination of length of life of trau,5fuscd blood corpuscles in man." Jour.
Exper. Med' 29, 267, I919.
J

BETllELL, F. H.: "The blood changes in normal pregnancy, and their relation to the iron and
protein supplied by the diet." lOtty. Amer. Med. Assn., 107} 564, 1936.
BETHELL, F. H., GARDINER, S. H" and MACKINNON, F.: 'IThe influence of iron and diet on the blood
in pregnancy." Ann. Int. Med., 13, 91, 1939.
BIRKELAND, I. W.: 'I lBothriocephalus Anemia': Diphyllobothrium latum and pernicious anemia."
Medidne~ 11, I, 1932.
BOMFORD, R.: "Anaemia in myxedema and the role of the thyroid gland in erythropoiesis." Quart,
Jour. Med.~ 31, 495, 1938.
CONNER, H. M., and BntKELAND, I. W.: (1 Coexistence of pernicious anemia and lesions of gastro-
intestinal tract: Carcinoma of stomach; Consideration of twenty cases: Eleven reported."
Ann. Int. Med., 7, 89, 1933.
DmcxMANN, W. J" and WEGNER, C. R.: "The blood in normal pregnancy. Blood and plasma
volumes." Arch. Int. Med., 53, 71, 19.34. ~
HESS; A. F., and FISH, M.: "Infantile scurvy: The ·blood, the blood vessels and the diet."
Am. Jour. Dis. Child" 8, 385, I9I4.
KEEFER, C. 5., HOANO, K. K., and YANG, C. S.: ((The importance of under-nutrition in the pro-
duction of anemia associated with chronic dysentery and tuberculosis of the intestine."
Nat. Med. Jou;. China, 15, 743, 1929.
KUNDE, M. M., GREEN, M. F., and BURNS, G.: "Blood changes in experimental hypo- and hyper-
thyroidism (rabbit)." Am. Jour. Physiol., 99, 469, 1:931,
LEICHSENRlNG, J. M., and HONIG, H. H.: "Blood regeneration studies. I. Changes in the volume,
number and size of the erythrocytes in hemorrhagic anemia." Am. Jour. Physiol., 98. 636,
1931.
LERMAN, J.~ and MEANS, J. H.: "Treatment of the anemia of myxoedema." Endocrinology, 16,
533, .1932.
LERMAN, J., and MEANS, J. H.: "Gastric secretion in exophthalmic goitre and myxoedema."
Jour. Clin. Invest., 11, 167, 1932.
METTlER, S. R., MINOT, G. R, and TOWNSEND, W. C.: "Scurvy in adults: Especially the effect
of food rich in vitamin C on bIood formation." Jour'. Amer. Med. Assoc., 95, 1089, 1930,
MILLER, D. K., and RlIOADS, C. P.: lIThe effect of hemoglobin injections on erythropoiesis and
erythrocyte size in rabbits rendered anemic by bleeding." Jour. Exp. Med., 59, 333. 1934.
RHOADS, C. P., CASTLE, W. B., PA"!lNE, G. C., and LAWSON, H. A.: "Observations on the etiology
and treatment of anemia associated with hookworm infection in Puerto Rico." Medicine,
13. 317, 1934.
 HYPOCHROMIC ANEMIA

ROBERTSON, O. H" and BOCK, A. V.: "Blood volume in wounded soldiers. II. The use of forced fluids
by the alimentary tract in the restoration of blood volume after hemorrhage." Jour. Exper.
Med., 29, 154, 1919.
STEELE, B. F.: liThe effects of blood loss and blood destruction upon the erythroid cells in the
bone marrow of rabbits." Jour. Exper. Med., 57, 88:1, 1933.
STRAUSS, M. B.: "Etiology and prevention of anemia in pregnancy." Ann. Int. Med., 9, 38,
1935.

TREATMENT

'" ApPERLY, F. L.: IlGastric acidity and its significance. A clinical and experimental study," Lancet,
1,5, 1936.
BARER, A. P., and FOWLER, W. M.: uInfluence of copper and a liver fraction on retention of iron."
Arch. Int. Med., 60, 474, I937.
BARKER, W. H., and MILLER, D. K.: "Clinical observations on the Whipple liver fraction (second-
ary anemia fraction)." Amer. loUt'. Med. Sci., 195, 287,1938.
BETHELL, F. H., GOLDHAMER, S. M., ISAACS, R., and STURGIS, C. C.: "The diagnosis and treatment
of the iron-deficiency anemias." Jour. Amer. Med. Assn., 103, 797, 1934.
BRIDGES, M. A.: "Dietetics for the clinician." 2d Ed., 6:1: and III, 1935. Lea and Febiger,
Philadelphia.
CASTLE, W. R., and MINOT, G. R.: "Pathological physiology and clinical description of the anemias."
Oxford Med, Publications, 1936. Oxford Univ. Press, New York.
ELVEHJEM, C. A.: "The biologi.cal significance of copper and its relation to iron metabolism."
Ph'ysiol. Reviews. 15, 471, 1935.
ELVEHJEM, C. A., HART, E. B., and SHERMAN, W. C.: uThe availability of iron from different
sources'for hemoglohin formation." Jour. Biol. Chem., 103, 61, 1933.
FULLERTON, H. W.: "The treatment of hypochromic anemia with soluble ferrous salts." Edinburgh
Med. Jour., 41, 99, 1934.
GIBSON, J. G., HAruus, A. W., and SWIGERT, V. W.: "Clinical studifS of the blood volume in anemia
and polycythemia." Jour. CUn. Invest., 18, 621, 1939.
HEATH, C. W.: "Oral administration of iron in hypochromic anemia." Arch. Int. Mea., 51, 459,
1933,
HEATH, C. W., STRAUSS, M. B., and CASTLE, W. B.: "Quantitative aspects of iron deficiency in
hypochromic anemia. Parenteral administration of iron." Jour. CUn. Invest., 11, 1293, 1932,
HEUBNER, W.: "Bcmerkung zur eiscntherapie." Ztschr. I. klin. Med., 100, 675, 1924.
HUTCHISON, J. H.: "The role of copper in iron deficiency anemia in infancy." Quart. Jour. Med.,
31, 397, 1938.
Jm:'IEPIIS, H.: "Treatment of anemia of infancy with iron'and copper." Bull. Johns Hopkins HosP.,
49, Z46, 1931.
KATO, K., and Jou, V.: HInfiuence of cobalt on iron transportation and storage: A chemical and his-
tological study." Amer. Jour. CUn. Path., 10, 751, 1940.
MCGHEE, J. L.: "Effects of copper in the diet of one hundred forty persons," Jour. Lab. and Clin.
M,d., 22, 356, 1937. .
MEULENGRACHT, E.: ULarge doses of iron in various types of anemia in a medical department."
Acta M ed. Scandinav., 58, 594, 1923.
MmOT, G. R., and HEATH, C. W.: (~Tbe response of the reticulocytes to iron." Amer. Jour. Med.
Sci., 183, IIO, 1932. .
MuRPHY, W. P.: uRational treatment of the anemic patient." Ann. Int. Med., 8, 939, ~934.
ORTEN. J. M., SMITH, A. H., and MENDEL, L. B.: "Relation of calcium and of iron to the erythro-
cyte and hemoglobin content of the blood of rats consullling a mineral deficient ration." Jour.
Nutrition, 12. 373, 1:936.
REzNIXOFF~ P., and GOEBEL, W. F.: liThe use of ferrous gluconate in the treatment of hypochromic
anemia." Jour. Clin, Invest., 16, 547, 1937. _
SlIEFTEI., A. G.: CUEffect of ingested sodium chloride on the concentration of hemoglobin." Amer.
Jour. CUn. Path., 9. 554, 1939.
228 THE ANEMIAS

SMITHBURN, K. C., MASTERS, J. M., and ZElUI'AS, L. G.: "The 'treatment of secondary anemiaj
especially with various preparations of iron." Jour. Lab. and Clin. Med' l 16, 858, 1931.
STRAUSS, M. E.: liThe pharmacopeia and the physician: The use of drugs in the treatment of
anemia.lI Jour. Amer. Med. Assn ... 107, 1633, I936.
TYSON, R. M., KONZELMAN, F. W., and LENNON, H. C.: "Nutritional anemia: Clinical and experi~
mental studies." Amer. Jour. Clin. Path., 9, 58, 1939.
WHJl>PLE, G. H., RODSCHEIT-RoBBINS, F. S., and WALDEN, G. B.: "Blood regeneration in severe
anemia. XXI. A liver fraction potent in anemia due to bemorrhage." Amer. Jour. Med. Sci.,
179, 628, I930.
WITTS, L. J.: '(The therapeutic action of iron." Lanat, 1, 1:, 1-936.
 CHAPTER 17
IDIOPATHIC HYPOCHROMIC ANEMIA

(Primary Hypochromic Anemia) (Achlorhydric Hypochromic Ane-

mia) (Idiopathic Microcytic Anemia)
Idiopathic hypochromic anemia may be defined as a clinical entity char-
acterized by profound microcytic hypochromic anemia of unknown cause, accom-
panied by gastric achlorhydria, and which responds to the administration of large
doses of iron.
It seems likely that this syndrome represents a severe type of the general
group of hypochromic anemias. Whether it should be regarded as a clinical
entity is open to question, but it is usually considered such because of one out-
standing difference from the ordinary types of hypochromic anemia, that is,
the presence of gastric achlorhydria. Therefore, it represents an iron deficiency
anemia with some gastric dysfunction, and is similar to pernicious anemia in the
latter respect ..

HISTORY

There is little doubt that the disease has existed for many years .. The syn-
drome may be identical with chlorosis or other anemias reported in older litera-
ture at a time when gastric studies were often omitted.
It was first described as a separate clinical syndrome by Witts who care-
fully reviewed 183 cases of all types of anemia seen in Guy's Hospital during a
five-year period and found that 47 of these were characterized by varying degrees
of hypochromic anemia associated with achlorhydria, that all were females, and
that cure was effected by the administration of iron. His description of the dis-
ease has been amply confirmed by many observers in the past few years and
none of these has added any essential information to that offered in his report.

INCIDENCE AND DISTRIBUTION

There seems to be no significant geographical distribution, as the disease

has been reported from various parts of the United States and from the different
countries of Europe as well as from the Orient.
It is largely a disease of tbe white race though negroes are not exempt.
Wintrobe has reported it in three negroes but two of these were mulattoes. We
have not seen the condition among the colored population of Atlanta, nor in the
colored division of the City Hospital of Atlanta and in that respect it resembles
pernicious anemia.
 THE ANEMIAS

A remarkab'le feature of the disease and one that is difficult to explain is the
fact that it is largely confined to women, particularly those in the child-hearing
age. Wintrobe and Beehe state that 96 per cent are women, and Clough gives 80
per cent as the incidence. In Witts' original 50 cases, 49 were women. There
were only two males in 25 reported by Wintrobe. It has been suggested that
unknown factors concerned with the stress of repeated pregnancy may be con-
cerned in the etiology. An interesting correlation is the frequent occurrence
of pernicious anemia in males and achlorhydric anemia in females.
The average age is between 40 and 50, and Witts' series ranged from 20 to
70 years. In Wintrobe's series the average age was 43 and the range from 22
to 74 years. Its occurrence in children is doubtful, although a temporary achlor-
hydria in children is not unusuaL
It has been reported that many of the patients are of a definite constitutional
type, of a slender asthenic build, but there is disagreement concerning this. How-
ever, both Witts and Wintrobe have commented on the "pernicious anemia like"
constitution, including a tendency to the blond, with'light blue eyes widely set
apart (75 per cent), prematurely gray hair and wide costal angles. Minot
states that idiopathic hypochromic anemia can be explained by the cumulative
effects of several factors, including chronic blood loss, being born with a poor
iron endowment, living on a poor diet leading to iron -deficiencies in early years
of life, onset of menstruation, subclinical type of infection, and gradual develop-
ment of achlorhydria and pregnancy. Therefore, the etiology is multiple and, in
a sense; is cumulative. He' points out that a woman requires about four times
as much iron as man up to the age of menopause, and this alone could account
for the frequency of this disease in women. Fowler and Barer stress menstrual
blood loss as the most important factor.
Since several members of one family may have idiopathic hypochromic ane-
mia, this has given rise to the belief that there is a decided familial incidence.
Several families have been described in which male members suffered from
pernicious anemia and the females from the achlorhydric hypochromic type
(Bartlett), (Health), (Witts), (Maclachlan and Kline). This suggests a pos-
sible inherited gastric dysfunction since this finding is common to the two
diseases. Also it gives rise to speculation as to the role of hormonal dysfunction,
because of the high incidence in women, the occurrence mainly in the active sex
period, and the analogy to pernicious anemia in men of the same family.

SYMPTOMS AND PHYSICAL FINDINGS

In the average patient the time of onset is difficult to determine, &ince its
beginning is insidious. Many patients complain of periods of prolonged ill health
which may be manifested only by excessive fatigue and an increasing pallor, or
other signs referable to any anemic state of unusual severity. This pe;iod may
cover several years before a physician is consulted, and they may state that they
have suffered from such indefinite complaints since childhood. In other patients
the disease may be discovered by accident during the course of treatment for un-
 IDIOPATHIC HYPOCHROMIC ANEMIA 23 1

related diseases. Acute infections may focus attention on the existing anemia
which has previously been unnoticed. On general examination most patients
show evidence of the severe anemia, manifested by an extreme pallor of the
skin and mucous membranes with blue white sclerae. Some patients show slight
degrees of pigmentation. The lemon yellow color is not present as in pernicious
anemia, and the patient may appear to be undernourished.
The usual symptoms are those of anemia, including easy fatigue, pallor,
vertigo, shortness of breath, palpitation, perhaps loss of weight, loss of appetite,
occasionally dysphagia (Plummer-Vinson syndrome), variable periods of diarrhea,
vague gastro-intcstinal disturbances, sore mouth and tongue, labial fissures, and
many others quite vague and indefinite. Some cases show only part of those
mentioned above, including various combinations of them. The following list
includes the signs and symptoms present in Witts' original series of 50 cases.

Anemia .................... 39 Acroparasthesia. . . . . . . . . . . . . 3

Edema ..................... 6'
Angina. ..................... 2
Dyspepsia ..... ............. IS
Diarrhea .. ................. I I
SOTe mouth or tongue. . . . . . .. IO
Dysphagia. . . . . . . . . . . . . . . . . .
Glossitis. . . . . . . . . . .. . . . . . ... 25
Splenomega1y. . . . . . . . . . . . ... 16
Koilonychia. . . . . . . . . . . . . . .. 4
Amenorrhea. . . . . . . . . . . . . . . . 5
Menorrhagia. . . . . . . . . . . . . . . . 7
6

Thus it will be noted that the symptoms most frequently encountered are
those of anemia, gastro-intestinal disturbances, diarrhea, sore mouth and tongue
and glossitis. The diarrhea probably results from the achlorhydria. The splenic
enlargement is usually only moderate, and oftentimes it is .not palpable. The
glossitis in the average patient is not so severe as that usually observed in per-
nicious anemia in relapse, or in sprue and pellagra. Koilonychia, consisting of a
spoon shaped depression of the finger nails, is observed rarely, and the nails are
brittle with longitudinal striations.
Much interest has centered about the significance of dysphagia, sometimes
known as the Plummer-Vinson syndrome, since it was described by Vinson after
Plummer had made the initial observation. Occasionally this may be the first
complaint and the only complaint bringing the patient to the physician. However,
it occurs infrequently (Witts' series, 6 out of 50 cases; Wintrobe's series, lout of
25 cases). Usually the development of dysphagia is preceded by an indefinite
period of complaints referable to anemia, and therefore, it appears to be a part
of the general picture of this disease. Often the onset of dysphagia is sudden,
the patients being unable to swallow solid food. It then becomes necessary that
they masticate their food slowly and thoroughly in order to swallow only small
portions. They may require many hours to ingest an ordinary meal, this resulting
in muclr embarrassment when eating with others, to such an extent that they
prefer to eat alone, since the presence of other people oftentimes aggravates the
condition. This supports the concept that the disorder has a neurogenic basis.
Various concepts for its occurrence include hysteria, a derangement of the
nervous control of the muscles of deglutition (Kelly), inflammatory reactions
in the nerve endings (Hurst), and the presence of bands or veils of atrophic
fibrous tissue in the esophagus, recently reported by Hoover in 17 cases. Dila-
23 2 THE ANEMIAS

tation of the esophagus has been advised for the correction of the dysphagia,
hut it ·is also necessary to give adequate amounts of iron, and since the ad-
ministration of iron alone produces relief of the dysphagia, the esophageal
manipulations seem entirely unnecessary. The dysphagia does serve to aggravate
and accentuate an already existing severe anemia, because of the inability of the
patient to consume an adequate diet. It may be snrmised that the dysphagia is
a developmental part of and secondary to the general disease and its presence ac-
centuates the anemia. Thus a vicious cycle is produced. Esophageal dilatation
does not correct the anemia, but administration of iron does correct the dysphagia
and anemia. McFee and Goodwin carefully studied the esophagus of one case at
antopsy and noted a definite inflammatory reaction in the upper esophagus, con-
sisting of lymphocytic infiltration and diffuse fibrosis. This would indicate a
definite pathologic change as the basis for the dysphagia.
Neurologic manifestations were found in 36 per cent of Wintrobe's patients
and consist mainly of numbness and tingling in the extremities, which might
be confused with pernicious anemia in some patients. The degree of these
changes is not nearly so severe or evident as those commonly seen in pernicious
anemia. The presence' of neurologic changes however, offers another point of
similarity to pernicious anemia and indicates a more complex disturbance in
hematopoietic factors than is suggested by a mere iron deficiency.
Associated diseases are not unusual and the type of associated disease may
affect the clinical and hematologic syndrome seen in different patients. Thus, in
Dameshek's series of 25.patients, he observed other disorders in II, these in-
cluding 6 recent pregnancies, 2 gastric operations, one with myxedema, and
one beef tapeworm infestation .. It is probable that such conditions as impaired
gastric function following operative procedures or beef tapeworm infestation
could actually be causative in some instances.
One of the most important findings that stamps the hypochromic anemia as
belonging' to this group is the achlorhydria, and it is questionable whether the
diagnosis should be established if some degree of gastric dysfunction cannot be
demonstrated. There should be achlorhydria after an Ewald test meal but
not necessarily after stimulation with histamine. In this respect it can be
differentiated from pernicious anemia which does not show hydrochloric acid
after histamine stimulation. We believe that it is permissible to make the diag-
nosis if there are only a few degrees of acid after the Ewald test meal, and an
occasional case has been reported in which the gastric acid was normal but in such
instances the diagnosis should be questioned. It seems illogical to adroit the
existence of a clinical syndrome including complete achlorhydria and deny that
less severe types may oCCur with hypochlorhydria.
Davies has pointed out that the gastric contents contain an excessive secre-
tion of mucus and decreased amounts of pepsin, while the secretion in pernicious
anemia showed no mucus and total absence of pepsin.
It seems that there is a definite gastric deficiency in this disease but one not
nearly so severe as that of pernicious anemia. Evidence indicates that the dif-
ferences are quantitative as well as qualitative, since Castle has demonstrated
in two cases the presence of the intrinsic factor, which is absent in pernicious
 IDIOPATHIC HYPOCHROMIC ANEMIA 233
anemia. Wintrobe and Beebe have classified the patients into four groups, in-
cluding: first, those showing no acid after histamine stimulation; second, those
showing no acid after an Ewald test meal; tbird, those of mild hypochlorhydria;
and fourth, those with normal gastric acid_

HEMATOLOGIC FINDINGS

The changes in the blood are those of a severe hypoci).romic anemia includ-
ing very low hemoglobin values. The red cells are usually not reduced markedly
in number and oftentimes the red cell count is at the normal level. Therefore
tbe color index is quite low, ranging from 0.4 to 0.6. Each red cell may carry only
about half of its normal content of hemoglobin. The leukocytes and blood plate-
lets are normal in number and types. The volume of red cells is low, and in the
hematocrit tube the cells pack to only 20 to 30 per cent compared to the normal 01
44 per cent, resulting in a volume index that varies from 0.6 to 0.8. Therefore,
the average red cell is much smaller than normal. In this disease then, there are
two factors contributing to the state of anemia. Not only does each cell fail to
carry its normal hemoglobin but the cells average only about two-thirds of normal
size.
A study of the stained blood shows that the average size of the red cells. is
smaller than normal, with the largest ones being normal and by comparison these
may appear as macrocytes to the untrained eye. However, there is no macrocy-
tosis in this disease. There may be varying degrees of anisocytosis and poikilocy-
tosis, but rarely polychromatophilia. Occasionally basophilic stippling is seen
in the severe grades of anemia_ There is no increase in the reticulocytes, except
when the patient is under proper treatment. .
Since there is no excessive hemolysis the blood serum is of normal color or
pale, the icterus index normal and van den Bergh reactions negative.
In summary, the hematologic findings of diagnostic interest are the low hemo-
globin, relatively normal number of red cells, a low color index, definite micro-
cytosis, and absence of regenerative or hemolytic factors. These findings, coupled·
with an achlorhydria and improvement of the anemia after iron therapy, con-
stitute the chief points of diagnosis.

TREATMENT

In nearly every patient the response to iron therapy is immediate and satis-
factory. When iron is given in adequate amounts, there is an increase in reticulo-
cytes if the red cell count is below normal, and a constant and progressive increase
in the hemoglobin content. Within a few days definite differences can be noted
in the hemoglobin readings and in severe cases a rate of increase of one per cent
per day is sustained for some weeks, gradually becoming less as the normal is
approached. Coincident with this there is improvement in the sense of well being,
increaserl strength and gradual disappearance of all signs and symptoms. The
appetite improves, and dysphagia, if present, disappears_ Irregular menstrual
bleeding is usually corrected. Achlorhydria is the one outstanding finding that
234 THE ANEMIAS

apparently is not corrected by the iron therapy, and the patient recovers fully
except for this deficiency. There may be persistence of the papillary atrophy of
the tongue.
There is general agreement that large doses of iron correct the disorder and
restore the patient to health, but the maintenance dose should be determined
and used indefinitely or a relapse will occur in a few months. The dosage of iron
should be unusually large. Minot states that cases of idiopathic hypochromic
anemia with achlorhydria require distinctly larger doses of iron than do cases of
hypochromic anemia without achlorhydria. We prefer to use ferric ammonium
citrate in doses of IOO to ISO grains daily in capsules, taken wilh or just after
meals. If this dose cannot be tolerated due to gastric distress, smaller doses can
be employed in the beginning and then gradually raised as the improvement
justifies it. After restoration to normal has been effected, the iron intake should
be reduced to a maintenance level. This is usually one-third to one-fourth the
former dosage, and some patients seem to maintain good health with 20 to 25
grains daily. Since the ammonium salt seems to be the irritating factor in this
preparation, we have used ferric sodium citrate with good results. The amounts
used are approximately the same. Another excellent preparation for the restora-
tion of hemoglobin in this disease is the use of ferrous sulphate in doses of 30 to
45 grains daily.
Other supportive measures can be employed in conjunction with the iron
therapy, including a transfusion in those extremely ill, and dilute hydrochloric
acid by mouth, but the latter seems unnecessary unless there is prolonged
diarrhea, in which case it may serve to correct it. There seems little reason to
employ the organic iron preparations because they contain so little iron as to
be practically worthless when such large doses are required. There is disagree-
ment as to the value of copper. Mills has reported that the daily addition of 5
mg. of copper carbonate to the iron seems to aid in remission of the more re-
fractory cases and Dameshek has had a similar experience, but the consensus of
opinion is that copper is unnecessary. On the other hand, there seem to be no
ill effects from its continued use. The use of liver extract and desiccated hog
stomach is considered ineffective, but Murphy has contended that the iron
therapy in this as well as other types of hypochromic anemia should be supple-
mented with an occasional parenteral dose of liver extract. We believe that this
procedure is valuable, and it would seem that the more refractory 'cases might
receive some benefit from liver therapy supplementary to the iron.
There seems to be no justification whatever for the parenteral use of iron.
It is granted that good results are obtained If given in sufficient quantity, but
the injections are oftentimes followed by disagreeable or dangerous reactions.
Injections should be used only when it is impossible for the patient to take iron
by mouth, and these are rare instances.

COURSE AND PROGNOSIS

If patients receive adequate treatment, the outlook is very good, since nearly
all of them are restored to health, but as pointed out before, it is important that
 IDIOPATHIC HYPOCHROMIC ANEMIA 235
they remain on a maintenance dosage of iron. So long as this is done there will
be no recurrence of the disease. If the disease is untreated it becomes progres-
sively worse, the symptoms more pronounced, the anemia more marked and death
may result from intercurrent infections which presumably can easily develop
because of the debilitated state. Apparently there are no spontaneous remissions.

BIBLIOGRAPHY
IDIOPATHIC HYPOCHROMIC ANEMIA

BARTLETTr C. J.: HFamily pernicious anemia." Jour. Amer..Med. Assn.) 60, 176, 1913.
CASTLE, \V. B., HEATH, C. W., and STRAUSS, M. B.~ HObservations on the etiologic relationship
of achylia gastrica to pernicious anemia. IV. A biologic assay of the gastric secretion."
Amer. Jour. Med. Sci., 182, 520, 1931.
CLOUGII, P. W.: "Idiopathic hypochromic anemia/' lnternatl. Clinics, 3, ser. 45, I, Sept., 1935.
DAMESIIEK, W.; "Primary hypochromic anemia (erythronormoblastic anemia)." A mer. Jour.
lYIed. Sci., 182, 715, 1931.
FOWLER) W. M., and BARER, A. P.: "The etiology and treatment of idiopathic hypochromic anemia."
Amer. Jour. Med. Sci., 194, 625,11937.
HEATH, C. W.: "The interrelation of pernicious and idiopathic hypochromic ..anemia." Jour.
Glin. Invest., 11, 80S, 1932.
HOOVER, W. B.: liThe syndrome of anemia, glossitis, and dysphagia. Report of cases." New
Eng. Jour. Med., 213, 394, 1935.
HURST, A. F.: ~'Thc Plummer-Vinson syndrome." Guy's Hosp. Rep., 76, 426, 1926.
KELLY, A. E.: "Spasms at the entrance to the esophagus." Jour. Laryn. and Otol., 34, 285, 1919.
MACLACHLAN, \V. W. G., and KLmE, F. M.: "The occurrence of anemia in four generations."
Amer. Jour. Med. Sci., 172. 533, 1926.
MCFEE, L. C:, and GOODWIN, T. M.: "The syndrome of dysphagia and anemia.", Ann. Int. Med.,
11,1498 ,1938.
MILLs, E. S.: "The treatment of idiopathic (hypochromic) anemia with iron and copper." Can.
Med. Assn. Jour., 22, 175. 1930.
MURPHY, W. P.: "Rational treatment of the anemic patient." Ann. Int. Med., 7, 939, 1934.
VINSON, P. P.: "Hysterical dysphagia." Minn. Med., 5, 107, 1922.
WINTROBE, M. M., and BEEBE, R. T.: "Idiopathic hypochromic anemia." Medicine, 12, 187,
1933·
WITTS, L. J.: {{Simple achlorhydric anemia." Guy's Hosp. Rep., 80, 253, 1930.
 CHAPTER 18
THE HEMOLYTIC ANEMIAS
The hemolytic anemias include a large group of anemic states that are caused
by a more rapid destruction of red blood cells th,m the bone marrow can pro-
duce. They may be either acute or chronic and vary widely in their severity,
depending upon the type of etiologic agent that produces them, the duration of
exposure to the agent and the ability of the bone marrow to compensate for the
increased destruction of cells. It is indeed remarkable that the bone marrow in
the normal adult produces about one trillion red cells each day; or about ten
million cells each second during the 24 hours of the day which actually amounts
to about I eU.mm. of packed red cells per second. During this time an equal
number of cells is destroyed by the normal destructive mechanism, mainly in
the reticuloendothelium of the spleen and other tissues. Production of red cells,
therefore, must keep pace with destruction in order to maintain the normal
cellular values in the peripheral blood.
Excessive destruction 01 red cells at any time is always followed by a Com-
pensatory increased output of the bone marrow and evidence of this is seen in
increased numbers of reticulocytes in the blood. Only when the marrow is unable
to compensate for the excessive destruction do the signs of anemia appear. There-
fore, there are many situations in which an individual may be suffering from
excessive hemolysis of red cells, and the condition will actually remain undis-
covered. It is only when the marrOW fails to produce a sufficient number to
maintain cellular equilibrium that the patient becomes anemic and the state of
anemia is brought to the attention of the clinician. The development of anemia
from excessive hemolysis means that marrow compensation has failed and thus
anemia represents an advanced stage of such an action. It logically follows that
an increase in the products of cell destruction would be an accurate index to the
degree of activity of any hemolytic agent. .
. Red cells are destroyed by the process of hemolysis. This may occur (I) by
the action of toxic agents on the cells in the peripheral blood, or (2) by phagocytic
activity of endothelial cells. When hemolysis occurS the stroma of the red cell is
broken, hemoglobin is released into the blood plasma, the iron component of
hemoglobin is set free, and bilirubin is formed in the plasma in excessive amounts.
After release of the hemoglobin, the porphyrin fraction. is transformed into
bilirubin. This takes place in the reticulo-endothelial system and in the hepatic
cells. There is then formed a so-called bilirubin protein complex which does not
pass the kidney and which has been referred to as bilirubin type I. This is carried
in the blood stream to the hepatic cells where it is changed to bilirubin type II
and is excreted into the bile ducts and excreted as bile. After passage through
the intestinal tract this type II bilirubin is converted by bacterial action into
urobilin, most of which is excreted in the feces but some of which is reabsorbed
into the blood stream. Of that portion which is returned to the blood, a part is
236
 THE HEMOLYTIC ANEMIAS 237
re-excreted in the bile, a part of it is converted into hemoglobin, and the re-
mainder excreted by the kidneys as urobilinogen. Under normal conditions the
amount excreted is a direct indicator of the extent of cellular destruction. Since
the endothelial system has a considerable reserve capacity it is possible for great
numbers of red cells to be destroyed with the pigment metabolized by the en-
dothelium with the patient showing no increase of pigment in the blood plasma.
Therefore, the quantitative test for urobilinogen in the urine is probably a more
sensitive indicator for excessive destruction of red cells than any single laboratory
test, sinre it may be positive even though the red blood cells are normal in num-
ber and no evidence of anemia exists.
Based upon this concept, hemolytic anemias can be divided into three large
groups from a functional standpoint: (I), That situation in which excessive num-
bers of cells are being destroyed but in which the reticulo-endothelial system is
capable of metabolizing the pigment without increased bilirubin in the blood
plasma. In such situations an anemia would not necessarily exist. (2) When
larger numbers of red cells are destroyed, to such an extent that the endothelial
system cannot metabolize the excessive pigment and consequently there is an
excessive amount of bilirubin in the blood plasma which can be detected by the
usual tests, such as the van den Bergh reaction and icterus index. Such a patient
may show a latent or clinical jaundice. (3) The same process whereby even
larger numbers are destroyed so that there is free hemoglobin in the blood plasma
which may be passing the kidney threshold and the patient therefore may have
hemoglobinuria. In any of the above conditions the bone marrow is usually
making every effort to compensate for the increased destruction of cells. There-
fore reticulocytosis is also an accurate index as to the number of cells that has to
be replaced.
In summary, then, there are two reliable methods whereby the extent of red
cell destruction may be determined. These are (I) the number of reticulocytes in
the blood and (2) the amount of urobilinogen and other blood pigments in the
urine. The former is an index of cell production and the latter an index of cell
destruction.

THE BLOOD FINDINGS

Based upon the above concept, the patient with clinically detectable hemo-
lytic anemia will show certain signs. There mayor may not be jaundice, depend-
ing upon the extent of the hemolysis and the capacity of the endothelial system
to metabolize 'pigment. If there is increased bilirubin, there will be a positive
van den Bergh indirect reaction and an increased icterus index. In severe cases
there may be hemoglohinemia and in others th~re may he hemoglobinuria as well.
The urine always contains an excessive amount of urobilinogen and bile pig.
ments of the feces are increased. Since the bone marrow is an accurate indicator
of the amount of cell destruction, there are varying degrees of reticulocytosis.
The degree of anemia will vary, depending upon the balance that is being main-
tained between cell production and destruction. Usually there is an associated
slight to moderate leukocytosis with any type of hemolytic anemia because of
 THE ANEMIAS

the associated leukoplliesis accompanying the erythropoietic stimulation. In

severe cases the leukopoietic stimulation may be so marked that the leukocyte
count will reach a bigh figure with immature cell" including myelocytes, in the
peripheral blood, resulting in a leukemoid reaction. If the anemia is one of long
standing and the endothelium has been maintained in a state of overfunction over
a long period there is likely to be enlargement of the spleen. After a time the
bone marrow in its sustained effort to compensate becomes a hyperplastic type,
even to the extent of changing from a normal inactive yellow marrow to a red active
form. At times there may be a functional collapse of the marrow, with develop-
ment of a so-called erythroblastic crisis with showers of nucleated red cells into
the peripheral blood and other signs of regenerative activity, including basophilia,
basophilic stippling, Howell-Jolly bodies, and Cabot ring bodies.

MECHANISM OF HEMOLYSIS

The mechanism by which red cells E.<'e destroyed varies with different hemo-
lytic agents. In the classic congenital hemolytic icterus it is known that the
red cells are thick, spherocytic, and biconvex in,tead of biconcave. This has
long been thought to be caused by the production of a peculiar and defective
cell in the marrow. In this disease, however, only the mature cells in the periph-
eral blood, but not the reticulocytes, are spherocytic. This has led Dameshek to
the conclusion that such cells are acted upon by a hemolysin after they have
reached the blood stream, and that the sphericity of the cells is probably an·
initial stage of cell swelling, being the first stage of hemolysis. This in turn
would cause such a cell to become more readily susceptible to the normal de-
structive processes. '
In certain other forms of hemolytic anemia the mechanism of action is dif-
ferent and probably involves the interaction of poisonous agents with chemicals
within the cell. For example, saponin and bile are thought to act upon the pro-
tein in the cell. It is believed that snake venom, which is a powerful hemolytic
agent, acts on the lecithin and produces lysolecithin, which is known to be a very
active hemolytic agent. This in turn causes the cell to become spherical and
consequently more easily hemolyzed. In certain cases of hemolytic anemia, as
those caused by excessive destruction of cells in incompatible blood with trans-
fusions, this hemolysis is preceded by clumping or agglutination of the cells, and
this in turn followed by ingestion of the clumps by the phagocytic mechanism of
the endothelium. Furthermore, it is possible that some cells are produced in the
marrow that are inherently weak or defective in II way that is unkp.own at this
time.

CLASSIFICATION'

The classification· of this group of anemias is rather difficult because of the

large number and wide variety of etiologic agents that are known to produce
hemolysis of red cells. However, any classification that is based on an etiologic
concept is always worth while and I consider the following satisfactory for clini-
 THE HEMOLYTIC ANEMIAS 239

cal purposes in light of present knowledge. It is based upon knowledge of possible

factors that are known to produce excessive hemolysis.
I. Hemolytic anemias caused by infections.
Tbese are seen in generalized septicemia, infections with the hemolytic streptococci, B,
welchii, in malaria, Oroya fever, and rare1y in syphilis.
II. Hemolytic anemias caused by drugs~ chemicals, and poisons.
These include those caused by lead J arseniureted hydrogen, phenylhydrazine, acetyIphenyl~
bydrazin, pyridine, potassium chlorate, sulfanilamide, sulfapyridine, sulfathiazole, phos..
pborus, dinitrobenzene, dinitrophenol, trinitrotoluene, saponin, ricin, snake venom, organic
arsenical compounds, and iodo-acetic acid .•
III. Hemolytic anerm'as caused by hemolysins and agglutinins.
a. Incompatible blood transfusions.
h. Dameshek's experimental bemolytic anemia.
c. Presence of possible auto-agglutinins in the blood.
IV. H emotytic anemias that are clinical entities.
a. Chronic familial hemolytic anemia.
b. Sickle cell anemia.
c. Lederer's acute hemolytic-anemia.
d. Erythroblastic anemias of childhood.
c. Acquired hemolytic anemia.
V. Paroxysmal hemoglobinuria.
a. Cold hemoglobinuria.
b. March hemoglobinuria.
c. Nocturnal hemoglobinuria.
d. Allergic hemoglobinuria.
e. Myoglobinuria.

HEMOLYTIC ANEMIAS CAUSED BY INFECTIONS

It has long been recognized that many types of infections will produce
varying degrees of hemolytic anemia. Such conditions as generalized septicemia,
caused by tbe staphylococci or streptococci will produce anemia with increased
output of urinary urobilinogen. Even localized infections with the hemolytic
streptococci will produce hemolytic anemia and this is particularly true in blood
stream invasion with this organism and notably in puerperal sepsis.
Infections with the Clostridium welchi/, or bacillus of gas gangrene, are said
to produce a marked degree of hemolytic anemia in a remarkably short period
of time. Because of the rapidity of cell destruction there may be hemoglobinuria,
the development of hemolytic jaundice with an indirect van den Bergh reaction,
an associated leukocytosis that may be as high as 50,000 cells per cU.mm. or more
with the presence of immature white and red cells. With control of the infection
the blood soon returns to normal. A transfusion may be necessary for the cor-
rection of the anemia.
Varying degrees of anemia may develop during infection with syphilis, par-
ticularly in the secondary stage when the Treponemata are invading the various
tissues. Although the anemia may not be particularly severe and the hemolysis
not sufficient to produce clinical jaundice, yet excessive numbers of cells may
be destroyed.
* Finland and his associates found no instance of hemolytic anemia in 446 patients treated
with sulfadiazine (Jour. Amer. Med. Assn. II6:2641 (June 14), 1941).
 THE ANEMIAS

It has long been recognized that infection with malarial parasites is likely to
produce severe hemolytic anemia. This is caused by large numbers of red cells
becoming parasitized and destroyed by rupture associated with chills. For more
detailed findings in the plood in malaria see Chapter 42. Knisely and his asso-
ciates at the University o~ Tennessee have been able to observe and photograph
the changes in the capillaries of living monkeys that are heavily parasitized with
malaria. They noted a clumping of the parasitized red cells in large groups
with these being destroyed by hemolysis. Associated with these changes is a
reticulocytosis, even up to IS or 20 per cent, to enable the bone marrow to main-
tain the red cells at the normal level. The most severe anemia occurs in the
estivo-autumnal type, and when there is passage of large amounts of hemoglobin
in the urine the condition is known as Blackwater fever. The hemolysis may be
so severe that the hemoglobin may block the renal tubules and the patient die
from anuria with retention of nitrogenous products. In order to avoid this the
patient should be alkalinized to prevent the formation of the acid hematin in the
renal tubules. In cases of chronic malaria there is a slight and constant destruc-
tion of red cells with a subclinical type of jaundice, with increased urobilinogen
in the urine and a slightly increased sustained reticulocytosis. Finally there is
a hypochromic microcytic anemia.
In Oroya fever, sometimes known as Bartonella fever, there are usually vary-
ing degrees of hemolytic anemia. This disea,se occurs mainly in South America
and is caused by the Bartonella bacillifarmis. The red cells are destroyed in
tremendous numbers and this is accompanied by clinical jaundice and severe
anemia, enlargement of the spleen, a hyperplastic bone marrow showing consid-
erable numbers of megaloblasts, marked reduction of hemoglobin and red cells,
increased reticulocytes, and frequently erythroblastic crises characterized by
normoblasts in the peripheral blood. There is usually an associated leukocytosis
with a marked shift to the left. Transfusions are frequently required to aid the
marrow in maintaining a sufficient number of red cells to be compatible with
life. In all types of hemolytic anemia that are chronic and extend over a long
period of time, there finally develops microcytosis and hypochromia in the red
cells.

HEMOLYTIC ANEMIAS FROM CHEMICALS, DRUGS, AND POISONS

Among the agents that are known to produce hemolytic anemia is lead. For
"details of the blood picture seen in this type of poisoning consult the section on
lead poisoning. In brief, this may develop from poisoning in industry, among
painters, paint workers and paint chippers, in children who ingest lead paint from
gnawing their cribs, among people who ingest the metal from lead pipes and in
, those who absorb lead from certain cosmetics. The blood findings include the
development of a hemolytk anemia with compensatory reticulocytosis and baso-
philic changes in the blood, particularly stippling of the red cells. Rapid hemoly-
sis of the cells is more likely to occur in cases of acute poisoning rather than the
chronic type.
Anemia may also develop by inhalation of arseniuretted hydrogen. This is
followed by severe abdominal cramps, vomiting, diarrhea, headache, and varying
 THE HEMOLYTIC ANEMIAS

degrees of jaundice, depending upon the' rate of red cell destruction. There is a
positive indirect van den Bergh reaction with high icterus index. There is com-
pensatory bone marrow activity in the form of reticulocytosis, and not infre-
quently a normoblastic reaction in the peripheral blood. Some cases may be so
severe as to produce hemoglobinemia and hemoglobinuria.
The toxic action on red cells of phenylhydrazine and acetylphenylhydrazine
has long been recognized. It is such an effective agent in this respect that it has
been used therapeutically in the treatment of polycythemia vera (see p. 494). Ex-
posure to this substance seldom occurs unless it is being used as a therapeutic
agent. It mUst be used with great caution since its action is cumulative. Other
chemicals that are known to produce varying degrees of hemolytic anemia are
bemedri128, potassium chlorate, phosphorus, dinitrobenzene, trinitrotoluene, pyri-
dine, toluene diamine, and all of the nitro compounds of toluol, benzol, and phenol,
and no doubt other similar preparations.
Parker at Emory University has noted severe hemolytic anemia in rabbits
that were receiving intravenous and subcutaneous injections of minute quantities
of iodo-acetic acid. This was accompanied by a marked reticulocytosis and evi-
dence of excessive cell destruction in the form of large quantities of uribilinogen
in the urine and even hemoglobinuria. It has been stated that acetanilid and
phenacetin are capable of producing hemolytic anemia, although there is no
reliable evidence to substantiate this. It is recognized that these drugs may
produce methemoglobinemia in occasional instances, but without destruction of
red blood cells.
Another class of agents tbat are known to produce severe grades of hemolytic
anemia includes sapon;", ricin, snake venom, and the ingestion of a certain type
of bean, Vicia Java, that is indigenous to southern Italy and Sicily. The ingestion
of this bean or inhalation of its pollen may produce marked degrees of hemolytic
anemia. The condition is known as fabism or fabismus. It is thought to be allergic
in type, because first exposure to the plant does not usually produce the anemia.

HEMOLYTIC ANEMIA FROM THE SULFONAMIDE GROUP (SULFANILAMIDE,

SULFAPYRIDINE, SULFATHIAZOLE, SULFADIAZINE)
In 1937 Harvey and Janeway reported that sulfanilamide and sulfapyridine
were capable of producing marked decreases in the red cells and hemoglobin and
in some instances a severe acute hemolytic anemia. They reported three cases in
which the red cells showed an alarming and precipitous fall in short periods of
time, in one case from 4.9 to 1.5 millions in five days, in another from 4.2 to 2.2
millions in 36 hours, and in another, an infant, a fall from 4.2 to 2.0 millions in
seven days. Also another interesting feature was the coincident marked leuko-
cytosis that accompanied the anemia, being 87,000, 30,000, and 32,000 white cells
per cu.mm. respectively.
An example of the rapidity of red cell and hemoglobin decrease in a susceptible patient may be
illustrated by the following case. A 40-year-old woman was admitted to the Emory Hospital for
sDlenectomy for familial hemolytic icterus. At the time of operation the red cell count was 3,400,000
cells per ell.mm. and immediately afterwards the same. The count remained stationary for two
days at which time a postoperative pneumonia deVeloped. Along with other treatment sulfapyridine
was employed in the usual therapeutic dosage. Within 36 hours the red cell count fell from
3,500,000 to 1,800,000 with the usual evidences of increased blood cell destruction. The presenting
242 THE ANEMIAS

problem was whether to continue or omit the drug. It was omitted and the red cell count gradually
rose to normal limits in a Jew weeks.
There is adequate evidence that these drugs in a small percentage of patients
are capable of producing the most severe anemic states. Wood reported 2 I cases
of acute anemia in 512 patients treated with sulfanilamide, an incidence of 2.4 per
cent in adults and 8.3 per cent in children. Also milder depressions of the red
cells are seen in many cases, but not sufficient in extent 1.0 classify as a true
hemolytic anemia. Watson and Spink have presented adequate evidence that
these drugs produce accelerated hemolysis in all patients.
The disorder is characterized by increasing pallor, usually with cyanosis,
nearly always fever, and a rapid drop in the red cells and hemoglobin. At the
same time there is evidence of increased red cell destruction, with reticulocytosis,
increased icterus index with an indirect van den Bergh reaction, urobilinogen, and
perhaps clinical jaundice. On the appearance of such signs, the blood picture
should be investigated and treatment with the drug discontinued.
Usually the anemia oCcurS early in the course of treatment, appearing in the
second to the fifth day, so that large amounts of the drug are not required for its
production. This is different from the appearance of agranulocytosis which devel-
ops only after the prolonged administration of large amounts of the drug.
The mechanism of action on the red cells is not known, but is certainly a
process of hemolysis. Whether or not this may be preceded by agglutination of the
cells is questionable. The fundamental difference between the cells of susceptible
and non-susceptible persons is not known. There is no evidence of depression of
the bone marrow as shown by the reticulocytosis. Morphologic studies of the
blood reveal no abnormalities. The anemia is usually normocytic in type and the
cell characteristics change -little from the picture before the anemia occurred.
Removal of the drug is followed by cessation of the hemolytic process.
This may be a serious complication, as shown by two fatal cases, one re-
ported by Wood from sulfanilamide and another by Ravid and Chesner from
sulfapyridine. In both instances the mode of death was similar and apparently
was caused hy blocking of the renal tubules with masses of hemoglobin released
during the hemolytic process, with the patients dying from uremia. This picture
is identical with that seen in the kidneys after transfusion of incQmpatible blood,
which again suggests that agglutination of cells may precede the hemolysis. The
damage to the kidney tubules is also identical with that described by DeGowin
and his associates who transfused dogs with canine hemoglobin and observed
uremic deaths because of kidney tubular blockage, and more important, observed
it only in those animals in which the urines were acid in reaction. They state that
this complication can be prevented by alkalinizing the urine. From these studies
it would appear that the best means of avoiding severe damage to the kidney is to
alkalinize the patient during administration of the drug. Fortunately, this is
usually done anyway to prevent the development of acidosis.
When acute hemolytic anemia develops to a sufficient extent to produce a
severe anemia with the hemoglobin falling below 60 per cent, the blood values
should be restored by transfusions. If possible, the medication should be stopped
at once. Sometimes it is not advisable to do this in suru cases as streptococcus
 THE HEMOLYTIC ANEMIAS 243
blood stream infections, meningitis, and other serious disorders where the life of
the patient depends on continuation of therapy. The problem then is to replace
blood with transfusions faster than it is destroyed, to keep the urine alkaline, and
study the patient for threatened kidney damage as manifested by suppression of
urinary output or rising nitrogenous products in the blood. Fluids should be
given freely.
There is adequate evidence that sulfapyridine is also capable of producing
acute hemolytic anemia, and it seems to be equally as dangerous as sulfanilamide.
In treating 446 patients with sulfadiazine Finland and his associates noted no
inst'lnce of hemolytic anemia, and only nine instances of mild leukocyte depres-
sion.
I have not observed either agranulocytosis or acute hemolytic anemia follow-
ing the use of neo-prontosi!. This may be because this compound is used relatively
rarely as compared with sulfanilamide and sulfapyridine. Sulfathiazole produces
acute hemolytic anemia rarely if at all. In 271 patients studied hy Long and his
associates it was ,not observed in a single instance, and early in 1941 only one
case of agranulocytosis had been reported (Kennedy and Finland) and even this
was questionable. Sulfathiazole has been so recently introduced that the reports
from its use have not accumulated in sufficient number to provide valuable infor-
mation as to its capacity to produce hemolytic anemia. Long has stated that
sulfanilamide produces hemolytic anemia in approximately 3 per cent of the cases,
and severe hemolytic anemia in 1.8 per cent of treated cases between the first and
fifth day of treatment; and that only 0.6 per cent of patients treated with sulfa-
pyridine develop hemolytic anemia which usually occurs between the first and
fifth day of treatment; and he further states that mild cases of hemolytic anemia
from sulfathiazole have not been reported. Polini and his associates found that
in 169 patients treated with sulfathiazole there were no cases of hemolytic anemia.
Regardless of the drug that produces hemolytic anemia the treatment should
be: immediate cessation of the drug, multiple blood transfusions, forced fluids, and
alkalinization. In an occasional patient the sulfonamide gronp of drugs produce
an astounding degree of leukocytosis, although this bears no relation to the produc-
tion of hemolytic anemia. Furthermore, the methemoglobinemia and cyanosis
seen in so many of these patients are not associated with the production of hemo-
lytic anemia, and the development of cyanosis seems to be without clinical impor-
tance. Acute hemolytic anemia usnally develops early in treatment and white
cell changes as a rule develop late in treatment. For the depressive effects of this
gronp of drugs on the white cells see page 151.

HEMOLYTIC ANEMIAS CAUSED BY HEMOLYSINS AND RELATED BODIES

This group of hemolytic anemias includes those in which there is rapid de-
struction of transfused red blood cells because of incompatible blood, in which
donor's cells are immediately clumped and subsequently destroyed by the process
of hemolysis. In addition to this there are numerous examples whereby blood is
known to be compatible by ordinary technical methods, bnt nevertheless there
is destruction of the donor's cells. For further details of these reactions consult
the chapter on blood transfusions. Also there is an occasional severe type of
244 fHE ANEMIAS

hemolytic anemia that is caused apparently by auto-agglutinins in the patient's

blood. We have studied one patient at tbe Emory University Hospital that
showed a profound anemia, with red cells only one million per CU.mm. in whom
the patient's plasma not only agglutinated blood cells of all four types, but
agglutinated her own cells as well. This resulted in prolonged blood destruction,
increased urobilinogen, increased biliary pigments in the plasma, a subclinical
jaundice, and efforts of the bone marrow to compensate with a sustained high
reticulocytosis and showers of erythroblasts.
Then there is a type of experimental hemolytic anemia repo'rted by Dameshek
and his associates. These workers studied three cases of familial hemolytic
anemia and concluded that the blood of each contained hemolysins. They ap-
proached the problem by injecting rabbits with guinea pig cells and producing,
therefore, guinea pig hemolysins in the rabbit serum. The subsequent injection
of this serum into guinea pigs produced various gradations of hemolysis and cell
destruction. By using different doses they were able to produce an acute hemo-
lytic anemia, sub-acute hemolytic anemia, or even the chronic form, witl, micro-
spherocytosis and increased fragility of the cells. They came to the conclusion
that spherocytosis is produced by the action of some type of hemolysin, and
tllat it is the first stage of cell destruction which is subsequently completed after
the cells reach the peripheral blood where they are acted upon by the endothelial
system. Tigertt and his associates produced anti dog cell hemolysins in rabbits,
which upon injection into I I dogs, produced spherocytosis, increased fragility,
and varying grades of anemia. The importance of this work would be the sng-
gestion that patients with congenital hemolytic icterus do not inherit a deformed
cell but do inherit an atypical type of hemolysin which produces sphericity of the
cells. Furthermore, the fact that these patients frequently develop severe reac-
tions following the transfusion of what appears to be perfectly compatible blood
may lend further support to that assumption.

ACQUIRED HEMOLYTIC ANEMIA

The congenital type of hemolytic anemia is often referred to as the Chauffard-
Minkowski type and the acquired form as the Hayem-Widal type, bearing the
names. of the investigators who first descrihed them. There is considerable
divergence of opinion among hematologists as to the existence of the acquired
type of hemolytic anemia. It is quite possible that it develops spontaneously at
various periods of life without an apparent hereditary factor and may be caused
by a number of etiologic agents, perhaps some of those that have been previously
discussed.
Acquired hemolytic anemia is usually characterized by a sudden onset in an
adult who has not shown evidence of hemolytic anemia during childhood. The
red cells fall to an extremely low level with the hemoglobin reduced in proportion.
Microcytosis and spherocytosis are not characteristic, although it has been stated
that the cell thickness is a little more than the average. The reticulocyte count
is usually quite high, but the fragility of the red cells is normal. The patient
with the congenital type is more icteric than sick, and in this form the patient is
more sick than icteric. There is evidence to suggest that certain infectious dis-
 THE HEMOLYTIC ANEMIAS 245
eases such as malaria, syphilis, tuberculosis, Hodgkin's disease, and various live!
diseases may be causative. Another possibility is that these patients may develop
unusual hemolysins in the blood either in the form of auto-hemolysins or auto·
agglutinins and this may account for the difficulty in transfusing them. The
diagnosis is not always easy to establish, but is usually done on the presence 01
over-activity of the bone marrow in the form of reticulocytosis and erythroblastic
showers in the peripheral blood, with the presence of increased quantities of
urobilinogen in the urine, and possibly either a clinical or a subclinical jaundice
with increased bilirubin in the blood plasma. The red cells may show varying
degrees of macrocytosis so that pernicious anemia may be suspected. The spleen
is usnally enlarged to a variable extent so that various splenomegalic diseases
must be considered in the differential diagnosis. If the patient is studied during
a crisis, the diagnosis of leukemia may be made incorrectly, because of the
extreme myeloid hyperplasia with the presence of myelocytes and perhaps myelo-
blasts in the blood. The fact that it fails to respond well to blood transfusions
would rule out the acute hemolytic anemia of Lederer.
The treatment of acquired hemolytic anemia consists of bed rest, particularly
during the crises, and the use of multiple transfusions provided no untoward
reactions are associated with them. Splenectomy has been tried in a considerable
number of cases, but in general the results are disappointing. Even if the spleen
is removed, the prognosis of the disease is not good. If the balance between
cell production in the marrow and cell destruction can be maintained, even
though ·it is maintained at a level of moderate anemia, the patient can live for
a good many years, half well and half sick. It is believed. that the earlier sple.
nectomy is attempted, the better the prognosis is likely to be.
To summarize the features of acquired hemolytic icterus: It seems to be a
disease that is acquired during middle or late life, accompanied by severe attacks
of red cell destruction with attempts of the bone marrow to compensate for this
destruction, that the prognosis is not particularly good but the patien't may live
on for a number of years in a fair to poor state of health. Splenectomy appar-
ently does not effect a cure, although it may to some extent be palliative. It
seems likely that the disease may be caused by the presence of some unrecog-
nized infection, notably syphilis or malaria.

ACUTE HEMOLYTIC ANEMIA OF LEDERER

This is a rare type of acute hemolytic anemia which develops suddenly with
the patient showing evidence of an acute infecti6us process and which reacts
dramatically to single or repeated blood transfusions. It was first described by
Lederer in '925., The causative organism is not known. Dameshek and Schwartz
believe that it may be caused by a hemolysin that develops quickly and subsides
in a short time. The disease is seen mainly in young people. The onset is usually
acute but there may be a period of a few days of premonitory symptoms followed
by development of the full blown disease, consisting of fever, vomiting, nausea,
various aches and pains in the muscles, particularly in the back, and a more or
less constant headache. There mayor may not be diarrhea and abdominal pain
is common. The fever is variable and seldom subsides elltirely to normal but
 THE ANEMIAS

ranges between 100 and 104 degrees F. and remains so until blood transfusions
are given.
Tbe blood picture usually shows a rather severe anemia in whicb the red
cells may be as low as one million per eU.mm. and the hemoglobin low in pro-
portion. Tbere is usually a marked reticulocytosis, sometimes as higb as So per
cent, and in the acute phase the peripheral blood is usually showered with
erythroblasts. 'rhey have been reported to be as high as '5,000 per CU.mm. In
some patients the anemia is a macrocytic one, in others normocytic, and in still
otbers there may be a slight microcytosis. Tbere is an associated marked leuko-
cytosis wbich may be as high as 40 to So tbousand cells per eU.mm. with a marked
shift to the left including a few myelocytes and an occasional myeloblast. It
presents, therefore, a distinct leukemoid reaction. There are the usual evidences
of excessive destruction of red cells in the form of a slight clinical or subclinical
jaundice, increased bilirubin in the blood plasma, and increased urobilinogen in
the urine. 'rhere mayor may not be a slight enlargement of the spleen.
The disease is treated with blood transfusions to which there is a dramatic
response. From the first transfusion there is improvement in the clinical course
of the patient and the hematological findings are soon corrected. Several trans-
fusions are usually required before complete recovery takes place. Since tbe
cause is unknown, tbere is no indication for further therapy. Sub-acute and
chronic forms of hemolytic anemia have also been described and are thought to be
more prolonged courses of the same disease. (See Giordano and Blum for a
thorough discussion of Lederer's Anemia.)

BIBLIOGRAPHY

:I.ANOTOPOL, 'v., ApPLEBAl.Tl.f, I., and GoWlLUr, L.: "Two cases of acute hemolytic anemia with
auto-agglutination following sulfanilamide therapy." JOUT. Amer. Med. Assoc., 113, 488,
1939·
2. DAMESREX, 'V" and SClIWARTZ, S.D.: liThe experimental production of various types of hemo-
lytic syndromes by hemolytic sera with especial reference to spherocytosis, increased fragility
test and reticulocytosis." Jour. CUn.Invest ~ 17, 501, 1938.
3. DAMESIlEX, W., and SCHWARTZ, S.D.: ('Acute hemolytic anemia (acquired hemolytic icterus,
acute type)." Medicine, 19, 231, 1940.
4. DEGOWDi, E. L.: "Grave sequelae of blood transfusions; a clinical study of 13 cases occurring
in 3500 blood transfusions." Ann. Int. Med., 11. 1'1'11. 1938.
5. D:r;GoWIN, E. L., WARNER, E. D., and RANDALL, W. L.: "Renal insufficiency from blood trans-
fusion. II. Anatomic changes in man compared with those in dogs with experimental. hemo-
globinuria.1I Arch. Tnt. }.fed., 61, 609, 1938.
6. GIORDANO, A. 5., and BLUM, L. L.: "Acute hemolytic anemia (Lederer type)." Amer. Jour.
lied. Sci., 194, 3Il, 193'7.
7. GREENWAI.D. H. M.: "Acute hemolytic anemia." Amer. Jour. Med. Sd., 196, r79, 1938.
B. HAvEN, R. L.: "The nature of hemolytic anemia." Symposium on DIood. Univ. of Wis. Pres.s.
- 1939; p. 83.
9. HAJ!.VEY, A. M., and JANEWAY, C. A.: liThe development of acute hemolytic anemia during thl!
administration of sulfanilamide (paraminobemenesulfonamide).11 Jour. Amer. Med. Assoc.,
I09, 12, 1937.
Kn"NEDY, P. C., and FINLAND, F.: "AgranulocytoSis from sulfathiatole." Jour. Amer. Med.
Assoc., 116, 29$, 1941.
 TBE HEMOLYTIC ANE:MIAS 247
JI. Lmm, P. H.: "The clinical use of sulfanilamide and Its derivati\'es in the treatment and prophy~
laxis of certain infections." Bull. N. Y. Acad. Med., 16, 732, 1940.
12. LONG, P. H" and BLISS, E. A : "The clinical and experimental use of sulfanilamide, sulfapyridine
and allied compounds" The Macmillan Co. 1939.
13 PEPPER, O. H P.: "A survey of the so·caJIed hemolytic anemias." Ann. Int., Med J 12, 796, 1938.
14. RAvlD, J. M., and CHESNER, C.: "A fatal case of hemolytic anemia and nephrotic uremia follo\ling
sulfapyridme administration." Ame,.. Jou,.. Med. Sci. 199, 380, 1940
IS. TXGERTT, W. D., and DUNCAN, C. N,: "Erythrocyte morphology in experimental hemolytit:
anemia as induced by specific hemolysln." Amer. Jour. Med. Sci, 200, 173, 1940.
16. WATSON, C. J.: "Hemolytic jaundice and macrocytic hemolytic anemia: Certain obserVations in
~ a series of 35 cases." Ann. Int. Med, 12, 1782, I939.
17 WATSON, C. J, and SPINK, W. W.: "Effect of sulfanilamide and sulfapyridine Qn hemoglobin
metabolism and·hepatic function." Arch. Int. Med., 65. 825. 1940.
18 WOOD, H.: "A fatality from acute hemolytic anemia which developed during the admini<;tration
of sulfanilamide." South. Med. Jour., 31, 646) 1938.
19 WOOD, W. B., JR: "Anetnia during sulfanilamide therapy." Jour. Amer. JIed. Assoc., 111,
1916, 1938
 CHAPTER 19
HEMOGLOBINpRIA
As stated before, hemoglobinuria is a manifestation of excessive destruction
of red cells in large numbers, to such an extent that the phagocytic mechanism
of the endothelial system is unable to metabolize the hemoglobin pigments. In
such instances there,is free hemoglobin in the blood plasma (hemoglobinemia)
and hemoglobinuria. The pigment exists in the urine as free hemoglobin without
the presence of red cells, which if present are few in number and chiefly shadow
cells. This is in contrast to hematuria in which red cells are numerous and free
pigment is absent or slight in amount. Hemoglobinemia arises from excessive
cell destruction and hel11aturia usually from bleeding in the urinary traCt, acute
nephritis, etc.
In r854, Dressler noted that the color of the urine was caused by hemoglobin
and not whole blood. Ponfick, in r883, determined that hemoglobin alone resulted
from a preceding'hemolysis in the blood stream.
The color of the urine varies from light red to black depending upon the
concentration of the pigment. Even when the urine is quite dark the color is
seen to be red when held before a strong light. In spectroscopic examination the
two absorption bands of oxyhemoglobin are seen, but if bands-of methemoglobin
are found it can be assumed that the hemoglobin has been reduced by some
substance in the urine or ;m agent in the blood which has caused not only an
intravascular hemolysis but has reduced oxyhemoglobin to methemoglobin as
seen from the action of such drugs as acetanilid, sulfanilamide, carbon monoxide,
etc.
Under normal conditions the blood plasma does not contain free hemoglobin,
since the reticulo·endothelial system has the capacity to metabolize blood pig-
ment liberated in the normal process of red cell destruction and even sufficient
reserve for moderate degrees of cell destruction.
The presence of hemoglobinuria depends on two factors; first, there must be
hemoglobinemia, regardless of the agent causing the intravascular destru~tion
of the erythrocytes; and secondly, the renal threshold for hemoglobin. The
plasma concentration of hemoglobin necessary for tbe appearance of this pigment
in urine has been found to vary considerably. In five cases studied by Ham
hemoglobin was absent in the urine when the plasma concentration was below
30 mg. per cent but was present when between 30 to roo mg. per cent, and marked
when between 100 and 300 mg. per cent. In a group of normal human subjects
Ottenberg and Fox observed that hemoglobin would appear in the urine of some
with a plasma level of 96 mg. per 100 cc. and be absent in others whose plasma
concentration rose to 288 mg. per 100 cc. However, in the average subject it
appeared in the urine when the plasma level was ISO mg. per cent. At the same
time a more constant renal threshold for human hemoglobin was demonstrated in
terms of dosage per kilogram of hody weight. Strangely, it varied in the two sexes.
248
 HEMOGLOBINURIA 249

The dosage in the female was 73 mg. and in the male 92 mg. per kilogram of body
weight. An explanation for the presence of hemoglobinuria in various plasma con-
centrations might be found in the "glomerular threshold" concept of Whipple
whereby it is assumed that hemoglobin passes through the glomerular filter and
is rapidly reabsorbed by the tubular epithelium of the kidneys. As the rate 01
filtration by the glomeruli exceeds the rate of absorption by the tubules, hemo-
globin appears in the urine. This concept could explain the various renal thresh-
olds that have been obtained for hemoglobin. Thus, patients with impaired
tubular function and power of concentration would demonstrate a lower threshold
and this might explain the low threshold in the cases cited by Ham, as those
patients had a chronic hemolytic anemia with hemoglobinemia and paroxysmal
hemoglobinuria, and undoubtedly, some degree of hemosiderosis of the tubulaI
epithelium.
CLASSIFICATION

Any classification of the hemoglobin urias is not without criticism and will
probably remain so until further clarification of the various types of hemolytic
anemia. They can be divided into two principal groups: (1) hemoglobinuria and
(2) paroxysmal hembglobinuria.
1. Hemoglobinuria.
A. Exogenous causes.
1. Chemical agents.
2. Febrile intoxications.
3. Parasites.
a. Malaria.
b. Oroya fever.
4. Transfusions.
5. Burns.
6. Cobra and spider venom.
7. Bay sickness.
B. Endogenous.
1. Familial hemolytic anemia in severe relapse.
2. Atypical hemolytic anemias.
3. Acute hemolytic anemia of Lederer.
4. Cooley's anemia of childhood and infancy.
5. Sickle cell anemia.
II. Paroxysmal Hemoglobinuria.
1. Cold hemoglobinuria.
2. March hemoglobinuria.
3. Nocturnal hemoglobinuria.
4. Allergic hemoglobinuria.
5. Paroxysmal myoglobinuria?
HEMOGLOBINURIA

This group can be divided into the exogenous and endogenous types.
250 THE ANEMIAs

EXOGENOUS TYPE
This follows the entrance into the body of some toxic chemical that acts as
a hemolytic agent for red blood corpuscles. In some instances these substances
are given for therapeutic purposes. They include those mentioned before that
produce hemolytic anemia (see p. 24Q). It should be borne in mind that the dye
neoprontosil retains its color when excreted by the urinary tract, and should not
be confused with hemoglobinuria. If necessary a spectroscopic examination of
the urine can be done for differentiation of the dye and hemoglobin.
In occasional, instances the intoxication accompanying severe bacterial in-
fections is sufficient to cause a dissolution of the red corpuscles as seen in such
diseases as typhoid fever, scarlet fever, and yellow fever.
Malaria, particularly the cstivo-autumnal type, is a common cause of hemo-
globinuria, and when it occurs the condition has been called black water fever.
The parasite invades the red corpuscle, matures, and bursts the cells, thereby
destroying the erythrocytes and liberating hemoglobin to be excreted in the urine
if the renal threshold is exceeded.
The most striking feature of Oroya fever is the severe rapidly developing
anemia that is caused by a small pleomorphic organism, Bartonella bacilliformis,
that gains access to the red cells and endothelial cells. The red cell count may
fall to 1,000,000 cells per cU.mm. within a period of 3 or 4 days. The mortality
rate in the febrile stage is 30-40 per cent. So far this disease has been confined to
South America.
Transfusion of incompatible blood is frequently followed by hemoglobinuria.
Bordley determined that the injection of from 20-100 cc. of incompatible blood
is sufficient to cause hemoglobinuria. He concluded that no case receiving less
than 350 cc. of incompatible blood died from urinary suppression and no case
receiving more than 500 cc. recovered. Of course, this would depend upon the
degree of incompatibility, kidney function, and probably many other factors.
In fact, I have observed the transfusion of 500 cc. of type A blood into a type B
patient with no reaction whatever.
In cases of incompatible blood transfusions death is caused by anuria and
the retention of nitrogenous products. The anemia is caused by the precipitation
of hemoglobin in the renal tubules in the form of hematin crystals which produces
a mechanical obstruction. There is also necrosis of the tubular epithelium and
interstitial edema. The tubular epithelium, if not too severely damaged, has
marked regenerative capacity and returns to normal. Baker and Dobbs demon-
strated that a solution of hemoglobin was excreted unchanged by the kidneys
without deleterious effect on the animal unless the urine was acid with a pH of
6.0 or lower, in which case the hemoglobin would be precipitated in the tubules.
DeGowin transfused dogs with canine hemoglobin and also demonstrated that
hemoglobin is precipitated in the renal tubules when the urine is acid but not if
alkaline. The' animals with acid urine died with uremia whereas those having
alkaline urine could be given larger quantities of hemoglobin without precipita-
tion in the tubules and with subsequent recovery of the animal. There was no
hemosiderin deposition in the tubular epithelium and he concluded that the
 HEMOGLOBINURIA

deposition of hemosiderin in the renal tubules did not influence the development
of an acute renal insufficiency. Therefore, it is important to alkalize the urine of
patients with reactions following transfusions of whole blood if any hemoglo-
binuria occurs or uremia is impending.
Hemoglobinuria is sometimes observed in patients suffering from extensive
bums. Cobra snake venom is also hemolytic and acts by removing the unsat-
urated fatty acid from the lecithin molecule and producing lysolecithin which
itself is a powerful hemolytic agent. Gotten cites the case of a three year old girl
who had a spider bite and developed hemoglobinuria with an accompanying drop
in the red cell count to 2,940,000. A transfusion was given and complete recovery
followed. The spider was not a black widow but an Amaurobius jevox.
Stoeltzner has described a disorder known as Bay Sickness that is indigenous
to the region of Konigsberg, ·Germany. It is characterized by pain in the muscles,
stiffness, limitation of movement, and hemoglobinuria which has a tendency to
appear as a paroxysmal type.
The cause of this condition is the ingestion of resinous acids that are by-
products of cellulose factories of Konigsberg. These acids are drained into the
near-by bay after whiclr the fish and eels of the bay ingest them, and if caught
and eaten, transfer their poison to the host. Cats fed with these fish become ill
and develop hemoglobinuria along with weakness and progressive impairment
of muscular movement. At autopsy the striated musculature of cats and humans
is greyish red and resembles fish flesh.
The urine is red and resembles that containing free hemoglobin. However,
spectroscopic examination of the urine and the blood plasma discloses absorption
bands of myoglobin rather than hemoglobin. Also, there is no accompanying
anemia or evidence of excessive erythrocyte regeneration. Therefore, bay sick-
ness is probably not a true hemoglobinuria.

ENDOGENOUS TYPE
This group of diseases are those characterized mainly by anemia, run a more
or less subacute or clrronic course, and at times may show hemoglobinuria only if
the destructive cellular process is sufficiently severe. They are described in detail
elsewhere. The most common one is familial hemolytic jaundice in a severe
relapse. This' type responds well to splenectomy after which the patient seldom
has further difficulty. Atypical hemolytic anemias of the acquired type are
occasionally encountered that have relapses accompanied by hemoglobinuria. In
this group, there may be reactions from intravascular hemolysis after well-
matched transfusions. The etiological agents are obscure. Treatment is sympto-
matic and splenectomy is of no value.
The severe cases of acute hemolytic anemia of Lederer may be accompanied
by hemoglobin in the urine and since its cause is unknown there is no specific
therapy, but it is usually cured by transfusions (see p. 245). Cooley's anemia of
infancy and childhood and sickle cell anemia may be accompanied by hemo-
globinuria in some of the more severe erythroblastic crises.
 THE ANEMIAS

PAROXYSMAL HEMOGLOBINURIA

This group is characterized by transitory, irregular, or sporadic appearance

of hemoglobin in the urine. There are four recognized types.
Paroxysmal Cold Hemoglobinuria. This is a rare disease characterized by
the transitory appearance of hemoglobin in the urine following exposure to cold
and by the presence in the plasma of an autohemolysin which unites with the red
blood cells only at a low temperature. This is the most common of the paroxys-
mal group. The disease is a late and rare manifestation of syphilis, either con-
genital or acquired. Most cases reported have been in children with congenital
syphilis or in adults in the tertiary stage. !\fast often there is a history or some
clinical evidence of syphilis and serologic tests are positive in over 95 per cent of
the cases.
The basis of this disorder is the presence of an autohemolysin which unites
with the red cells upon chilling and becomes activated on return to normal or
higher temperatures. It is not known why some syphilitics develop this auto-
hemolysin and others do not. It is thermolabile and is destroyed when heated
to 50 to 60° C. for thirty minutes. This lysin is a different suhstance from that
giving the Wassermann reaction; it can be absorbed by red cells from the serum
without changing the titer of the Wassermann reacting material. There is no
parallel in the titers of the autohemolysin and the Wassermann reacting substance
nor any correlation between the susceptibility of the patients to attacks and the
titer of the serum. However, ,when the phenomenon occurs, the higher the titer
of the serum the less chilling is necessary to provoke a reaction. The fall of the
titer corresponds directly with the intensity of antiluetic therapy, otherwise it
remains constant over a period of time.
Symptoms may appear within a few minutes after chilling or be delayed for
six to eight hours. They are usually headache, pain in the legs, back, and
abdomen, chilly sensations or a shaking chill, and transitory elevation of the
temperature. There is a wide range in the amount of chilling necessary for an
attack; most patients require an exposure of 40 to 50° F. Others are sufficiently
chilled merely by opening the door of an unheated room or they may have attacks
in a well-heated hospital ward, or even in the summertime. Mild attacks may be
accompanied only by symptoms and hemoglobinemia but without hemoglobinuria
if the renal threshold for hemoglobin is not reached. The red cell count may fall
to as low as 2,000,000 in a single severe attack. A mild icterus is frequently
present with an indirect van den Bergh reaction and a hypochromic anemia is
often present. There is no hemoglobinuria between attacks. There may be
clinical evidence of syphilis, especially in the congenital type, but physical signs
may be less pronounced in the acquired cases. Early neurological and pupillary
changes may be the first indication of syphilis. Nearly all patients have attacks
when the feet or hands are immersed in water at a temperature of' 30 to 40° F.
for a period of ten to fifteen minutes (Rosenbach test).
In 1904 Donath and Landsteiner demonstrated the presence of the auto-
hemolysin by a simple laboratory procedure. The serum and cells are separated;
a 5 to 10 per cent suspension of red cells in saline is made; equal parts of the
 HEMOGLOBINURIA 253
patient's serum and fresh guinea pig complement added; the mixture is then
chilled for 10 to 30 minutes. This facilitates the absorption of the lysin by the
red cells. The mixture is then warmed to 37 0 C. for thirty minutes whereupon
the lysin becomes active, if present, and hemolyzes the cells. Normal red cells
of the same blood group will be bemolyzed by the patient's serum, emphasizing
further that the lysin is present in the plasma, and that this condition is not an
abnormality of the red cells.
Various forms of therapy have been tried but the only one of value is intensive
antisyphilitic ,therapy. Untreated cases continue to have attacks for years. After
adequate therapy the \Vassermann reaction usually becomes negative and the
autohemolysin disappears from the blood.
Paroxysmal Jl,farch Hemoglobinuria. This variety appears to have a single
causative factor which is physical effort. It has been observed principally in
soldiers following long marches, and was first described by Fliescher in 1881 in a
soldier who suddenly voided hemoglobin after a long march. 'Walking, marching,
or any type of physical exertion seems to be the important factor responsible for
the onset of "'ymptom",. There i", no relation to syphilis, cold, food, drugs, or
abnormatfragility of red cells. Witts cites a case in which hemoglobinuria was
provoked at will by exercising 30 minutes on a bicycle, walking, climbing stairs,
or hard labor. Feigel observed that more than one-half of twenty-seven men
whom he examined after an army pack march of 35 kilometers demonstrated, by
spectroscopic and chemical methods, blood pigment in the serum and urine.
There are no seasonal, climatic, or racial features. Most of the patients are
males in the second decade of life.
The bloo~ 'pigment may appear immediately following exercise but is usually
observed after about two hours. There is an accompanying hemoglobinemia.
The pigment in the urine is hemoglobin and not myoglobin. Frequently a feeling
of general discomfort and cramping abdominal pains precede the hemoglobinuria.
Chills are infrequent, but the temperature may be 99 to 100 0 F. The blood
destruction is seldom sufficiently severe or prolonged to produce anemia. Red
cells may be found in the urine in small to moderate numbers. The susceptibility
to this condition spontaneously disappears without therapy or complications but
it may reappear at intervals throughout life.
Paroxysmal Nocturnal Hemoglobinuria (Marchia/ava-Micheli Syndrome).
This syndrome is an outstanding feature of a chronic hemolytic anemia and
hemoglobinemia of unknown etiology characterized by exacerbations of hemo-
globinuria that are provoked by sleep. It was first described as a separate entity
by Marchiafava and Nazari in 19", discussed again in '928 and 193' by Marchia-
fava, and in '93' by Micheli and is frequently called the Marchiafava-Micheli
syndrome. The paroxysms were first noted during the night but later were found
to accompany sleep, whether during the day or night. Food, posture, or fluids
have no effect on the syndrome and there is no familial tendency. About 70 per
cent of the cases have been in males and the average age is between ,20 and 40
years with the youngest IS and the oldest 47 years of age. Fortunately it is rare,
less than 50 cases having been reported. No harmful effects have been observed
after long exposure to the sun, chilling, or physical effort. A continuous intra-
254 THE ANlnirAs
vascular hemolysis and hemoglobinemia persists between the paroxysms of hemo-
globinuria but the most marked cell destruction occurs during sleep thereby
elevating the plasma hemoglobin to the renal threshold with a subsequent hemo-
globinuria. At times the intravascular destruction of the erythrocytes may be
sufficient to cause the appearance of hemoglobin in the urine during waking bours.
The mechanism of hemolysis is not known. Ham, in his study of five cases,
points out that the fundamental abnormality does not reside in the serum, but
in the red blood cells which are abnormally suspectibJe to hemolysis in plasma of
increased acidity, yet within the normal physiological pH range of 7-2-7.35.
When washed red cells of patients afflicted with this disease were suspended in
heparinized plasma or serum from the patient, or even from normal subjects,
hemolysis of the patient's cells frequently, but not always, occurred within the
normal pH limits. However, if the acidity of the plasma of the serum was
increased within or beyond the physiological range of variation in pH, hemolysis
always occurred. This essential factor for hemolysis in the serum of the patients
and normal subjects is destroyed by heating at 56° C. and is not restored by
adding guinea pig complement. From these observations and the physiological
fact that the blood stream is slowed and that respiration is slower anu more shal-
low during sleep, Ham maintains that there would be sufficient decrease in the
pH of the blood following the increase in the carbon dioxide content during
sleep, to destroy some of the abnormally susceptible cells, even though the pH
of the blood was still within normal limits. All of his cases did not reveal dif-
ferences in the pH of the blood when awake and asleep, and in the prolonged .tudy
of one case in the Emory University Hospital we have not found differences in
the pH of the blood during waking hours and sleep. The buffer system of the
blood should be adequate to compensate for slight increases in CO" during sleep
without disturbances of the pH if it is capable of maintaining the balance during
exercise.
The disease is characterized by no outstanding symptoms other than the
passage of red urine following sleep, and weakness corresponding to the degree
of anemia. Backache, abdominal and muscular cramps, malaise, and a dull
generalized headache are generally present and more severe after sleep. Hemo-
globinemia is greatest at this time. Physical findings are limited to pallor and
mild jaundice. The liver is usually slightly enlarged and the spleen normal in
size, although the latter has been reported to be slightly enlarged in some cases.
The blood picture is that of a marked normochromic, normocytic anemia with
leukopenia and reticulocytosis. The red cell count varies from 1,000,000 to
3,000,000 with a hemoglobin of 20 to 60 per cent. The color and volume indices
range around the normal levels. There is a constant reticulocytosis of 10 to
30 per cent and this is the outstanding evidence of the persistent erythrocytic
regeneration. Nucleated red cells are frequently observed in the stained smear.
The total number of leukocytes is usually from 3,000 to 6,000 with a normal
differential count and the platelets are..normal. There is no increased fragility
of the red cells in hypotonic saline solution. The bilirubin content of the blood
plasma is increased and gives an indirect van den Bergh reaction. Hemoglobin
is present at all times in the plasma, varying from 30 to 300 mg. per cent. The
 HEMOGLOBINURIA 255
plasma proteins are normal and the Donath-Landsteiner and Wassermann reac-
tions negative.
The urine contains variable amounts of hemoglobin after sleeping, although
smaller quantities may be present throughout the day. However, some patients
are free from hemoglobinuria at all times for a period of several weeks to a
month. Hemosiderin in the urine usually can be demonstrated and small quanti-
ties of albumin with a few red cells and shadow cells are often present.
The pathological changes are those following prolonged intravascular destruc-
tion of erythrocytes and throw no light on the possible causes. Slight to moderate
degrees of hepatic necrosis have been observed in the central zone of the hepatic
lobule and around the central veins and there may be thrombosis of the smaller
branches of the hepatic veins. Scott believes that the hepatic necrosis is mechan-
ical in origin and follows. the blocking of liver sinusoids by agglutinated erythro-
cytes and incompletely destroyed 'erythrocytic stroma. Some of the Kupffer cells
contain a small amount of iron pigment, but there are only small quantities of
hemosiderin. The spleen shows no characteristic change. The kidneys show
hemosiderin pigment in the tubular epithelium of the convoluted tubules but not
in the collecting tubules. The bone marrow is the red and hyperplastic type that
would accompany excessive erythropoietic activity in any type of hemolytic
anemia.
The treatment is symptomatic with adequate diet and regulation of physical
activities as indicated by the severity of the anemia. Repeated large doses of
liver have been administered without therapeutic effect. The ,course of the dis-
ease has remained unaffected after splenectomy. Transfusions may be followed
by mild reactions accompanied by fever, chills, and severe hemoglobinuria for
several days. Others suffer no ill effects from transfusions of whole blood and
seem improved for a short time. The disease is ultimately fatal after only a
few years.
Allergic Paroxysmal Hemoglobinuria (Favism) (Fabismus). This condition
is characterized by a sudden onset with fever, prostration, an acutely developing
anemia, jaundice, and hemoglobinuria following the inhalation of pollen from
the blossoming bean plant, Vicia Java, or the ingestion of the beans, either raw
or cooked.
These symptoms represent a hypersensitivity or an allergic reaction to the
protein of the bean or its blossom. The red cell destruction does not follow
the first contact with the bean but is seen only after subsequent exposure or
ingestion. No anaphylactic reactions have been obtained in animal experiments
with tbe bean or the pollen until the animals have been previously sensitized.
Some who have eaten the beans without trouble for long periods of time suddenly
de\'elop the disease.
The condition occurs principally in Sicily, Sardinia, and the provinces of
southern Italy, and is called favism by the Italians while in the German litera-
ture it is referred to as fabismus. Only two cases have been reported in the
English language and these two patients were born in Italy of Italian parentage.
Heredity is thought to be an important factor in 20 per cent of the cases and
 THE ANEMIAS

in some instances every member of a family for several generations has been
affected.
The symptoms include a sudden onset with fever, perhaps chills, weakness,
often vomiting, vertigo, and even unconsciousness in some instances. Pallor is
quite marked at the onset. Jaundice develops and hemoglobinuria occurs within
a few hours to one day after the exposure and may last 3 to 4 days, after which it
rapidly disappears. The spleen mayor may not be enlarged during the seizure.
The most serious feature is the rapidly developing anemia. The decrease in
red cells may be so rapid that death may occur with a cell count of 1,000,000 or
less and a hemoglobin of 20 per cent. Hemoglobinemia is marked. At first a
mild leukopenia may he present but tbis is soon followed by a leukocytosis. The
resistance of the erythrocytes to hypotonic salt solution is not altered and no
auto-agglutination has been observed. The urine contains large quantities of
hemoglobin and frequently large numbers of red cells.
In many patients the attacks are not sufficiently severe to require treat-
ment. Others develoR shock in the early stages and should be treated accordingly
with adrenalin, and intravenous infusions of blood or fluids. Iron is adminis-
tered during the period of convalescence until the hemoglobin and red cells return
to normal.
Paralytic (Hemoglobinuria) Myoglobinuria. This is a disease of unknown
etiology that is characterized by the sudden passage of dark urine, in which the
pigment "myoglobin" rather than hemoglobin can be demonstrated as the color-
ing agent, and by the rapid development of paralysis of the muscles. The
syndrome was observed in horses long before man and is known as equine
hemoglobinuria.
Hemoglobin exists in the body as hemoglobin of the red blood corpuscle
and as myoglobin in the muscle. This condition, therefore, is correctly called
myoglobinuria and the term "hemoglobinuria" should be abandoned. It is said
to occur in work horses that have been idle for several days, yet have been main-
tained on a working diet. The symptoms appear a few minutes after resumption
of work and have been attributed to the sudden release of lactic acid from the
excessive amounts of glycogen which have accumulated in the muscle during
rest. This lactic acid damages the muscle fibers and allows myoglobin to escape
into the plasma and urine. Death may follow in the course of a few days. At
autopsy. the muscle is described as pale, edematous, and of a greyish yellow color
like "fish flesh." Strangely, this is the same description given the striated
muscle in persons dying of Bay sickness. If the animal recovers a residual
paralysis with atrophy may remain for months or years.
In 19II l\1eyer-Betz described the case of a I3-year-old boy who exhibited
a peculiar type of "hemoglobinuria/, associated with widespread muscular
paralysis. The onset was sudden with collapse, abdominal pain, muscle paralysis,
and dark urine two days later. He gave a history of several similar episodes
since his tenth year. Only three similar cases have been reported since. Muscle
dystrophies frequently follow severe exacerbations if recovery is made from the
acute phase. The case of Gunther and also the one of Paul came to autopsy
and the muscles were found to be quite pale and to resemble "fish flesh," thereby
 HEMOGLOBINURIA 257

offering pathologic evidence as to the similari ty of equine and human paralytic

myoglobinuria .
.Clinically and pathologically equine "hemoglobinuria" (myoglobinuria) and
human myoglobinuria are similar to Bay sickness as observed in Konigsberg,
Germany, following the ingestion of poisonous resinous acids from cellulose fac-
tories. Perhaps these cases of paralytic myoglobinuria have a common etiologic
agent.
No hemolysis of the red corpuscles exists in this condition; therefore there
is no anemia or reticulocytosis. Spectroscopic examination discloses the presence
of myoglobin in the blood plasma and urine. The treatment is entirely symp-
tomatic.

BIBLIOGRAPHY

I. BAKER, S. L, and DODDS, E. C.: "Obstruction of renal tubules during the excretion of hemo~
globin."Brit. Jour. Exper. Path., 6, 247, 1925.
~. BORDLEY, J.:
"Reactions following transfusions of blood with urinary suppression and uremia"
Arch. Int. Med., 47, 288, 1931.
3. DE GOWIN, E. L, WARNER, E. D, and RANDALL, W. L: "Renal insufficiency from blood trans·
fusions. II. Anatomic changes in man compared with those in dogs with experimental
hemoglobinuria." Arch. Int. j[ed., 61, 609, 1938.
4. DONATH. J. and LANDSTEINER, K.: "Uber paroxysmale Hamoglobinurie." Munchen Med.
Wchnschr., 51, 1590, 1904.
5. DRESSLER: 4IEin fall von intermittirendf"r albuminuI"ie und chromatuI"ie." Virchow's Arch. Path.
Anat., 6, 264, 1854.
6. GOTTEN, H. B., and MACGOWEN, J. J.: "Blackwater fever (hemoglobinuria) caused by spider
bite." Jour Amer. Med. Assoc., 114, 1547, 1940.
7. GUNTHER, H.: "Kasuistische mitteilung uber myositis myoglobinurica." Vi"chow's Arch. Path
Anat., 251, 141, 1924.
8. HAM, T. H: "Studies on destruction of red blood cells." Arch. Int. Med., 64, u7I. 1939.
9. HAMBURCER, L P, and BERNSTEIN, A.: 44Chronic hemolytic anemia with paroxysmal nocturnal
hemoglobinuria!' Arne" Jour. Med. Sci., 192,301, 1936.
10. HunON, J. E: uFavism, an unusually observed type of hemolytic anemia" Jour. Amer.
Me-d. Assoc, 109, I6:18, I937.
II. LlCDTZ, J. A" JR, HAvn.L, \V. H., and WHIPPLE, G. H.: 4'Renal thresholds for hemoglobin in
dogs." Jour. Exper. Med., 55, 603, I932.
12. MARCHIAFAVA, E.: uAnemia emolitica con emosiderinuria perpetua." Policlimca (sez .Med.),
35, I09, 1928j 3S, 105, 1931.
13. MARCHIAFAVA, E., and NAZARI, A.: "Nuovo contributa aHo studio degli itteri croDid emolitici."
PoUclinicoJ 18, 2:4I, ~9II.
14. McCRAE, T., and ULLERY, J. C.: 4lFavism" Jour. Amer. Med. Assoc 1 101, 1389, 1933.
15. McKENZIE, G. M.· "Paroxysmal hemoglobinuria." j.[cdicine J 8, 159, 1929
16. MEYER-BETZ, F.' "Beobachtungen an einem eigenartigen mit muskellahmungen verbundcnen
fall von hiimoglobinurie." Deutsch. Arch. Klin Med, 101, 85, 191:0.
17. MICHELI, F.: 4'Anemia (splenomegaha) emolitica con emoglobinuria-emoSlderinuria tipo Mar-
chiafava." Ilaematologico J I Arch J 12, 10I, 1931.
18. NEWMAN, W. V., and \VHIPPLE, G. H.: "Hemoglobin injection and conservailon of pigment
by kidney, liver, and spleen; influence of diet and bleeding." Jour. Exper. Mcd. 55,
637, 1932.
19 Orl'ENBERG, R., amI Fox, C. L., JR: "The rate of removal of hemoglobin from the circulation
and its renal threshold in human beings." Amer. Jour. PhysJOl, 123, 516, 1938.
20. PAUL, F.: "Paralytic hemoglobinuria." Wien. Arch. Inn. Med., 7, 53I, 1924.
 THE ANEMIAS

:no PON.FICK, K: "Ueber Haemogtobinillemie und ilire FoIgern." Berl. Kli1l. W,hnschr., 20, 3891
1883.
2:2.ROSENBACI!, 0: "Beitrag ror lehre von der periodiscben hamoglobinurie." Berl. Klin.
Wchnschr, 17, 132, 1880; 17, lSI, 1880,
~3. Scon, R. BO f RreB-SMIm, A. H. T., aDd SCOWEN, E. F.: liThe Marthiafava-Mic.heli syndrome
of nocturnal haemoglobinuria with haemolytic anemia." Quart. Jour. Med., 7. 9$, 1938.
24. STOU.TZND, W.: "Untersuchungen Uber die hoffkrankheit." Deutsch. Med. Wchnschr" 58, part
II, I929, 193%.
25. 'VATSON, E. M., and FISCHER, L. C.: "Paroxysmal 'march' hemoglobinuria with a report of
a case" Amer. Jo'Ur. Clin. Path., 5, 151, 1937.
26. W:rrrs, L. J.: "The paroxysmal haemoglobin urias." Lancet, 2, IIS, 1936.
 CHAPTER 20
HEMOLYTIC JAUNDICE
(Acholuric Jaundice) (Hemolytic Icterus) (Chronic Familial Jaun-
dice) (Hemolytic Splenomegaly) (Microspherocytosis)
Hemolytic jaundice is an inherited familial disease characterized by pro-
longed or recurrent. attacks of jaundice witb variable degrees of anemia and
splenomegaly. It is a classic example of a true hemolytic anemia which is caused
by the destruction of excessive numbers of red corpuscles. This takes place
because a considerable number of the red cells are inherently defective. This
defect consists of an excessive fragility which renders the cells unable to witb-
stand the normal factors tending to produce hemolysis. The possible mechanism
of this is discussed in the following pages.

HISTORY

The disease was observed by Murchison in 1885 and by Wilson in 1890. In

1895 it was described in detail by Hayem. Its familial features were pointed
• )ut by Minkowski, and Chauffard demonstrated tbat the basis for tbe condition
iVas the increased fragility of tbe erythrocytes and showed furtber tbat it was char-
,cterizcd by increased numbers of microcytes and reticulocytes. Tberefore; tbe
:ongenital or familial form has been designated the Chauffard-Minkowski type,
in contrast to the acquired form, the existence of which today is doubted by
nany competent observers. It seems that many instances of acquired hemolytic
jaundice tbat are supposed to develop as a secondary reaction to such diseases
as syphilis, hepatic cirrhosis, malaria, tuberculosis, etc., are in reality latent cases
of familial jaundice which are precipitated by attacks of these diseases. This
discussion, tberefore, refers to tbe congenital or familial type of the disease, since
we agree tbat acquired forms are rarely, if ever, seen. (See p. 244.)

FAMILIAL TRANSMISSION

There seems general agreement that the familial type is inherited as a true
dominant character according to tbe Mendelian law of heredity. Naegeli is a
vigorous exponent of this view and cites numerous instances suggesting its cor-
rectness, including tbe observations of Meulengracht who observed over 50 cases
in 10 affected families, and tbose of Hattensen who studied 26 cases in on" family.
Indeed, Gnasslen reported that over 20 per ceut of the inhabitants of one village
were sufferers, due mainly to clanni;,b marriages and much inbreeding. Naegeli
further states that tbe inheritance is as definite as that of bemophilia, and that
in the average family about one-half the children will show clinical evidence.
The disease appears to become more severe on successive transmissions, the
'59
260 THE ANEMIAS

first generation showing only mild symptoms with a low grade anemia, and in
the second generation it is a fully developed disease, while tbose patients of tbe
third generation, as Naegeli expresses it, are "pitiful." The transmission of
increased virulence is limited probably by the death of those affected witb the
more severe types. It is important to remember that some members of a family
may have a latent or subjaundice type all during their lives and yet never show
evidence of the cellular abnormality. Such people often lead active and vigorous
lives but may have a peculiar color which is usually considered their natural color.
However, such people are capable of transmission, as well as those who have had
the disease and who have been treated by splenectomy. Concerning this, Naegeli
states that "the astonisbment of a physician on learning that his splenectomized
patient gives birth to a child with hemolytic icterus is in proportion to his
ignorance of biologic scientific facts."
Furthermore the frequency of its association with various types of embryonal
maldevelopment or endocrine disorders lends furtber support to its transmissible
character. Naegeli cites a report of three children who exhibited marked in-
fantilism, all evidence of which disappeared following splenectomy for hemo-
lytic jaundice. .Langston has reported such a case with marked improvement
of infantile characteristics after splenectomy and hormone therapy. Evidently
most infants suffering from the more severe type die in the first few weeks of
life, in most cases with the designation of jaundice of unexplained origin.

IN.CIDENCE

Hemolytic jaundice occurs in any race and either sex. There seems to be no
geographic limitntions. It may be found at any age, though most cases are
presented for diagnosis and treatment in early adult life. In a series of 28 cases
studied by Sharp, the ages varied from 3 to 63 years while Mandelbaum reported
a case of hemolytic icterus with the initial hemolytic crisis occurring at the age
of 75. Its occurrence in early adult life is because the active hematopoietic system
of the adolescent is sufficient to maintain cellular equilibrium in spite of the
increased erythrocytic destruction, and hence there are no signs of anemia nntil
early adult life. It bas been reported at 14 months and also in aged people. If
the patient is able to maintain the normal number of circulating cells for the
first few years of adult life, he probably can live out the remainder of his normal
life span without difficulty, except one wbo develops attacks after other diseases,
which is an example of the latent process being precipitated into the clinical
disease.

ETIOLOGY AND PATHOGENESIS

There is some disagreement concerning the basic etiologic factors produc-

ing hemolytic jaundice. All agree that the basic cellular change is the presence
of a peculiar type of red cell which is characterized by its size and shape, in that
its diameter is smaller than normal and its thickness is greater than normal.
Thus, instead of having the normal shape of a flattened bi·concave disc, it is more
 HEMOLYTIC JAUNDICE 261

spheroidal in type. This round red cell with its lessened diameter, is described by
the term microspherocyte, and the condition is called microspherocytosis. If one
can visualize the normal red cell standing on edge and look at it as though viewing
a silver dollar on edge, it will appear as a thin line with a slight bi-concavity.
The microspherocyte, however, would appear swollen in the center and therefore
biconvex, its diameter smaller, and hence, the ceIl more globular. Haden has
stressed this feature of the red celIs of this disease and the production of such
celIs seems to be the inherent constitutional defect.
The microspherocytes are more susceptible to hemolysis when placed in hypo-
tonic salt solution. Therefore, they are more fragile than normal. Based upon
this is the Giffin-Sanford test for fragility in which it is demonstrated that the
celIs are hemolyzed more readily than normal cells.
Even, though there is general agreement that microspherocytosis is the basic
disorder, it is not known why this occurs. It has been stated that the celIs are
produced in that form by the marrow because of an inherent defect; that a dis-
turbance of cholesterol metabolism may be responsible; or that the spleen may
produce the alteration by some unknown perverted function, The latter concept
seems untenable because the condition remains after clinical cure has been effected
by splenectomy, However, Levi and Bairati, in a recent report, state that
splenectomy was followed by a return to production of normal red cells in one
case but in their second case no such result was obtained. Naegeli states that
in many instances there is improvement in the spherocytic state, but :rarely does
it disappear after removal of the spleen, In our experience we have not noted
a decrease of microspherocytosis after splenectomy.
There seems to be no satisfactory explanation for microspherocytosis, but it
is likely that the activity of the spleen is a secondary change and it becomes over-
active with enlargement because of its increased function to destroy these in-
capacitated celIs and remove them from circulation. Removal of the spleen
probably removes merely a large part of the phagocytic mechanism and the
spherocytes then circulate longer, are partly functional, relieve the oxygen carry-
ing load, and permit the bone marrow to resume its normal output, In an excel-
lent article on the pathology of the spleen in various diseases, Klemperer expresses
the opinion that the spleen of hemolytic jaundice is characterized by marked
arteriolar hyperemia caused by vasodilatation, and since the erythrocytes are pri-
marily abnormal, they are unusually vulnerable to the destructive action of
reticulum cells. Even though there is no satisfactory explanation for the cause
of microspherocytosis, the work of Dameshek and Schwartz may prove to be
important in this respect. These authors have prepared anti-guinea pig hemo-
lytic serum by injection of guinea pig red celIs into rabbits, This serum, when
reinjected into the pigs, was capable of producing various degrees of micro-
spherocytosis and possessed all of the properties of iso-hemolysins of the immune
body type. The authors expressed the opinion that the hemolytic syndrome
including microspherocytosis, may be caused by hemolysins, that possibly these
are inherited in congenital hemolytic icterus, and that microspherocytosis repre-
sents merely the initial stage of cell swelling or cell destruction, Once the cells
 THE ANEMIAS

have hecome swollen they are abnormal, and as such are destroyed in excessive
numbers by the spleeri~
Ham and Castle have pointed out that the more nearly spherical the
erythrocytes the greater is their susceptibility to hemolysis by further swelling
in hypotonic saline solution. They have shown also that when blood is incu-
bated under sterile conditions with stasis in the test tube, progressive swelling
of the red cells with increased sphericity and osmotic fragility results. They
consider such _a situatiOil comparable to that in the spleen since intravascular
stasis in that organ is associated with increased destruction of blood, and that
cells that are already spherocytic to a certain degree are destroyed earlier than
normal cells. It is possible that in congenital hemolytic icterus there may exist
an inherited hemolytic agent that produces the initial swelling of red cells, and
that these cells in turn are more readily susceptible to destruction by the spleen.
It would appear necessary that the two factors be operative together in order
to produce the condition seen in this disease. Although removal of the spleen
cures the disease, the patient still is left with variable degrees of microcytosis.
When the enlarged spleen is destroying such excessive numbers of red cells,
there necessarily follows a compensatory increased output from the hone-marrow
in an effort to maintain the cellular equilibrium. This results in a hyperactive mar-
row with hyperplasia of the erythropoietic tissue and outpouring of reticulocytes
into the circulation. Also the granulopoietic elements are involved in the process,
resulting in a granulocytic leukocytosis. In some instances the demand on the
marrow is so great that it cannot maintain the high production rate and collapses
functionally, sending a shower of nucleated red cells, including normoblasts,
microblasts, and many immature white cells into the blood. There then occurs
a bone marrow crisis with the patient showing a clinical crisis, with severe
anemia, prostration, fever and accentuated jaundice. This may follow some
extra demand on the marrow such as an intercurrent infectious process, or strenu-
ous muscular activity.
In summary, the health of the patient will depend upon the number of
microspherocytes produced by the marrow, their rate of destruction, and the
ability of the marrow to compensate for the cellular loss. Thus, a vicious cycle
exists which may be present for years with no evidence of the disease so long
as the marrow can compensate but when decompensation occurs with a functional
marrow breakdown, clinical symptoms develop. '

SYMPTOMS AND PHYSICAL FINDINGS

These depend upon the severity of the disease. In the latent form the
patient may be leading a normal life in apparent good health. and the increased
fragility of red cells may be discovered only during the course of a routine exami-
nation. Others may notice only a slight yellow skin color or a questionable
tendency to icterus which may have been present throughout life and thought
to be normal. In some only careful examination will reveal the sclerae to be
tinged with yellow. We have discovered instances of the latent process in the
course of studies on blood serum for some unrelated disease. As pointed out
 HEMOLYTIC JAUNDICE

before, an acute illness may precipitate the symptoms with increased hemolysis
and jaundice. It has heen stated that removal to a high climate, vigorous exer-
cise or massage, exposure to radiation or a quartz lamp may precipitate the jaun-
dice in a latent case. We have studied a young athletic college student whose
initial attack of jaundice was precipitated by participation in an intercollegiate
swimming meet.
The patient with the latent or subclinical type may give a history of jaundice
in other members of the family, of att::lcks in the past, or a history of numerous
periods of ill health.
The patient may show no evidence of jaundice and may give a negative his-
tory of ever having suflered from it. The chief complaints may be weakness,
fatigue, and lassitude from an apparent anemia. Also he may consult the phy-
sician because of pain in the upper abdomen or a dragging sensation of weight
because of splenomegaly. Thus, one may see hemolytic jaundice without the
jaundice, or without the anemia, or without the splenomegaly, or without any of
these three findings that constitute the diagnostic triad, 50 that the only finding
may be an increased fragility of the red cells and microcytosis. Furthermore,
there may occur in the same patient one period when jaundice predominates, an-
other peri9d with anemia and no jaundice, and still others when splenomegaly is
the only finding, all of which illustrates the extreme variability of the disease and
the fact that microcytosis and erythrocytic fragility are the basic diagnostic
findings in all cases.
If seen in an acute crisis, the patient may be deeply jaundiced and pro-
foundly anemic, with the spleen markedly enlarged. There is usually fever,
much fatigue, headache and variable pains, mainly in the upper abdomen. They
usually appear well nourished or even corpulent and obese. The jaundice is char-
acterized by an absence of pruritis, which is common in obstructive jaundice.
Due to the high incidence of gallstones in the disease (some report 60 per cent),
this finding may assume considerable importance in some cases. Other acute
crises may be characterized only by an intense jaundice. Some patients are, as
Chauflard remarked, "more yellow than sick." In severe cases there may be
nausea and vomiting, marked prostration and coma. In women, the chief com-
plaint may involve irregularity of menstruation, and other complaints referable
to various endocrine disorders. Falconer studied three such patients. The liver
mayor may not be enlarged. Chronic leg ulcers may be seen in long standing
cases of hemolytic jaundice. Leger and Orr have reported two such cases. I ob-
served one patient at the University of Nebraska whose legs were scarred by
old healed ulcers. The etiology of this is not known but the ulcers heal after
blood transfusions and splenectomy.

LABORATORY FINDINGS

Examination of the blood of the average patient reveals a variable degree of

anemia with the red cells low and hemoglobin lowered to a color index below
onc. In remission, the anemia may be only slight while in crisis the rcd cclls
may be as low as 1,000,000 per cu. mm. with the hemoglobin down to 20 per cent.
 THE ANF..MIAS

In remission the leukocytes are at a normal level or slightly elevated with no shift
to cellular immaturity. During a crisis they are usually quite high, in some
cases up to 50,000 per cu. mm. with many myelocytes and juvenile forms, and this
blood picture coupled with the enlarged spleen, may simulate chronic myeloid
leukemia. The platelets are unaffected in both number and morphology. A study
of the stained hlood in the chronic case will show varying degrees of erythrocyte
pallor with pale cells showing much variation in size and shape and varying
degrees of polychromatophilia. It is usually apparent on the stained smear
that most of the cells are smaller than normal, and if much variation'in size is
present the normal ones may appear to be macrocytic. However, macrocytes
rarely occur in the blood. If examined during crisis the smear usually shows a
large number·of nucleated red cells consisting of microblasts and normoblasts.
Naegeli states that he has never seen a megalohlast in this disease. The presence
of such large numbers of normoblast. would indicate an acute bone marroW
crisis and this is borne out by the presence of a marked reticulocytosis which
may be as high as 50 per cent. One case has been reported with 95 per cent
reticulocytes. .
If the blood is examined in the unstained state it will be noted that many
of the cells are spherical and small. The average cell diameter is only five to six
micra. The volume index, however, is normal or only slightly below the normal.
Even though the diameter of the cell is small, its thickness compensates for it
so that the volume is decreased but little. Therefore, determination of volume
index is very important in this disease, since there is no other anemia that show;;
such marked microcytosis with a normal cell volume.
The characteristic finding of. most importance is the increased fragility of the
cells when subjected to the action of hypotonic salt solution. This is determined
by placing equal amounts of blood in a series of tubes containing various grada.
tions of percentages of sodium chloride (see section on technic). We use the
Giffin-Sanford method, although the fragility can be determined more accurately
by using a series of red cell pipettes and computing the number of cells actually
hemolyzed in the various dilutions. With normal blood hemolysis usually begins
at 0.42 per cent and is complete at 0.34 per cent. In hemolytic jaundice it usually
begins at 0.48 per cent and is complete at 0.42 per cent. Compared to the nor·
mal, which varies slightly, the hemolysis should be complete in the same tube
in which it only begins in the control of normal blood. The degree of fragility
varies widely and hemolysis may begin in salt solution as high as 0.8 per cent
wbich is very nearly normal salt solution.
Other laboratory findings include a high icterus index because of increased
bilirubin in the blood, a positive indirect van den Bergh reaction, a highly colored
urine due to excessive urobilin, and perhaps increased color of stools for the same
reason.

TREATMENT

The treatment varies in different cases and centers on the question as to

whether the spleen should be removed. During the cri~is, the patient should
 HEMOLYTIC JAUNDICE

have the benefit of general measures, including bed rest, adequate diet, proper
nursing, and symptomatic therapy, with transfusions if the degree of anemia
is sufficient to warrant it. The object of treatment at that time is to restore
the patient to a latent phase or into a remission and then decide whether
splenectomy would be advisable. On the other hand, Doan, Curtis and Wiseman
advocate splenectomy during the crisis hecause of the apparent necessity of im-
,mediate removal to save the patient, and point out that the procedure often-
times accomplishes a valuable auto-transfusion by discharging the blood of the
sple_en into the general circulation at the time of the removal. Curtis has found
that there is an immediate increase in red blood cells after splenectomy regard-
less of the cell level at the time of operation. He recommends removal of the
spleen during periods of hemoblastic crisis even though red cells are extremely
low, with many nucleated red cells in the blood stream. He has done nine such
operations and believes that this is muclI better than giving a transfusion before-
hand and building the patient up since this latter may only accelerate red cell
destruction. I am inclined to believe that more patients with hemolytic icterus
should have their spleens removed during crises, particularly because of the
danger of transfusion in these patients. Curtis stresses the importance of
prophylactic splenectomy early in life; first, to prevent formation of gall stones;
secondly, to prevent acute hemoblastic crises; and thirdly, to prevent the pro-
longed debilitating effects of the disease. .
After the patient has passed the acute crisis, iron by mouth should be used
in large doses to correct the anemia. Diet should be adequate and high in
calories. One or more transfusions can be given. It appears that the use of
liver would be contraindicated. Cholesterol has been used because of a clIolesterol
deficiency, but with little success.
There is general agreement that splenectomy is followed by permanent re-
mission in nearly every case. Most patients never show clinical evidence of the
disease again, although the tendency to microcytosis, sphericity, and fragility
still persists to a variable degree. After splenectomy the increased fragility of
red cells is corrected to a variable extent. Tsai and his associates carried out
experiments on splenectomized dogs and found that splenectomy caused a re-
duction of fragility. It became evident a few days after operation and persisted
for four months. They found a greater fragility of cells in splenic vein blood
than in splenic arterial blood. Haden states that after splenectomy the anemia
rapidly disappears. The spherocytosis persists, although it is much less marked
than before. The question to be determined is whether the patient has a fair
chance to continue through life without danger of a crisis and remain in fairly
good health. It may be assumed that if the patient has been through one crisis,
another could occur at any time that he undergoes additional stress, and that
each time a crisis occurs there is some clIance that he may not survive. There-
fore, splenectomy should be adyised. In the clIronic case producing a mild
jaundice, a slight reticulocytosis and perhaps' no anemia, the question to be
determined is whether suclI a state is compatible with good health. This can
be determined only by prolonged observation and repeated hematologic studies
to determine especially the bone marrow output of reticulocytes. If the bone
 PLATE XXI

ANEMIA OF HEMOI"YTIC JAUNDICE

(SBCONDARY ANEMIA)

~
\:C;V
1

I. Neutrophits.
,. Microcyte
3. Macrocyte.
4. Poikilocyte.
S. Polychromatocyte.

Blood Fmdings Differential:

HemoglobIn 78 gms. (Newcomer's method) Mye10cytes 0%
R.D.C. 2,600,000 per c.mm. Juveniles ., 2%
w.n.c. 7,800 per c mm Bands 60/'0
Platelets 306,000 per c mm. Segmenters . 57%
Total neutrophlls
Color Index 08 Lymphocytes
Volume Index 07 2 EosinophIls
Icterus Index !5·o Dasophl15
Rehculocytes .13.0% Monocyte!'.

FragIlity of erythrocytes in hypotonic salt soIutions Hemolysis beginning at 049% NaCI

and complete at 040%.
Erythrocytes; microcytic and hypochromic with marked anisocytoslS, pOikIlocytosIS and poly-
chromatoph1l1a.
Plate XXI
Plate XXII.
.68 THE ANEMIAS

marrow manufactures ten per cent reticulocytes over a period of months or years
in order to maintain the patient free from anemia and maintain the normal
erythrocytic level, it should be borne in mind that such compensation represents
twenty times the normal marrow output, and very likely such a patient would b~
much better off without his spleen. The question to be decided is not whether the
patient can afford to lose his spleen but rather, can he afford to keep it?

INDICATIONS FOR SPLENECTOMY

There are only a few indications for removal of the spleen. It should be re-
moved in cases of hemolytic icterus as ,previously discussed, particularly in those
cases where it has been necessary for the patient to produce excessive numbers
of red cells over a period of years. It should be removed in thrombocytopenic
purpura and in Banti's disease, provided the disease is diagnosed fairly early
before far-advanced liver changes have developed. It should be removed for
primary splenic tumors which, however, are quite rare. Curtis states that he
has performed ten splenectomies on cases of hypoplastic anemia, one of which
was caused by benzene poisoning. He believes it is indicated in this group of
anemias, not that it cures the disease, but because it removes the normal destruc-
tive mechanism for red cells, therefore permitting the patient to preserve a
better cellular equilibrium. Curtis further points out tha.t the spleen of hemolytic
icterus ordinarily does not show any adhesions but this is not true in the enlarged
spleen of Banti's disease. He prefers to employ transfusions before splenectomy,
particularly in infants with thrombocytopenic purpura, but not in cases of
hemolytic jaundice.

RESULTS OF SPLENECTOMY

In nearly every case of hemolytic jaundice in which the spleen is removed,

cliuical cure follows and the patient remains free from the disease. Splenec-
tomy is immediately followed by a rise in hemoglobin and red cells with continued
reticulocyte output until the normal cellular level is attained. Fragility and
microcytosis remain but not sufficient to cause increased marrow activity. Sharpe
and his associates studied the blood changes after splenectomy in eight cases.
In five patients there was a sharp rise in hemoglobin during manipulation of
the spleen, even before ligation of its pedicle. 'One hour after splenectomy the
hemoglobin had markedly increased in every case, from 10 to 35 per cent, this
being followed by a slow decrease which continued until about the third day but
in no case did it return to the pre-operative level. The red cells were also in-
creased during manipulation of the spleen before ligation of the pedicle. Whereas
the average leukocyte count was I I ,800 before operation, after splenic manipu-
lation and before pedicle ligation the average was .8,000 cells per cU.mm. The
leukocytes finally reached 45,000 per cU.mm. some eigbt 'hours after operation,
hut the differential cell count was normal. The authors state that the bemo-
globin, red cell and white cell increases persist for only a few hours because of
the splenic manipulation in the surgical procedure. They also studied splenec-
 HEMOLYTIC JAUNDICE

tomy for other diseases and noted no such increases. There are few blood dis-
eases in which such a dramatic and spectacular result can be obtained as that
following splenectomy in this disease. After a careful study of 45 patients,
Thompson states that "the symptoms of typical hemolytic' jaundice are promptly,
completely and permanently relieved by splenectomy."
The spleen is an organ that consists predominantly of phagocytic reticulo-
endothelium and lymphoid tissue. It has been estimated that the spleen con-
tains from one-third to one-half of the lymphoid tissue of the body and approxi-
mately one-fourth of the reticula-endothelium.
The function of lymphoid tissue and lymphocytes seems to be chiefly one of
resisting certain types of inflammatory processes, particularly those where it is
necessary to wall off chronic types of inflammatory foci. The function of the
cells of reticula-endothelial tissue appears to be similar and in addition, this type
of cell exercises an actual phagoctyic function. Therefore, it can be assumed that
the removal of a normal spleen removes a portion of these two types of tissue.
There still remains in other areas an adequate amount of both types to carryon
their function until compensatory overgrowth takes place. -
The immediate effects of the removal of the spleen would appear to be simply
a sudden decrease in the amount of lymphoid and endothelial tissue. As time goes
on, however, there no doubt occurs a slow and gradual compensation in the form
of a slight generalized lymphadenopathy so that within a few months the individ-
ual probably bas just as much {unctional lymphoid tissue and endothelial tissue
as hefore.
So far as is known, the phagocytic function of the spleen is taken over hy
other endothelial elements, particularly those of the capillary endothelium, lymph
glands, and sinusoids of the liver. We can find no record of a splenectomized
patient subsequently developing malaria, and if this should happen, how effi-
ciently could phagocytosis of the parasites be carried out?
Dominici believes that other sections of the endothelial system may also be
responsible for excessive blood destruction and states that splenectomy, however,
removes the principal site of blood destruction but does not remove all of the
Lissue having this function.
The spleen is not essential to life as has been well demonstrated following
surgical removal and in congenital absence of the spleen. In '9I4, Riches col-
lected 13 cases of congenital ahsence of the spleen from the literature. He Con-
cluded that these people showed no deviation from normal and that their splenic
absence in no way contributed to any type of ill health. It is not unusual at
autopsy to find spleens that weigh only a few grams in adults who have died from
some totally unrelated disease.
Ash-Upmark has collected roo cases in which the normal spleen has been
removed. Ninety-nine of these were traumatic. He followed these from one to
27 years after splenectomy. In addition he collected 94 cases from the literature.
He found no increased susceptibility to infections or to malignant tumors. He
noted a tendency to rapid exhaustion after exertion. He stated that people whose
normal spleens have been removed should be accepted for insurance on the usual
'7 0 THE ANEMIAS

terms. He concluded that "anatomically, physiologically and clinically, the spleen

is not necessary to life, and should be removed only on vital indications,"
Wollstein and Kriedel studied the blood after splenectomy in 44 cbildren,
including .3 for traumatic rupture, 20 for rheumatic disease, one for sp]eno-
megaly, 4 for bemolytic icterus, 8 for hemorrhagic thrombocytopenia. Tbe ages
ranged from II montlis to 12 years. Tbey found no immediate post-operative
increase of platelets in those patients wbose platelets were normal before opera-
tion (traumatic, hemolytic icterus, etc.) but in tbrombocytopenic purpura tbere
was an immediate postoperative elevation. Tbe peak of the rise occurred dUI-
ing the second postoperative week, usually reaching 1,000,000 per Cll. mm. which
was maintained for weeks or months. We have observed essentially the same
results in adults.
Since immediate effects of splenectomy on tbe blood constituents amount to
a sudden increase of blood platelets, leukocytes and red cells and this increase
gradually reverts to the normal level in periods ranging from a few weeks to
approximately a year, it seems likely that during this post-splenectomy period
the blood cells continue to be mannfactured at their normal rate but since part
of the destructive mechanism is gone some of them live to an older age in the
blood stream. Therefore, it might be surmised that the average age of the cellular
constituents after splenectomy is older than normal. This situation, however,
soon corrects itself when tbe endothelial function is gradually taken over by
other tissues. This compensatory hyperplasia would necessarily mean a slight
generalized lymphadenopathy, which, within itself, would be of no importance
and during this time tbere would be excessive production of lymphocytes result-
ing, therefore, in a relative and absolute lymphocytosis.

BIBLIOGRAPHY
HEMOLYTIC JAUNDICE
CmUFFARD, M. A: "The pathogenesis of congenital icterus in adults." La Semaine Medicale,
27.25,1907·
DAMESBEX. W., and Scn:WARTZ, S.D.: "Hemolysins as the cause of clinical and experimental hemo-
1ytic:: anemia.s." Amer. Jour. Med. Sci., 196, 769, :1938.
DOAN, C. A, CURTIS, G M, and WISEMAN, B. K.: "Hemolytopoietic equilibrium and emergency
splenectomy." Jour. Amer. Med. Assoc., 105, 1567, :1935. '
FALCONER, E. It.: "Familial icterus a5Sociat~d with endocrine dysfunction." Endocrinology~ 20~
:174) :1936.
GIFFIN, H. Z, and SANFORD, A. H.: "Clinical observations concerning the fragility of erythrocytes."
Jour. Lab. CUn. Med., 4, 465, 1918.
HADEN, R. L.: liThe mecllanisrn of the intreased fragility of the erythrocytes in hemolytic jaun-
dice." Amer. Jour. Med. Sci, 188, 441, :1934.
HAM, T. H'I and CASTLE, W B: "Studies on destruction of red blood cells" Proc. Amer. Phil: Soc I
82, 411, 1940.
HAYEM, G.: IIAn unusual type of chronic icterus. Icterus and splenomegaly with chronic infec-
tion" La l'resse Medicale, 24, 121, 1898.
KLEYP!RER, P.: "The pathologic anatomy of splenOmegaly." Amtr. Jour. ClinT Path., 6, 99, 1936.
LO\NGSTON, W.: "Hemolytic icterus with infantilism." South. Med. Jour., 28, 316, 1935.
LEGER, L. H I and ORR, T. G: "Chronic leg ulcerations in congenital hemolytic Jaunruce." South
Mtd. Jour., 33, 463, 1940.
 HEMOLYTIC JAUNDICE

LEVI, G. M., and BAlRAn, A: <'D.u.tribution of erythrocyte population in regard to diameters and
osmotic resistance in splenectomized cases of hemolytic icterus: contribution to understanding
of pathogenesis of disease." Amt,. Jou,. Med. Sci., 190, 6:10, :1935.
MANDELBAUM, II.: "Congenital hemolytic jaundice. Report of a case with initial hemolytic crisis
occurring at the age of 75." Ann. Tnt. Med., 13, 87z, 1939.
MmKOWSKI, 0.: Verhandl d deutsch. Kong. f. inn Med .• 18, 316, 1900. Quoted from Nt.lson
Loose Leaf Medicine.
MURcnISON', C., "Diseases of the liver3-'1 ed. 3, 481. London, :1885.
NAEGELI, 0.: "Blood diseases aDd bJood diagnosis," 293. Springer, Ber.tiD, 19J1.
SCOTl'. A. hI.: "Acholuric jaundice," Lancet, 2, 872, Oct" 1935.
TnOMPSON, \V. P.: "Hemolytic jaundice. Its rliagnoSlS. behavior and treatment: a review of
forty-five cases." Jou,. Ame,.. Jled. Assoc, 107, :1776, :1936.
TSAI, C, LEE, J. Sf and Wu, C. H .. "The role of'splenic action in altering erythrocyte fragillty.!'
Chinese Jour Physiol., 15, 165, 1940.
Wn.sON, C: "HeredItary enlargement of the spleen." Brit. Aled. Jour., 1. 782, 1890.

RESULTS OF SPLENECTOMY

Asn·UPJ"ullX, E.: "Remote effects of removal of normal spleen in man." Svenskll Lakaresalls.
kapets Handlingar., 61, I97, I93S.
CURTIS, G. M : "The rationale of splenectomy in the treatment of certain anemias," Jou;. Omalla
Midwest CJin. Soc .• 2. 3D, I94I,
DO:ML'1'ICl, G.: "Clinical hematological and biological recent and late effects after splenectomy in
six cases of constitutional hemolytic icterus." Hematologica, 17, :185, :1936.
HADEN, R. L.: "The nature of hemolytic anemia. A symposium on the blood," Univ. of Wisconsin
Press. 1939; p. 83. ,
PEARCE, R. M., KRUMBltAAR, E. B., and FRAZIER, C. H.: liThe spleen and anemia.1I 1918. J. B.
Lippincott Company.
RICIIES, R. G.: "A case of congenital absence of the !pleen." Jour. Ment. Sci., 60, 630, 1914.
5nAnPE, J. C., McLAucmm. C. W., JR. and CUNNINGHAM, R.: "Hemolytic jaundice; immediate
and delayed changes in blood after splen~ctomy." AreTl. Int. Med., 64, 268, 1939.
WOLLSTElN, M., and KlmmEL, K. V.: I'Blood picture after .splenectomy in children with special
reference to platelets." Amer. Jour. Dis. Child., 51, 765, 1936.
 CHAPTER 21
HYPOCHROMIC ANEMIA OF LEAD POISONING
Lead poisoning may occur when the metal gains access to the body tissues
in excessive quantity, whether it be by oral ingestion, ipjection, inhalation, or skin
contact. It is seen in industrial workers more often than in other sections of
the population. This usually is due to working in poorly ventilated quarters,
resulting in the inhalation or ingestion of the metal, with certain amounts acci-
dentally transferred to the mouth, with the daily ingestion of small amounts. It is
a common disease among painters, and particularly in the "paint chippers"
working aboard ship in close quarters.
It has been reported in infants who gnaw at the paint on their cribs and beds
while teelhing, and who finally ingest sufficient quantity for production of symp-
toms. Since the.metal may be excreted in the milk of the nursing mot.her, infant
poisoning may occur from that source. Excessive lead can be fpund in drinking
water or foods. in rare instances. Since the use of lead treatment in certain malig-
nant :states, instances of lead puisoning have been reported in that class of
patients. ~
Lead poisoning may be either acute or chronic, which depends on the amount
of metal ingested, the duration of exposure to it, the susceptibility of the indi-
vidual, and the rate of excretion in the urine.

SYMPTOMS
The outstanding clinical features of the acute form include a marked pallor
due to the anemia, abdominal cramps, marked constipation, and v.arious palsies
of the peripheral nerves, which, if the radial nerve is affected,.is manifested by
the~ well known "wrist drop." In severe acute cases there may be signs of
encephalitis or even convulsions may develop. In the acute form there mayor
may not be a "lead line" of the gums, and there may be no evidence of the
characteristic stippling of the red cells. Depending upon the rate of red cell
destruction will depend tbe degree of hemolytic jaundice that may be present.
Chronic lead poisoning is characterized by a history of prolonged exposure
to the metal in industry or by accident, prolonged constipation, the presence of a
thin blue line at the gum margins, the so-called lead line, marked pallor because
of the anemia, and "- variable degree of prolonged or intermittent attacks of gen-
eralized abdominal cramps. It is also characterized by the presence in the blood
of numerous red cells that exhibit basophilic stippling and varying degrees of
polychromatophilia.
Lead seems to act as a powerful toxic agent that has a peculiar affinity for
lipoid or fatty structures, this accounting for its action on the lipoid fatty cover:
ing of the red blood cells and on the same constituents of nerve trunks. It affects
not only the cells in the circulating blood but also those of the bone marrow,
272
 HYPOCHROMIC ANEMIA OF LEAD POISONING 273

and it has been shown by Key and by Whitby and Britton that the cells affected
mainly are the reticulocytes.
Aub and Reznikoff studied the red cell changes after lead poisoning and con·
cluded that the lead unites with the phosphates of the red cells, damaging the cell
membrane, resulting in cellular fragility. Since the cells affected are mainly the
young reticulocytes the chromatin material of the reticulum becomes distorted
and deposited in unique formations, resulting in the punctate basophilia (stip-
pling) and diffuse basophilia (polychromatophilia). Since the polychromatophilic
cells are usually large, this is further evidence that the reticulocyte is mainly
involved. These changes are thought to occur largely in the bone marrow.

HEMATOLOGIC FINDINGS
The degree of anemia is variable and depends upon the factors mentioned
above, including duralion of action of lhe metal, and ability of lhe patient to
excrete it hefore its action on the cells. Tn acute poisoning the anemia may be
only slight while in the chronic forms it may be quite severe. The red cells are
seldom less tpan two million per cu. mm. and the hemoglobin at least propor.
tionately as low. The rate of hemoglobin decrease exceeds that of the decrease
in red cells so that a true hypochromia usually exists. It is not unusual to find
patients in which the red cells may be ncar the normal level and hemoglobin as
low as 50 per cent.
A study of red cells on the stained smear shows a moderate. degree of central
pallor, slight variations in size and shape, and the presence of polychromatophilia,
with a variable number showing punctuate basophilia (stippling). These cells
contain 20 to 40 separate clumps of chromatin material. Their presence in consid-
erable number with hypochromic anemia should lead one to suspect lead poisoning
until it can be ruled out.
The reticulocytes are usually increased, especially in the chronic forms, the
increase being brought about by the necessary compensatory stimulation because
of the excessive destruction of the damaged red cells. If the rate of destruction
is sufficient there will exist increased bilirubin in the blood, manifested by in·
creased -icterus index, and a positive indirect Van den Bergh reaction of hemo-
lytic icterus. The degree of reticulocyte increase is a more reliable index of
toxicity than the number of stippled erythrocytes.
The leukocytes are usually slightly increased or at the normal level. In (he
acute types of poisoning there may be a marked increase of leukocytes with a
shift to granulocytic cellular immaturity, presumably because of irritation of the
centers of granulopoiesis. The blood platelets are little affected, although Brook·
field states that there may be a transient increase.
When the characteristic blood picture is found, further confirmatory evidence
may be obtained by finding the characteristic changes in the diaphyses of the
bones, especially in children, and the presence of lead in the urine.
Although basophilic stippling of red cells is highly suggestive of lead poison.
ing, the same changes are often noted in other diseases, in particular those accom·
panied by extreme degrees of hypochromic anemia. We have observed red cell
stippling in the hypochromic anemias of malaria, intestinal parasitism, and pro·
 PMTE xx_nr:
ANEMIA OF LEAD POISONJ,.NG
(SECONDARY ANEMIA)

~
~
1

1. Neutrophil.
2. Erythrocyte with coarse basophIlic ~tipplmg.
3. Erythrocyte with fine ba.sophilic stippling.
4. Polychromatocyte.
Blood Findings: Differential:
Hemoglobin .6.5 gms. (Newcomer's method). Myelocytes 0%
RB.C. 3,I5o,000 per c.mm. JuvenIles . 5%
W.BC 8,450 per c mm. Bands 11%
Platelets 295,000 per c,nnu Segmenters 60%
Total neutrophlls
Calor Index .06 Lymphocytes
Volume Index 08 EOSlDOphlhi
Monucytes .
Erythrocytes; hypochromic and microcylic with pronounced basophDic stippling.
Plate XXIII.
 HYPOCHROMIC ANEMIA OF LEAD POISONING

longed blood loss, in which lead presumably is not involved as an etiologic factor.
It may be seen rarely in pernicious anemia. In these instances the basophilic
changes are probably indicative of bone marrow fegenerative activity.

THE BASOPHILIC AGGREGATION TEST

There bas been proposed a test known as the basophilic aggregation test,
which appears to be widely used in the industrial areas where there is suscepti-
bility to lead poisoning. This test demonstrates the regenerative signs of blood
formation in the peripheral circulation, manifested by tbe presence of a basophilic
substance either in the form of polychromatophilia, punctate basophilia, or a
reticular design in the red cell. In the test this substance is aggregated into
masses that are readily visible (see page 672 for technic). It is claimed to be more
accurate than the counting of stippled cells in ordinary Wright's smears.
Pearlman and Limarzi have showll that there is a close correlation between
the aggregation test and the reticulocyte count. In '934, McCord and his asso-
ciates studied the blood of 6,900 workers, using the basophilic aggregation test,
and concluded that a basophilic aggregate count of 2 per cent in the lead workers
suggests lead ahsorption and possibility of lead poisoning. However, others
consider the test simply another way of doing a reticulocyte count. It probably
is not diagnostic of lead absorption or lead intoxication but merely is an index
of bone marrow regenerative activity.

BIBLIOGRAPHY

Aua, J. c.. and RuNIKOPF, P.: IILead studies: III The effect of lead on rcd blood cells; chemi~
ca.l explanation of reaction of lead with red blood cells." Jour. Exp. Med. 40, 189, 19'4.
BROOltFlELD, R. W.' "Blood cbanges occuning during course of treatment of malignant disease
by lead, with special reference to punctate basoprulia and the platelets" Jour, Path. and
Bact., 31, :il17, 1928.
KEY, J. A.: "Lead studies. III. Blood changes in l~d poisoning in rabbits with especial refer·
ence to stippled cells." Amer. Jour. Pllysiol, 70, 86, 1924.
MCCORD, C. P., HOLDEN, F. R., and JOHNSTON, J.: "Basophilic aggregation test in the lead poison-
ing epidemic of 1934-35." Amer. Jour. Public Health, 25. 1089. 1935.
PEARI.MAN, M. D., and Ln.£ARl.I, L. R.: "Correlation studies of basophiliC': aggregation and reticulo.
cytes in various clinical conditions II Amer. Jour. CUn. Path., 8, 608, ]938.
WIIlTBY, L. E. H., and BRITTON, C. J. C.: "The relation of stippled cell and polychromatic cen
Lo lcticulocyte." Lancet, 1, 1173. 1933.
 .CHAPTER 22
PERNICIOUS ANEMIA
(Addisonian Anemia) (Biermer's Anemia)
Pernicious anemia is a disease of unknown'etiology, characterized by absence
of hydrochloric acid in the stomach, variable gastro-intestinal and neurologic dis-
turbances, with a classical type of anemia resulting from dysfunction of erythro-
poiesis, and by periods of spontaneous remission and relapse. It is a fatal disease
unless treated with adequate and specific treatment in the form of stomach, Iivet
and other preparations that contain the antianemic factor_
Since pernicious anemia was first described by Addison nearly one hundred
years ago, many theories of causation have been advanced. These have induded
those of the earlier school led by Cohnheim and Ehrlich, who maintained that
the essential dysfunction was in the bone marrow, characterized by a reversion of
erythropoiesis to the embryonic type, and that of Hunter and j\fuir who held
that the fundamental process was excessive blood destruction with the marrow
changes being a compensatory physiologic process.
The second period of development was concerned with the recognition and
study of the gastro-intestinal changes of the disease, including gastric mucosal
atrophy, glossitis, and the resulting variety of gastro-intestinal symptoms. This
gave rise to the concept that the etiologic factor was elaborated or ingested, and
absorbed from the intestinal tract. From these studies developed the thought
that oral sepsis may be responsible and various bacteria were suspected as etio·
logic agents. These included the Bacillus welchii, an anaerobic gas producing
organism and its toxins, the Bacillus coli, and various types of streptococci.
The third phase of advanced knowledge of pernicious anemia evolved mainly
about the recognition and study of the neurologic changes, this leading to the
appreciation of the fact that this is not solely a disease of the blood and of the
gastro-intestinal tract. Because of this, there developed a broader concept of
the pathogenesis amI the di.ease today is recogoized to, affect mainly three general
systems of the body, these including the neurologic, the gastro-intestinal, and the
hematopoietic systems.
The fourth period of development from '920 to the present time, has re-
sulted from the work of Whipple in studying the regenerative effects of various
tissues in anemic dogs; from that of Castle who demonstrated the presence of an
intrinsic hematopoietic factor in the stomach; that of i\Hnot and Murphy who
established the curative influence of liver; and that of Sharpe and Sturgis and
Isaacs who demonstrated the curative potency of gastric preparations. All of this
work which has been done in the past 15 years, has resulted in a renewed interest
in the disease and in the factors that may he causative. From it has arisen the
present concept of the cause, which is to the effect that it is a nutritional de-
ficiency disease.
n6
 PERNICIOUS ANE!&IA-

PRESENT CONCEPT OF PATHOGENESIS

There is general agreement that the normal maturation of red cells is gov-
erned by the action of a so-called hematopoietic factor, sometimes called the
"anti-anemic factor." The absence or defIciency of this factor iu the bone marrow
re'sults in the arrest of maturation of erythrocytes at the megaloblastic level, or
as Isaacs expresses it, there results a "bone marrow block" at that level.
There is further agreement that the hematopoietic factor is formed by the
union or interaction of a substance present in the diet known as the extrinsic
factor, with a principle produced by the glands of the normal stomach and upper
duo.denum, known as the intrinsic factor. Aiter iormation of the .hematopoietic
factor in the stomach, it is supposed to be absorbed, then stored in the liver, kid-
neys and other tissues. Presumably it is released by tbese tissues and utilized
by the bone marrow for production of sufficient number of erythrocytes to main-
tain the normal level. (For detailed discussion see section on Factors Influencing
Erythropoiesis.)
In view of the above concept of normal erythropoiesis it now seems well estab-
lished that the pathogenesis of pernicious anemia results from a defIciency of the
intrinsic factor in the stomach. This is supported 9Y: fIrst, the well known gastric
and duodenal mucosal atrophy resulting in achylia; sec,ondly, by the fact that the
administration of normal stomach mucosa will effect a remission of the disease
(Sharpe, Sturgis and Isaacs); and thirdly, by the work of (;astle . showing that
a remission can be induced by feeding a mixture of normal gastric juice, and meat
which contains the extrinsic factor. Therefore, in the light of present knowledge,
pernicious anemia is primarily a disease of. the stomach and upper duodenum in
which there is atrophy of the glands of the mucosa from an unknown cause.
Schindler and Serby have made gastroscopic observations on 23 patients and
found that the untreated group showed variable mucosal lesions, including super-
ficial gastritis, atrophic gastritis, or botb, or patchy and diffuse mucosal atrophy.
Strangely enough, the condition is corrected only in some of the patients after
adequate therapy.
In those instances of total gastric resection which are not followed by the
development of pernicious anemia, the normal gland tissue in the upper duodenum
probably elaborates a sufficient amount of hematopoietic substance for the normal
requirements which is indicated by the work of Meulengracht who showed that
the hematopoietic principle is elaborated mainly in the pyloric glands of the
stomach and to a lesser degree by the glands of the upper duodenum. Meu-
lengracht states that the most common pathologic change is a lesion of tbe
gastric mucous membrane consisting of atrophy of the glandular portion of
the stomach, particularly the chief cells and the parietal cells. He points out
that the anti-anemic factor is secreted in the pylorus region by the large clear
pyloric gland cells and the same type of gland cell is also present in the duodenum.
Even though the glands are more numerous in the duodennm, the anti-anemic
output of the duodenal glands is slightly less. Meulengracht holds that1!ie
 PLA;JrE XXIV

PERNICIOUS ANEMIA IN RELAPSE

(PKIMARY ANEMIA)'

t. Hypersegmented neutrophil.
2. Megalolilast
3. Macrocyte
4. "Tailed" erythrocyte.
5. "Racquet" erythrocyte
6, Erythrocyte wIth Howell-Jolly bodies.
7. Polychromatocyte.
Blood Findings: Differential:
Hemoglobm . , ... 5.8 gms (Newcomer's method). Segmenters .. . . . ..... 49%
RBC. •• 1,100,000 per c,mm. ('5% bypersegmented)
W.llC. 3,900 per c.rum. LYlnphocytes . " ..•.•.. 500/0
Platelets 40,000 per c.mm. EosmophIls . ........•.•.. I %
Color Index '.
Volume Inde~.
Icterus Index
Reticulocytes .
. ..
'.
, 5
120
0.1%
FragilIty of erythrocytes in hypotonic salt solutions: Hemolysis beginning at 0 ..38% NaCI
and complete at 0.300/0.
Gastric Analysis-Achylia.
Erythrocytesj macrocytic and hyperchromic with marked poikliocytosis, moderate pol},chro~
matophilia, marked anisocytosis, cccasional Howe1I~Jolly body, and pronounced megaloblastemia.
Plate XXIV.
 PERNICIOUS ANEMIA 279

intrinsic factor of Castle found in the gastric juice and that found in the stomach
wall is all produced by the pyloric glands and Brunner's glands, which are iden-
tical. He calls all of this the pyloric gland 'system. It is a paradoxic situation
that the stomach in pernicious anemia shows atrophy of the parietal and chief
cells in the fundus portion, but in the pyloric portion and duodenum the normal
glandular structures are preserved. Meulengracht explains this by stating that
the fundus gland system under normal conditions acts as a starter or pacemaker'
for the pylorus gland system, and that the latter fails to secrete simply because
there is no excitation from the fundus gland system. A second possibility is that
the interaction between the intrinsic and extrinsic factors does not take place
in the stomach' but in the intestine. It is known too that the intestine has some
measure of anti-anemic activity. For that matter, various parts of the entire
intestinal tract have anti-anemic potency. A comparison of this is shown by the
following:
Cardia................... ........... 30
Fundus .... ......................... 2
Pylorus.. ............................. 120
Duodenum.............. .............. 35
Jejunum .... ...................•...• 24
Ileum................................ 44
Large intestine............. ......... 9

Meulengracht also points out that the intrinsic factor may be secreted to
some extent in the lower portion of the intestine, just as it is in the upper portion.
Furthermore, the completed substance 'may not be fully prepared until it reaches
that point. It may be necessary that a second factor low in the intestines must
unite with the one from the pyloric gland area All of these are points that still
remain unsettled.

DISTRIBUTION AND INCIDENCE

Pernicious anemia is said to occur mainly in countries of temperate climate

and to be quite rare in tropical and sub-tropical regions. There has been no
adequate explanation for this distribution, but it appears reasonable that the
Nordic and blonde peoples are quite rare in the tropical countries as compared
to other races. According to Cornell it is marc prevalent in Northern Europe,
North America and the British Isles, than in any part of the' world.
The disease is more common in men than in women. It occurs in late adult
life or in old age with the average age between 40 and 60 years. It is quite rare
under 30 years and is said not to occur in children. In Isaacs' group of 10DO
patients, the symptoms began in most of them between the ages of 35 and 65
years, with the peak at 5S years.
It is chiefly a disease of the white race, and in particular, of the NorrHc types.
According to Hunter it is rare in Jews. It is doubtful if it ever occurs in a full
blooded negro, but has been reported in mulattoes. For ten years we have watched
carefully for the disease at Grady Hospital in Atlanta among a large number of
colored patients, but have not yet seen it in that group. There is no evidence
.80 THE ANEMIAS

to indicate that occupation, station in life, or living conditions, play any part in
the development of the disease. It occurs in the rich and poor alike.
Draper has studied very carefuIly the constitutional type of person that is
the usual victim. He describes them as people with short, broad faces; large
mandibular angles; very short noses; short but deep wide chests; and especially
wide subcostal angles. He states that "the male of the pernicious anemia race,
therefore, is a medium to tall individual with short chest, high placed umbilicus,
long abdomen, with relatively long lower extremities." They may show feminine
tendencies as regards the secondary sex characteristics. Addison, in his original
description, pointed out that "the disease occurs in patients of a somewhat large
and bulky frame and with a distinct tendency to fat formation."
There is much evidence to indicate that there is a hereditary or family pre-
disposition to the disease. Numerous instances have been cited, (l\Ieulengracht,
MacIachan and Kline, Naegeli). It is also known that there exists a familial
predisposition toward achlorhydria, regardless of whether or not there is perni-
cious anemia in the family. It has been pointed out that there are families in
which the males have pernicious anemia and the females have idiopathic micro·
cytic anemia. Both have achlorhydria. This leads to the speculation as to
whether the stomach defect is the hereditary transmissible factor. Meulengracht
points out that one important factor in pernicious anenlia is the individual him-
self, with particular reference to the constitution with which he was born. He
assumes that perhaps the genes of the gastro-intestinal tract are deficient. He
studied one family in which there were four cases of pernidous anemia and a
number of others in the same family in whom there was achylia but no pernicious
anemia. He points out that the hereditary weakness may lie in the stomach.
Also, the gastric achylia may precede the pernicious anemia by 10 or 20 years
or more. Many patients give a history of peculiar dietary habits, especially an
aversion to the use of meat. This may have been present since childhood, or
may have developed in late adult life.

SYMPTOMS AND PHYSICAL FINDINGS

Minot has found that the average duration of symptoms in 100 cases was
1.36 years before the diagnosis was made, emphasizing the fact that the disease
is usually well advanced before the patient consults the physician, and also, that
the onset is gradual and insidious. The disease may develop with mild relapses
in the early stages, or with intercurrent periods of remission and a feeling of well
being so that the history may include recurrent attacks of symptoms over a long
period of time with intervening periods of relatively good health,
The initial symptoms are quite variable and may include any number or com-
bination of findings referable to the gastro-intestinal system, the neurologic sys-
tem, or to the hematologic deficiencies. It has been stated that the usual early
diagnostic triad consists of weakness, sore tongue, and numbness and tingling
of the extremities. According to Musser and Wintrobe tbe common initial symp-
toms in order of frequency, are fatigability, weakness and faintness, numbness, tin-
 PERNICIOUS ANEMIA 28r

gling and stiffness, headache, nausea, lack of appetite, vomiting, dizziness, dyspnea,
palpitation, diarrhea, loss of weight, pallor, abdominal pain, and sore tongue.
Anyone or any combination of the above may be the first manifestation of the
disease. Based upon his studies of approximately one thousand cases of perni-
cious anemia, Isaacs states that the first symptom and the commonest one is ease
of fatigue and generalized weakness. It was the presenting symptom in 85 per
cent of his patients. Other symptoms in order of their frequency included numb-
ness, over 80 per cent j tingling of fingers and toes, 74 per cent; shortness of
breath, 64 per cent; symptoms referable to the stomach, 62 per cent; constipa-
tion, 64 pcr cent; palpitation, 50 per cent; edema, 44 per ccnt; loss of appetite,
44 per cent; difficulty in walking, 44 per cent; symptoms referable to the bladder,
35 per cent; dizziness, 28 per cent, etc. Isaacs noted that pallor or a peculiar
yellow color was present in the disease during .relapse. Lymph nodes were pal-
pably enlarged in 7 per cent, glossitis was seen in 74 per cent, and atrophy of the
papillae was present in 70 per cent.
Since it is well known that any of the three major systems involved may be
affected at different times, the earliest signs of illness may be referable to the
anemia, to the neurologic system, or to the gastro-intestinal system, without
the other two being involved. Also it is well to bear in mind that the patient of
modern times, since the advent of liver therapy, may not show the progressive
development of symptoms since many of tEem have been taking "shotgun" medi-
cation at irregular intervals, consisting of various mixtures of iron, vitamins and
liver preparations without having had an adequate and thorough dia!Wostic study
beforehand. The clinical picture may be quite atypical in this type of "half
treated" patient, and the clinical and hematologic findings somewhat obscured
or atypical at the time of initial examination.
Addison's original description of the patient with pernicious anemia in re-
lapse has never been surpassed: "The countenance gets pale, the whites of the
eyes become pearly, the general frame flabby rather than wasted; the pulse per-
haps large but remarkahly s6ft and compressible, and occasionally with a slight
jerk, especially under the slightest excitement. There is an increasing indisposi-
tion to exertion with an uncomfortable fee1ing of faintness or breathlessness on
attempting it; the heart is readily made to palpitate; the whole surface presents
a blanched, smooth and waxy appearance; the lips, gums and tongue seem blood-
less; the flabbiness of the solids increases; the appetite fails, extreme languor
and faintness supervene, breathlessness and palpitation being produced by the
most trifling exertion or emotion; some slight edema is probably perceived about
the ankles. The debility becomes extreme; the patient can no longer arise from
his bed; the mind occasionally wanders; he falls into a prostrate and half torpid
state; and at length expires. Nevertheless, to the very last, and after a sickness
of perhaps several months' duration; the bulkiness of the general frame and the
obesity often present a most striking contrast to the failure and exhaustion observ-
able in every other respect."
The above description was of pernicious anemia nearly 100 years ago but
few patients are seen today that present the picture described, because therapy
 THE ANEMIAS

with liver preparations has usually been employed long before they reach this
state. Therefore, the diagnosis of pernicious anemia becomes increasingly diffi-
cult as the use of liver becomes more widespread, and the untreated patient in
relapse is seldom seen.
The symptoms may be entirely those of the gastro-intestinal tract. Among
these are sore mouth and tongue which is seen in over half of tbe patients. Tbis
usually involves the anterior balf of the tongue, and is especially pronounced on
tbe ingestion of hot or bighly spiced foods. Tbe tongue may be swollen and in-
dentations from the teeth can be seen. On examination it is red with slight evi-
dence of ulceration or formation of small blisters. These are usually seen about
the edges on the anterior half. Later there is a wasting of the papillae with a
resulting smooth and shiny surface. At this time there may be a loss of taste
sensation. This process of a sore mouth and tongue may seem to extend into the
upper part of the esophagus.
The patient may complain of vague digestive disturbances, including loss of
appetite, nausea, vomiting, a feeling of fulness, belching, passing of gas with
foul smelling stools, and intermittent periods of constipation and diarrhea. There
is nothing significant about the stools except excessive fermentation or putre-
faction. The symptoms referable to the stomach usually coincide with the peri-
ods of severe glossitis, and in remissions all of these disappear only to return in
another relapse. Many of the symptoms are caused by the achlorhydria and are
common to other achlorhydric diseases such as pellagra, sprue, achlorhydric
microcytic anemia, and carcinoma of the stomach. The teeth are usually in a
poor state of cleanliness and pyorrhea is observed in many patients.
The liver mayor may not be enlarged. In the later stages it is usually
slightly enlarged because of the changes of fatty degeneration. Bigg found the
spleen palpably enlarged in only 3 per cent of 200 cases of pernicious anemia,
and considers that an enlarged spleen is not part of the clinical picture. On the
contrary if an enlarged spleen is present this should militate against the diagnosis.
There is seldom evidence of clinical jaundice although the icterus index may be
elevated. There has long been noted the characteristic lemon yellow color of
the skin and irregular blotchy areas of brown pigmentation. This latter finding,
with low hlood pressure and extreme weakness might easily be confused with·
Addison's disease.
There may be nervous or mental symptoms before there is any evidence of
anemia and in one-fourth of the patients these cbanges are the first to appear.
They require some time to develop, occur rarely in an acute form, and are espe-
cially common in the elderly patients. The following description of the neuro-
logic changes is quoted from Castle and Minot: "In the majority of patients
the symptoms of neural involvement are confined to slight disorders of sensation,
persistent and usually symmetrical numbness and tingling of the hands and feet.
Among the earliest of the objective signs is a diminution of the vibration sense
at the ankles, usually hut not always symmetrical. The development of marked
spastic ataxia indicates degeneration of the lateral and posterior columns of the
spinal cord. When the former tracts are particularly involved, spastic gait.
 PERNICIOUS ANEMIA

increased reflexes and positive Babinski signs are usually present; with lesions
of the latter tracts loss of vibration and position sense and ataxia appear. Girdle
sensations occur but lightning pains, girdle pains and abdominal crises are un-
common. Due to peripheral nerve involvement, 'stocking' hypesthesia (decreased
sensation), or anesthesia may occur. OccaSionally hyperesthesia (increased
sensation) of the sales of the feet may be very marked. Disturbance of the
sensation of heat and cold is unusual. Finally, absent or diminished reflexes with
involvement of the sphincters of bladder and rectum, contractures and decubitus
result in the advanced clinical picture."
Symptoms of changes in the special senses are seldom seen. Optic atrophy
occurs rarely and the disturbances of vision and auditory sense which are often
present, result from the anemia or retinal hemorrhages. Kampmeier and Jones
reported three instances of optic atrophy in pernicious anemia patients and
advanced the opinion that the process was one of intrinsic nerve degen~ration
rather than one caused by severe ischemia. The mental symptoms are V'l.riable
and may include irritability, restlessne;,s, mild depression, confusion, delUSions,
hallucinations, and maniacal outbursts. Some patients may appear listless or
indifferent, and appear to be suffering with severe melancholia.
The circulatory disturbances are dependent upon the severity of the <tnemia
and may include dizziness, tinnitus, palpitation, some dyspnea, and excessive weak-
ness and fatigue. The heart rate is usually increased but no murmurs or irregu-
larities are heard. However, soft systolic bruits may be heard over the apex.
In general, the circulatory changes are identical with those present in any type
of severe anemia. Fever is usually present during relapse but not during remis-
sions. It may be quite high and septic in type for a short time, or there Dlay he
a relatively low elevation of temperature which persists for several months.

LABORATORY FINDINGS

BLOOD
In most cases of pernicious anemia the blood findings are of consi<1erable
diagnostic value. The picture is characteristic when the patient is in r~lapse.
The red cells fall to extremely low levels and counts are recorded as low as
300,000 cells per cu. mm. However, it is quite unusual to find such low figures
since the use of liver extract. In the early stages of a relapse there is a Slightly
increased size of red cells before there is diminution "f the number. The fall of
red cells is more rapid when the number is between 5 and 3,000,000 per Cll. mm.
and after it reaches the latter figure the rate of decrease becomes slower.
Examination at any stage will show the characteristic macrocytosis in many
of the cells. The stained blood of the untreated patient shows extreme varlations
in size and shape of red cells, many of which are definite macrocytes, oth~rs arc
normal size, while a few are microcytic. A considerable proportion exhit,it the
most bizarre shapes, being large and round, or oval and tailed, with inegular
shapes of all types. Some show marked polychromatophilia and others ar~ char-
acterized by stippling and other evidence of basophilic changes, includillg the
 PLATE X.XV

PERNICIOUS ANEMIA DURING REGENEFATION

~
\i&
I

I. Neutropbil
2. Lymphoryte
3. Reticulocyte
4 Normoblast.
s. POlkllocyte.
6 Macrocyte (polychromatophilic).
Blood Findmgs (same case as shown III plate 2.f. during DrlIerentiJ,1
r('generattOn foUov,nng hver therapy)'

Hemoglobm I~ 6 gros. (Newcomer's method), Neutrophils

RBC 3,400,°00 per c mm. Lympbocyte9-
WBC. 9,600 per c mm. Eosinophils
Platelets ~50,ooo per c mm.

Color Index
Volume Index
Icterus Index
Reticulocytcs
Erythrocytes; slightly hyperchromic and normocytic with amsocytosis, slight poi~:llo[ytosisj
polychromatophilia, occasional normoblast, and marked relicutocytotis.

Gastric Analysl:l-AchyIia.
Plate XXV.
 PERNICIOUS ANEMIA

presence of so-called nuclear remnants, Cabot ring bodies and Howell-Jolly

bodies_ Tbe large round cells are probably reticulocyte. while the typical macro-
cyte is usually an oval, large, intensely stained celL
Nucleated red cells are seen frequently, and many of them are megaloblasts.
This occurs so consistently that their presence was formerly regarded as a reli-
able diagnostic sign. It is true that they occur in no other disease so frequently
as in pernicious anemia hut the megaloblast is seen in any type of severe anemia
in which there may be disturbance of erythrocytic maturation at the megaloblastic
level. They are found in the blood of patients with leukemia, especially the
acute myeloid types. The presence of megaloblasts and other nucleated forms
indicates an effort on the part of the marrow to supply the badly needed red
cells, ~nd further indicates that the patient is not receiving the necessary hema-
topoietic principle that governs orderly maturation. They should not be seen
after normal hematopoiesis has been established with liver extract, nor are they
seen during remissions. When the nucleated cells are present in large numbers,
this has been called a blast crisis.
Although the cells may be markedly reduced in number they are well filled
with hemoglobin. Most of them stain intensely with the eosin component of
Romanowsky stains and the central pallor of hypochromic anemia is seen in very
few cells. The color index is consistently above one and often is 1.5 or more.
Therefore the anemia has been designated as a classic example of a hyperchromic
type, This and the macrocytosis constitute the two outstanding features of the
red cell picture. Although macrocytosis and hyperchromia are characteristic,
their presence is not diagnostic of the disease since there are other anemic states
with similar findings. Tbese include certain pbases of the anemias of pellagra,
sprue, Diphyllobothrium infection, pregnancy, carcinoma of the stomach, liver
damage, secondary aplastic anemia, and leukemic states. Furthermore, even
though a patient presents the classic picture of pernicious anemia there are
times when macrocytosis and hyperchromia are not present, and during ade-
quate treatment the cell size may be normal with a color index below one. The
average diameter of the red cells is between 8.0 and 8.S micra, and with the cell
thickness normal, this results in an increased cell volume which may be as much
as 1.5, compared to the normal of one.
The leukocyte changes are not characteristic. In relapse and in the un-
treated patient there is usually a variable degree of leukopenia with the granulo-
cytes reduced in number, resulting in a relative but not an absolute lymphO-
cytosis. The leukopenia may be as low as 2000 cells per cu. mm. There is a
shif~ to the right in the Schilling index since many of the circulating granulocytes
are not only large but exhibit hypersegmentation of the nuclei and some of them
have as many as eight to ten lobes in the nucleus (Briggs, Cooke, Heck). This is
not a consistent finding and in our experience, is not of considerable importance
in diagnosis. In a study of 40 patients Sharpe and his associates found that the
hypersegmented neutrophil is seen in severe relapse and that this type of cell is
seen both in the marrow and peripheral blood. Jones has also advanced the
opinion that the hypersegmented large neutrophil has an abnormal development,
286 TUE ANEMIAS

even in the bone marrow. In the patients with extremely low leukocyte counts,
there may be a shift to the left with immature types. This is usually seen in
the stage of blast crises. Eosinophilia is present in patients under treatment,
and according to l\.1eulengracht, the eosinophilia may be as high as 50.. per cent
in patients treated with raw calf liver. This finding is not so evident in patients
treated with injections of liver extract.
The platelets are usually reduced in number, the extent of decrease rougbly
corresponding to the decrease of red cells. Therefore, because of tbis in relapse
there may be bleeding, including petechiae, purpuric spots, and retinal hemor-
rhages. The platelets promptly rise to normal levels when treatment is adequate
and a remission is induced. When the platelets are low there is an increased
bleeding time, a normal coagulation rate, and delayed retraction of the clot.
Other blood findings are those resulting from excessive destruction of red
cells, these including a dark color of the plasma, increased plasma bilirubin, in-
creased icterus index to as high as twenty, and a positive indirect van den Bergh
reaction.

URINE
The urinary findings may indicate the presence of chronic nephritis and in-
clude fixation of specific gravity, small amounts of albumin, and a variable
number of hyaline and granular casts. Fouts and Helmer have found that the
urea clearance is low in patients during relapse but may be quite normal or
high during remission. During relapse and during periods of apparent excessive
red cell destruction the urine contains ·increased amounts of urobilin and
urobilinogen. At no time, however, does it contain appreciable quantities of
the hematopoietic factor, which, according to Wakerlin, is excreted in the normal
urine. Dobriner and Rhoads ha"e pointed out that urinary excretion of copro-
porphyrin I and urobilin is increased d.uring relapse and becomes normal after
institution of therapy. They believe that hemolysis is a factor in pernicious
anemia and state that this is not incompatible with the etiologic mechanism
described by Castle. The urine contains these products only in the presence of
a deficiency of the hematopoietic factor.

STOOLS
There are no characteristic stools, since there may be alternating periods of
diarrhea and constipation. During diarrhea, the stools are watery and mucoid,
but do not contain pus or blood. They are usually colored darker than normal
because of increased biliary·pigments. In associated gall bladder disease, which
seems to occur so frequerilly with pernicious anemia, there may be light colored
stools due to partial or total obstruction of the common duct with fibrosis.

GASTRJC ANALYSIS
Gastric findings are of the utmost importance, the chief one being the char-
acteristic achylia. In suspected pernicious anemia the gastric contents should
 PERNICIOUS ANEMIA

always be examined after stimulation witb histamine (injection of one·half to

one cc. of histamine hydrochloride subcutaneously). If the achlorhydria per·
sists after injection, it is then regarded as a true achylia, without which it is
questionable if the diagnosis should be made. Complete achlorhydria should be
present before it can be stated with assurance tbat a patient has pernicious
anemia and it constitutes the single most reliable criterion for diagnosis. It
is found at all times, in relapse and remission, and is the single pathological
state that seems unaffected when all other evidence of the disease disappears with
proper treatment. There should be absence of free HCI even on stimulation with
histamine, before it can be stated with assurance that the patient has pernicious
anemia. It is not unusual to see absence of HCI even with alcohol stimulation in
young healthy people, as shown by the report of Townsend, who studied 6,
healthy young adult students and found seven of these with no free Hel even
after an alcohol meal. However, six of them responded to bistamine with free
Hel.

DIAGNOSIS

The diagnosis of pernicious anemia is based largely on the development of

paIlor 1 recurring periods of fatigue and weaknes.s, sore tongue and glossitis with
variable gastrointestinal complaints, lhe neurological findings including the
parasthesias, decreased vibration sense and ataxia, a-total achylia of the stomach,
and the hematological findings of decreased red cells, macrocytosis, and increased
color and volume indices.
Recognition of the disease in the untreated patient is not difficult, but nearly
all patients have had some type of medication in the form of various mixtures
of iron and liver preparations, so that both the clinical symptoms and laboratory
findings may be obscured to the point where the exact nature of the process can·
not be ascertained. In such instances it is far more satisfactory to stop all medica·
tion and permit the disease to pursue its course until a definite diagnosis can be
made. When this is done and the type of anemia properly.studied and classified
it will save the patient from future periods of partial illness and will not result
in the half treated and half sick individual who goes along for years never in good
health. Also, it will save an untold amount of useless and perhaps expensive
medicinal agents that are of no value. There seems to be a deplorable tendency
among some physicians to treat anemic patients with transfusions, intensive liver
and iron therapy to relieve the immediate symptoms and then try to establish
. the diagnosis.
In making a diagnosis of pernicious anemia it is important to remember that
not all patients have anemia (Sanford), nor do all of them show neurological
changes, but practically all of them have symptoms referable to the gastroin·
testinal tract.
When the diagnosis is difficult to establish the injection of a single dose of
potent liver extract is usually followed by a marked reticulocytosis in 48 to 96
 288 THE ANEMIAS

hours. This response, which is called the therapeutic diagnostic test, is stated
by many to confirm the diagnosis. However, it should be pointed out that
in any anemia in which the pathology is dysfunction of erythropoiesis at the
megalohlastic level, such a response may occur. This is notably true of all
anemias characterized by macrocytosis. Therefore, the reticulocytosis after liver
injection is not absolutely confirmatory of the presence of pernicious anemia,
although it is a valuable indication as to the type of anemia present ami the
nature of the deficiency.

TREATMENT

DEVELOPMENT OF LIVER AND STOMACH PREPARATIONS

In his studies on anemic dogs, Whipple l"as the first to point out that certain
tissues of the body are more capable than others in the regeneration of blood and
hemoglobin, and among those studied liver was the most efficient. (See page
168 for list of tissues.) In 1926 Minot and Murphy demonstrated that the feeding
of Hver to patients had a remarkable curative effect, and in 1929 Sharp and
Sturgis and Isaacs demonstrated a similar action with the dried mucosa of pig
stomach. Gansslen Was the first to use injections of liver extract, and Cohn
and his associates have been largely responsible for the refinement and con-
centration of liver for parenteral use.
At this time many preparations of liver and stomach for oral and parenteral
use are available. These are prepared by many manufacturers and many of
them are sold under a variety of trade names with various claims of superiority
being made for each product. This has led to so much confusion among physicians
who wish to utilize the best product available in treatment of their patients,
that we shall first review the general principles of therapy with these specific
agents.
In the treatment of pernicious and other macrocytic anemias, liver and
stomach preparations are specific in that they seem to supply a nutritional
need for the maturation of red cells. The commercial preparations are derived
mainly from mammalian liver, chiefly that of heef, and one preparation (Chap-
pell) is of equine origin, being made from horse liver. Fish liver is also potent
but its use is not practical. It is also known that kidneys, brain tissue and
placenta are relatively high in the hematopoietic principle.
It has been shown by Castle and his associates that the principle in liver
is not the same as that in stomach preparations although hoth give good re~ults
in treatment. Also Walden and Clowes have shown that when the two prepara-
tions are mixed there is an increased therapeutic efficiency of the resulting prod-
uct and that a ;;maller amount of the mixture, when taken hy mouth, will obtain
a response equal to that of a much larger amount of either preparation. This
action, usually called "potentiation of liver by gastric tissue," is utilized in the
preparation of variou~ products containing a mixture of the two. (Extralin,
Lilly & Co.)
 PERNICIOUS ANEMIA

The main difference between liver and stomach preparations, from a prac-
tical standpoint, is the fact that the essential principle in liver is thermostable,
or heat resistant, while that in stomach preparations is destroyed if heated above
65° C. Therefore, stomach preparations are prepared at a low temperature
and are available only in a dry powdered form, from which no products suitable
for injection have been made available. Also, no stomach products in aqueous
solution are available 'because of danger of bacterial contamination. On the
other hand, liver preparations are available in the form of dry powdered extracts
for oral use, and concentrates prepared from them for purposes of injection. The
concentration of liver has become so refmed that preparations are available in
which onc cc. represents 100 Gm. of fresh liver. (Lederle, Liver extract-i:on-
centrated.)
Co~centrated extracts of liver have been diweloped mainly as the result of
the work of Cohn and his associates.. The various methods of commercial extrac-
tion and preparation are modifications of their original methods. Essentially
the process is as follows: The raw and fresh liver of cows are ground and ex-
tracted with water. The filtrate is acidified sligbtly and heated. The acid and
beat insoluble substances are precipitated in large quantity. The resulting con-
centrated filtrate is an aqueous extract, which is then mixed with sufficient alcohol
to result in a content of 70 per cent, at which point there is a second precipitate
which does not contain the active principle, and is removed by filtration. To
the filtrate is added sufficient absolute alcohol to result in 95 per cent solution
by volume, and at this point a third precipitate is formed which contains the
active principle. This is the so-called "fraction G of Cohn." This substance
(precipitate) is soluble in water, resistant to heat by boiling or autoclaving and
can be used either for oral ingestion or intramuscular injection. The fraction
G of Cohn is the product that is ordinarily employed for intramuscular. injection
in the treatment of pernicious anemia. Since its preparation in 1938, various
groups of investigators have 'attempted further to purify the potent material.
The results of these efforts are summarized in an excellent article by Jacobson
and Subbarow (I941). They point out that although the potent principle has
not yet been isolated much progress has been made. There are many difficulties
in the isolation, chief among which is the lack of a satisfactory method for assay
of the materials. The guinea pig method of assay is of some value but does not
serve as a reliable indicator of the entire therapeutic potency of liver. They
point out that as the liver principle is isolated to smaller and smaller amounts it
loses therapeutic activity as fractionation proceeds. These authors state that
this loss of activity is probably caused by the fact that the hematopoietic prin-
ciple is not a single agent but is of a multiple nature. Practically all workers
have begun their efforts with the fraction G of Cohn as the starting point.
In 1930 Cohn and his associates obtained a material, 140 mg. of which in-
creased the concentration of red cells in a patient by nearly one million per
cU.mm. in ten days. This was an amorphous substance and had the properties
of a nitrogenous base. In 1935 Dakin and West found that Cohn's fraction G
 THE ANEMIAS

could be precipitated by rheineke acid and in 1936 Dakin and associates showed
that the same material could be precipitated by saturated ammonium sulphate.
Using these steps with certain other purifying methods a product was finally
obtainea which bas the properties of a peptid that gave a fairly good therapeutic
response in doses of 40 to 100 mg. Also in 1935 an additional step was dis-
covered when it was found that charcoal absorbed the active liver principle, and
that this material could be eluted from the charcoal with phenol. Subbarrow and
his associates finally used charcoal for absorption and alcohol for elution of the
active material. This latest method has yielded a chemically crude extract active
in doses of approximately. ID4 mg. derived from 100 Gm. of whole liver. They
believe that this contains several different substances consisting of the major
fraction and the so·called acce.sory fractions. The latter are therapeutically
inert but appear to augment the activity of the primary factor. In 1938, there
had been isolated from crude liver extract three rna terials considered to be acces-
sory factors. These were l·tyrosine, or fraction N, found to th~ extent of 6 mg.
per roo Gm. of whole liver; secondly a complex purine fraction C, found to the
extent of II mg. per 100 Gm. of liver; and thirdly, a fraction F, yielding 14 mg.
per roo Gm. of liver. In 1936, Dakin and his associates reached the conclusion
that the hematopoietic substance in liver is associated with a peptid.possessing
many but not all of the properties of an albuminose. The ultimate goal, of
course, is tbe crystallization of the primary 'factor with the possibility that it
might eventually be prepared synthetically. .
The following table, taken from Castle and 1I1inot, shows the available prepa· ~
rations of liver and stomach, the amounts of fresh material from which they
are derived, and the method of use, each amount being that necessary to produce
maximal reticulocyte response in pernicious alleluia.
TABLE XVII
Original lVeight Prepared Weight Route oj
(Gram,) Of volumes A dm~nistra.tion
Liver of kIdney ,. .. ... ... . .... . 500 400 Gill Ora]
Desiccated hog stomach (Ventric) ... . 250 30 Gm. Oral
Liver extracts:
Aqueous concentrate ....... .., .. soo 65 ce. Oral
Precipitate 95% alcohol fraction "G" 600 27 Gm. Oral
Dllute solution fraction HG" .. . . . . 10 2 ce. Intramuocular inj-ectiou
Concentrated solution fraction "G".. 20 0.6 ce. Intramuscular injection
Liver stQmach preparation. . . . .. . . . . . . 4.5 Gm. Oral
A study of the above table shows that for a patient to obtain a maximum reo
ticulocyte response he can eat 400 Gm. (one pound) of liver, or 30 Gm. of
desiccated hog stomach or drink 6S ec. of aqueous solution of liver. concentrated,
or he injected with as little as 0.6 ce. of concentrated liver extract derived from
20 Gm. of fresh liver. The extracts have been further refined so that he can also
be injected with one cc. concentrated from roo Gm. of fresh liver, which on the
basis of the figures in the above table, should be five times the amount necessary
to produce maximum reticulocyte response.
The effectiveness of liver preparations when injected, is about 60 times of
 P1!RNICIOUS ANEMIA

that taken by mouth. Therefore, the injection of one cc. derived from 100 Gm.
of fresh liver should be equivalent to the effects obtained from the oral ingestion
of about 6000 Gm. (twelve pounds) of fresh liver. These relationships are

FIG I -Showing the amount of liver required to treat a patient With pernicious anemia in
relapse for one month. EIghteen p-ounds of fresh liver) or powdered extract from thirty-three
pounds, for the injectable concentrate In seven vials which bas been denved from seven hUbdred
gram3 of fresh hver. Courtesy, Lederle Laboratories.

FIG. 2.-A graphic, 11lustration of th~ amounts of hver preparations required to produce
remission in the average patlent With pernicious anemia. Showing the amount of fresh INer,
powdered extract, or injectable concentrat~. Courtesy> Lederle l Laboratories.

shown graphically in Fig. I in which it can be seen that the liver required to
treat a patient in relapse for one month would be IS pounds of fresh liver by
mouth, or the powdered extract derived from 33 pounds, or seven vials of con-
29 2 THE ANEMIAS

centrate derived from 700 Gm. of fresh liver. A similar relationship is shown
in Fig. 2.
It seems evident, therefore, that a patient can be more efficiently and eco-
nomically treated by injections of liver rather than by the oral administration
of it. Furthermore, it has the advantages of assurance of absorption and accurate
measurement of dosage since there is no way to determine the amount of liver
absorbed after it is given by mouth. It can be given to patients who are critically
ill and have difficulty in swallowing; the patient remains under the close observa-
tion of the physician; and the treatment is more economical. Injections of liver
extract are followed by few undesirable reactions, and their efficiency in pro-
ducing reticulocytosis has been definitely demonstrated.
If a patient requires an injection of concentrated liver extract (from 100 Gm.
fresh liver) every three weeks (the average) for maintenance purposes, the cost
is' slightly more than one dollar for the material at present prices. Also pMients
can learn to give themselves an injection at occasional intervals, just as the
diabetic does it thre~ times daily. We have demonstrated to our satisfaction
that a South Georgia farmer living 20 miles from a railroad can maintain his
hematologic values by giving himself an injection every two weeks.
The simplification and refinement of liver preparations may be illustrated by
the following table:

TABLE XVIII
AVERAGE DOSES FOR PERN~CIOUS ANEUIA PATIENTS IN RELAPSE*
Total dose
per month
~~;~~~~iiv~~it:!.~~~ 3~a~ymdOS~ ~;:5 G~;.. ·: : ~.:. ~'::.:',:.::::: .::':".::
Qooo Gm.
375 Gm.
Solution lIver extract for jnjection. Each 5 ce. from 100 Gm. liver. Average dose
seven vials monthly . . . ........ . ... , ... , . .. . . . . .., . 3S ce.
193 2 Concentra.ted solution liver extract. Each 3 ce. from 100 Gm. fresh liver. Average
dose seven vials monthly.. .. . .... ..,.. ,0 •• 0 •• 0 •••• 0 0 ••• • ••• 21 CC.
1935 Concentrated solution livcr extract. Each I cc. from 100 em fresh liver. Average
dose seven. vials monthly ....o. •••••• o. •••••••• .., .,. • • • • • • •• • ••• 7 CCo
* From the Lederle Laboratories, New York, N. Y.

It should be pointed out that additional refinement and extreme concentra-

tion does not necessarily mean that all of the potent material present in the
original liver substance has been preserved in the final product. There is reason
to believe that as the concentrate becomes smaller the loss of potent material be-
comes greater. The fact, therefore, tllat a certain preparation is derived from so
much original tissue, is no as:mrallce that all of the potent maierial is present
in the product. The efficacy of the extracts has been very difficult to evaluate,
mainly because of a lack of a testing method, the only one being the reticulo-
cytosis of it patient in relapse. Because of the increasing scarcity of such patients,
human testing material has become less and less available. Probably nle most
satisfactory measurement of the efficacy of these products is the use of the term
"unit" of potent material, that is, that amount required when given daily to
 PERNICIOUS ANEMIA

produce a satisfactory reticulocyte, red cell, and hemoglobin response in a patient

in relapse, regardless of tbe amount of fresb liver from which it was derived, or
the manner in whicb it is given.

TABLE XIX
INJECTABLE PREPARATIONS OF LIVER'
&tat'l Price
Units per ce. Price per Unit
PreparoJ,ion Manufacturer U.S.P. per 10 ce. (in cents) Remarks
Campolon \Vinthrop 1.0 $ 1·'5 12·5 About twice the price, and
has no superiority over
other preparations.
Liver Extract Abbott 50 300 60 Apparently a good prepara~
tion.
Liver Extract with
Vito B 1 • Abbott (n.s.) 1200 Ordinary liver extract with
vitamin 8 1• A very high
price for the vitarmn
Liver Extract. Abbott 10 per cc. 6 00 60 Arathpr high concentration.
Liver Extract Lederle 3 0 2.00 6.0 A good preparatJOn~not so
concentrated.
LIver Ext ract Lederle 15·0 1000 6 7 A highly concentrated ex~
tract-perhaps too much.
Liver Extract Lilly I per cc. 90 9 0 Not so concentrated.
Liver Extmct Lilly 2 per cc. 1.20 6 u Certamly le55expensl ve Lhau
the one above.
Liver Extract LIlly 15 per cc. 8 75 5 8 Too concentrated.
j'Reticulogen" Lilly (n.s.) II .80 Liver extract plus vitamin
6 t • Again a high-pnced
vitamm.
Liver Extract Parke, DaVIS (n.s) . 70 U,nit value not given .
Icc=5gm s.
liver
Liver Extract No.
171 Parke, Davis (n.s.) I IO Unit value not gIven.
1 ce. = 5 gms.
liver
n s.-Not stated. ,
• Only the more widely used preparations are li!';ted.
Note: No doubt all preparations above are effettive for restoration of blood values. For neurologic
changes the less conccntra.ted products should be used.

TREATMENT OF THE PATIENT

After the diagnosis has been established the treatment is influenced'to some
extent by the condition of the patient at that time. If the relapse is sevel e and
untreated, the erythrocytes and bemoglobin extremely low, and signs of circula-
tory failure evident with the patient at bed rest, a transfusion of 500 cc. of blood
is indicated. Tbis is usually adequate to tide the patient over the crisis of severe
anemia until further tberapy bas time to become effective.
Tbe 'patient then sbould be given liver extract by injection. Some prefer
to use that amount derived from three to four hundred grams of fresh liver during
.tbe first 24 bours. We prefer to give the concentrate derived from 100 Gm. of
liver twice .daily for three to four days. After this the injection of tbe same
amount, twice weekly for tbree to four weeks, is usually adequate and then
this is followed. by injections once weekly for several weeks. The schedule of
dosage varies in different patients, and is dependent upon the severity of the
294 THE ANEMIAS

anemia and other symptoms, the degree of reticulocyte response, the improve-
ment in clinical symptoms and the rate of red cell and hemoglobin increase. The
amounts given in each case should be adequate for that' particular patient.
The above outline is considered to be intensive therapy and should be
followed by definite signs of hematologic and clinical improvement. In the
average patient the reticulocytes are increased in 48 hours and reach the peak
of increase in four to seven days. During this time they may rise from less
than one per cent to as high as 50 per cent. The blood shows the pres-
ence of increased normoblasts and the red cells begin a sustained rise in
number.
The rate of red cell increase varies in different patients but ranges between
50,000 to 125,000 cells per eU.mm. per day. Examination of the blood shows grad-
ual disappearance of the atypical microcytes and macrocytes, the bizarre forms,
and olher evidences of lbe anemic slale, and after four to six weeks the picture
on tbe stained smear looks fairly normal. Usually in eight weeks there is com-
plete resloration of the cellular values to normal with clinical improvement to the
extent that evidences of the disease are no longer present, with exception of the
neurological changes. Even though intensive treatment is maintained after
normal figures are reached, there will be no further increases in cell va] ues.
If there is any reason why the patient cannot be treated by injections, he
should receive an adequate amount of potent material by mouth. For this pur-
pose either liver preparations or desiccated hog stomach is satisfactory. 1\Iore
material can be provided by using a mixture of the two preparations which can
be given in capsules (Stomach, Liver combination, Extralin, N.N.R.). The
amount given should he at least five Gm. daily.
Although it is usually done, there is no reason to supply an adequate amount
of liver by injection and also use various products orally at the same time. Due
to the malnourished state of many patients it appears more reasonable to utilize
the gastric capacity to provide other factors that will enhance the well being of
the patient, including a high caloric diet with sufficient meat, various vitamins,
and an adequate supply of iron. Beth~lI points out that other treatment of the
patient with pernicious anemia in acute relapse should include general measures,
such as restriction of activity, correction of any existing nutritional deficiencies,
adequate fluid intake, and competent nursing care. Bed rest is usually advised
if the red cell count is below 2,000,000 cells per cU.mm. and also in cases of pro-
found weakness, impending circulatory failure, and presence of severe combined
degeneration of the cord, particularly if complicated by cystitis. Patients should
be given a high protein diet and one that is low in fats, supplying at least 1.S
Gm. of protein per kilo., chiefly in the form of milk, meat and fish.
The use of potent hematopoietic material in liver extract is sufficient for
restoration of red cells but the rate of cell production may be so rapid that
there will develop the so-called "hemoglobin lag" due to a temporary iron de-
ficiency. In such instances it is necessary to give from 5 to IS grains of a suitable
iron preparation (ferrous sulphate) by mouth, with Or after meals, to avoid a
 PERNICIOUS ANEMIA 295
transient hemoglobin deficiency and a resulting hypochromic type of anemia.
This can be discontinued after the cellular values have reached tbe normal level,
but treatment with liver extract must be continued indefinitely, using the mainte-
nance dose. (See establishment of maintenance dose.)
There seems little indication for the use of intravenous therapy. Although
the product is effective when given intravenously, as first shown by Castle and
Taylor, and later ,by Fouts and Zerfas, the danger of reaction is consistently more
than when intramuscular injections ure used and the latter are equallY,as effec-
tive. However, Isaacs points out that he has seen few patients who are ahle to
go for three or four weeks between intramuscular injections, and the only route
by which injections can be given at monthly intervals is the 'intravenous one.
He stated that he had studied 138 patients who had been receiving twelve in-
travenous injections a year from one to eight years and that they appeared in
perfect health.
A few patients are allergic to liver or· to liver extract. \Vhen this occurs
it is necessary to try out various types of liver preparations and one may be
found in which allergic manifestations might not occur. If not, it should be
given intramusc.ularly in repeated small doses, or epinephrine can be given by in-
jection' at the same time, and if this fails, it is necessary to employ a preparation
for oral administration, either liver or ventriculin.
It is well recognized that an occasional person is refractory to the use of
adequate amounts of liver extract. This is seen in patients with certain com-
plicating diseases, such as various infectious processes, far advanced arterio-
sclerosis, and also in the extremely aged. In such patients, 'the cause or focus of
the infection should be sought and removed if possible, and the amounts of
liver increased in an effort to overcome these handicaps. If there is no response
to therapy, another type of liver should be used, and it should be given by various
routes, and finally, if there is no therapeutic response, the diagnosis should be
questioned and the patient re-studied from a diagnostic standpoint.
We feel that dilute hydrochloric acid should be used, from five to six cc.
daily with meals, although many do not believe it necessary. Some of the
older methods of treatment are now abandoned, such as the use of arsenic, re-
peated transfusions, and splenectomy.
In addition to the correction of the hematological deficiencies, the patient
with adequate therapy shows marked improvement of symptoms. The appetite
improves, glossitis and sore tongue disappear, the gastro-intestinal symptoms
are corrected, the mental outlook improves, the diarrhea is checked, and the sense
of well being improved to a marked degree. In some patients the restoration to
health is quite spectacular.
There is some variation of opinion concerning the effect of liver tberapy on
the neurologic changes, and the COD.sensus of opinion is to the effect that de-
generative cord changes are arrested but there is no improvement or repair of
the damage already done, because of the weJl known inability of the neurons
in the central nervous system to regenerate. There may be, however, restoration
296 THE ANEMIAS

of function and alleviation of symptoms in areas of peripheral nerve involve-

ment because of their capacity for possible regeneration. McLester has expressed
the opinion that the neurologic changes may be more completely arrested if
the dosage, of liver preparations is excessive and many times that required for
correction of hematological deficiencies. Strauss and his associates reported the
treatment of 2 I patients with advanced spinal cord degeneration and 64 others
who had little or no neurologic disturbances. All patients were treated with
more than sufficient liver to maintain normal blood values. After treatment
there was complete arrest of the neurological lesions for seven years, with con-
siderable improvement in strength and in the group that had no neural lesions,
none developed during this period of adequate treatment. They state that recur-
rence of any neurologic signs during treatment is an indication for immediate
doubling of the dosage of liver.
Adequate therapy does not affect the achylia of the stomach, except to a
minor degree. According to Goldhamer there is increased output of the quantity
of gastric juice in patients with adequate treatment, but no return of hydrochloric
acid. Jones and his associates have studied the gastric mucosa of patients in
relapse and remission by gastroscopic methods, and found that the mucosal
atrophy disappeared after adequate treatment.
Many years ago Congo red was recommended for the treatment of pernicious
anemia. This was based upon the assumption that it protects the circulating
erythrocytes from hemolysis by forming a film of protective material around the
ceIl. Taliaferro and Haag found that the intravenous injection of this dye de-
creases coagulation time as measured in capillary tubes, but if given in ex-
tremely large doses, the coagulation time is markedly increased. In intravenous
injections in ten patients, in doses of IO to IS cc. in a one per cent solution, they
noted a definite decrease in coagulation time. Barker gave daily intravenous
injections of the dye to seven patients with pernicious anemia and to two with
sprue, and came to the conclusion that it was totally ineffective in the treatment
of these diseases.
Richardson at Stanford University studied the effect of the dye on blood
and found that a concentration of I :1000 protects red cells against hemolysis by
a hypotonic saline solution, hypertonic urea, sodium taurocholate in saline solu-
tions, and saponin in dextrose and saline solutions. He believes the effect to be
caused by a film over the red cells. He found that intravenous injection of the
dye in rabbits produced no significant effect on hemoglobin, red cells, platelets,
or reticulocytes and there was no decrease in coagulation or bleeding times regard-
less of the size of the dose. On the contrary, very large doses produced anti-
coagulating effects. He concluded therefore, that the hemostatic action of Congo
red in normal individuals lacks experimental evidence.
Heinle and Miller treated two patients with ordinary dried brewers' yeast,
following the suggestion of Wintrobe that pernicious ~nemia patients would
respond to such treatment. They obtained some degree of hematopoietic re-
sponse but it was not as satisfactory as when patients were treated with a minimal
 PERNICIOUS ANEMIA '97
amount of ventriculin or with daily injections of liver extract. The use of yeast
as a therapeutic agent_will require further confirmation.

ESTABLISHMENT OF THE MAINTENANCE DOSE

An important feature of the treatment and control of the patient is the
establishment and regnlation of the amount of liver necessary to maintain him
free from symptoms and hematologic changes. This can be done only by the
trial and error method. After the patient has been restored to normal from a
state of relapse and is in complete remission, the amount of liver required is
not considerable. In the average patient it is well to begin with an injection of
extract derived from 100 grams of liver, at intervals of two weeks for three
months, then check the blood findings and examine for any symptoms or signs
of retrogression. If none is present the injections may be given at intervals
of three weeks for another period of three months, and if the patient is still
maintained in good health, the interval between injections may be lengthened
tOr four weeks.
The average patient requires the injected equivalent of 100 Gm. of liver
every three weeks to maintain a remission., but this. is quite variable, and it is
important that this be carefully evaluated in each patient. Although it is true
that excessive dosage of liver is probably not harmful, this is no excuse for giving
more than necessary since the patient has to pay for both the liver and the in-
jections. Strauss recommends the use of ten units weekly for six months and
a cautious reduction of the dose after that time at six or twelve month in-
tervals. Since the unit dosage is not expressed on many of the commercial
liver preparations, for the present it is necessary to r~gulate dosage on the basis
of material derived from stated quantities of fresh liver.
We believe that the patient can be maintained more satisfactorily, more
economically, under better observation, and with better results, using parenteral
therapy than by using oral preparations. After a study of 176 patients under
long time observation, Murphy and Howard state that the intramuscular in-
jection of I cc. of liver extract containing IS U.S.P. units, administered at periods
of 30 weeks, is sufficient to maintain a normal state of the blood and to prevent
neural damage.
Bethell states that after ohserving nearly a thousand cases of pernicious
anemia it has been demonstrated conclUSively that the parenteral administration
of liver extract is more successful than the use of oral preparations of any type,
and furthermore, that no instances of relapse have been observed in patients re-
ceiving parenteral liver extract in recommended doses, whereas this statement
can not be made of those taking the material by mouth. In the 2 per cent of
patients who develop sensitivity to parent~ral liver extract he recommends
desiccated stomach or ventriculin by mouth as the treatment of choice.
It is important to rememher that the patient will always have the deficiency
responsible for the disease and that the antianemic material will always bave to
be provided for him. Maintenance therapy, the~efore, -is a permanent under-
2g8 THE ANEMIAS

taking, and as long as it is adequate, the patient will remain in good health with
no recurrence of symptoms.

BIBLIOGRAPHY
PERNICIOUS ANEMIA
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Thomas Publisher, Springfield, III, 1932 .
.. BARKER, W. H ; "Congo red in the treatment of pernicious anemia and sprue." Amer. Jov' Med.
Sci., 194, 293, 1937.
BETHELL, F. H.: '(The treatment of "pernicious anemia." Jour. Omaha Mid-West CUn. Soc. 2,
23, 194I.
BIERMERJ A.: "Ueber progressive perniciose anamie." Correspondez-Blatt. /. Schweiz. A~rzte, 2,
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ErGG, E.: "Spleen size in pernicious anemia.1! Ann. Int Med., 14, 277, :1940.
BRIGGS, L. H.: "The neutrophilic blood picture in pernicious anemia." Anter Jour. Med Sci, 148,
413, 1914·
CASTLE,.W. B • "Observations on the etiologic relationship of achylia gastriea to pernicious anemia.·
I. The effect .of administration to patients with pernicious anemia of the contents of the
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CASTLE, W. B . "The etiology of pernicious anemia and related macrocytic anemias." Ann. Int.
Med., 7, :2, 1933.
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CASTLE, W. B., and MmoT, G. R.: "Pathological physiology and clinical description of the
anemias." Oxford Univ. Press, New York, 1936.
COHN, E. J., MINOT, G. R., ALLES, G. A, and SALTER, W. T.: "The nature of the material in liver
effective in pernicious anemia." Jour. Bioi. Chem, 77, 325, 1928.
'COOXE, 'W "i..: ~~'run'neT oDseTVa'tl.OnS on 'tue macropti'lycy'te:' Bril:.. Mea. JD"»T., \_, ~oo, "111"-')1.
CORNELL, B. 5: "The etiulogy of pernicious anemia." A-fedicine, 6, 375, I927.
DOBlumm, K., and RHOADS, C. P.: "Metabolism of blood pigments in pernicious anemia," Jour.
CUn. Invest., 17. 95. 1938.
DRAPER, G.: "Human constitution." W. B. Saunders Co., Philadelphia, 19:24.
FOUTS, P. J., and HELMER, O. M.: "Urea clearance in pernicious anemia." Arch. Int. Aled, 61,
87, '938.
FOUTS, P. J., and ZERFAS, L. G.: ('Use of liver extract intravenouslY Report of teJl cases."
Arch. Int. Med., 50, 27, 1932.
GXNSSLEN, M.: "Ein hocbwirksamer injizierbarer Ieberextrakt" Klin. JVt:hnsch".~ 9, 20991 1930.
GOLDHAMER. S. M.: "Presence of intrinsic factor of Castle in gastric juice of patients with per-
nici_ous anemia" Amer. Jour. Med. Sci, 191, 405, 1936.
HECK, F. J., and WATKINS, C. H.: "The neutrophil in pernicious anemia." Amer. JoUr. Clin.
Path, 3, 263, 1933.
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Jour. Clin. Inv.cst J 18, 257, 1939.
HUNTER, W.: "Is pernicious anemia a special disease?" The Practitioner, 41, 8T, J88
ISAACS, R.: "The bone marrow in anemia. The red blood cells." Amer. Jour. Med. Sci J 193,
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JACOBSON, B. M., and SUBBARROW, Y.: UStudies of the principle iit liver effective in pernicious
anemia." Jour. Amer. Med. Assoc, 116, 367, 1941.
 PERNICIOUS ANEMIA 299
JONES, C. M' l BENEDICT, E. B., and A. 0.: "Variations in gastric mucosa in pernicious
HAMPTON,
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596, 1935
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Med, 60, I002, 1937.
KAMPMEIER, R H, and JONES, E.: "Optic atropby in pernicIous anemia." Amer. Jour ..Med. Sd,
195, 633, 1938
MACLACHLAN, W. W. G, and KLINE, F. M' "The occurrence of anemia in four generations"
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Soc. Exp. Bioi. and jfed .• 32, 160'/, 1935.
3 00 THE ANEMIAS

WALDEN, G~ B., and CLOWES, G. H. A.: "On a method whereby the principle which is effective in
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 CHAPTER 23
DISEASES CHARACTERIZED BY MACROCYTIC ANEMIA
Although pernicious anemia presents the outstanding example of macrocytdsis ,
there are other diseases which may be characterized by this same type of red
cell picture. Th.ese include the anemia of sprue, pellagra, c.e:t:tain aneu\\as Qt
pregnancy, those of extreme liver damage, the leukemic states, intestinal (lys-
functions as the chronic diarrheas, iodiopathic steatorrhea (Gee's disease), intes-
tinal anastomosis, advanced carcinoma of the stomach, Diphyllobothrium latum
infection, and many of the anemias of childhood.
SPRUE
This is a disease seen most frequently in the tropics and sub-tropical wun-
tries. It is not rare in the Southern United States. It is characterized by milny
signs and symptoms similar to tllOse of pernicious anemia, including sore tongue
and glossitis, various evidences of indigestion, intermlltent and severe diarrllca ,
with characteristic stools which are large in amount, putty colored, frothy, fer-
mentative, and filled with gas. Neurological changes are rare and if present, do
not attain the severity of those in pernicious anemia.
The cause of the anemia seems to be a defIciency of the extrinsic facto! in
the diet, since many of these patients have no evidence of gastric dysfunction.
ThIS would result in a failure to elaborate a suffIcient -amount of potent hejIla-
topoietic principle for red cell maturation. Therefore, the red cell picture may
"",\So
'>:Ie ~<C."''''\.'''-'''\ 'P"""""-'' ' ' '
\.'ll.,.\. ,,"''''''' '''' 'I>.",,,,w,\'I>..
The most effective treatment is the intensive use of liver extract by injec-
tion. It may be necessary to give I cc. of liver concentrate daily (from roo Om.
fresh liver) for several weeks. Ashford states that many patients respond well to
liver and stomach preparations by mouth. The injection method is preferred
because of aSsurance of absorption and probable utilization. Other treatment
should include the use of an adequate -diet, consisting of liberal quantities of
meats, mil~, and eggs. If the hemoglobin is low and the color index below nonllal ,
supplementary iron therapy by mouth is advisable. The dietary regime should
be continued indefinitely and the use of liver continued until the blood findings
are restored to normal along with remission of symptoms. It may be necessary
to place the patient on a maintenance dose of liver extract by injection, but this
is not required in all patients as it is in pernicious anemia. Since the extriflS ic
factor is present in large quantity in autolyzed yeast, the use of this product by
mouth may correct the deficiency, especially in patients in the early stages. We
have found stomach preparations (ventriculin So to lOa Gm. daily), when ground
to a fine powder, of considerable value in· checking the diarrhea. If it is not
ground to a fine powder it is likely to come out in the stools in the form of the
same coarse particles that were administered, and the diarrhea may be accen-
tuated instead of relieved.
30.
3 02 TIlE ANEMIAS

PELLAGRA
This disease occurs in many parts of the world and in particular, those areas
where there is intense radiation from sunlight. It is very common in the rural
sections of the United States. It seems to be associated with chronic alcoholism
in many patients, and in the South is seen often in a class of farmers who sub~
sist on diets deficient in protein material, especially in the form of meats, eggs,
cheese, milk, and other dairy products. This deficiency occurs so consistently that
Goldberger elaborated his theory of a deficiency of the heat stable vitamin B.
Recently Sydenstricker and his associates at the University of Georgia
have shown that there exists a prohable gastric deficiency as well, since they
were able to produce complete remissions by the daily administration of normal
human gastric juice, even though the patients were being maintained on a so-
called pellagra producing diet.
The symptoms vary widely from the mild cases to those severely affected.
In general they involve four systems, including the skin, the gastro-intestinal
tract, the neurological system, and the blood.
The skin manifestations consist of symmetrical areas of erythema, vesicula~
tion and exfoliation, with varying degrees of pigmentation, occurring usually in
those areas exposed to sunlight. The parts most often showing these changes
are the dorsal surfaces of the hands and forearms. Gastro-intestinal symptoms in-
clude sore tongue and glossitis, loss of appetite, abdominal discomfort, and in
the severe stages, prolonged and profuse diarrhea, and in general, they are quite
similar to those observed in sprue. The stools, however, are not characteristic.
Neurological findings may be the outstanding feature, particularly the
mental changes. These range -from irritability, depression, and melancholia, to
violent maniacal outbursts. Many patients have to be restrained over intermit-
tent or long periods of time_ There may be various parasthesias of the hands and
feet, or signs of ataxia.
The findings in the blood are not always those of macrocytosis and hyper-
chromia. These variations seldom occur to the extent seen in pernicious anemia.
In some patients the red cell picture is one of normocytosis and hypochromia.
The volume and color indices are usually above one, and signs of active cellular
regeneration are seldom observed. The leukocytes are not appreciably involved
but a leukopenia may be present in an occasional patient. Absence of gastric
hydrochloric acid is seen in less than 50 per cent of the patients and in some
of these the achlorhydria is corrected after adequate treatment.
Although pellagrins usually have a multiple vitamin deficiency, treatment
should consist in adequate amounts of nicotinic acid and an adequate diet rich
in proteins. Nicotinic acid, nicotinic acid amide or sodium nicotinate can be
used. A dose of 500 mg. daily is usually adequate; some workers also give from
three to five mg. of riboflavin daily for the usual associated riboflavin deficiency.
The diet should provide a liberal amount of milk, eggs, lean pork, and other
meats.
After proper treatment, the macrocytic anemia and other hematological defi-
ciencies are no longer present. Other symptoms rapidly improve, glossitis dis~p;
 DISEASES CHARACTERIZED BY MACROCYTIC ANEMIA 303

pears, there is return of hydrochloric acid in the stomach, a sense of improved

well being, and usually complete restoration to health.
The amounts of substances required to restore full health vary widely and
depend on the stage of the disease when treatment is instituted. The success
of Sydenstricker in daily feeding of normal gastric juice is quite important, not
only from the standpoint of etiology of the disease, but also by the addition of
a type of treatment that may be of especial value in refractory cases.
PREGN~NCY

Anemias of pregnancy are usually of the hypochromic type, in some instances

associated with microcytosis. In such patients the anemia develops because of
an inadequate intake of iron, and can be corrected by the administration of
large doses of a suitable preparation of iron. However, a small percentage of
the anemias are of tbe macrocytic type and develop apparently because of a
failure to utilize an adequate amount of hematopoietic principle. In a series
of 38 patients described by Strauss and Castle, eight of these were definitely
of the macrocytic type, with a blood picture indistinguishable from that of
, pernicious anemia. Parker has reported 100 pregnant negro women and found
none that showed the macrocytic pernicious anemia-like blood picture, although
a few had achlorbydria. Since the cause of this anemia is obviously an inade-
quate amount of hematopoietic principle available for red cell production, the
reasons for its absence are multiple and the causes are varied. Thus, there may
exist a deficiency of the extrinsic factor in the diet j or a failure of interaction
of intrinsic and extrinsic factors. in the stomach, because of achlorhydria or per-
nicious nausea and vomiting, or failure of absorption of the principle, even though
it is formed; or incapacity of the liver for storage, synthesis and distribution, as
might conceivably occur in the damaged liver of the various toxemias of the
pregnant woman. Therefore, the cause of macrocytic anemia may be the result
of the pregnancy, or more likely, from one of its complications. A possible
vitamin B deficiency being responsible for macrocytic anemias of pregnancy
has been stressed by Elsom who studied eight pregnant women who had devel-
oped severe macrocytic anemia. These patients had glossitis, ulceration of the
tongue, impairment or loss of vibratory sense, tachycardia, various gastro-
intestinal symptoms, and easily induced fatigue and paresthesia. The blood
findings were those of typical macrocytic anemia. The patients responded very
well to the administration of brewers' yeast or to liver extract intramuscularly.
The authors believe that these were brought about by vitamin B deficiency.
The patients may present variable degrees of the same symptoms seen in
pernicious anemia, induding nausea and vomiting, diarrhea, sore tongue and
mouth, pallor, excessive fatigue and loss of 'strength, and even the neurological
findings of numbness and tingling of extremities, and ataxia.
The blood picture shows a reduction in the number of red cells, lowered hemo-
globin values with a color index that is high, varying degrees of macrocytosis
with variations in size and shape of the red cells. The volume index is increased
above normal and signs of cellular regeneration are usually absent. The leuko-
cytes are seldom affected and the platelet values are normal. Achlorhydria or
 IILATE XXVI

ANEMIA OF LIVER DAMAGE

.
,'''-.'''-,
\,_-_)
3

o 2

~. N<t\\\.O;~}>s,\\.
2. Lymphocyte.
3 Macrocyte.
4. Poikilocyte.

Blood Findmgs: Differential:

Hemoglobin 12.1 gms. (Newcomer's method). Neutrophils .
R.RC. 3,000,000 per c mm Lymphocytes
WBC. . 6,800 per c rom. Eosinophlls
Platelets . 260,000 per c mm. Basophtls
Monocytes
Color Index ., I2
Volume Index I.J
Icterus Index 750
Reticulocytes . 020/0

Gastric Analysis-Normal. •
Erythroc-,'tes; macrocytic and hyperchromic with anisocytosIS and slight polychromatopJliJia.
Plat< XX\-I.
 DISEASES' CHARACTERIZED BY ]\:[ACROCYTIC ANEMIA 305

hypochlorhydria may he present, hut not complete achylia. Usually some degree
of hydrochloric acid can he demonstrated after stimulation with histamine. The
anemia is not always hyperchromic, and if there is an associated inadequacy of
iron intake the color index may be low and the anemia hypochromic. The con-
dition and blood picture is quite similar to the tropical anemia of pregnancy
that has been descrihed hy Wills and usually called the tropical macrocytic
anemia of pregnancy, which is prevalent in certain parts of India, affects pregnant
women mainly, but also occurs in males, and is thought to develop because of
protein and vitamin dietary deficiencies.
Macrocytic anemias of pregnancy can usually he entirely corrected hy placing
the patient on the same regime of treatment as though she had pernicious anemia
in relapse, except that the use of hydrochloric acid is unnecessary unless there is an
achlorhydna and diarrhea. The treatment includes the daily injection of liver
extract (derived from 100 Gm. of fresh liver) or in anemias of less severity less
frequent injections can he used. They should also he given a diet that is ade-
quate with particular reference to protein content, which should include liberal
amounts of eggs, meats and dairy products. Bethell states that the treatment of
the more severe ,macrocytic anemias of pregnancy should include I.S Gm. of
animal protein per kilogram of body weight, that is, about 50 Gm. of animal
protein daily. Large doses of yeast are effective in those in whom the deficiency
is in the rliet, hut since this cannot be determined with certainty the safest form
of treatment should be the liberal use of liver extract by injection. If there is
evidence of hypochromia, iron should be given by mouth (~ to 15 gr. daily of
ferrous sulphate).
After the institution of liver therapy there is a prompt reticulocytosis, and
a gradual return of cellular values to the normal level with a consistent correc-
tion of symptoms. The presence of a severe anemic state in pregnancy is no
indication for therapeutic abortion, since the condition is amenable to treatment,
but just as in all anemias, it is highly important to ascertain the deficiency,
properly classify the anemia, and treat the patient only witb that material
effective for that type of anemia.

GASTRO-INTESTINAL DYSFUNCTIONS

If it is true that a deficiency of hematopoietic principle and a resulting macro-

cytic anemia develop from a lack of intrinsic factor in the stomach, then it is
reasonable to expect a macrocytic anemia to follow total or partial gastrectomy,
or other operative procedures in which the pyloric portion of the stomach and
upper duodenum have become removed from contact with food material. There
is some evidence to indicate that this occurs in a sufficient nnmber of instances
to be more than mere coincidence. Although it is generally accepted that the
antianemic factor is elaborated mainly in the lower stomach, Uotila has prepared
extracts from the ileum and produced remission in two patients with pernicious
anemia and Schemensky has obtained good results with oral administration of
the powdered colon of hogs. Dussaharger and associat~s performed gastrectomy
3 06 TIlE ANEM1AS

on eight monkeys. None of these showed any evidence of pernicious anemia after
two and a half years of observation. Some of them '<lid show a slight tendency
toward macrocytosis. Dreher studied the hematological findings in IOI patients
operated upon at the University of Kiel in a period of IO years. In all of these
either partial or total gastrectomy had been performed. In only 13 cases was
there evidence of anemia, macrocytosis, increased color index, or reticulocytosis.
She concluded too, that hypochromic anemia was just as likely to develop as the
hyperchromic type.
Finney and Rienhoff found that gastrectomies in nine patients were followed
by macrocytic anemia in two of these. Gastroenterostomy has been followed by
pernicious anemia in ten patients, according to Hurst. Macrocytic anemia is
a frequent finding in patients with advanced gastric carcinoma. Sturgis and
Goldhamer point out that several factors probably influence the development of
macrocytic anemia after resection of the stomach. The anemia may not develop
for some years afterwards because it is dependent to some extent on impairment
of the gastric and intestinal function, and secondly, by the amount of reserve
hematopoietic substance that is stored in the liver and perhaps elsewhere in the
body. Furthermore, the age of the patient and presence of infection would
influence this.
Macrocytic blood pictures have been observed in patients with prolonged
diarrhea of amebic and bacillary dysentery, ulcerative colitis, intestinal strictures,
and stenosis of varying degrees in the small intestine. It has also been observed
in idiopathic steatorrhea (celiac disease) and infestation with the fish tapeworm.
In such cases it seems likely that there is interference with the normal capacity
for absorption of the hematopoietic material, due either to pathologic changes
in the absorptive areas, or to rapidity of food passage, or both.
Sturgis and Goldhamer have reported 10 cases showing the variable types
of gastro-intestinal dysfunction that may be followed by macrocytic anemia.
Their cases included gumma of the stomach, two carcinomas of the stomach, one
infiltrating type of carcinoma of tile stomach, multiple anastomoses of the bowl,
ileitis, ileo-colic fistula, two cases of cirrhosis of the liver, and one case of acute
hepatitis.
Plum and Warburg have reported four case, of ileitis, in three of which
there was development of a macrocytic anemia. They review tbe literature of'
157 other cases. The cause of this is unknown although a dietary deficiency
may have been responsible. .
Disease of the pancreas has been suggested as a possible cause for a rare
type of macrocytic hypochromic anemia. Six cases of pancreatic disease were
studied by Cheney and he noted that all of them presented a marked macrocytosis
with hypochromia. He suggests that hypochromic macrocytic anemia may be
characteristic of certain forms of pancreatic disease. In such instances it is
possible that associated liver disease could be responsible for some of the
macrocytosis.
These anemic states do not differ from the hematologic picture seen in other
 DISEASES CHARACTERIZED BY MACROCYTIC ANEMIA 307

macrocytic anemias previously described They respond to the administration

of liver extract in adequate quantity, which in most patients, should be supple-
mented with iron. Because of the severity of many of the associated and causa-
tive disease processes, the respon~e to therapy is not as striking as that observed
in the anemias of pellagra, sprue, and pregnancy.

ACHRESTIC ANEMIA

In 1935, Wilkinson and Israels in England described a type of megalocytic

anemia that was apparently closely associated with pernicious anemia yet dif-
ferent from it in several important ways. It is characterized by a blood picture
identical with that of pernicious anemia with a bone marrow that shows a true
megaloblastic hyperplasia. However, there are three important differences from
pernicious anemia. First, free HCl is found in the gastric content; secondly,
the patient shows a complete failure to respond to antipernicious anemia treat-
ment so that the course of the disease is always progressively worse leading
finally to death; and thirdly, adequate amounts of the anti-anemic principle
have been shown to be present in the liver. Therefore, they bave suggested
that the basic cause of the disease is a failure of the bone marrow to utilize the
principle or to mobilize it from the tissues even though the store of hematopoietic
principle in the liver is quite normal. The disease is a chronic one with an in-
sidious onset. There is no difference in the sexes. The age of onset is more
variable than that seen in pernicious anemia, since achrestic anemia is found at
all ages. Furthermore the disease is not characterized by spontaneous remissions
as seen in pernicious anemia.
The blood findings include the development of a true macrocytic anemia
with the mean diameter of red cells being much above normal. The red cells vary
between two and three million per CU.mm. and the hemoglobin correspondingly
high with a color index greater than one. Nucleated red celIs, including megalo-
blasts, are seen occasionally, and there is a varying degree of anisocytosis and
poikilocytosis. The reticulocytes are low even after intensive liver therapy is
instituted. There seems .. to be no evidence of excessive hemolysis. The icterus
index and van den Bergh values are normal. The red cell fragility also is normal.
There is frequently a leukopenia with a relative Iympbocytosis. There is usually
no disturbance of the HCI content of the stomach. They use the term achrestic,
based upon the concept that the anemia develops because of a failure of the
bone marrow to utilize the hematopoietic principle.
'There are no disturbances of the gastro-intestinal tract or central nervous
system. Wilkinson has noted the occurrence of this disease in about one to
every hundred cases of pernicious anemia. He considers it to be a distinct
clinical entity. In a careful study of the bone marrow in six cases they report
typical megaloblasts in all stages of the disease. However, tbis same picture
may be classified as one of five groups of aplastic anemia as described by Rhoads,
see p. 313.
J08 THE ANEMIAS

LIVER DAMAGE

We have observed repeatedly macrocytic anemia in patients in whom there

are varying degrees of damage and destruction of liver substance. This may
occnr particnlarly in instances of long standing disease and far advanced destruc- .
tion of liver tissue followed by fibrosis, as seen in nodular atrophic cirrhosis and
Ranti's disease. In one instance we advanced a diagnosis of Banti's disease on
the presence of a macrocytic anemia on the stained blood film and the information
that the patient had a massive enlargement of the spleen, the correctness of
which was later verified.
It appears that the liver may be so altered by pathologic change and 50
incapacitated in its function to store, synthesize and release hematopoietic mate-
rial that macrocytic anemia can develop in spite of the known ability of other
tissues as the kidneys to perform this function. Higgins and Stasney have
consistently produced macrocytic anemia in experimental portal cirrhosis in dogs.
The blood changes are variable in degree and not all patients with liver
damage develop the anemia, this being due, in our opinion, to the capacity of
other tissues to assume the function of storage of the hematopoietic factor.
In Banti's syndrome there is an associated leukopenia at the expense of the
granulocytes. In any instance there may be a hemoglobin deficiency to the exteni
of a hypochromia, and iron is necessary for its correction. These macrocytic
anemias do not respond to the injection of liver extract and this has been advanced
as a reason that the concept is untenahle, but this is evidence that the liver stores
injected potent liver extract and in cases of severe damage it is unable to exercise
this function. .

TROPICAL 1IlACROCYTIC ANEMIA

This is a form of macrocytic anemia that is seen only in tropical Countries.

It exists apparently on the basis of a dietary deficiency since it responds very
well to the administration of marmite and crude liver extracts. It does not
respond to purified liver extract or any of the concentrated products.
The disease usually occurs in women and has been noted mainly in India.
In that country a large percentage of pregnant women show evidence of this
particular type of anemia. The symptoms are those of a severe anemia j there
is usually a marked weakness, and pallor of the conjunctiva. There is not the
sore tongue seen in pernicious anemia and apparently no neurological disorders.
The blood shows the presence of the typical macrocytic anemia with reduced
red blood cells and hemoglobin relatively higher, with a color index above one.
The volume index also is above one. There is some anisocytosis and poikilo-
cytosis. There is usually a mild leukopenia. The platelets apparently are not
involved. The HCI of the stomach shows no abnormality and there is no increase
of the biliary pigment in tbe blood plasma.
The treatment of this type of anemia consists of the use of large quantities
of liver by mouth or the use of crude liver extracts. Marmile has been recom-
 DISEASES CHARACTERIZED BY l\lACROCYTIC ANEMIA 309

mended by Lucy Wills in a daily dose of 30 Gm. and it cures the anemia rega;'dless
of the presence of pregnancy.

LEUKEMIA

In many patients with chronic myeloid leukemia, there exists a macrocytic

type of anemia, in which the color index is relatively high, the average cell volume
above the normal, with many macrocytes and poikilocytes. This is seldom ob-
served in the acute' forms of either the myeloid or lymphatic types. We have
seen this type of red cell picture in many patients with chronic myeloid leukemia
and it is present in nearly every recorded case of monocytic leukemia on file in the
HematQlogical Registry. Also, after the cells of lymphatic leukemia begin to
infiltrate the bone marrow and crowd the erythropoietic centers, a macrocytosis
may develop.
This finding may be of some diagnostic importance in differentiation be-
tween aleukemic myeloid leukemia, and aplasti~ anemia, in which no such changes
are observed.

BIBLIOGRAPHY
MACROCYTIC ANEMIAS
ADRAlJAMSON, L., and THOMPSON, A.: "Achrestic anemia." Irish Jour. Med. Sci., Ser.,6, 66, 1937.
ASHFOlU>, B. K.: "An evaluation of liver extract in the treatment of the anemias of sprue." Jour.
Amer. Med. Assn, 91, 242, 1928.
BUSSAI$ARGER, R. A., Ivy, A. C., WIGOWKY, H. S., and G\JNN, F. D •• "Effect of gaSlrectomy on
the monkey." A.nn. Int. Med., 13, 10l8, 1919
CHENEY, G.: "The megalocytic hypochromic anemia of pancreatic di.sease." Folia Haem., 56,
28, 1936.
DREHER, M.: uVeranderungen des rotem blutbildes nach magenresektion." Ztschr. Klin. Med.,
136, 525. 1939.
ELSOY, K. 0.: "Macrocytic anemia in pregnant women with vitamin deficiency." Jour. Clin.
Invest., 16, 463, 1937.
FINNEY, J. M. T .• and RIENHOFF, W. F, JR.: "Gastrectomy.'l Arch Surg, 18. 140, 1929.
GOLDBERGER, J., and WHEELER. G. A.: "Experimental pellagra. in the human subject brought
about by a restricted diet.".. Publtc Health Reports, 20, 3336, 1915.
GROEN, J., and SNAPPER, I.: "Dletary deficiency as a cause of macrocytic anemia." Amer. Jour.
Med. S"., 193, 633, 1937.
HIGGINS, G. M., and STASNEY, J.: "Macrocytic anemia in experimental cirrhosis." Proc. Staff
Meet., Mayo Clinic. 10. 429, 1935.
HURST, A. F.: "Achlorhydria and achylia gastrica, and their connection with the Addison's anemia-
subacute combined degeneration syndrome and simple (non-Addisonian) achlorhYdnc anemia."
Quart. Jour. Med., 25, IS7. 1932.
MOGENSEN, E.: "The anemia of gastric cancer." Folia Haem, 56. 206, 1936.
PARKr:R, F. P.: "Anemia. of pregnancy in the Negro. Report of IOO cases." Read before South-
ern Interurban Clinical Club, Atlanta, Ga., Feb., I937.
PLUM, P., and WARBURG, E.: "Hematological changes, especially megalocytic anemia, in regional
ileitis." Acta Aled. Scandinav., 102, 449, 1939.
SCHEMENSKY, W.: IIPatbology of pernicious anemia. Therapeutic results with oral administration
of powdered colon of hogs. Therapeutic considerations of cohtis gravis." Ztschr t. Klin. Med.,
128, 4,8, [935.
3'0 THE ANEMIAS

SPIES, T. D., CHINN, At and McLESTER, J. B.: "Treatment of endemic pellagra," South. Med. Jour.,
30, 18, 1937.
STRAUSS, M. B., and CASTLE, W. B.: ~'Studies of anemia in pregnancy. III. The etiologic relation-
ship of gastric secretory defects and dietary deficiency to the hypochromic and macrocytic
(pernicious) anemias of pre£D,ancy and the treatment of these conmtions." Amer. Jour. Med,
Sci., 185, 539. 1933.
STURGIS, C. C .• and GOI.DIIAlnR. S. M.: "Macrocytic anemia other than pernicious anemia, associ-
ated with lesions of the gastrointestinal tract." Ann. Int. Med, 12, 1245, 1939·
SYDENSTlUCXER, V. P., ARMSTRONG, E. S., DERRICK, C. J, and KDsp, P. S.: "On the existence of an
intrinsic deficiency in pellagra," Amer. Jour, Med. Sci, 192, I, I936.
SYDENSTRICKER, V. P., 'and THOMAS. J. W.: "Some factors in tbe etiology of pellagra." South.
Med. Jour I 30, 14. 193'1.
UOTILA, U.: liOn the presence of antianemic actiVity in the ileal part of the small intestine." Acta
Med. Scandinav., 89, So, 1936.
Wn.lClNSON', J. F., and ISRAELs, M C. G.: "Achrestic anemia." Brit. Med. Jour., 1, 139, 1935.
Wn.XINSON', J. F., and ISRAELS, M. C. G.: "New observations on the aetiology and prognosis of
achrestic anaemia." Quart. Jour. Med., 9. 163. 1940.
Wn.r.s, L., and MEHTA, M. M.: "Studies in 'pernicious anemia' of pregnancy. I. Preliminary re·
port." Ind. Jour. Med. Research, 17, 1'17, 1930.
 CHAPTER 24
APLASTIC ANEMIA
(Aregenerative Anemia) (Hypoplastic Anemia) (Bone Marrow Insuf-
ficiency) (Toxic Paralytic Anemia)
Aplastic anemia is (he term applied (0 a group of anemias characterized by
a partial or total inhibition of bone marrow output resulting in severe anemia,
leukopenia, and thrombopenia, followed by a progressively fatal course in most
patients.
The term, aplastic anemia, although in wide clinical use, is somewhat mis-
leading because it has been customary to include within this group any type of
anemia in which there is deficient cell production in the marrow, regardless of
the cause, but it is applied particularly to any condition in which the peripheral
blood is characterized by decreased numbers of erythrocytes, granulocytes, and
thrombocytes from an unknown cause. Naegeli is probably correct in stating that
it is not a clinical entity but is a hematologic expression denoting the charac-
teristic blood picture that may follow a number of causes.
The term, aplastic anemia, carries the implication that the bone marrow
has become non-functional to the point that it is no longer able to supply the
requisite number of cells to the peripheral blood, this resulting in the concept
that the marrow is "aplastic." That such a concept has to be revised is indi-
cated by the fact that, although a patient may show the clinical and peripheral
blood changes of aplastic anemia, the marrow is not necessarily "aplastic," as
pointed out by Thompson and his associates who found aplasia in only one of thir-
teen cases.
Isaacs has recently stated that "the bone marrow is said to be hyperplastic,
hypoplastic, aplastic, depleted, stimulated, depressed, exhausted, irritated, in-
jured, recovered, normoblastic, megaloblastic, myelophthisic," in the various blood
diseases. Therefore, before the marrow is said to be aplastic, the exact pathology
should be described and the term defined. In current usage, the aplastic marrow
refers to one that is quantitatively hypocellular or acellular. Thus, in some
cases of aplastic anemia the marroW is characterized by decreased hematopoietic
tissue, which is largely replaced by f,at and fibrosis, while in other cases it may
actually be hyperplastic, showing an actual increase of hematopoietic tissue,
a decrease in fat spaces, and a conversion of fatty marrow of long bones into an
active state. In such instances the reason why cellular output is stopped may be
because of a "maturation arrest" of the red cells, or as Isaacs expresses it, "a bone
marrow block." Thus, there may occur a physiologic paralysis of marrow func-
tion, yet in the histologic sense it is not "apla.stic." In other cases of aplastic
anemia, there may be encroachment on the marrow tissue from extraneous sources'
as seen in osteosclerosis, metastatic tlJmors, multiple myelomata, tubercles, and
septic processes as in osteomyelitis, all resulting in a decreased, marrow output, yet
3I l
 PLATE XXVII

APLASTIC ANEMIA

, ,-',\
\ . _./
·Z

I. Lymphocyte.
2. Normocyte
Blood Findings: Differential:
Hemoglobin 3 gms (Newcomer's method) Neutrophils 6%
RB.C. 900,000 per c mm. Lymphocytes 94%
W.B.C . . 1,650 per C.mm.
Platelets 8,000 per c mID.

Color Index . 1.0

Volume Index 10
Reth:ulocytes . None
Erythrocytes; normocytic and normochromic.
Note absence of platelets and characteristic halo around red cells.
Plate XXVII,
 APLASTIC ANEMIA

the remaining marrow tissue may be functioning quite efficiently and is not
aplastic.
It seems advisable to consider aplastic anemia as a hematological and clinical
syndrome, resulting from a variety of types of marrow patbology and from varied
causes, and because of tbis tbere results different types of peripheral hlood pic-
tures. In view of tbis it is illogical to maintain rigid diagnostic criteria that a
patient should fulfill in order to be placed in the category of aplastic anemia.
It has been claimed that the diagnosis should not be made if tbere exists any
evidence of blood regeneration, and it has been further suggested that the process
be divided into two classes; namely, that of primary aplastic anemia of unknown
cause that shows no evidence of marrow activity, and secondary aplastic anemia
that does show some evidence of cellular regeneration. Furthermore, it has been
proposed that those patients with no regenerative activity be designated as
aplastic anemia, and those that show evidence of regeneration as hypoplastic
anemia. In view of the varied causes and the varied pathology in the bone mar-
row, these are largely matters of academic interest but not of practical importance.
For several years the chief research interest of Rhoads at the Rockefeller
Institute was the study of that group of refractory anemias usually known as
aplastic anemias. Rhoads states that those characterized by hypocellularity of
the bone marrow comprise only a small number of all cases of anemia that are
refractory to treatment. He prefers the term refractory anemia for this group.
Some of these are secondary to other disorders, such as malignancy, tuberculosis,
Hodgkin's disease, nephritis, cirrhosis of the liver, subacute bacterial endocarditis,
leukemic infiltration, etc. He studied SO cases of which biopsies were made either
at autopsy or during life or both, and based upon these findings he classifies the
bone marrow picture of refractory anemias in five different types: (I) a hypo-
cellular marrow with aplastic anemia, (2) an immature cellular marrow, (3) an
active cellular marrow, (4) sclerosis of the marrow, (5) megakaryocytic marrow.
Rhoads found no characteristic race, sex, age, or familial distribution. II
quite a large number there was definite exposure to benzene in a variety of ways
Three patients who had the disease were given amidopyrine and tbis was followec
by serious exacerbations. There was no suggestion that these anemias were causec
by excessive hemolysis of the red cells, in spite of the fact that the marrow ap
peared to be hyperplastic. Some of the bone marrow findings were identical wiU
those seen in cases of agranulocytosis after amidopyrine poisoning and the marro,""
findings in known cases of benzene poisoning could not be differentiated bisto-
logically from the usual idiopathic type of aplastic anemia. He advanced the idea
that some of these of unknown etiology were manifestations of benzene poisoning
to which the individuals were exposed unknowingly, or to benzene-like compounds
formed endogenously, presumably in the intestinal tract. His group included
cases designated as myelosclerosis. Some of these cases of refractory anemia just
before death developed evidences of leukemia, and certain types may be consid-
ered to be preleukemic conditions. No treatment was of value in any of this
group. Transfusions bad only a temporary effect and in some instances were
decidedly harmful because of hemolysis of transfused cells. He concluded that
there are several types of refractory anemias and that the varied bone marrow
 THE ANEMIAS

pathology is the result of exp05ure to poisonous compounds of the benzene ring

group, of either exogenous or endogenous sources.
January and Fowler of the University of Iowa have commented upon the
diversity of bone marrow pictures seen in aplastic anemia as reported by Rhoads
and Miller. From this they say that "one is forced to the conclusion that either
aplastic anemia is not a separate entity but rather a group of refractory anemias
with similar clinical features." They report '9 cases which present the accepted
diagnostic criteria for aplastic anemia. In all of their cases studied by bone
marrow biopsy, either during life or at autopsy, tbere was evidence of definite
bone marrow aplasia or hypoplasia. They found none of their cases with a normal
or hyperplastic bone marrow and believe that aplastic anemia is a distinct entity
with hypoplasia of tlie marrow and that the term aplastic anemia should be re-
tained for that particular syndrome.

SECONDARY APLASTIC ANEMIA

BENZENE
Among the agents capable of damaging the bone marrow to the point of
aplastic anemia, benzene (Benzol C6H6) is outstanding. This product should
not be confused with benzine, a product of petroleum distillation. Since benzene
is the starting point in manufacture and serves as the central structure for many
thousands of organic chemical products in daily use to which people are ex-
posed, it should always be regarded as a potential source of bone marrow damage.
In 1897 Santesson in Sweden observed a group of workers .with aplastic
anemia that had developed as a result of exposure to benzene by inhalation during
the course of their work in a bicycle tire factory where the chemical was used
as a solvent for rubber. In '9'0 Selling observed four young women with signs
of marrow aplasia who had been engaged in similar work. A striking feature
of these cases was the development of the disease in some of these patients long
after they had been exposed, thus illustrating the possible delayed or cumulative
effects of benzene. Since that time the dangers of exposure to benzene in industry
have heen more fully recognized and appreciated, especially in tbe industrial
field. Alice Hamilton presented an excellent review of this subject in '93'.
Benzene is widely used as a starting point in the manufacture of drugs and
dyes, as a solvent in the manufacture of rubber, and as a fuel. It is also ·widely
used as a dry cleaning agent in small shops and in homes.
Instances of poisoning are the result of inhalation of the fumes especially
when workers are exposed to high concentrations in the air, due to inadequate
and poorly ventilated working quarters. There is no evidence that marrow
depression can be produced by absorption through the skin. Also benzene can
exert its depressant action when it is taken by mouth or when it is injected sub-
cutaneously or intramuscularly. Therefore, it is capable of producing varying
degrees of marrow damage when it is (1) inhaled, (2) ingested by mouth, or (3)
injected into the tissues. Intravenous injection of minute quantities produces
instant deatll (Kracke).
Danger from oral ingestion is negligible since it is no longer used in the
 APLASTIC ANEMIA 31 5
treatment of myeloid leukemia. However, many patients with leukemia were
treated with the product many years ago and it was observed repeatedly that
it exerted a marked depressant influence on marrow output, that the action was
delayed, uncontrolled, and oftentim.es cumulative, so that it was abandoned
mainly for that reason.
Man is never subjected to the injection of benzene but it has been demon-
strated by Weiskotten and his associates that the intramuscular injection of one
cc. daily into rabbits is capable of consistently producing severe marrow aplasia
and a resulting blood picture of aplastic anemia. We have shown that the intra-
muscular or subcutaneous injection of small quantities into rabbits is followed
by a selective action on the granulopoietic tissues with resulting severe granulo-
penia, and if the dose is increased the action involves the entire cellular output
of the marrow.
Exposure to benzene is followed by the development of marrow aplasia in
only a small number of the people exposed. In general, women seem more sus-
ceptible than men, young people more than older ones, and white people more
than negroes. These variations in susceptibility have never been explained.
Therefore, most instances of chronic industrial poisoning are seen in young white
women. Mallory and his associates at the Massachusetts General Hospital have
described in detail the bone marrow pathology from '9 patients wbo had heen
exposed to benzene over long periods of time. This.material included 14 autopsies
and five biopsies. There was considerable variation in the type of bone marrow
pathology. Not one of these showed complete aplasia of the matrow; six showed
a marked hypoplasia, three showed a normal marrow" three'others had a definite
increase in the marrow cells, and five showed, an extreme grade of hyperplasia.
One of the patients presented findings that were characteristic of acute myelog-
enous leukemia, and another showed what appeared to be a lymphatic leukemia,
It is evident then that the bone marrow picture in cases of benzene poisoning
will vary greatly, depending upon the extent and duration of exposure to ben-
zene. Some will show aplasia, others hypoplasia, others no changes and still
others hyperplasia and perhaps in an occasional one there may be the actual
development of leukemia. These observations are in agreement with Rhoads'
findings in which he describes five types of bone marrow pathology in his group
of refractory anemias, all of which he either proved or suspected of being poisoned
on benzene.
The pathologic changes in chronic benzene poisoning bave been thoroughly
studied by Hunter at Massachusetts General HospitaL In a study of 89 people
that were chronically exposed to b~nzene fumes, 10 of whom terminated fatally,
he found a wide variety of pathologic changes in the marrow and also in the
peripheral blood. He did not find a single instance of leukopenia without some
other cellular change. Seven showed increased red cell counts, one showed a normal
red cell count, and 29 showed decreased counts. Some showed increased leukocyte
counts, others showed normal leukocytes and still others showed leukopenia.
Unfortunately the blood platelets were not studied. Some showed a macrocytic
anemia, others normal sized cells and still others microcytosis. It is emphasized
that blood changes may occur some time after exposure takes place.
3 16 THE ANEMIAS

There seems little relation between the time and duration of exposure and
the initial appearance of symptoms. In 54 cases collected from the literature
by Selling and Osgood, nearly all of these worked in benzol-rubber factories,
and the duration of exposure varied from two weeks to 35 years with the average
duration less than one year.
The symptoms and blood findings are quite variable and depend upon the ex-
tent of marrow damage and on the type of cells involved. Most patients complain
of a gradually progressive development of excessive fatigue, weakness, headaches,
increasing pallor and perhaps the presence of purpuric lesions in the skin. Fever
mayor may not be present. The blood picture is cbaracterized by a decrease in
the number of circulating granulocytes, erythrocytes and thrombocytes. In
some patients the anemia may be most pronounced, in others the leukopenia, and
in others the decrease in the platelets will be the outstanding feature. The clinical
symptoms depend upon the type of cells involved and the extent of marrow
damage. Thus, in the thrombocytopenic patients, there may be a grave syndrome
with generalized purpura, epistaxis, tarry stools, or uterine bleeding. In the in-
stances of severe leukopenia the ,findings may be those of secondary infection with
ulcers of the soft tissues of the mouth, and the condition confused with agranulo-
cytosis. In all patients, however, there is some degree of thrombocytopenia and
anemia. The more chronic the disease the more pronounced is the anemia, and
the more acute phases show tho:. most marked decreases in leukocytes and platelets.
The white cells may be only a few hundred with granulocytes absent, the platelets
a few thousand"r entirely absent, and the red cells at less than a million per
cu. mm. in the severe cases.
In the acute and fulminating type the red cells show little change in size
and volume and the hemoglobin decrease is equal to the red cell depression
while in the chronic forms there is usually seen variation in size and shape of
red cells, and in some a tendency to macrocytosis. The presence of cellular
regeneration is seldom seen except in patients in the early stages of recovery. This
is characterized by nucleated red cells, reticulocytosis, polychromatophilia, and
basophilic stippling.
Treatment consists of removal of the patient from the source ,!f exposure
and repeated transfusions to supply a sufficient amount of blood until the
marrow begins to regenerate enough cells to maintain the cellular equilibrium.
In the more chronic phases of recovery the administration of suitable iron and
liver preparations, a high caloric diet, and other general measures may be of some
benefit. It is important to bear in mind that even though the patient is removed
from the source, of poisoning, the chemical may exert a prolonged influence on
the marrow and the uisease may not even develop until long after the patient
has had contact with the no;dous agent.

ARSENIC PREPARATIONS
A state of marrow aplasia with the resultjng picture of aplastic anemia may be
seen rarely after the administration of organic arsenical compounds. It is not
known whether the arsenic or the benzene ring structures in these compounds are
responsible for the toxic action. Most of the arsenical preparations used in the
 APLASTIC ANEMIA 31 7
treatment of syphilis contain two benzene rings united with a double bond to
which is attached the molecule ot arsenic. We agree with Farley that the benzene
ring structure is responsible because boncl marrow depression is seldom if ever
seen in patients from poisoning with inorganic arsenic preparations 5uch as
Fowler's solution and on the known depressant activity of the benzene ring
structure. However, 'Vheelihan has reported a case of severe granulocytic
aplasia of the marrow in a patient following the use of inorganic arsenic (Fowler'.
solution).
Fortunately, aplasia of the marrow following the nse of organic arsenicals j.
quite rare. In '9.,0 Farley found only 39 cases in the literature and added 7 addi-
tional ones seen at the Pennsylvania Hospital in seven years. McCarthy and
Wilson collected only 79 cases of marrow aplasia from the literature in '932. We
have seen three patients with aplastic anemia following arsphenamine administra-
tion at Grady Hospital in Atlanta in the past ten years. In view of the frequent
use of the drug in large numbers of people the toxic action seems to be due to
an unexplained type of marrow susceptibility, since it often develops quite sud·
denly after only one or two doses. Stephenson and Wingo reported only "
blood dyscrasias, most of which were of the aplastic type, during the administr'l-
tion of approximately one million doses of neoarsphenamine in the U. S. Navy
during the ten years ending in '935.
Although salvarsan was introduced by Ehrlich in '910, it was not until ten
years later that marrow aplasia was reported from its use. This gives rise to
the speculation that the products in use now may vary in quality, and that some
batches of the material may be unusually susceptihle to disintegration. The same
may be true of amidopyrine which was introduced in the form of the German
product, Pyramidon, in ,898 and agranulocytosis was not observed until '922.
Arsenical marrow aplasia may occur at any age, in either sex, and'in any
race. Those we have seen were in negroes. l\1ost patients are young adult~
but this is probably because the incidence of arsenical administration is higher
in that group. Smith and Lyon observed aplastic anemia in a seven year old girl
after antisyphilitic treatment with stovarsol. .
The onset of symptoms is usually relatively acute, with weakness, fatigue,
bleeding from the gums, nose or uterus, purpuric spots, fever and headache,
and in some instances dyspnea and palpitation because of the anemia. The
blood picture includes varying degrees of anemia of the normocytic type, leuko-
penia, and thrombocytopenia and the findings arc similar to those described
under benzene poisoning. If the platelets are low there will be a prolonged
bleeding time, positive capillary resistance test, normal coagulation time, failure
of the clot to retract, and various hemorrhagic disorders. If the leukopenia pre-
dominates, tncrc is usually fever, with oral ulcerations and perhaps other evi-
dences of an infectious state.
Treatment consists of withholding further antisypbilitic therapy until the
marrow is able to resume its function. The intravenous use of sodium thiosul-
phate may serve to neutralize the remaining arsenic in the tissues. Repeated
transfusions are indicated until the marrow begins- to show signs of regen era lion.
The prognosis depends upon the early recognition of tbe disease and its
3 IB THE ANEMIAS

cause. If the patient is not seen until the cells are markedly depleted, hemor-
rhages are pronounced and infection has supervened, then the chances of re-
covery are lessened. If seen before these sequelae have taken place the patient
can usually be kept '!clive and supported with sufficient blood from transfusions
until the marrow again begins to function. In our experience, the outlook is
fairly good if the diagnosis is made early before complications occur.

RADIATION
The marrow depression of radiation has been recognized for years. The
extent of the pathological changes and the severity of the cellular depression
depend upon the duration and intensity of exposure to the radioactive substances.
It has been reported after exposure to both roentgen rays and the alpha rays of
radium or mesothorium.
In a comprehensive review of this subject in 1929, Martland cited numerous
instances of alteration of the hematopoietic tissues after exposure to these sub-
stances, including reports of aplastic anemia, severe leukopenias an occasional
J

leukemia and various mild anemias that were apparently traceable to contact
with radioactive agents. He cited the report of Rud who found leukopenias in
seventeen nurses, physicians, and technicians handling radioactive substances,
and that of Jaulin who reviewed seventeen deaths attributed to contact with
radiation.
Martland states that "the opinions of different authorities all agree that
contact with radioactive substances and x-rays frequently produces, aside from
lesions in the external organs such as the skin, deep and often profound altera-
tions, of which the principal objective symptom5 are usually a leukopenia, more
rarely a leukemia, an anemia usually of the aplastic type, a fall in systolic blood
pressure and sterility."
Naegeli refers to the aplastic anemia of radiation and cites several instances.
Martland emphasizes the constant danger to workers in contact with these sub-
stances in stating that, "even wjth the most modern methods of protection, such
as proper screens, frequent shifting in work, vacations and proper ventilation,
all workers with x-rays and radioactive substances are exposed to the deleterious
effects of radiation and are pote~tial candidates at any time for the development
of a serious and fatal anemia or leukemia." It seems logical that the action
of radiation on the marrow would depend not only on the amount of exposure but
on the susceptibility of the individual. There is little doubt but that nearly all
radiologists go through their entire life with no effects on the marrow function,
and this is especially true in recent years with newer and more adequate methods
of protection.
Marrow depression from rad,ium has been seen mainly in t.hose people who
are subjected to this agent in industrial work, as seen in the group of over 50
girls studied by Martland. These patients developed varying degrees of anemia,
leukopenia. thrombocytopenia, and necrosis of the bones. In these cases the radio-
active substance was swallowed and then deposited in the bones close to the
hematopoietic tissue. In such instances the deposits of insoluble radium salts
remain there permanently giving off a constant bombardment of electropositive
 APLASTIC ANEMIA 319
charges which enter the negatively charged colloid structure of the red cells, pro-
ducing the most damage in that series of cells in the erythropoietic centers.
Unfortunately there is no way to rid the bones of these depositions of
radium salts so that the damage becomes permanent with little likelihood of the
marrow being able to escape the constant barrage from these rays, if disintegra-
tion of the radioactive substance ooes not occur in a few days, and this is quite
unlikely since the disintegration -period of mesothorium is about twelve years
and that of radium 1750 years (Martland).
The resulting blood pictures after marrow damage by radioactive substances
are quite variable but are characterized mainly by leukopenia, erythropenia, and
thrombocytopenia or any combination of them. Therefore, the clinical picture is
also quite variable. One patient may show the results of severe leukopenia with
its complicating septic processes, another the signs of severe anemia, while an-
other may present various hemorrhagic disorders because of the thrombocyto-
penia. In still others the bone changes are most marked, with areas of necrosis,
pathological fractures, and secondary infectious states, while the blood findings
may not be an olltstanding feature. Rosenthal and Grace have been able to repro-
duce the human picture of radium poisoning in rabbits by the oral administration
of radium sulphate. They 'state that the bone marrow changes in tlrese animals
included first, a hyperplasia of the erythropoietic elements, followed by de-
creased erythroblast maturation, replacement by myelocytes, and finally fibrous
tissue replacement. They also noted extramedullary myelopoiesis in the lymph
nodes and spleen, and point out the possible development of leukemia instead
of aplasia.
The treatment, as outlined by Martland, is removal from the poisonous sub-
stance and symptomatic tlrerapy: including repeated transfusions, the care of the
mouth, the prevention of sepsis, the use of any agent that may aid in promoting
erythropoiesis, including liver extract, sunlight, high altitudes, quartz lamps
and general hygienic measures. The outlook is poor but the longer tire patient
survives, the better is the chance that recovery will ensue for the reason that
the radium in the marrow slowly undergoes disintegration.

IN OTHER DISEASES
A blood picture that is characteristic of aplastic anemia may be seen at
various times in other diseases, particularly in the terminal stages. Thus, in
pernicious anemia in relapse, the red cells may be quite low, the hemoglobin
slightly higher, the color index above one, with only a moderate degree of macro-
cytosis, all of which may be accompanied by a marked decrease in the number
of leukocytes and platelets. The marrow may be quite fatty. Such pictures are
seen only in the long standing cases that have received no treatment_ It is cor-
rected, of course, by the proper anti-perniciolls anemia therapy.
In the terminal stages of the leukemic processes of the myeloid type marked
erythrocytic aplasia may occur with low values for hemoglobin, thrombocytes and
red cells. Usually the red cell picture is characterized by varying degrees of
anisocytosis, poikilocytosis, and perhaps polychromatophilia. There may be
slight macrocytosis, and evidences of regeneration in the form of erythroblasts
32 0 THE ANEMIAS

may be seen in the peripheral blood. This aplasia is brought about by marked
encroachment of the rapidly proliferating myeloid tissue on the erythropoietic
tissue of the marrow. Strictly speaking, it is not aplastic but overcrowded with
granulopoietic elements.
Marked aplasia may be seen in long standing infectious processes which
terminate by overwhelming sepsis. The bone marrow is not "aplastic" but the
peripheral blood may be characterizedby a decrease in the numbers of circulating
granulocytes, erythrocytes, and thrombocytes. In such instances the marrow is
usually highly cellular but is subjected to a physiological paralysis whereby it is
unable to ei ther produce or deliver the normal number of red cells. Such a
condition may follow periods of marked marrow stimulation over a long period
of time.
Naegeli states that he has observed instances of aregenerative or atrophic
marrow with the blood picture of aplastic anemia in the following conditions:
Radium poisoning; x-ray overdosage; thorium poisoning; polonium poisoning;
in severe intoxications, including hemolytic sepsis, poisoning with lye, saponin,
arsenic, collargol, bichloride of mercury, and salvarsan; in typhus, sprue, and
arthritis; in severe carcinomata; in senility and at the menopause; after pro·
longed hemorrhages; in fish tapeworm anemia; in osteosclerosis; and in bone
marrow tumors. He points out that in these instances one cannot state with
absolute assurance that all of the marrow is aplastic since it is obvious that
it all canDot be examined.

PRIMARY IDIOPATHIC APLASTIC ANEMIA

This is apparently a disease entity of unknown cause, occurring mainly in

young adults, characterized by extreme weakness and pallor with severe grades
of erythropenia, leukopenia, and thrombocytopenia, and the complications reo
suIting from these cellular deficiencies.
The disease occurs most frequently in young adult life after the period of
adolescence, though it has been reported in young children and infants. Smith
described it in a child of six years and collected 64 cases in the literature up to
1918. The youngest of these was two, the oldest 68 and the average 30 years.
In our experience, the disease has been in young adults" equally distributed as
to sex, and we have not observed it in a negro. The cause seems to be entirely
unknown.

SYMPTOMS AND PHYSICAL FINDINGS

The clinical findings depend largely on the extent of the cellular depression
and the type of cells involved. The onset is usually insidious. and consists in a
gradual development of increasing pallor, weakness and fatigue. By the time
the patient consults the physician these findings are apparent. The patient is
usually weak, and perhaps dyspneic. There may be palpitation and a hemic
murmur if the anemia is profound. The precipitating onset may be a syndrome
of hemorrhagic disorders, with an outcropping' of purpuric spots or excessive
uterine bleeding, with oozing from the gums and soft tissues of the mouth. The
 APLASTIC ANEMIA 32 I

onset may be characterized by the development of oral ulcers and the presence of
various septic states with an irregular febrile course. Always present is the
clinical evidence of severe anemia.
In those patients in whom there are no complications such as hemorrhages or
infection, there is an absence of physical findings of significance. There is no
lymphadenopathy or splenomegaly.

LABORATORY FINDINGS
The peripheral blood is characterized by marked decreases in the number
of erythrocytes, granulocytes, and thrombocytes. In Smith's reviewed series of
64 cases, the red cell average was 1,000,000 per cu. mm., the hemoglobin 17 per
cent, and the leukocytes 3000 cells per cu. mm.
In this disease the red cells reach the lowest figures seen in any condition.
The hemoglobin is reduced in proportion so that the color index is usually about
normal. We have studied one patient whose red cell count was 800,000 per cu.
mm. and the hemoglobin 15 per cent.
A study of the stained smear shows the red cells to be of normal size and
shape but few in number. In stained blood there seems to be a characteristic
zone about the cell that appears as a faint pink staining halo. This has been
said to be due to increased albumin globulin concentration about the cell. There
is no evidence of regeneration in the form of reticulocytosis, normoblasts, or stip-
pling. In this type of aplastic anemia the cells are normal in size, in contrast to
the secondary types which may exhibit varying degrees of macrocytosis.
The leukocytes are characterized by a marked decrease in the number of
granulocytes with a relative lymphocytosis. In some patients the granulocytes
may be entirely absent and in the terminal stages the lymphocytes and other
cells are also decreased in number.
The platelets may be entirely absent or there may be only a few thousand
per cu. mm. The few platelets seen in the stained smear are usually large and
cellular. Because of the decreased number there is prolonged bleeding time, a
normal coagulation time, delayed retraction of the clot, and a positive tourniquet
test of capillary resistance.

TREATMENT AND PROGNOSIS

After the diagnosis is established the treatment is directed mainly toward
efforts to stimulate the marrow to increased activity and to supply the patient
with a sufficient number of red cells to maintain life. Therefore, the treatment
should include repeated transfusions which may have to be given every ten days
or two weeks for a long period of time. Practically all measures designed to
stimulate hematopoiesis have been tried. These have included the intensive use
of liver and stomach preparations by mouth and liver extract by injection, and
nucleic acid products, bone marrow extracts, various dietary measures, and x-ray
stimulation of the marrow, but all of these seem useless since the patient is never
able to restore the marrow to its normal function. Therefore, the prognosis
seems hopeless, and treatment only palliative. The outlook for the patient
depends upon the length of time that it is possible for him to live on "borrowed
3 22 TUE ANEMIAS

blood" in the form of transfusions. This is variable, because there is the ever
present danger of the development of renewed hemorrhages, or intercurrent in-
fections hecause of the depleted granulocytes. Osgood reports a case of a 19 year
old girl that received 42 transfusions in 52 days. In addition this patient was
given 18 cc. of sternal bone marrow which was mixed with SOD cc. of blood and
given intravenously. No effects were observed from it. The case terminated
fatally. Since that time olher inslances of removal of sternal marrow from
normal donors and transfusion into patients have beeu recorded but in general
these procedures have been without effect.
Smith reports the average duration of illness after diagno~is in 64 cases to
be three months, the shortest four days, and the longest one and one-half years.
In our experience the disease has invariably terminated fatally in a few weeks.
Primary aplastic anemia may be 'placed in the class with the secondary
types if the cause is ever discovered, aud even if this is doue there remains little
hope for an efficient therapeutic agent because in the regime of hospital treatment
at this time they probably are removed from the noxious agent that may pro-
duce the disease. The diagnosis should be made as a last resort because of its
serious prognostic import. In our experience it is sometimes difficult to differ-
entiate aplastic anemia from the so-called aleukemic leukemia which is charac-
terized by the presence of immature hematoblasts in the peripheral blood. The
differentiation is of little practical importance, however, since. the outlook for
the patieut with aleukemic leukemia is equally as bad.

OSTEOSCLEROTIC ANEMIA

This disease was described in 1904 by Albers-Schonherg, a radiologist of Ham-

burg, Germany. He called it marble-bone disease. According to Wortis who
reviewed it in 1936, less than 80 cases had been reported up to that time
under various names including Albers-Schonberg disease, osteosclerosis fra-
gilis generalisata, osteopetrosis, congenital osteosclerosis, chalky bones and
marble bones. The disease is characterized by increased thickness and density
of the skeletal system caused either by proliferation of connective tissue or by
the deposition of solid compact bone toward the medullary cavity; therefore, the
crowding of the hematopoietic tissue results in severe grades of anemia. The most
striking changes are seen in the vertebrae, pelvic bones, ends of the femur and
distal ends of the tibiae. There is an increase in opacity and density in X-ray
examination with partial or complete obliteration of the medullary cavity.
The cause of the increased proliferation of fibrous structures and bone
structures is unknown. There is some evidence that a predisposition to this may
be inherited and consanguinity of parents has been reported in a number of cases.
In the case of Lamb and Jackson, the patient's great-grandfathers were brothers.
It has been reported in father and son. There are no sex, racial, or geographic
features of significance. The diaguosis is made upon the finding of opaque bone,
either by accident or in connection with pathologic fractures, or by finding
severe grades of anemia that are ultimately proved to be on that basis. In
children there is usually combine<t anemia, retardation of growth, with enlarged
 APLASTIC ANEMIA 323

liver, spleen, and lymph nodes. Some writers have described two forms of the
disease; one being the marble bone type, seen usually in youug adults, with depo-
sition of bone in the diaphyses and secondly, that known as myelosclerosis, seen
more often in adults with anemia the most striking clinical feature.
The hlood picture is characterized by changes usually seen after depression
of hone marrow actidty. There is usually a gradually progressive anemia with
the red cells finally reaching extremely low levels and hemoglobin reduced in
proportion. This may be accompanied by a few immature erythrocytes in the
form of normoblasts. Leukopenia may also develop and the platelets have been
variously estimated from normal figures to below. The white cells may show
scattered myelocytes in the peripheral blood. The disease has to be differentiated
carefully from idiopathic thrombocytopenic purpura.
There is no treatment that seems to be of any value in osteosclerotic anemia.
Various measures have been tried to influence the mineral and vitamin metab-
olism, and various endocrine treatments have been employed but all of these
appear to be without effect.
Spontaneous bone fractures should of course be treated by· the usual ortho-
pedic methods: Blood transfusions are frequently employed, mainly for prolonga-
tion of life, although they do not affect the course of the disease.

BIBLIOGRAPHY
N>LASTIC ANEMIA

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Med. Sci., 186, 353, 1933.
FARLEY, D. L: "Depressed bone·marrow function from the arsphenamines, including a type of
so-called agranulocytosis." Amer. Jour. Mea. Sci., 179. 2I4, 1930.
HAMILTON, A.: "Benzene (benzol) poisoning." Arch. Path, 11, 43-4, 601, 1931.
HUNTER, F. T.: "Chronic exposure to benzene (benzol): clInical effects" Jour. lndust Hyg. and
T()xicol., 21, 331, 1939.
ISAACS_, R: "The bone marrow in anemia. The red blood cells." Amer. Jour Med. Sci~ 193, 1:81,
1937·
JANUARY. L. E., and FOWLER, W. M.: "Aplastic anemia." Amer. Jour. CUn. Path, 10, 792, 1940.
KRACKE, R. R.: "The experimental production of agranulocytosis." Amer Jour. Clin. Path, 2,
II, 1932.
KRACKE, R. R., and PARKER, F. P.: liThe etiology of granulopenia (agranulocytosis): With particular
reference to the drugs containing the benzene ring." Jour. Lab. and CUn. Med., 19, 799, 1934.
MALLOP.,Y, T. B) GALL, E. A., and BRICKLEY, W. J.: "Chronic exposure to benzene (benzol): Patho.
logic results." Jour.1naust,. Hyg. and Toxieol., 21. 355. 1939.
MARnAND, H S.: "Occupational poisoning in manufacture of luminous watch dials; general review
of hazard caused by ingestion of luminous paint, with especial reference to the New Jersey cases
.. (concluded)." Jour. Amer. Med. Assn~ 92. 552, 1929.
McCARTHY, F. P., and Wnsow, R, JR.: "The blood dyscrasias following the arsphenamines"
Jour. Ame,.. Merl. Assn, 99, 1557, 1932.
McCOlm, C. P.: "The present status of benzene (benzol) poisoning." JOllr. Amer. ]"Itd. AJ'sn J 93,
28-0) 1929.
NAEGELI, 0 . "Blood diseases and blood diagnosis." 5th ed, 288-291. J. Springer, Berlin, 1931.
OsooOD,E. E., RIDDLE, M. C., and MATHEWS, T. J.: "Aplastic anemia treated with daily transfusioIl!
and intravenous marrow; case report." Ann. Int. Med .• 13, 357, 1939.
 THE ANEMIAS
,
RHOADS, C. P.: "Aplastic anemia. A symposium on the blood." Univ. of \Vlsconsin Press 1939,
p·51.
ROSENTHAL, M., and GRACE, E. J.: IlExperimentaI radium poisoning. I Bone marrow and lymph
node changes in rabbits, produced by oral administration of radium sulphate." Amer. Jour.
Med. Sci., 191,607. 1936.
SANTESSON, C. G.: "Ueber chronische vergiftungen mit steinkohlentheerbenzinj vier todesfalle."
A,.ch. ,. Hyg., 31~ 336. 1897.
SELLING, L.: "A preliminary report of some cases of purpura hcmorrbagica due to benzol poison.
mg." Bull. Johns Hopkms Oosp., 21, 33, 1910
SELLING, L I and OSGOOD, E. E.: "Chronic benzol poisoning.~' lnternatl. Clin., 3.z ser. 45, 52, 531
1935
Swm, L. W.: ({Report of an unusual case of aplastic anemia." Amer. Jour. Dis. Child., 1'7, 174,
19"19·
SMITH', D. L., JR., and LYON, R. A.: "Aplastic anemia following stovarsol (acetarsone) therapy."
Jour. Ped., 6, 624, 1935.
STEPHENSON, C. S., and WINGO, E. H.: 14Toxic effects of arsenical compounds employed in the
treatment of diseases in the United States Navy, 1935." U. S. Nav. Med. Bull, 35, III, 1937.
THOHPSON, \V. P., RICHTER, 1\1. N., and EOSALL, K. S.: "An analysis of so-caUed apJastIc aI1emia."
Amer. Jour. Afed. SCi, 187, 77, 1934.
WEISKOTIEN, H. G: j'The normal life span of the neutrophile (amphophllc.) leucocyte (rabbit).
The action of benzol. IX." Amer. Jour. Path., 6, 183, 1930.
WHEELIIIAN, R. Y.: "Granulocytic aplasia of the bone marrow fonowing the use of arsenic,a Amer.
Jour. Dis. Chtld, 35, 1032, 1928.

OSTEOSCLEROTIC ANEMIA

ALBERS-SCHONBERG, 11.: "R6ntgenblldel' einer seltenen knochener-krankungt' Aert.z;e: Verlin.ald,

Hamburg, Feb. 9, 1904.
- - : Munchen Med. Wchnschr, 51, 365, 1904.
- - : HEine hisher nieht benschreibene allgemeinerkrankung des skelettes in rontgenbild" FC)rtschr.
Geb. Rimtgenstrahlen, 11, 261, 1907.
--~ uEme seltene, bishernicht becannte struktur-anomalie des skelettes" Fortscllr, Geb
Rontgenstrabien., 23, 174, 1915~
CHAPMAN, E. M.: "OsteoscIerotic anemia." Amer. Jour }'led. Sci., 185, 171, 1933.
THOMPSON, W. P., and ILLYN~. C. A.: liThe clinical and hematologic pic::ture resulting from bam
marrow replacement." Med. Clin N. Amer., 24, 841, 1940.
WO'RllS, H.: uQsteopeUosis II Amer. Jo'Ur. Dts. Childre_n,52, Il48, 1936
 CHAPTER 25
SICKLE CELL ANEMIA

(Sicklemia) (Meniscocytosis) (Drepanocythemia)

(Herrick's Syndrome)
This disease is a true hemolytic anemia which is hereditary and translqissi_
hie according to the Mendelian law of heredity. It is characterized by the pres-
ence of bizarre, crescent.shaped or sickle-shaped red corpuscles, which ar~ de-
stroyed in excessive numbers. All of the clinical symptoms are the result ot this
peculiar red cell deformity.
HISTORY
Sickle cell anemia was first described as a clinical entity in 19I~ by
Herrick, who observed "peculiar elongated and sickle-shaped red blood corP4scles
in a case of severe anemia," but the report attracted little attention at the time.
In 1922 Mason gave the disease its name of "Sickle Cell Anemia" and in 19 2 4
Sydenstricker centered attention on the disease when he reported the Sickling
trait in a considerable percentage of negroes even though they presented· no
evidence of anemia. He referred to this as the "latent phase" of the disease, "'hich
is the same phase that has been called "meniscocytosis" by Miyamoto and
Korb and by Graham.
Huck was the first to demonstrate that the sickling phenomenon is ah in-
herent trait of the red cells rather than one of the plasma, as previously sUPPosed,
and Hahn and Gillespie demonstrated that the sickling could be produced more
,."..mIiS· wl!tlr ,'tie" far' ,;mll;- Wt!l.". ,,,,II\NW &crlIr CUlnm.1.' mi.fr u;cygel1:
DISTRIBUTION
The disease has been observed in practically all parts of the world in
which there is a negro popUlation. It is almost entirely confined to the l\egro
race, though a few reports have cast some doubt as to whether or not it can
occur in other races. Thus, one case in an Arab was reported by Archibald; one
in a Cuban by Stewart; one in an Italian child by Castana; and one in a o,reek
child by Cooley and Lee. Rosenfeld and Pincus described the disease in a ':hild
of undoubted Mediterranean ancestry, in whom the possible admixture with
negro blood C0l1ld absolutely be ruled out. Wallace and Killingsworth f~und
sickling.in women of the same Mexican family, and Cooke and Mack studied a
white American family in which there was sickling in two children and the
father. Killingsworth and Wallace, who have studied the disease extensively in
the Southwest United States, state that "a careful review of the literature shows
several cases in which there is little or no possibility of, Negro blood."
* For an excellent summary of Sickle Cell Anemia, consult article of Bernhard Sternberg, Arcb.
Path. 9, 876, 1930.
32 5
 THE ANEMIAS

In spite of these rare reports sickle cell anemia is essentially a disease of

negroes. The table reproduced below, prepared by Killingsworth and Wallace,
gives the percentage of negroes who possess the sickling trait as reported by vari-
ous observers.

TABLE XX
INCIDENCE OF SICKLE CELL ANEMIA IN NEGROES

(Anemic and Non-anemic Phases)

No. Number""(;J.ith p", CenI
I nvesligaJor and Place Eromined Sidling Sicklmg
Sydell.$trickcr, ]'1:ulhctin and Houseal, Georgia. . . . . . . 300 '13 4·3
Cooley and Lee, Michigan...... .................•. 400 30 7·5
Miyamoto and Korb~ Missouri............ ........ 300 19 6·3
Wollstein and Kreidel, New York................... ISO 13 8 6
Josephs, Maryland... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 250 16 6.4
Smith, Louisjana. ......... . . . . . . . . . . . . . . . . . .. . 5 5. 0
Dolgopol and Stitt, New York. .. ........ ... ..... 77 4 5 ,
I..evy, New York. ........ ...................... 213 5·6
Graham and McCarty, Alabama .... ,.............. 1500 U2 8.1
Brandau, Texas .................... _., '" .... l50 10 6 7
Sydenstricker. Georgia.. ....... ................. 1800 99 5·5
Diggs, Ahmann and Bibb, Tennessee ...•.... ...... 2539 21I 83
Ahmann, Florida .. .. ............ . . . . . . . . . . . . 674 6S 9 6
Killingsworth and Wallace, Texas ........... ~....... 1205 6S 5·3
Totals, ten states.. .......................... 9658 684 707 U. S. Av

It will be noted that approximately 7 per cent exhibit this trait, but 'these authors
point out that only one out of 's of these develops the disease, which emphasize5
the fact that a tendency to sickling may be present in many people and yet they
never develop any evidence of anemia.
The sickling trait has been reported at all ages (average age 13 years) and
it apparently predominates in females. Young adults are more likely to develop
the anemia after puberty, and the older the person the less likely is the disease
to develop. This has given rise to the concept that sickle cell anemia is pre-
dominately a disease of childhood and this is generally true, but it bas been ob-
served in very elderly people. Warrick studied 1570 negro patients for sic~ling
of red cells and found over 9 per cent who showed this trait. He correlated the
group that showed sickling with studies for agglutinogens M and N and blood
groups. He found that there was no relationship between the sickling trait and
the various blood groups. He showed further that sickling of the cells occurred
regardless of the medium in which the cells were placed and that serum was not
necessary for sickling.

FA~TORS PRODUCING SICKLING

As stated before the sickling phenomenon is the basis of the disease and there-
fore, many theories to explain it have been proposed. First, it has been assumed
that there is an heredi tary defect in bone marrow function, manifested by the
production of these deformed cells. This concept is supported by the definite
familiar tendency and to the frequency of associated diseases that are known to
be the result of maldevelopment. Secondly, some believe that the sickling is
 SICKLE CELL ANEMIA

brought about by changes in the blood plasma concerned with oxygen tension,
because of the tendency for the sickling to occur in the absence of oxygen, the
variation in sickling in different degrees of oxygen content, the restoration to
normal shape in carbon monoxide, and the fixation of the sickled cells in oxidizing
agents such as formaldehyde gas, Zenker's solution, ferrous ammonium sulphate
solution, neutral dyes, etc. From these observations it has been suggested that
the hemoglobin undergoes chemical alteration, and tbat the crescent shape ill
stable when the associated hemoglobin is in the reduced state. Also the work
of Hahn and Gillespie indicates that the variations in hydrogen ion concentra-
tion, associated with oxygen changes, may affect the cellular shape, since th,
sickling is facilitated in acid suspensions.
Graham believes that the anemia is caused by an unknown peculiar weak·
ness inherent in the physical components of the cell, mainly the cytoplasmic
network, and that certain physical surroundings, such as pressure and la.terai
traction, by fibrin strands in the plasma, may produce the cellular deformity
Since many people with the sickling trait are known to develop the diseas,
following some infectious process, there seems to occur a disturbance, presum·
ably in the blood plasma, that precipitates the acute attack of the anemia
When this occurs more cells become sickled and are more susceptible to th,
normal destructive processes, so that the reticulo-endothelial system enters ,
phase of increased activity, particularly in the spleen. This results. in excessivE
liberation of hemoglobin with larger amounts converted into .bil<;·.pigment, thi:
being followed by the hemolytic jaundice that is seen SO o"fteri in the acutE
attacks. Then the concept has been advanced that the primary site of the dis
ease is in the overactivity of the spleen, but this would appear to be a secondar)
change, mainly because the removal of the spleen does not correct the sicklin!
since the patient has just as much after the splenectomy as before.

SYMPTOMS AND PHYSICAL FINDINGS

The average patient, which is usually a young negro adult, presents a his
tory of recurrent attacks of weakness, ill health, anemia, easy fatigue, and othe
signs and symptoms referable to attacks of anemia. He may state that othe
members of the family have a similar type of illness. The symptoms are variabll
to an extreme degree, being dependent upon the severity of the disease. Most 0
the symptoms may be referable to rusturbances in the intestinal tract, includinl
upper abdominal pain, nausea and vomiting,or to the joints with variable type
of arthritic pains. Therefore, if the patient is seen for the first time in a sever<
acute attack, the rusease may be confused with acute surgical conditions of th
abdomen, or with various types of arthritic syndromes. The upper abdomina
pain is thought to be due to the recurrent infarctions which can be seen so well il
the removed spleen. A common finding in those cases that have a prolongel
course is the presence of ulcers on the legs near the ankles. These may hav,
developed because of an initial trauma, coupled with lowered resistance, un
hygienic surroundings, inadequate diet, and other similar factors seen so often il
the cla~s of people who are the most frequent victims.
 PLATE XXVIII

SICKLE CELL ANEMIA

(MENISCOCYTOSIS)

1. Juvenile neutrophil
2. Segmented neutrophils.
3 NOrInoblasts
4. Microblast.
5. SICkle cell.
6 Erythrocyte with Cabot ring body,
7 Erythrocyte with basophtlic sttppling.
8. Macrocyte (note endogiobular degeneration).

Blood Fmdmgs. Differential:

Hemoglobin 74 grus. (Newcomer's method). Myelocytes I%
RBC. 2,000,000 per c.mm Juvemles 8%
WBC. 19,000 per cmm. Bands. I60/0
Platelets 550,000 per C.mm. Segmenters 60%
Total neutropru.ls
Color Index , 1.1 Lymphocytes
Volume Index 10 Eosinophils
Icterus Index 350 Basophils •
Reticulocytes :[4 0 0/0 Monocytes
Erythrocytes; slightly hyperchromic wIth marked anisocytosis, polychromatophilia, nj.lme:·
aus macrocytes, sickle cells and norma blasts. Wet preparation shows sicklmg phenomeI]L)n ill
40% of erythrocytes.
Plate XXVIII.
 SleRLE .CELL ANEMIA

The physical findings depend upon the acuteness or chronicity and the
duration of the disease. The sclerae are usually greenish yellow hecause of
jaundice. There may be slight generalized lymphadenopathy, with a slightly en-
larged liver and spleen. The mucous membranes are pale, and there may be in-
creased cardiac dulness with a hemic murmur, and increased temperature with a
fast pulse and respiration rate. The patient may appear acutely ill and near pros-
tration. Leg ulcers mayor may not be present, though they are common
in old cases, or scars may be evident. All of the above findings may be ahsent
in the mild type with the only symptoms referable to a moderate or mild grade
of anemia. Grinnan reported four cases showing thickening of the bones of
the skull with thinning of the outer and inner tables. This, however, may be
present in any type of severe anemia in which there is marked over activity of
the bone marrow.

LABORATORY FINDINGS

The diagnosis can he made on the characteristic findings in the blood. The
erythrocyte count is usually low, ranging from 1,000,000 to 4,000,000, and the
hemoglobin is even more reduced, this resulting in a low color index. If the
patient is seen in the stage of acute bone marrow crisis, the blood will show signs
of active regeneration, including large numbers of nucleated red cells, marked
leukocytosis, increased platelets, the presence of megaloblasts, and reticulocytosis.
Apparently at this stage the bone marrow is releasing all types of young immature
cellular elements in a final effort to maintain an adequate number of cells, with
regeneration similar to that seen in the crises of acute hemolytic jaundice. The
degree of granulocytic immaturity in those patients showing high leukocyte
counts may be so marked as to be confused with myeloid leukemia, particularly
if the spleen is enlarged. It should be emphasized that in estimating the number
of white cells, proper allowance should be made by careful computation for the
large number of nucleated red cells present (see section on technic for method).
If the patient is in the chronic stage, the anemia is less marked, the red cell
count only moderafely reduced with the hemoglobin in proportion, and the color
index at normal or below. The leukocyte count may he normal, the platelets unaf-
fected, and in tbe smear the red cell picture shows a moderate cellular achromia,
some polychromatophilia, perhaps signs of basophilic degeneration including erytiI-
rocytic stippling, and variable degrees of macrocytosis. The latter may be de-
pendent upon tiIe degree of liver damage. Eosinophilia is seen in many instances,
and Huck has called attention to the increased monocytes which may exhibit
a tendency toward phagocytosis of red cells.
The characteristic finding is the sickling of the red cells, which in extreme
cases may be evident on the routinely stained smear. However, the patient may
have an extremely acute attack and the sickling not be noted in stained prepara-
tions. If present the sickled cells appear as crescents or sickle shaped cells
in variable proportions. As many as 50 per cent may show the change while in
other patients they may be quite infrequent. Sickling can be demonstrated by
the method of Hansen-Priiss, by taking one drop of blood and treating it as
33 0 THE ANEMIAS

for a reticulocyte count, using cresyl blue dye. This and other similar dyes
seem to act as rapid oxidizing agents so that when tbe blood is examined in the
wet state a short time later the sickling is pronounced (see section on technic).
The usual technic is to place a drop of blood on a clean slide under a cover glass,
seal the edges with paraffin or vaseline, allow to stand for 24 hours and ex-
amine for sickling at various intervals. The method of Hamen-Prliss, however,
using intravital dyes, brings out the sickling several hours earlier. The method
of Beck and Hertz is also valuable, in which blood is placed in a tube under a
layer of mineral oil, and then fixed with formalin. It is said that the method
reveals the sickling trait in about twice as many people as the technic in ordi-
nary use.
Other laboratory findings that are useful as additional information, include a
positive indirect van den Bergh reaction, a high icterus index, and increased
urobilin in the urine. The sedimentation rate may be increased. The blood
chemistry findings are normal. Hypoacidity or achlorhydria may be present in a
considerable number of patients. •

PATHOLOGY

The chief finding has been thoroughly described by Diggs, and this involves
the various changes seen in the spleen in different stages of the disease_ Based
on a study of the literature and a study of nineteen spleens, he states that the
earliest lesion consists of congestion of the reticular spaces with sickled erythro-
cytes, this resulting in a gross lesion that is enlarged, dark purple, and soft. After
this the picture is variable. The lesions do not progress at the same rate and
therefore there may be seen in the same organ the changes of congestion, hemor~
rhages, infarcts, organization, and early stages of fibrosis. As the latter change
becomes more pronounced the spleen becomes smaller, its surface nodular and
harder with depressed scars and elevations, and the entire organ atrophic and
fibrotic. Scattered through are small rustlike granules, from one to two Mm.
in diameter. These are connective tissue nodules that contain masses of pigment
infiltrated with iron and calcium saIts and hence tbe process is called sidero-
fibrosis, this being the characteristic pathologic change in the spleen. The entire
process seems to come about because of the markedly increased load placed· on
the organ in an effort to destroy and dispense with its enlarged load of sickled
cells, and in the process of doing this, it destroys itself. It would be well to con-
sider that if the spleen is removed fmm a patient with this disease there is reason
to assume that one may remove the only organ that serves as a destructive agent
for the sickle cells.
Other pathologic changes include those that are characteristic of any type
of severe hypochromic anemia, including chiefly fatty degeneration, and hemo-
siderosis because of the release of large amounts of pigment. Sydenstricker and
associates have reported the finding of sickle cells in the marrow removed at
autopsy. The regenerative blood picture would suggest that the marrow would
be markedly hyperplastic, this depending upon the time the marrow specimen is
removed and the severity of the anemia.
 SICKLE CELL ANEMIA 33 1

TREATMENT

All forms of therapy are generally unsuccessful and so do not affect the prog-
ress of the disease_ Treatment, therefore, should be directed toward the alleviation
of symptoms, and if possible correction of the anemia. Since the course of the
disease is characterized by relapses and remissions and the patient is seen
usually during a relapse, it is obvious that if he survives the acute attack, im-
provement is to be expected and one should be cautious in attributing such im-
provement to some specific agent or procedure. Because of this it seems that
splenectomy was thought to be of benefit, but there is now general agreement
that it does not affect the course of the disease. The only time splenectomy
should be done is in the rare instance when the organ is so enlarged that it pro-
duces marked discomfort. Transfusions should be used, especially in the acute
crisis. These should relieve the strain on the bone marrow in its effort to main-
tain sufficient cells for life. The use ot various hematopoietic agents such as
liver extract, ventriculin, iron preparations, etc.," has been followed with little
success, although these agents would appear useful after the acute crisis has
passed. In this respect they would be considered as part of general measures
to aid the marrow in rebuilding the normal blood picture. Treatment is largely
symptomatic, and this is usually adequate to restore the patient to temporary
good health. This, coupled with the natural tendency to remission and to the
fact that as the patient grows older the attacks become less in frequency and
severity, seems to be about all that can be done for the patient with sickle
cell anemia.

PROGNOSIS

Some patients die in acute attacks, particularly in childhood. Many patients

go through adolescence and the young adult period with repeated attacks and
remissions, which become more infrequent and finally all evidence of the disease
disappears except the remaining sickling trait which is present for life even though
there is no further clinical evidence of it. However, these patients are known to
be unusually susceptible to infectious processes of various kinds and they should
carefully avoid exposure to the various infectious diseases.

OVALOCYTOSIS

In 1938 Florman and Wintrobe of the Johns Hopkins Hospital reviewed the
question of ovalocytosis or elliptical red corpuscles in the human being. They
state that the disease was described by Dresbach in 1904 and since that time at
least 134 additional instances have been reported in approximately 30 families.
These authors reported eleven new cases of this anomaly in members of <three
families. They point out that ovalocytosis cannot be considered to be present
unless at least 40 per cent of the erythrocytes are elliptical and at least 10 per
cent are rod forms. Apparently the trait bears no relation whatever to sickle
 THE ANEMIAS

cell anemia. The cells show no evidence of sickling, even under conditions of
oxygen exclusion. The condition may be seen in either the white or colored race
and it is extremely rare that there is any degree of anemia whatever.
The condition is recognized to be an inherited anomaly and appears to be
transmitted as a Mendelian dominant character. It is unrelated to sex, age, or
blood group. It occurs consistently in successive generations. It seems to be
generally agreed that the anomaly in shape is a property of the cells themselves
and is not the result of any peculiar or unique environment. According to
McCarty the elliptical erythrocytes are bi-concave and tbey are rounded at both
ends (5 X 13 1'). They react as do normal cells to hypotonic salt solution, dilute
acetic add, Hayem's solution, etc. The elliptical cells have not been seen in
nucleated forms in the bone marrow and only a small percentage of the reticulo-
cytes are found to be elliptical. Huck and Bigelow's patient was used as a donor
to transfuse elliptical cells to measure the life of such ceIIs and after two months
they were found in the blood of the recipient. The presence of this trait in families
should not give rise to any serious concern, since anemia seldom develops and
symptoms do not arise from their presence.

BIBLIOGRAPHY
SICKLE .CELL ANEMIA
ARCHIBALD, R. G.: "Sickle cell anemia in the Sudan.'t T,.. Roy. Soc. Trop. Med. and Byg J 19.
389. 19 26.
BECK, J. S. p" and IhltTz, C. S.: It Standardizing sickl-e cell methcd and evidence of sickle <ell
traiL" Amer. Jaur. Clin. Path., 5, 3:1-5, 1935.
CASTANA, V.: "Gigantocytes and sickJe celIs.'~ PediatritJ, 33, 431, 1925.
CnZNEY, G.: "Elliptic human e:rythrocytes." Jour. AmeT. Med. Assoc., 9&, S,8, 1932
CooK, J. V., and MACK, J. K.: "Si.ckle-cell anemia in a white American family." JOUT. Ped., 5.
601, 1934.
COOLEY, T. B, and LEE, P.! "The sickle cell phenomenon." AmeT. Jour. Dis. Child., 32, 334,
1926. .
COOLEY, T. B., and LEE, P.: "Sickle ce]] anemia'in a Greek family." Amer. Jour. Dis. Child, 38,
103, 1,)2.<}
DIGGS, L. W.: "Siderofibrosis of the spleen in sickle cell anemia." Jour. Amer. Med. Assoc. 104,
538, 1935.
FLORMAN, A. L., and WINTROBE, M. M.: "Human elliptical red corpuscles." Bun. Johns Hopkin:;
Hosp., 63, 209, 1938.
GRAHAM, G. S., and McCARTY, S. H.~ "Sickle cell (meni~ocytic) anemia." S01dh lrled. Iour, 23,
598, 193(),
GRAHAM, G. S.: "Sickle cell anemia with necropsy." Arch. Int. Med J 34, 778, 1924.
GREENWALD, L., and BUltRETT, J. B.: uSickle cell anemia in a white family." Amer. Jour. Med.
Sci., 199, ']68, 1940.
GlmmAN', A. G.: "Roentgenologic bone changes in sickle cell and erythroblastic anemia." Ame-r.
Jour. Roentg. and Radium Ther., 34-, 291, 1935.
HAHN, E. V., and Gn.LE.Sl"IZ, E. B.: "Sickle tell anemia. Report of a c.a~ greatly improved by
splenectomy. Experimental study of sickle cell formation." Arch. Int. Med., 39, 1233 , 1921.
HANSEN-PRUSS, O. C.: "Experimental studies of the sickling of red blood cells." JUUY. Lab. CUn.
Med., 22, 3n, 193-6.
IIaJuCE, J. B.: uPeculiar elongated and sickle-shaped red blood corpuscles in a case of .severe
anelPia." Arch. Int. Med., 6, 511, 1910.
HUCK, J. G.: "Sickle cell anemia!' Bull. Johns Hopkim Hosp-., 34-, 33S, 1923..
 SICKLE CELL ANEMIA 333
KlLuNGSWORTH, W. P., and WALI.ACE, S. A.: ~'Sicklemia in the Southwest." South. Med. Jour." 29,
941, 1936 .
LAWRENCE~ J. S.: ItHuman elliptical erythrocytes." Amer. Jour. jled. Sci., 181, 240, 193:1.
MCCARTY, S. H.: "Elliptical red blood cells iJ1 man." Jour. Lab. an.d CUn. Med., 19. 612. 1934.
MASON, V. R: "Sickle cell anemia." Jour. Amer. Med. Assoc., 79, 1318, 1922.
MIYAMOTO, K., and KORB, J. H.: "Meniscoc::ytosis (latent sickle cell anemia); its incidence in St.
Louis," South. AJed. Jou, J 20, 912, 19n.
Ros:tNFELD, S., and PINCUS, J. B.: liThe occurrence of sicldemia in the white race," Amer. Jour.
Med. Sci, 184, 674. z932.
STEINBERG, B.: "Sickle cell anemia." Arch. path., 9, 876, 1930.
STEWART, W. B.: "Sickle cell anemia. Report of a case with splenectomy." Arner. Jour. Dis.
Child., 34, 12, 1921.
SYDENSTRICKER, V. P.: "Sickle cell anemia." South. Med. lour, 17, J77, 1924.
'SYDENSTRICXER, V. 'P.: ~~Furtber observations un IDt'.&'J.-e u!iJ.1ffieniia." Jvur. A'IfI1ff. M"tn.. A~~Dt., %~,
12, 1924.
WALLACE, S. A, and KILLINGSWORTH, W. p,: "Sicklemia in the Mexican race." Amer. Jour~ Dis.
Chtld ~ 50. noS, 1935.
WARRICK, W.: "Immunologic studies of sickle cell anemia." Arch. Int. Med., 60, 623, 1937.
 CHAPTER 26
THE ANEMIAS OF CHILDHOOD
In children, hematopoietic reactions to stimuli are more severe, rapid, and
embryonic than in adults. The "erythron" of chi1dren is immature, widely
scattered, and the threshold of cellular delivery is delicately balanced. The
mechanism of the anemias of childhood differs from the adult only in degree.
The immaturity of the "erythron" renders it more susceptible to anemia-pro-
ducing factors in some instances, while in others it is more resistant to such fac-
tors. Some diseases, sttch as pernicious anemia, are rarely found in children
and the few cases reported have not been proved authentic. Normal children
seem to be born with sltfficient liver storage of the hematopoietic factor for the
normal maturation of red cells. On the other hand, the demands of the rapidly
growing infant, concomitant with the change from embryonic to extrauterine
activity, often predispose a child to an anemia of the deficiency type unless the
food intake, especially iron, is suffIcient to meet these demands.

PHYSIOLOGIC ANEMIA OF FIRST MONTH

Parsons and Smallwood have presented an excellent summary of factors

influencing the erythron in late embryonic life and early infancy. During fetal
life, hematopoiesis is not limited to the bone marrow but the liver, spleen, IYtn-
phatic tissue, and even the kidneys are active sites of blood production. Shortly
after birth these extramedullary sites of blood formation relinquish their hemato-
poietic function but when there is need for blood regeneration these organs may
revert to embryonic activity. Under normal conditions, however, the bone m'lr-
row carries the load of erythrocytic production. Throughout infancy all of tbe
bones in the body are Packed with red active marrow, in contrast to adults in
which a majority of the long bones contain only inactive yellow marrow.
The oxygen tension is low in the fetal circulation, resulting in insufficient
aeration and a compensatory increase in erythrocytes. This probably accounts
for the high erythrocyte counts and hemoglobin values as well as the presence of
immature, nucleated red cells in the blood of infants at birth. Red blood cells
vary from 5 to 8 million per cu. mm. and the hemoglobin varies from 9S to 140
per cent. Within the first two weeks the excessive red cells are partially destroyed
and an increase of bilirubin in blood plasma occurs, resulting in the so-called
"physiological icterus" <If the newborn. The jaundice is mild, transitory, atld
recedes without tberapy.
During the first thtee or four months after birth the red cells gradualIy
fall to normal or slightly below and the hemoglobin continues to decrease to a
level often as low as 65 to 7S per cent, resulting in the so-called "physiologkal
anemia" of early infanty which disappears spontaneously at about the fifth
334
 TIlE ANEMIAS OF ClIILDHOOD 335
or sixth month. This anemia is attributed to the failure of the bone marrow to
adapt itself to the requirements of the rapidly developing infant. No type of
therapy is entirely efficacious in preventing this physiologic decrease of hemoglobin.

SYMPTOMS OF CHILDHOOD ANEMIA

Parsons and Smallwood have Qutlined the outstanding symptoms and physi-
cal findings of children with anemia. As a rule infants and children do not exhibit
symptoms until an anemia has progressed to a severe degree. The presence
or absence of pallor is deceptive; the nutrition of the skin is often excellent during
marked anemia and, on the other hand, pallor is frequently present in the ab-
sence of anemia. The color' of the skin differs in the various anemic states; it
is usually icteric in hemolytic anemias, marble-white in iron deficiency anemias,
and a waxy-gray in leukemias. Prominent physical findings are heart murmurs,
tachycardia, elevated temperature, and in severe cases, heart failure and edema.
Purpura and hemorrhages are prominent manifestations when the blood platelets
are diminished. Irritability, lethargy, restlessness, and sleeplessness may be the
first signs of an anemic state in atchild.
Enlargement of the spleen and lymph nodes may be the first· indication to
the physician that a child is suffering from some type of blood disease which is
accompanied by an anemia. 'When these organs are enlarged in the absence of
infection and both the erythrocytes and hemoglobin are markedly reduced in a
full-term child, a cause other than simple nutritional deficiency of iron should be
strongly suspected. Therapy should never be instituted until an effort is made to
classify the type of anemia. Iron deficiency is accompanied by hypochromia
of the red cells, and marked reduction of erythrocytes occurs only in the most
severe cases. An iron deficiency may exist when the diet is entirely adequate,
the Source of trouble being faulty absorption of iron due either to achlorhydria or
intestinal dysfunction.

RELATION OF ANEMIA TO AGE LEVEL

Mackay has shown that iron deficiency anemias are prevalent in children
especially during the first four years of childhood. In order to facilitate an under-
standing of the anemia-producing factors operating during infancy and childhood,
she has divided the first few years of life into arbitrary age levels:

BIRTH
It is extremely rare for a child to be anemic at birth. A nutritional iron de-
ficiency in the mother is not reflected in the blood of a child at birth, but it does
result in an inadequate storage of iron in the liver, rendering the child less
capable of handling emergencies and, therefore, more susceptible to anemia at
a later date.

FIRST MONTH
, In the first month, there are two main causes of anemia, namely, bleeding
 THE ANEMIAS

and hemolysis. (These anemias are discussed in detail in the section on the
hemolytic and erythroblastic' anemias of the newborn.) From the first until
the fourth month a normal full term child undergoe", a physiologic diminution
of hemoglobin which is not related to dietary factors and cannot be prevented
by iron therapy, although the degree of diminution may be somewhat lessened
by iron prophylaxis. True anemia may occur at thi", period in premature or im-
mature babies and in small twins. When an anemia appears it is associated with
a low birth-weight and is prohably an accentuation of the normal physiologic
decrease in hemoglobin (see the anemia of prematurity).

FROM FOURTH MONTH TO FOURTH YEAR

Anemia in children at this age level is caused largely by nutritional iron
deficiency. Almost all children show a gradual decline in hemoglobin beginning
at about the fifth or sixth month, since by this time, the iron store in the liver
is depleted and the child is dependent entirely upon dietary consumption for its
needs. The folla,ving groups of children are especially prone to iron deficiency at
this age: (a) babies born of anemic mothers bave an inadequate iron store
and generally become anemic during the first year of life; (b) babies of low
birth-weight have probably had the earlier anemia of prematurity resulting in an
iron deficiency; when their rate of growth is quite rapid and their diet inade'luate
for their needs, they may develop an anemia of severe grade; (c) a baby rendered
anemic from infection, from bleeding, or any cause is likely to develop an iron
deficiency; (d) a normal baby kept too long on a milk diet will not receive suffi-
cient iron to prevent the development of a nutritional anemia.

SCHOOL AGE
Iron deficiency occurs rarely in children of the school age and when present
is usually the result of bleeding, infection, systemic diseases, or blood diseases
such as hemolytic jaundice, Banti's disease, sickle-cell anemIa, leukemia, etc.

EFFECTS OF IRON DEFICIENCY

Mackay has shown the results of iron deficiency from a study of a large
number of cbildren wbich were divided impartially into two groups, one group
being given iron medication and tbe other used as untreated controls. She con-
cluded that an iron deficiency results in a mild hypochromic anemia in a majority
of children, which begins at about the fourth month, and may be prevented by
iron medication. The anemia is in itself relatively unimportant, but it is suffi-
cient to lower markedly the resistance of the child to infectious diseases. In
the series of cases treated with iron to prevent nutritional anemia, she found the
incidence lower and the duration of infection considerably sborter than in the un-
treated group. In addition, iron deficiency retards the rate of growth since
babies receiving iron averaged one pound heavier than those of the control
group. She concluded that "anemia is so important a factor in predisposing to
infection than no anemia, even of slight degree, is insignificant."
 THE ANEMIAS OF CHILDHOOD 337

CLASSIFICATION

A comprehensive discussion of the anemias of childhood would entail a con-

sideration of every type known to exist, with the possible exception of pernicious
anemia, since children are susceptible to practically every anemic state found
in adults. Anemias associated with other diseases such as chronic hemolytic
jaundice, sickle cell anemia, leukemia, purpuras, malignancies, parasitic infections
and chemical poisons are considered in greater detail in other chapters.
A classification of these anemias is difficult. In gena-al the hypochromic
iron deficiency anemias are more prevalent in childhood than the macrocytic-
erythroblastic types. A child reacts differently to iron deficiency at certain age
levels. The etiology and mechanism of many of the hemolytic and erythroblastic
anemias are obscure. An erythroblastosis in childhood does not have the same
interpretation as in the blood of an adult. The immature erythropoietic tissues
are crowded with young megaloblasts and normoblasts and a stimulation or a
need for new red cells causes a liberation of the immature cells into the circulating
blood.
I. HypOCHROMIC DEFICIENCY ANEMIAS
A. Iron deficiency anemias.
I. Anemia of prematurity and twin gestation.
2. Nutritional anemia of infants . .
3. Hypochromic anemia of coeliac rlisease.
4. Iron defiCIency anemias in older children.
II. MACROCYTIC HYPERCHROMIC ANEMIAS
A. Idiopathic anenna of the newborn.
B. Macrocytic anemia of coeliac disease and fish tapeworm infestation.
III. HEMOLYTIC (ERYTHRONOCLASTIC) ANEMIAS
A. Hemolytic anemias of the neonatal period.
I. In the newborn.
(a) Erythroblastosis foetalis.
(b) Erythroblastosis of the newborn.
(c) Hemolytic anemia of the newborn without icterus gravis.
2. Anemias later in the neonatal period.
B. Hemolytic anemias of late infancy and childhood.
I. Acute hemolytic anemia (Lederer type).
2. SubchIonic hemolytic anemia.
3. Chronic hemolytic anemia (familial icterus).
4. Sickle cell anemia.
IV. ANEMIAS OF BONE MARROW DYSFUNCTION (ETIOLOGY UNKNOWN)
i. Von Jaksch's Anemia.
B. Cooley's erythroblastic anemia.
C. Banti's disease (splenic anemia).
V. ANEMIAS ASSOCIATED WITH OTIIER DISEASES
A. Anemias associated with infections.
B. Anemias associated with hemorrhagic diseases.
C. Anemias associated with leukemia.
 THE ANEMIAS

ANEMIA OF PREMATURITY

The mother's condition plays an important role in the causation of nutri-

tional anemias of infancy. The offspring of an anemic mother has normal hemo-
globin and red cell values at birth (Davidson and Leitch) since the mother sup-
plies adequate iron for fetal spleen and liver storage during the last few months
of pregnancy regardless of the cost to herself (Ochsenius). This storage may be
inadequate for later requirements. l\1aternal deficiency is reflected in the blood
picture of the child at about the sixth month; children of such parentage de-
velop anemia more frequently than the offspring of normal mothers (Davidson
and Leitch). The high incidence of anemia in premature infants and in twins is
explained on the basis of inadequate liver storage. The amount of iron is inade-
quate for twins because the supply is insufficient especially from an anemic
mother, and the storage is incomplete in an infant born before term.
The anemia of prematurity, occurring generally during the first three
months, differs from the nutritional anemia of infants only in its earlier appear-
ance. During the first few days after birth, the blood of a premature infant shows
the normal high values found in full-term babies (Abt and Nagel). The cellular
elements, however, are more embryonic. The anemia of prematurity has been
called "physiological" because it occurs at the same time that normal infants ex-
hibit a physiologic decrease in hemoglobin content, which is at about the end
of, the first two or three weeks after birth. The difference, however, is one of
degree and extent, the premature infants showing much lower values for hemo-
globin and red cells. The more premature the infant, the -more severe is the
anemia. Other factors, such as infection, syphilis; and inadequate diet, must
be excluded before an anemia in a premature infant can be classified as that
type which depends upon the factor of prematurity alone (Abt and Nagel).
The exact mechanism of the anemia of prematurity is s till unknown. The
most tenable theory is the assumption that the anemia results from insufficient
prenatal storage of iron in the liver, in contrast to deficiency anemias in full-term
children of healthy mothers who possess adequate iron storage but receive an in-
adequate iron diet during the first m-;)jl_ths of life. Dietary factors probably have
little influence on the causation of the anemia of prematurity because of the CJ:jual
incidence of this anemia in lower and higher social strata (Abt and Nagel). One
argument against the iron storage theory is found in the work of Abt and Nagel.
They were unable to prevent the development of this anemia by the administra-
tion of iron or any other type of prophylaxis.
The hematologic picture of this anemia varies little from the later nutri-
tional anemias of infancy, other than a tendency to become more severe and
a delayed response to iron therapy, once the anemia has begun.
Merritt and Davidson have studied the anemia in a series of premature and
immature infants. They conclude that the early decrease in red cells and hemo-
globin cannot be prevented entirely, but the anemia is less severe and recovery
begins earlier in treated infants; that iron as well as anti-rachitic vitamins and
orange juice should be administered to all premature children from the second
 THE ANEMIAS OF CHILDHOOD 339
week until the blood findings are normal; that such treatment may obviate the
necessity for transfusion; that infants treated with liver and iron show no better
response than those treated with iron alone; that treatment after the anemia
begins is not as effective as therapy ins tituted soon after birth; that a majority of
premature infants develop anemia, and the more immature the infant the more
marked the anemia. They recommend therapy in the form of iron and ammonium
citrate (50 per cent aqueous soL) in 0.3 Gm. (0.05 Gm. of reduced iron) per
kilogram of body weight.
Abt and Nagel have made a comprehensive study of the efficacy of the various
prophylactic agents in the anemia of prematurity. If used alone, neither iron nor
liver proved effective but favorable prophylactic results were obtained through
the use of a dried liver fraction combined with ferrous ammonium citrate. Their
method is as follows: "The liver in this preparation was in the form of the dried
liver fraction suitable for secondary anemia, made according to the formula of
Whipple and Robscheit-Robbins. The iron in this preparation was in the form
of ferrous ammonium citrate. Each level teaspoonful of this empiric formula
contains approximately 3.75 Gm. of liver fraction and 0.65 Gm. (10 grains) of
ferrous ammonium citrate. The initial dosages started with from I to 2 Gm.
twice daily and were increased up to 4 Gm. twice daily as the infants grew
older. This preparation, which was started after the first week of life, was easily
and well tolerated in these doses and caused no disturbance in the infants' diges.
tion, feedings or stools. The amount fed twice daily was mixed with I drachm
(4 cc.) of the milk mixture and was given at the end of a feeding. As the infants
grew older the liver-iron preparation was given in from one-half to one ounce of
milk formula."

NUTRITIONAL ANEMIA OF INFANTS

The earlier experiments of Bunge and Aberhalden demonstrate that the iron
content of the liver and spleen is highest at birth and decreases gradually until
the end of the normal lactation period. They produced an anemia in animals by
continuing a milk diet for a period longer than normal.
Helen Mackay has classified the nutritional anemia of infants as an iron de-
ficiency state, and has recognized its analogy to the anemia of experimental ani-
mals produced by a diet limited to milk.
She states that nutritional anemia occurs more frequently after the fourth
month of life, at the time when the iron store in the liyer is beginning to be de-
pleted. If the diet contains an inadequate amount of iron at this stage, as is
frequently the case in prolonged milk feeding, an iron deficiency anemia will in-
variably de,·elop.
The clinical and hematologic features of this anemia have been described
in~ detail by Parsons and his associates. The child may appear normal in size
and weight. The degree of pallor is variable. Mucous membranes are pale rather
than icteric, although in rare instances the skin may exhibit the brownish dis-
coloration characteristic of idiopathic hypochromic anemia of adults. The
340 THE ANEMIAS

spleen is often mildly en!;uged, but the lymph glands are not involved. Infec-
tion and rickets may be secondary sequelae.
The hematologic picture is characterized by hypochromia and microcyt~sis.
In mild cases the red cell count may not fall far below normal but in the more
severe types there may be a marked reduction of both red cells and hemoglobin,
and a low color index. The platelets are normal or slightly diminished in number.
In the presence of infection, the leukocytes are altered according to the type of
organism involved. Inl ants show a high leukocyte count in response to infections.
In response to iron therapy there may occur varying degrees of anisocytosis
and polychromatophilia. The microcytosis disappears as iron therapy institutes
improvement. A majority of these infants respond to iron therapy alone, but
in some instances there is a deficiency of copper and some other factor that is
found in yeast. When iron administration fails to bring about a rapid recovery,
copper and yeast are necessary supplements.
Since this anemia has a low mortality, autopsy studies are scarce. Von
Haam has studied the pathology of albino rats suffering from goat's milk and
cow's milk anemia which is similar to that seen in nutritional anemia of infants.
The most characteristic findings were atrophy of the spleen, fatty degeneration
of the liver, and hypertrophy of the heart muscle. Bone marrow of the animals
red on cow's milk showed a normoblastic reaction with normal composition of
myeloid cells. Animals with goat's milk anemia exhibited a significant ab-
senCe of mature leukocytes, which, according to Von Haam, is analogous to the
reported leukopenia in human cases of goat's milk anemia. These findings indi-
cate a close relationship of these anemia, to the iron deficiency states of childhood.
,z, Von Haam and Beard have studied the pathologic changes in the liver and
spleen in nutritional anemia in rats. The pathology in the liver resembled chronic
congestion with secondary atrophy of the liver parenchyma. In the spleen there
were fewer and smaller lymph follicles than normal and the cells showed signs
of marked degeneration.
Faber and his associates have noted the similarity between idiopathic micro-
cytic hypochromic achlorhydric anemia of adults and the "iron deficiency," nutri-
tional anemia associated with a marked secretory defect of the stomach after
histamine stimulation. The latter anemia in every case was relieved by the admin-
istration of soluble iron. These authors believe that during the first two years of
life children secrete small amounts of hydrochloric acid in the stomach and,
therefore, are incapable of absorbing a sufficient quantity of iron to prevent ~he
devel<lpment of a mild degree of anemia. They consider this inadequacy partially
responsible for the prevalence of hypochromic anemias in early infancy.

PROPHYLAXIS AND TREATMENT

Mackay has shown that nutritional anemia between the fourth month and
fourth year can be prevented by adequate prophylaxis which may be accom-
plished to a limited degree by providing the pregnant mother with adequate iron.
Treatment of the nursing mother with iron is of little benefit, since such a pro-
cedure does not appreciably ·increase the quantity of iron in the milk.
She recommends the routine administration of iron directly to the baby as
 THE ANEMIAS OF CHILDHOOD 341
the best prophylactic measure. The dosage is 4.5 grains of ferric ammonium
citrate daily, beginning about the second month and continuing until the child
is from 12 to IS months old. The iron mixture is given gradually, requiring
about a week or more to reach the full dose. This procedure usually prevents
the development of colic and intestinal symptoms which frequently occur when
a large dose is given to an infant too suddenly. A mixture is given to a breast-
fed baby, but a combination of dried milk and the-iron salt may be given to a
bottle-fed baby.
Mackay administers larger doses of ferric ammonium citrate (9 to IS
grains daily) in the treatment of an iron deficiency anemia. She concedes that
ferrous sulphate (4.0 grains daily) gives a more rapid response, but oxidation of
this preparation must be prevented to insure its therapeutic potency_

HYPOCHROMIC ANEMIA OF COELIAC DISEASE

Coeliac diseases in both infants and older children are frequently accom-
panied by an iron deficiency anemia which is the result of faulty iron absorp-
tion (Parsons and Smallwood). The condition is characterized by pallor and
a mkrocytic, hypochromic anemia which disappears slowly after cessation of
the coeliac disease. Gastric fmdings are normal and splenic enlargement is only
rarely an accompaniment of the anemia. The respon5e to iron is satisfactory but
quite slow. Vitamin therapy is recommended. A macrocytic anemia is some-
times associated with coeliac disease. (See discussion below.)
Parsons has reported the bone marrow findings of hypochromic anemia asso-
ciated with coeliac disease as follows: (I) the replacemen t of a large part of the
marrOW by a red jelly-like material; (2) the predominance of normoblasts and
the absence of megaloblasts in the foci of erythropoiesis; and (3) a slight imma-
turity of myeloid elements. On the basis of these findings, he helieves that this
anemia is closely allied to tbe idiopathic, hypochromic anemia of adults.

IRON DEFICIENCY ANEMIAS IN OLDER CHILDREN

Hawksley, Lightwood and Bailey have investigated the relationship of achlor-

hydria, gastro-intestinal disease, and hemorrhage, to the iron deficiency anemias
of childhood. Their studies were concerned chiefly with the OCCurrence of these
anemias in children who have passed the age of infancy and in whom factors
other than those causing nutritional iron deficiency play an important part.
They have reported 12 cases of iron deficiency anemia associated with achlor·
hydria. In 3 patients, achlorhydria was present for more than a year after a
hematologic cure by iron therapy and was considered of probable significance in
the etiology of the anemia_
They reported 2 instances in which an iron deficiency anemia, responding
to iron therapy, was associated with an intestinal polypus unexpectedly dis·
covered at autopsy. These cases resembled the ordinary nutritional anemia of
childhood. They found a similar anemia in a case of the so-called ulcerative
342 THE ANEMIAS

colitis and 2 cases associated with coeliac disease. These anemias were the result
of faulty iron absorption.

SUMMARY AND TREATMENT OF IRON DEFICIENCY ANEMIAS

In a summary of the current ideas concerning the etiology of all iron de-
ficiency anemias in childhood, the following predisposing factors are well estab-
lished: (I) prematurity; (2) twin gestation; (3) anemia in the pregnant mother;
(4) prolonged milk feeding; (5) defective diet; (6) achlorhydria; and (7) faulty
absorption of iron as the result of various intestinal disorders.
~ According to Hawksley the sequence of events in a patient"s response to iron
therapy is: (1) an initial reticulocytosis which subsides as recovery progresses;
(2) a rise in erythrocytes with a return to normal cellular size; (3) a slow but
steady increase of hemoglobin to a normal level; and (4) a slight reduction in the
red celJs after the maximum rise. When other factors are deficient in addition to
iron, the therapeutic response is incomplete.
Ochsenius agrees that iron should be administered in all cases and recom-
mends ferrum reductum as the best preparation. He emphasizes the advis-
ability of adding vegetables and fruit to a child's diet as early as the sixth month.
He states that orange juice is of value in the absorption of iron, that liver is a
necessary adjunct to iron in many cases of anemias of childhood, and that trans-
fusions are indicated in severe cases. Neither breast milk nor cow's milk contains
sufficient iron to prevent a deficiency in a young infant, but of the two, breast
milk is decidedly superior (Parsons, et al.).
" The addition of copper to iron in the treatment of milk anemia in rats has
been established by Hart and many authorities have advocated the combination
in the treatment of the iron deficiency anemias of childhood. Josephs, Hart,
Steen bock, Waddeli,.Elvehjem and Sherman are among those who have presented
clinical and experimental evidence to show that copper is essential for actual
hemoglobin synthesis and have recommended copper supplement to iron medi-
cation in nutritional anemias. Helen Mackay believes that iron alone is effica-
cious. Others believe that the iron compounds used in her experiments were con ..
taminated with sufficient copper to produce the desired effect. Parsons obtained
excellent results with the administration of ferrous sulphate containing less than
0.1 mg. per c.ent of copper, OT 1/25 of the amount of copper claimed to be a
necessary supplement to iron. He concluded that the nutritional anemia of
childhood is an iron deficiency anemia, that copper is an unnecessary supplement
to iron therapy in a majority of cases since copper is usually stored in infants in
adequate amounts, and that the instances are rare in which the anemia is caused
by a dual copper and iron deficiency.
What preparation to choose in the treatment of these anemias is a problem
which confnmts the clinician. According to Parsons and associates there is some
evidence that iron must be broken down from the ferric into the ferrous state be-
fore it can be utilized; however, when the ferrous salts are administered glucose
must be added to the solution to prevent mddation to the ferric state. Parsons
and Smallwood state that inorganic iron is more effective in the treatment of the
 THE ANEMIAS OF CHILDHOOD S43
nutritional anemia of infants than organic combinations. They recommend the
following preparations: (I) Ferrum reductum, one grain three times daily, mixed
with two or three times its weight of sugar; (2) ferrous sulphate,s grains, three
times daily, mixed with syrup to prevent oxidation; (3) "Ferrofax," a preparation
containing iron, copper, yeast and other minerals which is used when the .anemia
fails to respond to iron therapy alone.
l\Iackay recommends 9 to IS grains daily of ferric ammonium citrate. We
have found this preparation highly effi<:adous in the treatment of aU iron de-
ficie.ncy anemias in children.
Parsons has reviewed the, effect of yeast in the treatment of the nutritional
anemias of infants. That yeast will effect a cure in the milk anemia of rats is
admitted, but here again the analogy does not hold good for infants. He was
able to correct nutritional anemia in only one infant by yeast administration alone.
In two other infants a complete recovery was not accomplished until iron was
supplemented with yeast. He considers these cases rare exceptions to the rule
that iron therapy alone corrects the nutritional anemia of infants.

ANEMIA OF VITAMIN DEFICIENCY (SCURVY)

Scurvy is a deficiency disease which may alter the blood picture in child-
hood. The anemia which may accompany scurvy is due to a deficiency in both
the ante-natal supply and the post-natal storage of vitamin C. The anemia pre-
sents a hypochromic and microcytic blood picture and is inseparable hematologi-
cally from the other nutritional anemias of childhood. When the child fails to
receive adequate storage of tbe vitamin from tbe mother, the anemia appears
within the first six months of life, but it generally develops later than that time
when the causal factor is a post-natal dietary deficiency.
The anemia of scurvy is corrected by the administration of vitamin C al-
though it is not surprising that iron therapy is often necessary, since a vitamin
deficient diet almost invariably contains insufficient quantities of irun. Copper is
contra-indicated as a therapeutic adjunct since this metal destroys vitamin C.

ANEMIA OF THYROID DEFICIENCY

Cretinism in children is accompanied by a hypochromic type of anemia which

is due presumably to a deficiency of thyroid secretion, since an anemia of a
severe grade develops in animals after total removal of the thyroid gland. -

IDIOPATHIC MACROCYTIC ANEMIA OF THE NEWBORN

An idiopathic, hyperchromic, macrocytic anemia of newborn infants was re-

ported in '919 by Ecklin and by Donnally in 1924. Abt, in 1932, reported a case
and analyzed the case records in the literature. At that time 15 cases had been
reported. The appearance of an unexplained severe anemia during the 6,rst two
weeks of life in an otherwise normal infant justifies its inclusion here, despite its
infrequency.
344 THE ANEMIAS

An excellent description a!ld summary of this anemia are found in the mono-
graph of BIackfan, Baty and Diamond. They differentiate the anemia from the
hemorrhagic disease of the newborn, from the hemolytic erythroblastic anemias,
and from infectious processes. There is no history of bleeding, jaundice or any
other signs of hemolysis, and there is no evidence of congenital syphili3, tubercu-
losis, or any type of infection. Children affected with this anemia are born at
term from healthy parents~ Pallor is the first sign of an abnormality and may be
present at birth, or may develop suddenly within the first two weeks after birth.
The red cells decrease rapidly to an extremely low level. The anemia is maCro-
cytic and hyperchromic. Reticulocytes are usually diminished, and normoblasts
are seldom found in the circulating blood. There is no sign of excessive destruc-
tion of red cells by hemolysis since there is no jaundice or bilirubinemia, and the
fragility of erythrocytes is not increased.
The mortality rate is low with recovery beginning within one or two weeks.
At this time the blood shows signs of regeneration with the reticulocytes con-
siderably increased. Transfusion is recommended when the anemia is marked,
one or two transfusions generally initiating recovery. In addition, Blackfan and
his associates recommend 2 to 4 grams (30 to 60 grains) of iron ammonium
citrate daily.
The etiology of this anemia is unknown. The explanation has been offered
that for some unknown reason the hematopoietic activity of the child is diminished
at birth or shortly after birth, resulting in an anemi.a which disappears, either
spontaneously or after therapy, when normal regeneration is renewed. Pars()ns
and Hawksley question the occurrence of this anemia which is neither nutri-
tional, bemorrhagic nor hemolytic. They state that in many of the cases re-
ported hemolytic factors have not been sufficiently excluded. They point out that
Abt's description of the sudden appearance of a sl:reet·white pallor is hard to ex-
plain except by blood loss, either from hemorrhage or hemolysis.

MACROCYTIC ANEMIA OF COELIAC DISEASE

Deficiency of the gastric intrinsic factor, as manifes ted in pernicious anemia

of adults, probably does not have its exact analogy in childhood. Presumably,
this factor is present before birth or is produced in a majority of infants in
sufficient quantities to supply the need during the early periods of life. There is,
however, a growing tendency to classify many of the erythroblastic, hemolytic
anemias as deficiency states similar to pernicious anemia, hut differing from the
adult syndrome in the more labile hematopoietic reaction of the immature
erythron. 'We are in accord with this tendency, but the limited knowledge of
etiologic factors does not permit a radical departure from the older classification.
Parsons and Smallwood have offered a brief description of the macrocytic,
hyperchromic anemias of childhood occurring occasionally in association with
celiac diseases of long standing and, more rarely, with D,pltyllobolltrium lat,.m
infestation. These anemias are similar hematologically to pernicious anemia hut
differ somewhat in pathogenesis; the syndrome in childhood is probably caused by
a faulty absorption and utilization of the "extrinsic" factor in the food.
 THE ANEMIAS OF' CHILDHOOD 345

The blood picture is characterized by macrocytosis, hyperchromia, and varia-

tion in size and shape of erythrocytes, with nucleated forms in severe cases. The
most effective therapy is the control of the diarrhea and ,the administration of
marmite (autolyzed yeast) in doses of two to four grams daily.

HEMOLYTIC ANEMIAS OF CHILDHOOD (GENERAL DISCUSSION)

Parsons, Hawksley and Gittings, who have made an extensive study of the
,hemolytic anemias of childhood, consider "erythronoclastic anemia" a more cor-
rect term since hemolytic anemia is a destructive disease, not only of the periph-
eral blood, but of the entire blood forming system. They justly insist upon the
use of the term "erythron" to designate the blood system as a unit, including
both fixed hematopoietic tissue and circulating blood.
These anemias, especially during the neonatal period, are frequently hyper-
chromic and macrocytic with a megaloblastic-erythroblastic red cell picture,
polychromatophilia and marked variation in size and shape of the red cells.
These immature forms do not have the same significance as in the deficiency
anemias of adults. In the latter, megaloblasts appear in the peripheral blood
because of arrested maturation which is due to a deflciency of the "X" hema-
topoietic factor. In the "erythronoclastic" anemias these cells are sent into the
circulation because a severe stinlulu5 renders the hematopoietic system markedly
active.
Parsons and Smallwood have offered an explanation of the mechanism in-
volved in the hemolytic or "erythronoclastic" anemias. Excessive red cell de-
struction with the consequent liberation of hemoglobin, results in bilirubinemia
which gives a positive indirect van den Bergh reaction and an elevated icterus
index. When the destruction is marked, hemoglobin, urobilinogen, and urobilin
may be present in the urine and bile pigments are often found in the stools. The
erythropoietic elements of the bone marrow respond to hemolysis by an increased
activity to supply the loss of red cells, and regenerative forms (reticulocytes and
erythroblasts) are usually circulating in increased numbers. There is a concomi-
tant stimulation of myeloid leukocytes, especially in children, which results in a
leukocytosis and an increase of immature granulocytes. Osteoporotic changes
frequently accompany the bone marrow hyperplasia. In some instances, the bone
marrow is damaged and fails to react with a compensatory hyperplasia and a pic-
ture similar to aplastic anemia may develop. Prolonged hemolysis may be evi-
denced clinically by an icteric tint to the skin. The splenic enlargement is due,
presumably, to an increased phagocytic activity of the organ in an effort to re-
move damaged and non-functional cells.
Other authorities do not consider the hemolytic feature to be of major eti-
ologic importance but believe that hemolysis is the result of a compensatory effort
on the part of the reticulo-endothelium to remove abnormal cells which are pro-
duced by a deranged or immature hematopoietic system.
The description of the hemolytic anemias of the neonatal period and the !'lcute
hemolytic anemia of Lederer is summarized from the extensive and numerous re-
ports of Parsons and his collaborators. At this point we diverge from their classi-
 THE ANEMIAS

fication since they include von Jaksch's and Cooley's anemias in their list of
hemolytic anemias. Since the etiology of these anemias is unsettled we prefer to
regard them as separate entities.

HEMOLYTIC ANEMIAS OF THE NEWBORN

ERYTHROBLASTOSIS OF THE NEWBORN (IcTERUS GRAVIS NEONATORUM)

This disease is often familial and usually fatal. It is characterized by icterus
gravis, petechial hemorrhages, slight enlargement of the liver and spleen, pro-
found hyperchromic anemia, erythroblastosis and megaloblastosis of variable
degree, and eosinophilia during the phase of recovery. Occasionally, an infant
with this anemia is slightly jaundiced at birth but, in a majority of cases, jaundice
occurs a few hours after birth and gradually becomes intense at about the third
day. The child may become drowsy and cyanotic; hemorrhage and convulsions
may develop and death may occur at about the fourth or fifth day. If the child
survives the period of jaundice, recovery is spontaneous and the blood returns to
normal. This severe, mahogany-brown jaundice should be distinguished from the
icterus associated with syphilis and other infections_ Intramuscular injection of
10 cc. of parental blood' serum, given daily for several days, is a highly successful
treatment when instituted early. Frequent small transfusions are also recom-
mended for this type of anemia.

HEMOLYTIC ANEMIA WITHOUT ICTERUS GRAVIS

This rare form of anemia occurs during the first week of life and is an acute
hemolytic type, similar hematologically to erythroblastosis neonatorum but differ-
ing in the absence of an icteric skin. According to Parsons and Smallwood, the
anemias have the same pathogenesis, the only difference being the ability of the
liver to excrete the bilirubin as rapidly as it accumulates in the plasma Eryth-
roblastosis (increased number of erythroblasts in the entire blood system) is
found at birth but erythroblastemia (increased number of erythroblasts in the
circulating blood) is absent' in a majority of cases.
It now appears that erythroblastosis of the newborn, sometimes called eryth-
roblastosis foe talis, is a severe manifestation of a general group of diseases,
among which is that known as universal edema of the fetus, congenital hydrops
with erythroblastosis, icterus gravis neonatorulll, and congenital anenlia of the
newborn with erythroblastosis. In all of these the infant in the first few days of
life may show from 200 to 2000 nucleated red cells per CU.mm. There is usually
hydramnios with an enlarged and edematous placenta, and the fetus is usually
quite pale and edematous. There is generally icterus with an enlarged spleen and
liver. In this entire group frequent small transfusions should be used and hema-
topoietic agents seem to be of little value. .

HEMOLYTIC ANEMIA LATER IN THE NEONATAL PERIOD

Only a few cases of this type of anemia are on record. The infants vary in
age from a few weeks to two or three months. Mild jaundice' may be present
but never to the extent seen in icterus gravis. The spleen is usually enlarged,
 THE ANEMIAS OF CHILDHOOD 347

and there is progressive pallor due to a severe, hyperchromic anemia. Recovery

is spontaneous and eosinophilia is usually present during convalescence.
The anemias of the hemolytic types differ from the nutritional anemias of the
same period; the former are characterized by (I) a hyperchromic anemia; (2)
evidences of hemolysis, as shown by a positive indirect van den Bergh and excess
urobilinogen and urobilin in the urine; (3) spontaneous recovery in many cases;
(4) erythroblastemia and megaloblastemia; (5) frequent eosinophilia; and
(6) failure to respond to iron therapy. The absence of clinical jaundice in some
of these types does not exclude hemolysis as the causative agent. The appear-
ance of icteric skin and sclera is dependent upon the ability of the liver to excrete
the bile pigments.
According to Parsons there is evidence that excessive hemolysis stimulates
an erythroblastosis which should be regarded as a symptom and not a cause of
the disease. He states that the nature of the hemolyzing agent is unknown and
that hemolysis occurs to a mild degree continuously before and after birth, but
it is prevented from becoming excessive by some anti-hemolytic agent in normal
blood serum. The mother's supply of the anti-hemolytic factors may be inade-
quate and this deficiency may be responsible for the production of the fatal icterus
gravis. When a child is unable to produce this factor after hirth, a hemolytic
anemia may follow. These suppositions are partially substantiated by the fact
that jaundice and hemolytic anemias are more severe in premature than in full-
term babies. The successful therapy of these anemias is based on this concept.

ACUTE HEMOLYTIC ANEMIA (LEDERER TYPE)

A form of acute hemolytic anemia was described by Lederer in 1925. The

syndrome is considered a rare clinical entity occurring during late infancy, in
childhood, and in young adults. O'Donoghue and Wilts have recently reviewed
the cases reported in the literature. The outstanding physical findings are ab-
dominal pain, diarrhea, vomiting, and enlargement of the spleen, liver, and lymph
nodes. The onset is sudden and the patient soon becomes desperately ill. In
severe cases pallor, jaundice, and hemoglObinuria appear, and petechial hemor-
rhages are not uncommon. The clinical and hematologic features, as described
by Parsons and Hawksley, are: marked hyperchromic anemia characterized by
polychromatophilia, anisocytosis, poikilocytosis, basophilia, megaloblastosis, and
reticulocytosis; usually an intense bone marrow reaction with a high leukocyte
count and myeloid immaturity; a mild thrombocytopenia; a positive indirect van
den Bergh reaction in some cases; an elevated icterus index; and a norma]
fragility of red cells. In some instances the child has had sufficient time to excrete
the excess pigment. In some cases the bone marrow appears to be damaged
resulting in leUkopenia and thrombopenia, findings which make the differential
diagnosis from aplastic anemia difficult. In these cases, prognosis is unfavorable.
The majority of patients with acute hemolytic anemia recover when mul-
tiple small transfusions are given early in the dise4se. Relapses are not infre-
quent. It is believed that the donor's blood supplies "an anti-hemolytic factor
which institutes the cure." Slight relapses may be brought on by infection, but
recovery is permanent. In some instances; a nutritional anemia develops which
 THE ANEMIAS

requires iron therapy. These anemias are easily confused with the early stages
of acute leukemia and a diagnosis should not be made until leukemia is ruled
out. The etiology and the nature of the hemolytic agents are unknown. Lederer
considered infection to be a prominent factor in the production of the anemia.
Parsons and Hawksley descrihe a rare, suhacute, hemolytic anemia which
differs from the Lederer type in a more gradual onset and more chronic course.
The anemia seems to be milder and patients frequently recover without the
necessity of transfusions. It has been called subchronic hemolytic anemia.

CHRONIC HEMOLYTIC ANEMIA (FAMILIAL HEMOLYTIC

ICTERUS)

Children are subject to this form of anemia, but their reaction differs in no
striking way from that of adults. For the sake of differential diagnosis from
other childhood anemias, it should be emphasized that there is an increased
fragility of red cells in this condition and that splenectomy institutes a cure in a
large majority of cases. For the details of the clinical and hematologic find-
ings, see chapter 20.

SICKLE CELL ANEMIA

This anemia occurs exclusively and frequently in the negro race, in h'lth
children and adults. When a negro presents himself with a marked anemia,
splenomegaly, and leg ulcers, the most probable diagnosis is sickle cell anemia.
It is a chronic familial anemia of long duration, with intermittent attacks of
weakness, leg ulcers, and pains in the joints. Other clinical characteristics are
pallor, icteric tint to the sclera, fever, vomiting, abdominal pain and tenderness,
eulargement of the liver, splenic enlargement in the early stages of life, and
atrophy of this organ after the disease has progressed. Lymphoid enlargement
occurs occasionally but not consistently.
The most striking hematologic feature of the anemia is the unique quality
of the erythrocytes to exhibit a sickling phenomenon when examined in the ab-
sence of oxygen. The percentage of sickle cells varies with the severity of the
anemia. Sickle-shaped erythrocytes may not be apparent on the stained smear.
The blood picture is characterized during relapse by a marked anemia, a reticulo-
cytosis and normoblastosis, an elevated leukocyte count with a shift to imma-
turity of tbe cellular elements, and usually a thrombocytosis. The icterus index
is elevated, but the resistance of erythrocytes to hypotonic salt solutions is normal
or increased. It should he emphasized that none of these hematologic features
may be present during remissions.
Nothing is known of the etiology of the anemia and no type of therapy is
effective. The anemia is usually classified as a hemolytic type because of' the
high icterus index. Splenectomy has been reported to be of some benefit in
patients with splenomegaly by Corrigan and Schiller, but the operation is not
recommended by aU authorities. (Further details of clinical and hematologic
lindings In sickle cell anemia may be found in chapter 25.)
 T:aE ANEMIAS OF CHILDHOOD 349

VON JAKSCH'S ANEMIA

In 1889, von Jaksch described an anemia of early childhood, "psendo-Ieu-

kemica infantum/' characterized by an enlarged spleen and liver and extremely
high leukocyte counts. He failed to stress the occurrence of large numbers of
nucleated erythrocytes, a finding which has been emphasized in subsequent re-
ports of the disease. In later years it became evident that various investigators
interpreted von Jaksch's syndrome to include a number of miscellaneous condi-
tions.
There seems to be little unanimity of opinion concerning the nomenclature,
etiology, and pathogenesis of the anemia; and there is considerable dispute con-
cerning the question of whether or not the syndrome represents a disease entity.
Numerous authorities are in favor of discarding the term entirely since a wide
vari~ty of childhood anemias have been incorrectly designated as von Jaksch's
anemia for lack of proper identification.
In 1927, Cooley summarized the various theories regarding the identity of
this disease and according to his summary, there are three different concepts held
by those authorities who believe that von Jaksch's anemia is secondary to some
other disease and does not represent a clinical entity: first, the anemia is con-
sidered one of the ordinary secondary anemias of childhood which are char-
acterized by atypical and varied manifestations; secondly, the anemia is thought
to be the result of the metabolic disturbance causing rickets; and thirdly, the
anemia is a "biological variant" of numerous childhood diseases such as rickets
and syphilis, and represents an "extreme reaction" which is conditioned by a con-
genital ahnormality of the hematopoietic mechanism (Naegeli).
In contrast to these ideas, numerous authorities believe in the existence of a
distinct clinical entity similar to, but not identical with, the original description
by von Jaksch (Eppinger, Cooley). The adherents of this idea emphasize the
splenomegaly, the marked leukocytosis with myeloid immaturity, the normo-
blastosis and megaloblastosis, and the chronicity of the disease. Von Jaksch
stressed the tendency to recovery, while some of the later authorities consider
the disease grave and usually fatal. For the most part, the sponsors of the dis-
ease-entity concept believe that the anemia is hemolytic in origin and admi t that
numerous cases reported as von Jaksch's anemia have been diagnosed erroneously.
A large number of "cured cases" fall in the group of "false diagnoses." Accord-
ing to Cooley's critical review of the case reports, those cases with prompt re-
covery following splenectomy suggest a confusion with familial hemolytic jaun-
dice, and others with Banti's "splenic anemia" group. A considerable number of
recovered patients obviously were suffering with rickets, syphilis, and other infec-
tions. In other cases reported cured, the patients were not followed sufficiently
to eliminate positively the possibility of recurrence.
In regard to the etiologic relationship of rickets to von Jaksch's anemia,
Cooley states that a majority of patients with rickets have only a mild degree
of anemia; definite cases of von Jaksch's syndrome have been reported without
rickets; and recovery from the two conditions, when they occur together, is not
350 THE ANEMIAS

simultaneous. Rickets, therefore, has been excluded as an etiologic agent in von

Jaksch's anemia.
Syphilis seems to playa more important role, since in a number of cases
syphilis has been demonstrated in the parents and, more infrequently, in the
child. Von Jaksch's syndrome, however, is not cured with antisyphilitic treat-
ment and the anemia occurs in the absence of syphilis. It appears, therefore,
that syphilis is not the etiologic agent, but may be an aggravating factor which
predisposes to the development of this peculiar anemia.
The theory that von Jaksch's anemia is merely a marked reaction of an indi-
vidual with a congenitally inadequate hematopoietic apparatus to a numher of
stimuli has not been proved.
Cooley has suggested the inclusion of additional crileria, such as derangement
of pigment metabolism and the reaction to splenectomy, in order to define this
disease more c1early.* He analyzed a number of cases that exhibited a response to
splenectomy by an "erythroblastic crisis" in the peripheral blood which tended to
persist following the operation and appeared to be more pronounced in younger
patients. He noticed that every case exhibiting the peculiar erythroblastosis fol-
lowing splenectomy belonged to one of the Mediterranean races, Syrian, Greek or
Italian. In later reports, he has separated the anemia occurring in Mediterranean
stock from von Jaksch's syndrome and has established the former condition as a
clinical entity which he has called "erythroblastic anemia of childhood." In addi-
tion, he has expressed the opimon that this is probably the only true disease entity
il). that miscellaneous group of anemias classified as "von Jaksch's." Many au-
thorities have accepted the separation of the erythroblastic anemia of Cooley
from von Jaksch's syndrome and, for this reason, the disease is discussed in the
following pages under a separate title.
There is a tendency on the part of American writers to discard entirely the
term "von Jaksch's anemia." Blackfan, Baty, and Diamond omit the disease
from their discussion, giving as a justification the opinion that a number of
known morbid processes may produce the clinical and hematologic features typical
of "von Jaksch's" syndrome.
British writers admit that their idea of the condition differs from the
original description of von J aksch and conforms more closely to the features
described by Luzet, a pupil of Hayem. The following description of von Jaksch's
syndrome is given by Parsons and Hawksley as representing the concept of British
pediatricians. They believe that the syndrome differs from Cooley's anemia in
its rarity, its tendency to recovery in many instances, and its appearance in
Anglo-Saxon people.

CLINICAL FEATURES
The condition has decreased in frequency during the last tW,enty years. It
is a sub-chronic disease of late infancy and childhood. The affected child shows
a marked waxy pallor, frequently an edema of the feet and ankles, and petechial
hemorrhages in a number of instances. The most striking clinical feature is the
extensive enlargement of the spleen. The liver may be moderately enlarged, but
the lymph glands usually are not palpable. The anemia is not uncommonly asso-
• In the early study of the disease, splenectomy was tried but was found to be of no value.
 THE ANEMIAS OF CHILDHOOD 351
ciated with ricket; and syphilis, but both conditions have been ruled out as
pos5ible etiologic agents since the' anemia 5hows no re5pon5e to antirachitic or
antiluetic therapy. The marked bone changes, an outstanding feature of Cooley's
erythrobla5tic anemia, do not figure prominently in tbe British description of
yon J aksch's anemia.
HEMATOLOGIC FINDINGS
The blood picture is characterized by a profound anemia, frequently of the
hyperchromic macrocytic type; an absence of increa5ed fragility; an extreme
degree of erythroblastemia; numerous megaloblasts; marked anisocytosis; poi-
kilocytosis; polychromatophilia; reticulocytosis; and basophilic stippling. A con-
spicuous feature is a leukocytosis with numerous myelocytes and other immature
myeloid lorms, although the leUKocyte count may be normal during some stage 01
the disease. The clotting and bleeding times are normal. The platelets are usu-
ally normal in number but may vary from a high to a moderately low level. In
a majority of patients, there is an increased amount of urobilinogen in the urine
and bilirubin in the blood plasma.
TREATMENT
Treatment is entirely symptomatic. Proper therapy should be directed to-
ward the alleviation of dietary deficiencies, syphilis or rickets, when these condi-
tions are present. Transfusions are given when the anemia is se'(ere. Splenectomy
has been recommended but appears to be more drastic than the benefits warrant.
PROGNOSIS ,
Parsons and Hawksley state that the prognosis is good and that recovery is
spontaneous, unless the patient dies from intercurrent illness. The disease is
chronic and may result in bone marrow failure. Numerous authorities disagree
with British writers concerning the good prognosis, since the chronicity of the
disease makes it difficult to study tbe patient over a sufficient period of time.
DISCUSSION
Parsons and Smallwood offer considerable evidence that von Jaksch's syn-
drome is a sub-chronic hemolytic anemia. Since von Jaksch (ailed to descrihe
the syndrome fully, these authors recommend dropping his name from the disease.
We are in accord with British authorities in believing that a syndrome similar
to von Jaksch's original description occurs in children other than those of Medi-
terranean stock. We have seen the syndrome of anemia, leukocytosis, erythro-
blastemia and splenomegaly in the negro, unassociated with rickets, syphilis, in-
fections, poisons, or leukemia. The most striking feature in negroes is the
marked leukocytosis, often reaching 75,000 to IOO,oOO cells per cu. mm., with
numerous eosinophilic, neutrophilic, and basophilic myelocytes. The anemia is
macrocytic and hyperchromic. The patients improve with tram;(usiom and we
believe that a majority recover.
No conclusive proof has been offered that the anemia is hemolytic in origin.
There is excessive embryonic blood formation in the bone marrow and numerous
extramedullary sites of hematopoiesis. The liver and spleen often contain
 PLATE XJGX

VON ]AKSCH'S ANEl\II~

(PSEUDOLEUKEMIA OF CHILDHOOD)

I. Neutrophilic myelocyte. 7 Normoblasts..

2. BasophlllC myelocyte. 8. Macroblast in division.
3. Juvenile neutrophll. 9. Erythrocyte WIth Howell-Jolly body.
4. Band neutrophil. IO Erythrocyte with basophilic stippling.
5. Segmented neutrophils. II. Poikilocytes.
6. Lymphocyte.
Blood Fmdings. Differential:

Hemoglobin 9.4 gms. (Newcomer's method) Premyelocytes , r 0/0

R.Be . . 2,800,000 per c mm. NeutrophIlic myelocyles 6%
W.B.C 15,500 per c.mm. E051nophtlic myelocytes 3%
Platelets 580,000 per c mm. BasophilIc myelocytes 30/0
Juveniles 20%
Color Index Bands . 25%
Volume Index Segmenters . 26%
Icterus Index Total neutrophils 840/0
Lymphocytes 6%
Eoslllophtls 30/0
~a~~~~::es . ~~,
Erythrocytes j normochromic and sbghtly macrocytic with marked anisocytosis, polychro-
matophiba, pOikilocytosis and normoblastemia.
Plate XXIX.
 THE ANEMIAS OF CHILDHOOD 353
numerous erytbropoietic foel. Tbis indicates a defect of blood formation
or an arrested development of the blood forming organs. The evidence of
hemolysis, such as an elevated icterus index and a positive indirect van den Bergh
reaction, may be explained as the result of a compensatory phagocytosis by the
spleen in an effort to destroy and remove immature, atypical ceJls from the cir-
culating blood.
The etiology of this defect is unknown. Cooley emphasizes the similarity
to pernicious anemia in adults. Whipple and Bradford believe that the erythro-
blastic anemias of childhood are the result of an inherited defect whicb can be
explained by a lack of some unknown factor. Tbey stress tbe similarity of the
bone marrow picture to that of pernicious anemia. An attractive theory is that
von Jaksch's syndrome represents a deficiency of some factor necessary for ade-
quate blood formation. Possibly, the liver retains its embryonic activity to such
an ,extent !bat it cannot assume the adult function of storing the hematopoietic
factor. A just criticism of !bis theory is !be presence in !be blood of reticulocytes
and intermediate forms between !be megaloblast and !be mature erythrocyte.
These forms are absent in those conditions characterized by a deficiency of the
anti-anemic factor. In addition, the anemia is not cured by the administration of
Hvcr extract.
Despite the objections to !bis theory, the etiology of the anemias in which
!be liver reverts to its embryonic blood forming activity may become clearer
when more is known concerning !be 'nature, storage, and utilization of the anti-
anemic factor.
COOLEY'S ERYTHROBLASTIC ANEMIA

The erythroblastic anemia of Cooley has been separated from von Jaksch's
syndrome and is given !be specification of a distinct clinical entity. Diamond
points out !bat this anemia occurs only in children of Greek, Italian, and Sicilian
parentage. More tban lOa cases have been reported. Tbe average age of onset
is 16 months, and !be younger the child the more severe is !be disease. There is
no known instance of direct hereditary transmission. The familial tendency
however, is very striking.
Whipple and Bradford have suggested !be name "thalassemia," from the
Greek word meaning great sea, or "Mediterranean anemia." They consider
Cooley's "erythroblastic anemia" an inadequate term since erythroblastosis is
Iiot limited to !bis condition, but is a characteristic feature of many diseases.
Cooley bas described the unusual bone changes which later writers bave
emphasized as an almost constant feature of the disease.
CLINICAL FINDINGS
The anemia originates in late infancy, generally in the latter part of the
first year. This anemia is characterized by pallor, gradually enlarging abdomen,
splenomegaly, hepatomegaly, excessive fatigue, anorexia, vomiting, and oftentimes
fever. The mentality is usually normal. There is generally a mongoloid facies
with high malar eminences, a short nose, prominent upper teeth, a peculiar yellow
skin, and enlarged head wi!b prominent frontal bosses. The disease runs a chronic
course and progresses slowly to a fatal termination. In 1932, Koch and Shapiro
354 THE ANEMIAS

reviewed the case records in the literature. Of 23 cases reported at that time, 7
were known to terminate fatally, and none of the remaining patients was reported
as having recovered when last observed. In this group, the shortest course was
IS months and the longest, 10 years. Toward the end of the illness many of the
patients developed anorexia, fever, and edema.
HEMATOLOGIC FINDINGS
The blood picture is characterized by a moderate to severe anemia usually
with a color index below unity. Nucleated erythrocytes and reticulocytes appear
in excessive numbers and there is a considerable degree of anisocytosis, poikilo-
cytosis, and polychromasia. The leukocytes are usually elevated in number, al-
though some cases have been reported with normal leukocyte counts. Immature
myeloid cells may be present or absent. The platelets are I!sually normaL The
fragility of erythrocytes is not increased. The icterus index is usually elevated
and the blood serum gives a positive indirect van den Bergh reaction. As a rule,
urobilinogen is found in the stools and urine.
BONE CHANGES
The bone changes have been described in detail by Koch and Shapiro. These
changes may not occur in the early stages but are almost invariably present in
the advanced stages of the disease. There is an extensive thickening and widen-
ing of the cranial and malar bones. Upon roentgenographic examination, the long
bones have a porous appearance with sharp trabeculations and thinning of the
cortex. The flat bones of the skull show medullary thickening, radial striations,
and the tahles are usually thin. Similar changes are found in the sternum. Cooley
believes that the bone changes are the result of extensive bone marrow hyperplasia.
Whipple and Bradford disagree witb this concept and suggest that the changes are
radalor are due t<> a metabolic disturbance th",t may be associated with gl",ndu!ar
dysfunction of various types. Nittis of the University of Michigan states that
the pathological changes 'and the appearance of the bones in erythroblastic anemia
are not peculiar to this syndrome but may occur in any type of prolonged marrow
hyperplasia. He points out that erythroblastic anemia starts in early infancy
and is a continuous process but in other forms the process may start later and
be characterized by many remissions and relapses. In continuously stimulated
hematopoiesis, the marrow in the bones of the skull, can expand only outward,
therefore the trabeculae connecting the two plates are elongated, which produces
a bristle·like roentgenologic appearance. If on the other hand the process becomes
arrested, calcification takes place with formation of transverse trabeculae. There-
fore the thickened diploe and the honeycombed or "pepper and salt" appearance
sometimes seen on the x-ray plate may indicate a previously abnormally stim-
ulated hematopoiesis in infancy from which the individual may have recovered.
PATHOLOGY
Whipple and Bradford have studied the pathology of the bone marrow,
spleen, and liver in histologic sections. In their cases, the bone marrow was deep
red and frequently chocolate-colored. There was an extreme hyperplasia of all
cellular elements. The erythroid marrow was similar to that of pernicious anemia
and suggested a lack of some material essential for the normal maturation of red
 THE ANEMIAS OF CHILDHOOD 355
blood cells. There were present nUmerous large phagocytes with foamy cyto-
plasm similar to those described in Gaucher's disease.
They found pigment deposits, similar to hemochromatosis, in the spleen,
liver, pancreas, stomach and mucous glands, thyroid, parathyroid, and pituitary
glands. They attribute considerable importance to this type of pigmentation since
it suggests a disturbance of pigment metabolism.
In the late stages of the disease, the spleen was fibrotic with small malpighian
>odies, and there was an increase of connective tissue throughout tbe pulp. The
,;nusoids were dilated and thickened and the endothelial cells contained large
,mounts of pigment deposits giving a stain for iron.
rHERAPY
No type of therapy has been found successful. Transfusions are only pallia-
tive. Koch and Shapiro have shown that hematopoietic stimuli do not alter the
course of the disease. Liver extract and iron are ineffective. Splenectomy has
been advocated but the only effect of the operation seems to be a rapid and
persistent increase of nucleated red cells in the peripheral blood. Cooley con-
siders the reaction to splenectomy to be a characteristic phenomenon. He ex-
plains the reaction on the assumption that before operation the immature nucle-
ated red cells arc destroyed by the spleen, and after operation they remain in the
peripheral blood until the reticulo-endothelium in other parts of the body assumes
the destructive activity.
ETIOLOGY
Parsons and Hawksley place this anemia in the hemolytic group. Cooley,
in early reports, classified the anemia as a hemolytic type, but be considered the
hemolytic process of less importance than other features and suggested the sig-
nificant similarity to pernicious anemia of adults. In his later reports he no
longer believes the anemia to be hemolytic in origin but rather a deficiency
similar to that of pernicious allemia.
yaughan has studied the occurrence in other clinical conditions and the
etiologic factors concerned in the leuco-erythroblastic anemias (anemias charac-
terized by immature erythrocytes and leucocytes). From her studies the incidence
is shown in the following table:
TABLE XXI
INCIDENCE OF LEUCO-ERYTHROBLASTIC ANEMIA
Leuco-"ythr(}blastic
Disease No. of cases Bone Lesions anemia
Carcinoma. ... ........ 55 24 8
Myelomatosis. .......... _ 21 U
Myelosclerosis ....... .. 3 3
Cooley's Anemia. . . . . . . . . I :r :r
Hodgkin's disease.... . . . . 37 IS 0

She concludes that neither hemolysis of erythrocytes, altered calcium metab-

olism, mechanical blocking, nor stimulation play any part in the production of
leuco-erythroblastic anemia. She believes that erythroblastosis is the result of
abnormal blood production, has features akin to those found in pernicious anemia,
and is caused by a deficiency of a factor or factors necessary for normal hemato-
poiesis.
 THE ANEMIAS

SPLENIC ANEMIA (BANTI'S DISEASE)

There has been a great deal of controversy over the syndrome characterized
by anemia, splenomegaly, neutropenia, and liver atrophy which has been called
Banti's disease. The disease may occur in childhood but is not limited to any
age level. In view of the claims that splenectomy is an effective remedy, the
pediatrician would be certain that all other causes for anemia are non-existent
before a diagnosis of this condition is made. It is said that if pyogenic infec-
tions, syphilis, malaria, leukemia, thrombopenic purpura, hemolytic icterus, von
Jaksch's anemia, Cooley's anemia and deficiency anemias are definitely ruled out,
and the patient exhibits the above clinical and hematologic syndrome, Banti's
disease may be considered as a probable diagnosis.
The anemia is macrocytic and hyperchromic unless an iron deficiency results
in a hypochromia. The red cells are large or normal in size. Volume and color
indices are increased.
The neutropenia may be marked, sometimes as low as rooo to 2000 cells per
cu. mm. The platelets mayor may not be decreased.
The diagnosis is based on the enlarged spleen, small atrophic liver, neutro-
penia, macrocytic anemia, and hemorrhages in some patients. Treatment is only
palliative. The value of splenectomy has not been proved.
ANEMIAS ASSOCIATED WITH INFECTION
Deficiency and hemolytic factors are not the causative agents in a majority
of the anemias of childhood: Infection probably plays a greater part in pro-
ducing anemias than any other single factor and may institute a variety of
changes in the blood picture, which include a diminution of erythrocytes, bone
marrow depression, destruction of blood platelets, and hemolysis. The resulting
picture may resemble aplastic anemia, granulocytopenia, thrombocytopenic pur-
pura, or hemolytic anemia (Blackfan, et al.).
The anemia of infection is usually hypochronllc and microcytic, but in severe
cases the toxic action of bacteria may institute a severe grade of bone marrow
depression with a blood picture of granulopenia, thrombopenia, and erythropenia
and result in a normocytic, normochromic red cell picture. On the other hand,
an infectious process may act as a myeloid stimulant and an erythroid depressant.
In this type of reaction, there is an anemia of varying severity and marked leuko-
cytosis which may appear almost leukemic in degree. \Ve have observed un-
usual leukocytoses in patients with Whooping cough complicated with pneumonia
in which the leukocytes sometimes rose to roo,ooo per cu. mm. An anemia ac-
companying these conditions is usually microcytic and hypochromic a~d normo-
blasts are not infrequently found.
Kugelmass and Lampe have analyzed the mechanism of anemia 'associated
with infection in infancy and childhood which appears quickly in acute infections
and gradually in the chronic types. The anemia is produced by three mechanisms:
(I) a diminished production of red cells through the depressive action of infection
on the bone marrow; (2) a destruction of mature erythrocytes; and (3) the pro-
duction of hemorrbage. The anemia may be the result of one or a combination
 THE ANEMIAS OF CHILDHOOD SS7
of all three mechanisms which are represented by these authors in the following
diagram:
+---- Hemolysis
Hematogenic - - - - )..
Inactivity t
ANEMIA

Hemorrhage
These authors have presented an extensive study of the types of anernia
occurring in childhood as a result of various infections. A summary of their
findings is given below.
Pneumonia produces a mild grade of anemia with the hemoglobin diminished
to a greater extent than the erythrocytes. The anemia is more pronounced in
children than in adults and infants are more anemic tban children. The anernia
appears at about the time of convalescence. Normoblasts and reticulocytes are
slightly increased. A majority of cases show little evidence of increased blood
destruction, but some cases with positive blood cultures have high plasma pig-
ments.
Otitis media is accompanied by a mild degree of hypochromic anemia as a
result of bone marrow depression with no evidence of blood destruction.
COItgenital syphilis produces a hypochromic anemia, the severity of anernia
being dependent upon the degree of syphilitic manifestations. Severe anernia
is associated with visceral and bone syphilis and is slow to resporid to anti-luetic
therapy. The anemia is due to an inhibition of the regeneration of hemoglobin
and red cells. The contention that congenital syphilis presents a specific type of
anemia is not substllntiated.
Tuberculosis does not produce a severe grade of anemia unless there is
tuberculous involvement of the bone and peritoneum. A mild degree may occur
as a result of toxle injury to the hematopoietic system. Normal hemoglobin val-
ues aTe frequently found in patients with active miliary tuberculosis. Monocytes
are increased in active tuberculosis in children and lymphocytes are increased
during the healing stage.
A mild hypochromic, microcytic anemia may be associated with chronic
nephritis of long standing. There is no evidence of excessive blood destruction.
The mechanism involved is depression of erythropoietic function. Hydremia does
not influence the degree of anemia.
A septic process predisposes to the rapid development of a hypochromic
anemia. Hemolytic streptococcic septicemia causes toxic hemolysis of erythro-
cytes. The streptococcus viridans in subacute bacterial endocarditis depresses
bone marrow function and produces liver damage of sufficient extent to cause
increased plasma bilirubin of hepatic origin.
A majority of the anemias associated with infection are the result of damage
to the bone marrow, rather than injury to the circulating cells, and are, therefore,
myelopathic. The demand for blood cells exceeds the supply, and anemia
develops.
TREATMENT
The TIrst consideration should oe the removal of the cause of infection.
Therapy should be directed toward the correction of the anemia when it becomes
 THE ANEMIAS

seyere. Transfusions are recommended as an immediate procedure in grave ane-

mias, iron when the hemoglobin is markedly reduced, and liver in combination
with iron when there is evidence of bone marrow insufficiency.
ANEMIAS ASSOCIATED WITH HEMORRHAGIC DISEASES
Anemia is invariably associated with thrombopenic purpura of any type and
with any disturbance of the clotting mechanism which causes blood loss. Authen-
tic idiopathic purpuric diseases of childhood are limited in general to a single
condition, morbus hemorrhagica neonatorum. This disease is called "purpura of
the newborn" when the skin manifestation is accentuated; "melena)) when the
bleeding is chiefly intestinal; and "omphalorrhagia" when tbe bleeding is from
the umbilical cord.
It should be emphasized tl1at a l1emorrhagic dia.tl1esis, bleeding, pete<.niae,
or ecchymoses in older children may be the first clinical manifestation of
leukemia. A blood examination is indicated in a child with any type of bleeding.
ANEMIAS ASSOCIATED WITH LEUKEMIA
The anemia invariably associated with leukemia in a child is of a serious
nature and may lead to death before the leukemic process is far-advanced or
even recognizable with any degree of certainty. The anemia is usually myM
elophthisic in type due to the encroachment upon the erythroid elements by leu-
kemoid hyperplasia (see section on leukemia).
A large number of cases of acute leukemia in both children and adults are -
accompanied by an unusual type of anemia. From an examination of the stained
smear the erythrocyte picture presents a striking similarity to pernicious anemia,
particularly in those cases of aleukemic leukemia in which there is a progressive
anemia, leukopenia, and thrombopenia.
The anemia is of the hyperchromic, macrocytic, and megaloblastic type with
anisocytosis and poikilocytosis. There may be a marked diminution of platelets
with the reSUlting hemorrhagic diathesis. 'Vhen the leukocyte count is normal
or below, the anemia then presents diagnostic difficulties and is often inseparable
from other hyperchromic, megaloblastic anemias. The differential count alone
may be the only clue to the existing pathology.
The presence of large numbers of leukoblasts in a child with ane!llia, thrombo-
penia, enlargement of spleen or lymph glands should always lead one to suspect
leukemia. Naegeli has described a state of the leukocytes, "hiatus leukaemicus,"
which is the appearance in the peripheral blood of leukoblasts and mature leuko-
cytes with no intermediate stages between the two. The persistent existence of
this condition is regarded by him as practically pathognomonic of leukemia. A
recognition of these immature cell types will often reveal the marked anemia
to be an accompaniment of the leukemic state.
ANEMIA AND SPLENOMEGALY IN CHILDREN
Hypertrophy of the spleen does not have the same diagnostic import in chil-
dren aS'in adults since splenomegaly is associated frequently with numerous unre-
lated childhood diseases. Ellis has given a list of the conditions of childhood
 THE ANEMIAS OF CHILDHOOD 359
which are characterized by anemia and splenomegaly. The following chart is
prepared from his discussion:
D~sc(JSe Splenomegaly Type of Anemia
Icterus Gravis Neonatorum ................ _. Marked Hemolytic
Acholuric Family Jaundice .............•.•. Marked Hemolytic
Leukemia. ..•. . ..... . .........•. Slight or marked Myelophthisic
Lymphadenoma and Lymphosarcoma .......•. Moderate Aplastic
Albers~Schonberg's Disease . . . .. . ...•. Marked Myelophthisic
Nutntional Anemia .................... . Mild, occasionally Iron Deficiency
Von Jaksch's Anemia .•........ Marked Hyperchromic
C(901ey's Anemia. . •. . . • • . •. . •••••.. , .•• Marked Hypochromic
Banti's Splenic Anemia. ............•. . Marked HypochrOmic
Generalized Infections... . ....•.•••....... Moderate Hypochromic
Syphilis, Tuberculosis, Malaria. . . . . .. . ... Moderate HypOchromic

BIBLIOGRAPHY
ANEMIAS OF CHILDHOOD

ABT, A. P.: "Anemia of the new-born." Amer. Jour. Dis. Child., 43, 337, 1932.
MT, A. F, and NAGEL, B. R.: "Prophylaxis of premature infants; Use of various agents in at-
tempt to prevent development of the so-called physiologic anemia of premature infantsi
PrelIminary study." Jour. Amer. Med Assoc., 98, 2270, 1932.
BLACKFAN, K. D, BATY, J. M, and DIAMOND, L: K.: "The anemias of childhood"; Oxford
Monographs on Diagnosis. and Treatment, 9, Supplemented to 193L ~
BUNGE, G.: "Text book on physiol, and pa.th.," 1890; and ABDERHALDEN, E.: Ztschr. ,. Physiol.
Chem, 26, 4Q3, 1339. (Cited by Parrons.)
COOLEY, T. B.: "Von Jaksch's anemia." Amer. Jour. Dis. Child, 33, 786, 1927.
Coo:r..EV, T. B, and LEE, P: "A series of cases of anemia with splenomegaly and peculiar
bone changes." Tr Amer. Pediat. Soc, 37, 29, :1925.
CORRIGAN, J C, and SCHILUR, I. W.: "Sickle cell anemia; Report of eight cases, one with
necropsy." New England Jour. Med J 210, 410, 1934.
DAVIDSON, L. S P., and LErrcn, I.: "The nutritional anemias of man and animals" Nutr. Ali-str.
and Rev ~ 3. 1}01, 11}34
DIAMOND, L. K.: "The erythroblastic anemias, or anemias associated with erythroblastemia. A
symposium on the blood." Univ. of Wisconsin Press. 11}39, p. 57.
DONNALLV, II. II: "Anemia in the new-born." Amer. Jour. Dis Child, 27, 369, 1924.
ECKLIN, T.: "Ein Fall von Anamie bei einem Neugeborenen." Monatschr. f. ·Kinderh .• 15. 425,
1919.
ELLIS, R. W. B. "Anaemia associated with splenomegaly in chlldhood." PractitionerJ 134, 317,
:1:935.
ELVEHJEM, C. A, and SHERtrAN, W. C: "The action of copper in iron metabolis.m." J 01/.1'.
BioI. Chern J 98, 309, 1932.
FABER, H. K., MERMOD, C., GLEASON, A L., and WATKINS, R. P.: "MicrocytiC, hypochromic
(iron deficiency) anem~a in infancy and childhood: its relation 'to gastric anaCidity and to
simple achlorhydric anemia of adults." J. Pediat J 7, 435, 1935.
HART, E. B, STEEN~OCK,'H, WADl)ELL, J., and ELVEHJEM, C. A.' "Iron in nutrition. VII. Cop-
per as a supplement to iron for hemoglobm building in the rat" Jour. BioI Chern., 77.
797, 1928; and WADDELL, J, STEENBOCJ{, H, and HART, E. B, "Iron in nutrition, VIII. The'
ineffectiveness of high doses of iron in curing anemia in the rat." Ibid., 83, 243, I1}29, and
lbtd., "X. The specificity of copper as a supplement to iron in the cure of nutritional
aneInia," 84, lIS, 1929.
HAWKSLEY, J. C, LIGHTWOOD, R., and BAILEY, U. M.: "Iron deficiency anaemia in chil-
dren; Its association with gastro-intestinal disease, achlorhydria and haemorrhage." Arch.
Dis. ChIld, 9, 359, 1934
JAKSCH, R., VON:' "Ueber Leukamie und Leukocytose im Kindesalter." Wien kUn. Wchnschr., 2,
435 •• 899
JOSEPHS J H.: "Mechanism of anemia in infancy." Bull. Johns Hopkins Hosp., 51, 185, 1932.
360 THE ANEMIAS

JOSEPHS, H., W.: "Mechanism of anemia in infancy; Physiological anemia." Bull. Johns Hopkins
Hasp J 55, 335, 1934.
KOCH, L. A., and SHAPIRO, B.: f'Erythroblastic anemia, review of cases reported showing
roentgenographic changes in bone, and five a.dditional cases." Atne,. lour. Duo eIuU., 4,(,
318, 193':1.
KRACKE, R. R t and GARVERJ H. E.: "Hypocytic leukemia (aleukemic leukemia)." 1nternat. Clin.,
4, 37, 1935.
KUG1~.U£ASS> I. No, and LAMPI!!, M.: "The mechanism. of anemia with infection in infancy and ill
childhood." Amer. Jour. Dis. Child., 43, 291, 1932.
LEDERER, Y.: "A form of acute hemolytic anemia probably of infectious origin," Amer. Jour,
!Jfed. Sci. 170, 500, 1925.
LUZET, C.: "Etude sur Ies Anemies de la Premiere Enfance et sur L'anemie Infantile Pseudo-
leucemique." Paris, 1891. (Cited by Parsons and Hawksley.)
MACKAY, H. M. M.: "Normal hemoglobin level during the first year of life. Revised Figu!ea.t'
Arch. Dis. Child., 8, 2:lI, 1933.
MACKAY, H. M. M.: "Copper in the treatment of nutritional anaemia in infancy." Arch. Di,.
Child., 8, 145, 1933.
MACKAY, H. M. M., and GOODFELLOW, L.: Med. Res. Council, London, 1931. Sp. Rep., Series
No. 157.
MERRITT, K. K., and DAVIDSON, L. T.: "The blood during the first year of hfej The anemia of
prematurity." Am. lour. Dis. Child., 47, 261, 1934.
NUTIs, S.: "The mechanism of the production of bone changes in the plastic anemias/' Univ.
Hosp. Bull. Uni". oj Mieh, 5, 27, 1939.
OCBSl:NlUS, K.: "Uber Prophylaxe und Tberapie der kindlichen Anamien in der Pra.XJ.s.!1 Deutsch.
med. Wchnschr., 58~ 1015, 1932; Abstr. Brzt. Med. Jour., 2. Epitome, 48, 1932.
O'VONOGHUE, R. J. L., and WITTS, L. J.: "The acute haemolytic anaemia of Lederer." Guy's
Hosp. Rep., 82, 440, ::1932.
ORDWAY, R, and GORHAM, L. W.: "Diseases of the blood." Oxford ,Monographs on DiagnosiJ
and Treatment, 9~ 516, 1930. (Supplemented in 1931.)
PARSONS, L. G.: "Studies in the anaemias of infancy and early childhood. t. Introduction."
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Arch. Dis. Child., 8, 85, 1933; PARSONS, L. G. t and HICKMANS, EVELYN M' Ibid., un.
The effect of yeast on nutritional anaemia in ra.ts." [bid., 8~ 95. 1933; PARSONS, L. G. and
HAWKSLEY, J. C.: Ibld., "III. The anhaematopoietic anaemias (deficiency diseases of the
erythron): Nutritional anaemia. and the a.naemias of prematurity, scurvy. and coeliac dis.-
ease." Ibid, 8, II'l, 1933.
PARSONS, L. G., HAWKSLEY, J. C., and GITTINGS, R: "Studies in the anaemias of infancy and early
childhood. IV. The hemolytic (eryilironoclastic) anaemias of the neonatal period; With
special reference to erythr(lblastosis of the newborn." Arch. Dis. Child. 8. 159. 1933.
PARSORS, L. G., and SMALLWOOD, W. C.: liThe anaemias of infancy and childhood." Practi-
tione1', 134, 298, 1935.
ROYSTER, L. T.: uHemorrhagic disease of the new-born." Virginia Med. Monthly, 51. 693,
1925.
TuO)'{pSON, W. P.· "The pathogenesis of Banti's disease." Ann. Int. Med, 14, 255, :1940.
VON HUM, E.: "The pathology of rnllk anemia." South. Med. Jour., 28, 2:3, 1935.
VON lIAAM, E., and B:&AlID, H. H.: 'iPatboIogical changes in liver and spleen in nutritional
anemia in rat." Proc. Soc, EXp. BioI. and Med.) 31, 639, 1933-1934.
VAUGHAN, JANET !'L: tiLeuco-erythroblastic anemia!' Jour. Path. and Bacteriol., 42, 541, 1931}..
VON JAKSCH, R.: "Ueber Leukamie und Leuk(lcytose in Klndesalter." Wten. klin. Wchns~
2~ 435, :1899.
WIIIPPLE, G. H.: "II. HemorrhagiC diseases; Antithrombin and prothrombin factors." Arch.
lnt. Med.} 12, 63'7, '1913.
WHIPPLE, G. H, and BRADFORD, W. L.: "Racial or familial anemia of children associated with
fundamental disturbances of bone and pigment metabolism (Cooley, von Jaksch's}." Amer.
lour. Dis. Child., 44., 336, 1932.
 SECTION FIVE
THE LEUKEMIC STATE
CHRONIC MYELOGENOUS LEUKEMIA
CHRONIC LYMPHATIC LEUKEMIA
THE ACUTE LEUKEMIAS
MONOCYTIC LEUKEMIA
ATYPICAL TYPES OF LEUKEMIA
THE ROENTGENOLOGIC TREATMENT
OF LEUKEMIC STATES
 SECTION FIVE
THE LEUKEMIAS

CHAPTER 27
THE LEUKEMIC STATE
Leukemia is a progressively fatal disease, characterized by a widespread
hyperplasia of the hematopoietic tissues, resulting in the production of excessive
numbers of immature white blood cells, which either circulate in the blood stream
or ,become deposited in the fixed tissues, or both. There are three forms of
leukemia, depending upon the hematopoietic tissue that is involved, these includ-
ing the myelogenous, the lymphatic and the monocytic types.

HISTORY
This disease was first described in 1845 by Bennett, who recognized it as a
new clinical entity cbaracterized by "suppuration" in the blood, accompanied by
enlargement of the spleen. He called the condition leukocythemia, which is a
more correct term than that employed by Virchow in the same year, who called
it leukemia. Virchow also recognized that one type was lymphatic and another
myelogenous.
In 1900 Naegeli described myeloblastic leukemia and was able to show that
most of the acnte forms were characterized by the presence in the blood of vari-
ous forms of myeloblasts. In 1913 Reschad and Schilling called attention to the
third major group, the monocytic form. Only in recent years has the existence
of plasma cell leukemia been noted.
Although much progress has been made in the recognition and classification of
leukemia, there has been little improvement in the methods of treatment, since
the life span of the patient with leukemia today is little more than it was one
hundred years ago.

DISTRIBUTION AND INCIDENCE

Leukemia has wide distribution throughout the entire animal kingdom. It
has been reported in avians and mammals, including chickens, mice, pigs, horses,
cattle, birds and dogs. When it occurs spontaneously in these animals there is
little to distinguish it from the disease that is seen in man, and apparently it is
the same disease.
Fortunately it is relatively rare in the human. According to Ordway and
3 63
 THE LEUKEMIAS

Corham, the incidence is from one to three cases per one thousand hospital
admissions. In six thousand autopsies at Cook County Hospital in Chicago, there
were fifty-eight cases of leukemia (Levine).
Males are more frequently affected than· females, the ratio being about three
to Olje. In tbe series of Rosenthal and Harris the ratio was 1.5 to one. In 86 cases
of leukemia reported by Wintrobe and Hasenbush, the males predominated in both
myelogenous and lymphatic types. There seems to be no racial susceptibility,
since the disease has been reported in all types of people. We have seen many
cases of acute and chronic myeloid and lymphatic types among negroes.
The review of 9'9 cases by Ward shows that the disease is rale in early
childhood, although it docs occur, usually in the acute form. Chronic leukemia
is a disease of middle life, and in patients under 25 is usually either acute or
subacute. The disease also occurs in the aged. Naegeli cites an instance of the
lymphatic type in a patient 84 years of age. The average age in the chronic
types is between 40 and 50. We have observed a woman of fifty years with the
acute myeloblastic type, and a young boy with the chronic myeloid form.
Occupation or station in life seems to play no part in its development. It
occurs in rich and poor alike. It is possible tbat the incidence is increased in
workers in industry who are exposed to tbe action of benzene, or to excessive
action of radiation.
There is no definite evidence to indicate a familial tendency, although rare
instances of its occurrence in members of the same family have been reported.
Curschmann states that there are a few instances of familial leukemia. Those
reported are mainly of the lymphatic type, hut the relationship is doubtful.
Leukemia has also been reported in rare instances in the newborn. Rhamy re-
ported such a case that was combined witb erythroblastosis. Death occurred
from traumatic rupture of the spleen during delivery.
Kelsey and Anderson have reported one case of congenital leukemia. They
reviewed the literature of all such cases and state that in not a single instance was
leukemia found in any of the parents. There is no suggestion, therefore, that this
condition is inherited~
The possible relationship of pr\'Ceding trauma to development of leukemia
has been brought up in an occasional case. Yaguda and Rosenthal reported three
such instances of leukemia. All of these patients were previously in good health.
One was struck on the shin by a shovel and twelve days later sbowed an acute
myeloblastic leukemia. The second patient had a heavy object to fall on his
foot and ankle and six weeks later showed 85,000 white cells per cu.mm. chiefly
lymphocytes. The third fell and injured his left hip with a fracture of femur.
Two weeks later he was found to have aleukemic myelogenous leukemia. They
point out that in 1930 Lutham had collected 40 cases in which trauma had pre-
ceded the development of leukemia. Such instances are quite rare considering
the total number of cases of leukemia, and the assumption of a relationship be-
tween trauma and the disease is only problematical at best.

NATtmE OF LEUKEMIA
At the present time most evidence favors acceptance of the theory that
leukosis in all its forms is neoplastic in nature. Isaacs summarizes the features
 THE LEUKEMIC STATE

which suggest its relationship to cancer: (I) uncontrolled growth; (2) secondary
foci (metastasis); (3) fatal termination with cachexia; (4) neoplastic type of
metabolic rate of celis; (5) maturation of cells with irradiation; (6) failure to
transmit the disease in humans by inoculation with the blood of infected indi-
viduals; (7) absence of bacterial etiology; (8) birth of normal children from
leukemic mothers.
Piney bases his contention that all leukemias are neoplasms upon the presence
in all forms of leukemia, of medullary and extramedullary foci of cells of a single
immature type. He considers this focal arrangement of masses of leukohlasts
(100 to 200 cells in a cluster), surrounded hy simple hyperplastic tissue to be the
essential feature of leukemia and to represent a definite type of microscopic tumor
formation and metastasis. He presents evidence that the blood picture in leu-
kemia is composed of cells emigrated from two sources: first, from these foci
of tumor cells, and secondly from the surrounding hyperplastic tissue. In the
acute leukemias these foci of leukoblasts are more extensively distributed than
simple hyperplastic tissue and the blood picture is compos~d mostly of leukoblasts
wbile the reverse is true in the chronic forms in which the cells are derived
mostly from the hyperplastic tissue and from tbe foci to a lesser degree, until the
terminal stages when the foci increase at the expense of hyperplastic tissue. In
this event, the patient dies with a myeloblastic exacerbation since the acute
forms are more malignant than the clIronic.
Other evidence in favor of the neoplastic theory is the occurreJ:lce of recog-
nized tumor formations with a leukemic blood picture, as seen in chloroma and
leukemic myeloma, and furthermore, localized tumors of the blood forming organs,
as in aleukemic myeloma, myeloblastoma and lymphoblastoma in which the cells
do not enter the blood stream. A quotation from Piney illustrates this point:
"There seems to be no gap in the series described; a continuous gradation be-
tween single myelomas on one hand and leukotic processes on the other. It seems
difficult to escape from the view that the leukotic process is a truly neoplastic
one."
Neal states that leukemias should be considered as true malignant diseases
in that they begin and progress, as do carcinomas and sarcomas, with a multiplica-
tion of atypical and immature cells to the detriment of the host. These cells
grow locally, invade the tisues of their origin, spill over into the hlood stream
and form metastatic nodules in various other tissues and organs. He considers
the leukemic process no different from a sarcoma except that the fluid tissue of
the blood is'invaded during some stage of the disease.
Naegeli believes, however, that the leukemic process cannot be classified as a
malignancy and cites the prolonged course of the disease in some patients, which
may be as long as ten years or more. Secondly, malignant processes never develop
so quickly as do some of the acute leukemic states and the various organs, al-
though infiltrated with enormous numbers of leukemic cells, usually remain func-
tional until the termination of the disease. The presence of the so-called "hiatus
leukemic us," in which~the circulating cells may he extremely immature with no
intermediate forms between them and the most mature types, is unlike tumors.
Although leukemic processes have a tendency toward tumor formations, as seen in
 THE LEUKEMIAS

chloroma, histologic examination of these nodules shows that they are composed
in many instances of eosinophils and megakaryocytes.
Naegeli also opposes the idea that leukemia is an infectious disease on the
basis that leukemic infiltrations may be bighly differentiated cells; that many
leukemic states are characterized by certain pathologic cells that are never seen
in infectious states j that no organisms have been isolated in leukemia, even by
the most competent bacteriologists; that the disease is never transmitted from a
leukemic mother (0 a child in utero; that the oral infectious states usually occur
late in the discase and not early; that tumor-like proliferatious do not occur in
infectious states; and that inflammatory cellular response of infection never
shows the hiatus leukemicus. Since he does not helieve the process to be either
that of a tumor or of infection, he believes it to be one caused by dysfunction of
the glands of internal secretion.
Furth and his associates, on the basis of extensive studies in the production of
leukemia in mice, have concluded that practically all types of leukemia, including
the acute and chronic forms, are true neoplastic diseases.
Much of this controversy concerning the nature of the leukemic process is
largely an academic question since it is generally agreed that the mechanism of
leukemia includes a marked, widespread and uncontrolled hyperplasia of the
hematopoietic tissues, accompanied by the discharge of these immature cells into
the circulating blood, and finally by their deposition in clumps and masses in dif-
fuse or tumorlike masses in various tissues of the body. The main question that
remains unanswered is, "What is the nature of the agent that initiates this uncon-
trolled hematopoiesis?" It seems unlikely that this question will be answered
until the exact nature of the substance that initiates uncontrolled growth in all
cells can be determined.
If the above concept expresses the true sequence of events in the leukemic
process, it does not explain why leukemia may at one time show an extremely
high leukocyte count in the peripheral blood and at another time show almost a
complete disappearance of these cells, resulting therefore, in the aleukemic forms.
Neither does it explain why one patient may have an extremely high leukocyte
count throughout the entire course of his illness while the blood of another will be
characterized by extremely low leuk9cytc counts. We believe that this is depend-
ent largely upon the type of cell that is circulating in the blood stream. Isaacs has
shown that normal mature cells are excreted mainly in the gastro-intestinal tract.
It is apparently the function of the spleen to remove and destroy cells that are im-
mature and probably non-functional. In those instances in which the majority of
the circulating cells are immature forms, the spleen removes these as speedily as
possible and becomes enlarged in the process. If the destructive activity of the
spleen is sufficient to compensate for the increased hematopoietic activity, such a
leukemic patient will probably show a normal or decreased leukocyte count. But,
if hematopoiesis is so excessive that the spleen is unable to destroy the cells, the
leukocyte count becomes elevated and the patient presents the typical leukemia
with the high leukocyte count. Therefore, the treatment of leukemia should be
tempered with caution concerning measures directed toward the spleen since, its
 THE LEUKEMIC STATE

removal or destruction may take from the patient one of his major safeguards
against excessive numhers of circulating leukocytes.
EXPERIMENTAL PRODUCTION OF LEUKEMIA
Since it is known that leukemia occurs in various lower animals, it logically
follows that the disease might be produced experimentally. Mice have been used
mainly as experimental animals in efforts to produce the leukemic state by the
administration or injection of various chemicals. For example, Bungeler admin-
istered small doses of indole to a series of mice over a long period of. time and
produced a wide variety of blood pictures of typical leukemic processes with
high blood counts and extremely low counts, in addition to various types of
lymphadenoses and lymphosarcomata. Bernard has injected small amounts of tar
into the femoral marrow of young white rats and was able to produce a state of
the blood that he designated as erythroleukemia. Lignac injected 54 white mice
with small doses of benzene and in eight of these there developed what appeared
to be a true leukemic process.
The possible role of benzene and radiation in the production of leukemia has
been further emphasized by the occasional occurrence of the disease in workers
exposed to the action of benzene, as illustrated by the report of Delore and
Borgomano who observed leukemia in a worker who had spent five years extract-
ing pyramidon with benzene, and the case of Falconer who reported an instance
of the development of lymphatic leukemia in a worker who had recovered from
acute benzene poisoning.
In '928, Opie presented an excellent review of the experimental study of
leukemia. According to him there has been one attempt to transfer leukemia
from one human being to another. In '905, Schupfer injected leukemic blood into
four patients who had carcinoma, with no development of the disease. Many
efforts have been made to transmit leukemia into monkeys, dogs and rabbits by
the intravenous injection of human leukemic blood but all of these have been
unsuccessful. There is one recorded instance of the production of leukemia in a
hen which had received an intravenous injection of leukemic blood. In this in-
stance, however, the fowl could have been suffering from spontaneous leukemia.
LEUKEMIA IN FOWLS
Most of the work done in experimental leukemia has been the transmission
of the disease from one fowl to another of the same species. According to Eller-
mann and Bang, leukemia in fowls can he transmitted by cell-free material and
the causative agent is a filter passing substance. They have shown that this sub-
stance is destroyed by freezing and is somewhat susceptihle to the action of heat.
Wallbach has concluded that all of this research permits the assumption that
leukemia in fowls is a disease similar to human leukemia but the major que,tion
remains whether or not the disease produced in fowls is identical with that of the
human.
LEUKEMIA OF MICE
At the University of Wisconsin symposium on blood diseases in '939, Furth
summarized the current knowledge of experimental leukemia. He stated that
 THE LEUKEMIAS

experimental leukemia in the guinea pig, the mouse and the rat is essentially the
same. Leukemia is a neoplastic disease readily transmissible from one animal to
another under proper conditions and the successful transmission depends on the
presence of living leukemic cells in the inoculum. The inoculated cells multiply
in the new host without restraint, and reproduce themselves without completing
their normal maturation cycle. Therefore these are malignant celis, and he desig-
nates them as malignant myelocytes, lymphocytes, and monocytes, depending
upon the type of cell involved. The different strains have their individual char-
acteristics and these are maintained through the course of numerous successive
passages. These characteristics include (,) their morphologic character, (2) abil~
ity to invade the blood stream or keep out of the blood stream, (3) the ability to
produce localized tumors or diffuse infiltration, (4) a tendency to localize in
different organs, and (s) the production of secondary anemia, hemorrhagic
diathesis, and other complications of leukemia. Furth also states that when
malignant lymphocytes from leukemic mice showing a high cell count are intro-
duced into other mice, the aleukemic form of leukemia never develops, and the
reverse is true, that is, if the inoculum is taken from aleukemic mice they never
produce leukemia with a high cell count. Also leukemia from high cell count
blood is more easily transmitted by the intravenous than by the subcutaneous
route. Such cells may produce the disease if introduced intravenously but if put
in subcutaneously they either perish or form only slight infiltrations at the site of
injection. Or they may produce tumors without production of the systemic dis-
ease, and such tumors in some instances may be indistinguishable from lympho-
sarcoma. But when the celis of such a tumor are injected into other mice they
produce a high cell count leukemia; hence, lymphosarcoma and leukemia would
appear to be rather closely related. The type of disease depends upon the" portal
of entry. He further points out that different strains differ widely with respect
to their ability to localize in different organs of the host, and that they are fairly
constant in their affinity for specific organs.
The pathogenesis of transmitted leukemia can be explained by assuming that
the introduced malignant cells grow without restraint in the susceptible host, and
there is little evidence that the reticular cells of the host are stimulated to produce
malignan t cells.
There is not sufficient evidence to show that mouse leukemia can be produced
by a filterable agent. Furth also points out that neoplastic tissue may lose its
ability to transmit the disease after it has heen exposed to approximately 5,000
roentgen units, which is a dose that ordinarily does not injure a virus. Although
the death of the cells is not immediate when they are transferred into a new
animal they may live for a short til1le, but they do not set up the disease.
The following agents can be regarded as having produced leukemia under
controlled experimental conditions: x-rays or radium radiations, benzol, indol,
methyl cholanthrene, benzpyrene, and among these benzol and x-rays have been
thought to cause human leukemia in occasional instances. Benzpyrene is also
known to be an estrogenic compound, and from this it might be inferred that
. some of these compounds can produce leukemia. Furth believes that the leukemia
of mammals and that of man are very similar and· practically indistinguishable,
 THE LEUKEMIC STATE

since in both forms there are acute and chronic forms, high and low cell counts,
diffuse infiltrations, and tumor formations with various transitional forms between
lymphosarcoma and myelosarcoma. He concludes that leukemia is a neopl::tstic
disease. The use of radium and x-rays in leukemic animals, although producing
prolongation of life, has failed to cure transmitted leukemia under experimental
conditions. Colchicine brought about regression of subcutaneous leukemic tuntors,
but failed to control the systemic disease.
Slye has pointed out that heredity is a factor in the leukemic diseases of ,nice
for the following reasons: First, every type of leukemia has been definitely con-
fined to certain cancerous strains during the 19 years she has observed the disease
in mice; secondly, there are many strains completely free from any forrrl of
leukemia; thirdly, when a double leukemia parentage is obtainable, it is possible
to derive strains showing lOa per cent of these diseases.
From this experimental work certain conclusions at this time seem to be war-
ranted. These are: first, that fowl leukemia can be transmitted by cell-free fil-
trates from the organs of the leukemic fowls and tbat the transmissible agent is
possibly a virus, with the process probably infectious in nature; second, that
leukemia in mice is quite similar to that of man and probably is of neopl3stic
nature since it is transmitted by living cells only; third, that the relatiort of
leukemia to heredity is similar to tbat of neoplasms and that frequent tumor
formations are found in the process i fourth, that such agents as radiation and
benzene products are capable of producing leukemia in mice and may playa role
in the development of the leukemic process in an occasional person.

CLASSIFICATION OF LEUKEMIA
The leukemic states can be classified on either a clinical or hematologic basis
or botb; tnat is, on (he basis of fhe tissue involved in the process and on wbether
or not the clinical course is acute or chronic. Moon states that all c1a5'ifications
of leukemic disorders and similar pathologic conditions should be based upon
three fundamental criteria: first, the type of cell that is involyed; secondly, the
presence or absence of leukemic blood; third, the presence or absence of local
neoplastic growth or cell infiltrations. Usually the chronic forms are characterized
by more mature cell types. If the process develops because of excessive hemato-
poiesis in granulocytic cells in the bone marrow it is of the myeloid type and if
the hyperplasia is in the lymphoid system, it is lymphatic, and if in the reticulo-
endothelial system, it is probably the monocytic type. Based upon these con-
siderations, therefore, the following classification of leukemia seems to be prac-
tical: •

1. Myelogenous leukemia.
(a) Chronic.
(b) Acute.
2. Lymphatic leukemia.
(a) Chronic.
(b) Acute.
370 THE LEUIUlMlAS

3. Monocytic leukemia.
(a) Chronic.
(6) Acute.
4. Plasma cell leukemia (rare).
5. Megakaryocytic leukemia (rare).
It will be noted that the so-called "aleukemic leukemia" is not considered in the
above classification. This is mainly for tbe reason that it is well recognized that
any of the above leukemic states may at various times show decreased numbers
of circulating leukocytes so that the aleukemic stage may develop in any of them.
It should be empbasized, theref<>re, that aleukemic leukemia is not a sel,arate
disease but is a phase of any type of leukemic process.
If certain rare forms 01 the above leukemias should develop, more detailed
terminology can be used for descriptive purposes; for example, myelogenous
leukemia can be divided into neutrophilic, eosinophilic, and basophilic cell types.
Doan and Rinehart have reported four cases of basophilic leukemia occurring in
about lOa instances of the chronic myelogenous type.
 CHAPTER 28
CHRONIC MYELOGENOUS LEUKEMIA
(Chronic Myelosis)
Chronic myelogenous leukemia is a progressively fatal disease characterized
by ,marked hyperplasia of the granulocytic elements of the bone marrow, and
usually by the presence of extremely high numbers of leukocytes in the circulat-
ing blood with infiltration of these cells into the various tissues. It is rarely seen
before adult life and is primarily a disease of middle age, the average being
between 30 and 40 years.

SYMPTOMS AND PHYSICAL FINDINGS

The onset of the disease is insidious and the symptoms develop gradually over
a long period of time. The early symptoms are those caused by a slowly developing
anemia, including fatigue, weakness, and increasing paIlor. In some patients
the first signs may be those referable to the enlarged spleen, including a sense of
fulness and pressure in the left upper abdomen and some complain of a sense of
weight and dragging in the abdomen. If the patient is not seen until the late
stages, there may be marked fatigue, extreme pallor, and a persistent cough, with
variable degrees of fever and sweating. In the advanced stages the patient may
complain of shortness of breath, palpitation, loss of appetite, night sweats, loss
of weight and extreme emaciation. Wintrobe and Mitchell have stressed the wide
variety of clinical symptoms that may develop in the various leukemic states.
They divide them into four groups: first, those that suggest some acute inflamma-
tory condi tion; secondly, those with symptoms suggesting abdominal and cardiac
disease; thirdly, those suggesting bone and joint disease, and fourth, those char-
acterized by skin changes. They point out that clinical findings which may sug-
gest a leukemic process include deep bone pain, arthritic symptoms, putrefaction
of the gums, slight enlargement of the lymph nodes and spleen, unexplained fever,
bone tenderness, and purpura.
If the platelets have become decreased in the process, the original symptoms
may be those of hemorrhage, including nose bleed, purpura and sponginess of
the gums. Certain patients also complain of distnrbances of sight and hearing,
which are due to hemorrhages in the retina and to cell infiltrations about the
auditory nerve or in the labyrinth. There may be generalized itching. Some
complain of bone pains which occur spontaneously or may be elicited On pressure.
The disease in many patients may be accidentally discovered during the course of
a routine physical examination or during the process of examination for some
other disease. In one month at the Emory University Hospital we have found two
cases of chronic myelogenous leukemia in patients who were being treated for
genito-urinary disturbances. .
Physical examination may reveal a patient who appears to be in good
371
 PLATE XXX

CHRONIC MYELOID LEUKEl\lIA

I. Myeloblast 6 Juvenile neutrophHs.

2 Premyelocyte. 7. Band neutrophlls
3 Neutrophil1c myelocyte:.. 8 Segmented neutrophil::..
4 £tJhlIWI.niult- fDYeIULyd;- <i~ lihhruthhbt~
5 Basophilic myelocyte 10. PoikIlocyte.

Blood Fmdings' Differential:

Hemoglobin IO.r gm'i (Newcomer's method) Myeloblasts .,
RBC 3.0$0,000 per c mm. Premyelocytes .
WB.C 270,000 per c.mm. NeutrophIlIc Dlyelocytes
Platelds 190,000 per c.rum. EosinophIlic trlyelocytes.
Basophilic myelocytes
Color Inde,;: 10 Juvemle neutrophIls
Volumt: Inde.x 09 Band neutrophil::.
Reticulocytes 120% Segmented neutrophils
Lympbocytes
Eosmophi.15
Basophils
Monocytes
Erythrocytes; normochromic and slightly microcytic with pOIkIlocytosis, anisocytosis, poly-
chromatophlha and occaslOnal nQrmoblast and macroblast.
Plate XXX .
Plate XXXI.
 PLATE )LXXI

CHRONIC MYELOID LEUKEMIA

(PEROXIDASE STAIN)

I Non.granular myeloblast
21 Non·granular cell (eHher a lymphocyte or mlcroroyeloblast).
3. Famtly granular cells (typical reaction of premyelocytes).
4. Heavily granulated myeloId ceUs (myelocytes, juveniles, bands and segmenters).
5. Erythrocyte
6, Cluster of platelets
Thh plate represents a peroxidase stam of the case shown in Plate 30 A differential count
shows 97% peroxidase·positn'e gr:mular cell~ and a% of tbe non-granular types.
374 THE LEUKEMIAS

health, or with the signs of a long continued illness. Most of them have 10s1'
weight and are considerably emaciated. The skin is usually pale. Some describe
it as a greyish type of pallor. There may be purpuric spots over the body. Ex-
amination of the abdomen shows a variable degree of splenic enlargement and the
lower border of the spleen may be as low as the crest of the ilium. The spleen
is usually smooth and quite firm. The characteristic shape is retained 'and the
splenic notches can be felt in a thin subject.
The liver is usually not enlarged although in some instances there may be a
temporary enlargement due to the presence of degenerative changes following in-
fectious states.
Examination of the heart may shO'IV an enlargement in all diameters because
of hypertrophy due to severe anemia. Systolic murmurs may be present which
are hemic in origin. In severe cases there may be fluid in the peritoneal or
pleural cavities. Lymph glands may be enlarged to a variable extent. It is not
unusual to find a marked and generalized lymphadenopathy which may give rise
to the suspicion that the process may be lymphatic leukemia. Lymphadenopathy
probably develops because of the compression of the lymphoid tissue in the spleen
with its ultimate destruction, with compensatory hyperplasia of the entire lymph-
oid system. Or, in some instances, there may be infiltration of the lymphoid
tissues with masses of immature granulocytic cells. The genito-urinary findings
may include those of renal disease because of infiltrating cells in the kidney sub-
stance. In women disturbances of menstruation are Common. The occurrence of
priapism, which was stressed many years ago, is a relatively uncommon- finding.
Naegeli states that this is brought ahout either by thrombosis or leukocytic stasis
in the corpora cavernosa.-
Tenderness in the bones can be elicited usually by pressure on the sternum.
This tenderness apparently develops because of increased calcification in some
areas and decalcification in others. Daub and Hartman have studied this in a
large series of cases and found punched out areas of bone destruction in several
diseases, including myeloid leukemia, chloroma, Hodgkin's disease and lympho-
sarcoma. Therefore, the finding is not peculiar to myeloid leukemia alone.
The nervous system may be involved to a variable degree with a resulting
variety of symptoms. These are due in most part to hemorrhages, or infiltra-
tions of leukemic cells. Schwab and Weiss studied the records of 334 patients
with particular reference to the neurologic findings and found them in 20 per cent
of the group. These included, in order of frequency: palsies of the sixth and
seventh nerves; absence of deep reflexes; paresthesias of various types; and signs
of meningeal irritation. In an effort to explain the meningeal irritation they
studied the spinal fluid in thirty-four patients and noted almost consistently,
marked increases in spinal fluid pressure, protein and cell content.
There may be present decreased or impaired vision, but seldom to the point
of complete blindness. The ophthalmoscopic examination reveals in some pa-
tients, small, white, irregular spots lying adjacent to the retinal vessels and a pale
fundus because of the anemia. Bonnet reported that fifteen per cent of the
patients showed changes in the retina and that these occur nearly always in
the myeloid type. They included leukocytic infiltrations in the retina about the
 CHRONIC MYELOGENOUS LEUKEMIA 375
vein walls, retinal hemorrhages of various types, and a reddish stippling of the
temporal fields.
It has long been noted that there is an increased metabolism in patients
with leukemia, as shown by their frequent preference for a temperature too low
for the comfort of other people, and also by their increased basal metabolic rate,
which, according to Riddle and Sturgis, is roughly proportional to the severity
of the leukemic process. This develops, apparently, hecause of the increased
oxygen consumption by the large number of white cells and tire general eleva-
tion of metabolism coincident with the high rate of destruction of cells. This find-
ing has been seen so consistently that patients have been treated with iodine,
and Dameshek reported the removal of the thyroid gland in a case of chronic
lymphadenosis, with good results. Israel, however, found no effect on the
metabolism after treatment with iodine and does not recommend such a radical
procedure as thyroidectomy.

LABORATORY FINDINGS
Examination of the blood usually reveals the characteristic picture that per-
mits the correct diagnosis. On taking the blood specimen it may appear to be
unusually thick, sticky, and dark, because of the increased viscosity, this being
dependent upon the extent of white cell increase. The red cell picture shows
the changes characteristic of anemia. The red cell count may be as low as one
to two million cells per cu. mm. and the hemoglobin reduced in proportion to 30
or 40 per cent. In cases of prolonged illness with inadequate food intake, the
hemoglobin may be even lower and the color index below one. We have observed
some instances in which the color index was above one. A study of the stained
red cells shows them to vary considerably in size with the presence of many dis-
torted forms. They usually are of average size and the volume index is normal.
In some instances there is a tendency to macrocytosis with a small percentage of
atypical macrocytes, intensely stained, and well filled with hemoglobin. Poly-
chromatophilia is frequently seen and signs of regeneration in the form of baso-
philic stippling, increased reticulocytes, and nucleated red cells, may be found.
This is brought about, no doubt, by the stimulating effect on the erythropoietic
centers by the rapidly expanding areas of myelopoiesis in the bone marrow.
The red cell picture in some instances may closely resemble that seen in per-
nicious anemia and if the leukocyte count should be quite low, the hematologic
picture could easily be mistaken for pernicious anemia.
The leukocyte picture is usually characterized by a tremendous elevation in
the number of cells. These may range from the normal of eight to ten thousand
to as high as a million or more per cu. mm. We have recently studied one patient
in whom the number of leukocytes exceeded the number of red cells, being
1,600,000 and 1,200,000 per cu. mm., respectively. The differential count shows
all varieties of immature leukocytes, ranging from myeloblasts through the most
adult forms of segmented neutrophils. These constitute from 95 to 99 per cent
of the cells, and in general, the degree of cellular immaturity is an indication to
the acuteness of the process.
In some patients the leukocyte count may not be more than ten thousand
 PLATE XXXII

ATYPICAL MYELOCYTES IN SUBACUTE MYELOGENOUS

LEUKEMIA

2 3

4 5
Q 6

7 8
8 9

[0 [[

Cells drawn front a hlood film front a case of subacute myelogenous leukemia. Duration of hfe
five months from time of diagnosis Cells are atypical small stram mydocytes. Note the tendency
toward breakIng up and fragmentation of nucIa. Cells Nos. i, 7, 8, 1I, and 12 ha"'e small dear
oval structure.." suggestive of nucleoli. Cytoplasmic granulation varies considerably_
Plate XXX 1I .

[f a 13akA
 CHRONIC MYELOGENOUS LE.UKEMIA 377

cells per cu. mm., but in such instances there is usually an extreme shift to the
left with the majority of the cells quite immature. In the older literature it has
been stated that a patient must present a leukocyte count of at least fifty thou-
sand before a diagnosis of leukemia can be established. It should be emphasi~ed
that the number of circulating Jeulrocytes is of relatively little importance in
arriving at the diagnosis but the immaturity of the cells is the criterion upon
which the diagnosis must be made. The cells of the greatest diagnostic importance
are the neutrophilic myelocytes, which may constitute as many as 50 per cent of
the total number of leukocytes. Also, there is a definite increase in the percentages
of eosinophilic and basophilic myelocyte., and in an occasional case these cells
may predominate, as illustrated by the case of basophilic leukemia reported by
Groat, and one of eosinophilic leukemia observed by Stephens. Thomsen -!lnd
Plum have reported one case of eosinophilic leukemia and collected 15 other
cases from the literature.
Because of the fact that some infectious processes may give rise to extremely
high leukocyte counts characterized by large numbers of neutrophilic myelocytes,
it should be realized that the presence of these cells alone is not adequate for
diagnosis. We consider the presence of basophilic and eosinophilic myelocytes
of more diagnostic importance than any other finding in the examination of the
blood. These cells seldom, if ever, occur during resistance to an infectious process,
regardless of its severity.
The blood platelets are usually decreased in number, and in extreme cases
may be difficult to find-on the stained smear. Their absence results in a pro-
longed bleeding time and delayed retraction of the clot, causing the various hemor-
rhagic manifestations that may be present. In some instances the platelets Ilre
entirely normal in number and no hemorrhagic findings are present.

DIAGNOSIS
The diagnosis is based upon the history of a gradually developing weakness,
increased fatigue and pallor, usually occurring in a middle aged or elderly per-
son, combined with physical findings of an enlarged spleen and the hematologic
findings of excessive numbers of circulating leukocytes of immature types.
In the diagnosis of myeloid leukemia, it has to be differentiated from severe
infectious states accompanied by an extremely high leukocytosis; from the anemic
states, including pernicious anemia; from von Jaksch's anemia with an extremely
high leukocyte count which may be as high as 100,000 cells per cu. mm. of
blood; from the acute crises of hemolytic icterus in which the red cell'fragilitJl is
increased; from diseases characterized by splenomegaly, and occasionally from
infectious mononucleosis with its high leukocyte counts. In occasional instances
there may be confusion with metastatic carcinoma of the bone marrow, Banti's dis-
ease, and Hodgkin's disease.

TREATMENT
The treatment of chronic myeloid leukemia is concerned' mainly with the
judicious use of radiation and arsenic. It seems to be fairly well agreed tllal
radiation therapy does not prolong life but it does serve to reduce the number of
 THE LEUKEMIAS

circulating leukocytes, to reduce the size of the spleen, and to make the patient
more comfortable. Many forms of therapy have been used for leukemia in the
past, including splenectomy, hut all seem of little value except radiation and
possibly the arsenical preparations.
Naegeli has long advocated the use of arsenic. He believes that patients
should be given arsenic by mouth over a long period of time; that the use of
radiation to the spleen should be avoided as long as possible and that it should be
used as infrequently as possible, consistent with the comfort of the patient. He
prefers to use an injectable preparation of organic arsenic rather than Fowler's
solution. Stephens and Lawrence have recently treated seven patients with Fow-
ler's solution, the period of treatment extending over a period of several months
t~ three years. They were given gradually increasing doses to the point of tol-
erance. This was followed by marked decreases in the number of circulating
leukocytes, and still more important, by decreased numbers of immature cells,
by increased numbers of platelets and with improvement of the anemia. Omis-
sion of the drug resulted in a relapse of the hematologic and clinical pictnre to its
former state. They point out that Fowler's solution should not be increased, but
somewhat decreased after the appearance of toxic symptoms, which include diar-
rhea, anorexia, nausea, vomiting and itching. They recommend its use in alter-
nating periods with radiation to the spleen. Wintrobe has reported that he
found potassium arsenite solutions without value in lymphatic leukemia and of
less value than radiation in the myelogenous type.
Castle and his associates have treated patients with desiccated hog stomach,
powdered hog intestine, hog pancreas, and with nearly all of the fresh organs of
new-born rabbits, but obtained no significant results. Iodine therapy seems to be
useless.
General measures should be employed, these to include as much rest as pos-
sible, with adequate food and symptomatic treatment as indicated. Blood trans-
fusions are of considerable value but they should be used only when the anemia. is
severe. The details of radiation treatment are discussed in Chapter 30.
In evaluating the progress of the leukemic patient, it is well to consider the
effects of other agents, particularly intercurrent infections. Naegeli points out
that leukemia may be temporarily improved because of the presence of inter-
current infections and he has observed a return of a normal blood picture in
leukemic patients who developed erysipelas, sepsis of various types, influenza,
localized suppurative processes, miliary tuberculosis, and carcinoma of the stom-
ach. We have observed a patient with chronic myeloid. leukemia who developed
pneumonia and after re~overy from the pneumonia showed marked improvement
in not only the blood picture, but in the clinical symptoms. Naegeli believes that
the infectious state elaborates a hematopoietic principle which can supplant the
one that is produced in leukemia. Evaluation of the progress, therefore, may b~
difficult, and spontaneous improvement due to association with another disease
may be falsely attributed to the treatment.
The treatment of the anemia of leukemic patients is equally as important as
efforts directed to reduce the number of white cells. There is no reason why a
patient can not be ~perfectly comfortable if the leukocytes number from 100,000
 CHRONIC MYELOGENOUS LEUKEMIA 379
to 200,000 per cu. mm. There seems to be a tendency to gauge the therapy on the
number of circulating leukocytes but it is far more important to regulate therapy
on the number of circulating immature cells and on the clinical findings.
In summary, the object of treatment of the patient with leukemia is to keep
him as comfortable and as free from clinical symptoms as possible; to maintain
the normal number of red cells; to prevent the development of anemia and to keep
the circulating leukocytes as mature as possible. It seems that this can be done
by the careful and judicious use of arsenic preparations, alternated with radia-
tion, with an occasional transfusion when indicated. There should be careful and
frequent checking of the blood picture during this time. Even though treatment
is properly carried out it is questionable if the duration of life i& extended to any
considerable degree.

PROGNOSIS
The course of myeloid leukemia is steadily progressive to a fatal termina-
tion with intermittent periods of fairly good health and spontaneous remissions.
The average duration of life after the diagnosis has been established, according
to Naegeli, is from three to four years. In a series of 455 patients reported
by Rosenthal and Harris, the average duration was two to three years. In that
series 35 per cent died in less than a year, 45 per cent lived from two to four
years and a few lived from five to eleven years.
Naegeli states that recovery never occurs and that leukemic patients with
high leukocyte counts succumh earlier than those with low leukocyte counts. He
cites an instance of one patient who is living 25 years after the diagnosis was
made. We have recently studied a patient who died 18 years after the diagnosis
was established. However, these are rare instances.
The immediate prognosis is good as long as there is no development of in ter-
current complications such as severe infectious processes and extreme hemorrhages.
The prognosis becomes poor when the patient becomes severely anemic and when
there is a definite trend toward increased immaturity of leukocytes, to such an
extent that we have become impressed with the truth of the following statement:
"The life expectancy of a leukemic patient is roughly proportional to the maturity
of the circulating leukocytes; the more immature the leukocytes, the earlier a
fatal termination; and the more mature the leukocytes, the longer does the patient
have to live."
 PLATE XXXIII

Cll.RGNK L'lMI'BKnC t;~::~J"K.~MI;\'

1. Lymphocytes.
2. Lymphocyte with azure granules.
3. Nuclei without cytopla5M
4. Smudges. (degenerating lymphocytes).
Blood Findings. Differential'

Hemo.glof;lin 84 gms. (Newcomer's meth.od). Large lymphocytes ,

RBe. 3,700,000 })er c.mm Small lymphocytes
WB.C. 390,00-0 per c rom. Segmented neutrophils
Platelets 24-5,000 per c,mm. Numerous smudge forms.

Color Index , 06 Peroxidase Reaction:

Volume jndex 07'
Re-ticuJoc}'te5 .35% Granular tells .
Non~granular cells
Erytllrocytes.; hypochromic and microcytic.
Plate XXXIII.
 CHAPTER 29
CHRONIC LYMPHATIC LEUKEMIA
Chronic lymphatic leukemia is a fatal disease of unknown cause, char-
acterized by widespread hyperplasia of the lymphoid tissues, accompanied by
generalized lymphadenopathy, increased numbers of lymphocytes in the blood,
and their deposition in the various tissues.
Eti~logical factors include those discussed· under the general heading of
leukemia. There is no occupational, racial, seasonal, or geographic incidence of
significance. The disease occurs in middle and late life and predominates in
males. It accounts for about 20 per cent of all cases of leukemia.

SYlIIPTOMS AND PHYSICAL FINDINGS

The disease has a gradual and insidious onset. It is usually difficult for the
patient to recall the first symptoms or to give the exact date of onset of ill health.
The first signs may be increasing weakness, fatigue, and pallor. Some pa-
tients note painless enlargement of. the cervical lymph glands as the first sign.
A slowly developing period of ill health with loss of weight' and occasionally a
slight afternoon temperature may be responsible for the patient seeking medical
aid. As in other leukemias, the disease is often found during the course of some
medical examination, or treatment of some unrelated disease.
In far advanced cases the chief signs may be those referllble to the anemia
and include dyspnoea, palpitation, vertigo, tinnitus, cough, and general lassitude.
Hemorrhagic states are rarely seen although they may occur.
General examination may ·reveal some emaciation and a decided pallor of
the skin and mucous membranes, although there may be no pallor, either because
of lack of anemia or because of capillary dilatation.
The most important findings are the changes in the lymph glands. There
is usually a general and eXtensive glandulnr enlargement of all of the superficial
lymph nodes, including those of the cervical, axillary and inguinal groups. The
cervical enlargement involves both the anterior and posterior groups. A supra-
clavicular node may be noted. The glands vary in size from those barely pal-
pable, to massive enlargements several centimeters in diameter. They are found
in definite chains, are discrete and hard, close together, but not matted and
continuous unless secondary inflammatory changes have occurred. They are
freely movable and not attached to the skin. The glands are usually painless
and are not tende, on pressure.
Other lymph glands are involved in the process, including those of the thorax,
mediastinum, and abdomen. These can be demonstrated only by x-ray examina-
tion. Careful examination may reveal others enlarged, such as the epitrochlear
and submaxillary glands, and the lymphoid tissue of the tonsils.
The spleen is usually enlarged and can be palpated below the costal margin.
The enlargement does not attain the size of that seen in myeloid leukemia, but
381
 THE LEUKEMIAS

it is present in practically every case. This appears to he a part of the general

process of lymphoid hyperplasia. We have recently studied a patient who had
fully developed lymphatic leukemia (300,000 cells per cu. ",m.) but who had no
glandular enlargement except a moderate splenomegaly due to lymphoid hyper-
plasia.
There is usually some enlargement of the liver but this does not occur to the
extent and consistency that makes it important for diagnosis. It is probably
brought about by cell infiltrations and the degenerative changes associated with
the leukemic process ..... Liver damage is not sufficient to cause jaundice except in
extreme cases.
Lesions in the skin occur frequently in lymphatic leukemia but rarely in the
myeloid type, with exception of the acute forms. The skin lesions are generalized
and consist of small discrete nodules of infiltrated leukemic cells, with bronzing,
vesicles, and rarely pustules. When the skin manifestations are predominant the
disease is referred to as "leukemia cutis." As a rule skin changes occur more fre-
quently in the aleukemic types but this phase is seldom seen in the chronic forms
of lymphatic leukemia. The extent and variety of skin lesions may be so exten-
sive as to give the "leonine fades." and be confused with mycosis fungoides.
Bone changes may be the same as those seen in the myeloid types with a
greater tendency to the formation of tumor nodules, and they occur less frequenily
than in the myeloid types.
Disturbances of vision are rare and if present) are due to retinal hemorrhages~
Deafness may be present because of tumor infiltrations in or about the labyrinth.
The tonsils may be considerably enlarged.
There are no findings in the heart and lungs that are of specific aid in diag-
no.sis. The findings present are usually those of severe anemia, including tachy-
cardia, palpitation, and hemic murmurs. In the terminal stage, there may be
compression of lung areas by mediastinal enlargement, pleural effusions and ter-
minal bronchopneUlnonia.
There may be a mild elevation of temperature over a long period of time
or periods of high fever resembling that of a septic origin.

LABORATORY FINDINGS
The findings in the blood are typical and offer little difficulty in diagnosis.
The leukocyte count may reach extremely high levels (from 50.000 to over a mil-
lion cells per cu. mm.). The predominating cells are small, adult lymphocytes
mixed with fewer numbers of large lymphocytes. The lymphocytes may comprise
as many as 99 per cent of all white cells. Some of them show atypical nuclear
formations, as apparently dividing or lobulated nuclei (Reider cells), and the
a
nuclear material occupies practically all of the cell space, leaving only thin rim
of cytoplasm. Some seem to have nQ cytoplasm at all.
A characteristic feature of the blood is the presence of large numbers of so-
called "basket cells" or degenerated forms. These cells, sometimes called
"Gumprecht's Shadows" or "Smudge Cells," are found consistently in this type
of leukemia but are also found in other types. We believe these to be cells that
 Plate XXXIV.

XXXIV
KIDNEY Of' ACUTE LYMPHATIC LEUKEMIA
_:';Ole that the surface of the kidney is pale with irregular dark areas representing: areas of
hemorrhage and masses of infiltrating leukemic cells. The cut surface is lighter than normal and at
the petipbe.ry of the cort~:x are flame s.haped hem-orrhag1c areas. Thnmgh-out the: k\dn-ey is a diffus-e
cellular infiltration. with Iyrnphoblasts.
 Plate XXXV.

Showmg maSSlVe ccllu1.u Inflltra

tion of If'ukcmlc cell!'. 10 the liver
from a case of acute m)elogenous
lC\Jh.ernla. In the tcrmma\ stage5 of
hl~ l11nrss the patIent prr~('nterl
aleul..eJY>ic blood pIcture

Section of th~ kidney from the

same case as above showll1g masses of
leukemiC: cells. about the glomeruh.

xxxv
LEUKEMIC CELL I!,\FJLTRATIO~ I~ I.I\'ER A);D KID::-.JEY
 CHRONIC LYMPHATIC LEUKEMIA

are excessively fragile to the extent tbat tbe nuclear material is smeared out in
strings on the glass slide.
The red cells are reduced in number and the hemoglobin in proportion, with
the color index below one. There is little variation in size and shupe of tbe red
cells and little evidence of macrocytosis. There may be evidence of regeneration
in the form of stippling, reticulocytosis, and normoblasts, these changes being de-
pendent upon the extent of bone marrow invasion and irritation of erythropoietic
centers.
The platelets are usually reduced in number but not to the extent seen in
myeloid leukemia. Minot and Buckman have found the platelets to be low in
practically all cases. In those instances there is increased bleeding time and de-
layed retraction of the clot, followed by hemorrhages.
The urinary findings are of no significance, although in damaged kidneys due
to cell infiltrations, there may be albumin, a few hyaline and granular casts, but
rarely impairment of renal function.
Krantz and Riddle have studied the basal metabolic rate in 3 I cases and
found it to be increased only when the leukemic process causes systemic symptoms,
and if the cell count is high with a predominance of immature cells the basal
metabolic rate is also high. As pointed out before, some patients have been
treated with iodine and others with thyroidectomy, but with little effect.

PATHOLOGY
The essential pathologic changes are those of the proliferating lymphoid
tissue and the organs into whicb the excessive cells have become deposited. The
lymphoid structures are characterized by hyperplasia and crowding with small
lymphoid cells. This is also seen in the splenic pulp to the extent that the entire
spleen resembles a solid mass of lymphocytes. The same cells are seen in nodular
and diffuse infiltrations in practically any tissue of the body, but particularly in
the liver, kidneys, and spleen. Cell infiltrations may occur in the testicle, epi-
didymis, ovary, pancreas, esophagus, bronchi, choroid conjunctivae, nerve trunks,
meninges, sweat glands, salivary glands, and often in the epicardium (Naegeli).
Grossly, the bone marrow is gray or grayish. red, not only in the flat bones,
but the long bones as well. There is a decided increase in cellularity at the ex-
pense of the fat spaces. On microscopic examination, however, the cellularity is
seen to be due to infiltrating lymphocytes at the expense of the normal gran-
ulopoietic and erythropoietic centers. This is responsible for the development
of the anemia, the presence of erythrocytic regeneration, and the granulocytic de-
creases that are often seen in the disease.

DIAGNOSIS
The diagnosis of chronic lymphatic leukemia is based on the slowly develop-
ing anemia with fatigue and weakness, the generalized lymphadenopathy J and
the presence of excessive numbers of lymphocytes in the blood stream.
In the typical case the diagnosis offers no difficulty, but in the early stages
before the leukocyte count has reached large numbers there may be confusion with
those diseases causing cervical lymphadenopathy, including glandular tubercu-
 THE LEUKEMIAS

losis, Hodgkin's disease, secondary inflammatory lymphadenitis, Iymphosartoma,

infectious mononucleosis, and syphilis. Also the lymphocytosis may be confused
with that occurring in other conditions, such asthat seen in whooping ~ough
of children, the lymphocytosis of infectious states (the so-called lymphatic reac-
tion), and that of miliary tuberculosis. If the leukocyte count is low, there may
be confusion with the relative lymphocytosis of the leukopenic diseases, sUch as
intluenza, agranulocytosis, and the acute exanthemata of children.

TREATMENT
As in myeloid leukemia, treatment is directed toward improvement of the
well-being of the patient, the alleviation of symptoms, reduction in number of
white cells, and correction of the anemia. Radiation with Roentgen ray is the
best method of treatment and should be directed to the enlarged lymph glands.
They immediately decrease in size, the leukocyte count is reduced and the P'ltient
shows marked general improvement. The same caution and frequent chel:king
of the blood should be employed in this type of treatment, as in myeloid leukemia.
Lymphatic leukemia does not improve with arsenic therapy although NIlegeli
believes it to be of some value. No agent has yet been used that seems to offer
any specific deterrent effect on the progress of the disease. General treatment
should include a maximum amount of rest and vacation, an adequate diet, and
symptomatic therapy. The disease is characterized by spontaneous remissions
during which the leukocytes decrease, the lymph glands and spleen decreflse in
size, and there is marked improvement in general health. There periods may at
times be attributed to the type of treatment in -use.
Transfusions are valuable in prolonging life, and should be used only when
the patient is anemic. Repeated transfusions are reported to be of much value
in correcting the anemia, 6ut nave no etrect on tne white ceIl picture.

COURSE AND PROGNOSIS

With no exceptions the disease is ultimately fatal. The duration of ' life after
diagnosis is slightly longer than that of myeloid leukemia and averages from
five to six years. The higher the leukocyte count the more unfavorable is the
prognosis. In some instances of low leukocyte counts patients may go ()n in
fairly good health for ien to fifteen years and continue their work.
In some patients there may oCCur what appears to be a permanent remJssion
of the disease, in which the leukocyte count remains stationary at a low level,
the lymph glands do not enlarge further, a sense of good health is present, and in
such cases there may be the delusion that a cure has been effected. Finally, how-
ever, signs of relapse will develop, these usually consisting of a gradual elevation
of the leukocyte count, enlargement of 'Iymph glands. and a slowly devel~ping
anemia, all or any of which is an unfavorable sign. Wintrobe has reported that
intercurrent infections failed to produce remission ,in physical signs or hlood
picture in either myelogenous or lymphatic leukemia. Death finally occur. be-
cause of hemorrhagic disorders or because of sepsis, which the patient is UIlable
to combat properly, due to impairment of bone marrow output and decr ..ased
numbers of neutrophiles.
 CHAPTER 30
THE ACUTE LEUKEMIAS
(Acute leucosis)
Discussion of the acute leukemic states can well be placed under one heading
since there is little difference in the clinical picture and hematologic findings with
exception of the predominating immature cells.
Acute leukemia is a rapidly fatal malady of unknown cause, characterized
by sudden ons.et, a febrile course, necrosis and ulcerations of the oral tis5Ueg,
hemorrhages from skin and mucous membranes, the presence of a severe anemia,
with predominating numbers of immature leukocytes in the blood stream, infiltra-
tion of organs with these cells, a progressive course unaffected by treatment, and
death in every patient.
The discussion presented here refers to all types of acute leukemia, including
those of myeloid, lymphatic, and monocytic origin. These are often called
acute myeloblastic leukemia, acute lymphatic leukemia, and acute monocytic
leukemia. Piney refers to the entire group as "acute leucoses," and since the
exact type is largely an academic question of no considerable practical impor-
tance, the diagnOSis of "acute leukemia" is usually adequate.
Although the acute leukemias have been recognized for over 75 years, it was
not until the description of the myeloblast by Naegeli in 1900 that their classifica-
tion became relatively. accurate. Before that time it was thought that practically
every case was one of the lymphatic type, but in recent times the pendulum has
swung in the other direction and now it is believed that nearly all caSes are of
myeloblastic origin. Naegeli states that he has studied 40 cases in 'S years and
that all of these were of the myeloid type, and he questions whether or not acute
lymphatic leukemia ever occurs in an adult.
Rosenthal and Harris reviewed their 455 cases of leukemia and reported
that 38 per cent of these were of the acute type. It appears then that one out
of three cases of all forms of leukemia is acute. In this same series, 30 per cent
were of the myeloid and only 7 per cent of the lymphatic type. According to
modern reports of the distribution it would appear that the lymphatic forms are
seen less and less frequently. However this is because of the more careful and
detailed hematologic studies and recognition of bizarre types of myeloblasts. We
bave studied over 20 cases of acute leukemia in the last two years and, in our
opinion, all but one were of the myeloblastic variety.
The disease is rare in adults, especially in later life. The oldest patient we
bave studied was 35 years. Most all occur in children and in young adults. Acute
leukemia occurs with the same frequency in childhood as does the chronic types
in late adult life. In a period of fifteen years Cooke analyzed 50 cases of acute
leukemia in the St. Louis Children's Hospital, and during the same period only
three chronic types were seen in children.
 PLATE XXXVI

ACUTE MYELOID LEUKEMIA

(MACROM~LOBLASTIC TYPE)

r. MacromYelorldlsts
2. Normomyeloblast.
3 Normomyeloblast with Auer bodies In cytoplasm.
4 NeutrophilIc myelocytes (young).
S. Mlcromyeloblasts.
6. Myeloblastic nucleus without cytoplasm.
7 Smudge (degencratmg myeloblast),
S. Normobla.3ts with bal>ophilic stippling in cytoplasm.
Blood Fmdings' DIfferential;
~ Hemoglobin 47 gms. (Newcomer's method) Myelohlasts . 90 %
R.Be. 1,450,000 per c mm. Premyelocytes 7%
W.BC. 105,000 per c.mm. Myelocytt's 3%
Platelets 74,000 per c.mm.
Peroxidase Reactton.
Color Index 1.0
Volume Index 07 Non-granular celIs
Reticulocytes . 150% Granular cells .
Erythrocytes; normochromic, microcytic, With anisocytosis, polychromatopblha, poikiloCYtoslS
basophIlic st1pphng and numerous normoblasts. '
Plate XXXVI.
Plate XXXVII.
 PLATE XXXVII

ACUTE MYELOID LEUKEMIA

(MICROMYELOBLASTIC TYPE)

I Normomyeloblast.
2. Normomyeloblasot wltb vacuole in cytoplasm.
3. Mrcromyeloblasts
4. Myeloblastic nuclei wIthout C) toplasm.
5. Smudges (degeneratillg myeJoblasts),
6, Macroblast.
7. Premyelocyte
8. Neutrophilic myelocyte.
Blood Finrlmgs' Differential:

Hemoglobin 45 gros. (Newcomer's method). Myelohlasts .95%

RBC. 1,200,000 per c mm. PremyeIocytes 1%
IV.BC 78,000 per c mm. Mye!ocytes • • 10/0
Platelets IO,OOO per c mm. Segmented neutrophils .. 3%
Color Index II PeroxIdase ReactIOn:
Volume Index 10
Reticulocytes .. 100% Granular cells . 18%
Non-granular cells 82 %
From the peroxidase reaction it is assumed that some of the cells cIassified as myeloblast! were
early premyelocytes
Erythrocytes; slightly macrocytic and byperchromic.
 PLATE XXXVIII

ACUTE MYELOID LEUKEMIA

(MICRO-MYELOBLASTIC TYPE)

0 0 Q
2 2l

0 4
0
5
G
6

00 7 -8
0
9

0 0 10 11 I
0 12
Cells No I, 4. 7. and 10 are intermediate size myeloblasts showing variable nucleolar structures
ranging h'oDl. oue to three in number) with only a slight fringe of cytoplasm. Cells No.2, 5) 8, and
II are micromyeloblasts showing a slight cytoplasnuc frmge WIth nuc1eoh from one to three in
number. Cells NO.3, 6, 9, nnd u are atypical small myeloblasts sometimes confused with small
lymphocytes.
Plate XXXVIIl,
Plale XXXIX,
 PLATE XXXIX

ACUTE ALEUKEMIC MYELOID LEUKEMIA

(CONCENTRATED BLOOD)

t, Myeloblasts. 7. Sm.udge.
z Premyelocytes. S LymphQcyte.
3. Early neutrophilic mye1ocyte. 9. Lymphocyte with azure granules in Cyt04
4. Late neutrophihc myelocyte (advanced alw plasm
most to the juvenile stage). 10. Large cellular thrombocyte.
5. Segmented neutrophil!>. n. Normoblast
6. Nuclei without cytoplasm 12. Polycbromatocyte wIth basophllic ~tippiiDg.

Blood Findings' Differential'

Hemoglobin .. 4 gms. (Newcomer's methoo). Myeloblasts 50%
RBC. t ,030,000 per C.mm Premyelocyte;; .. 4%
WB.C. . ............ . 1,100 per c mm. Myelocytes ..... . .10%
Platelets . ........... . 26,000 per C.mm. Segmented neutrophils 200/0
Lymphocytes 160/0
Color Index .... . .. .. I.~
Volume Index . . . .. . . ... 1.1 Peroxidase Reaction:
Reticulocyte5- ..... . . . . . . . 9.0%
Granular cells .
Non-granular cells
Erythrocyte!;j sligtltly hyperchromic and macrocytic, anisocytosh;, poikilocyto!;is. polychrcma-
topbilia, basophilic stippling and normoblastemia.
 PLATE XL

ACUTE LYMPHATIC LEUKEMIA

t. Lymphoblasts.
2 Large lymphocyte.
3. Small lymphocyte . .
4. Nucleus witbout cytoplasm.
5. Smudges.
Blood Findings: Differential:
Hemoglobin 46 gms. (Newcomer's method). Lympboblasts 50%
R.BC•.. 1,350,000 per C.IDm. Lymphocytes .. 49%
W.B.C • . . . . 60,000 per c mm. Segmented neutrophils 10/.
Platelets . 19,000 per c mm.
Peroxidase Reaction:
Color Index .. . " ...... 1.0
Volume Index .. 09 Granular cell! . .. 1%
Reticulocytes .. 60% Non-granular celIs .... 99%
Erythrocytes; normochromic and normocytic.
Plate XL.
 THE ACUTE LEUKE1<IAS 39 1
It predominates in males in a ratio of two to one, is seen in all races, and
has no peculiar geographic or' occupational incidence. Some have observed what
appears to be an epidemic tendency in certain areas. A recent observation of in-
terest is that of Sydenstricker who studied seven cases of acute myeloblastic leu-
kemia in young people from a very limited section in South Georgia. These oc-
curred rapidly one after the other, and comprised more cases of leukemia than had
been seen from the same area in 25 years. He could establish no causal factors
in this group.
The acute onset of acute leukemia has led many to suspect that the process
may be different from the chronic types. Therefore, the proponents of an infec-
tious theory of causation are more numerous than those who believe tbis to be a
neoplastic process. The sudden onset, the actively febrile course, and the periods
of leukopenia alternating with leukocythemia, would suggest the action of an
unknown toxic agent, possibly bacterial in origin. However, no organisms have
been isolated in the disease that appear to bear a causal relationship. In the late
stages after cellular resistance is low and the patient becomes a prey to all types
of invading bacteria, there results varied types oi infections with many different
kinds of bacteria, which can be isolated from the local infected areas and from
the blood stream. These changes, however, are sequelae and apparently follow
the leukemic process.
There is little reason to separate the fundamental changes in the acute forms
from those of the more chronic types because hyperplasia of the hematopoietic tis-
sues is present in both; in the chronic types the level of cell maturity is more
advanced than that of the cells of the acute forms. In both types the cells
maturate rapidly, excess numbers are found in the circulating blood and these be-
come infiltrated in the various tissues. Furthermore, the fact that chronic leu-
kemia of years duration may suddenly become converted into the acute form is the
most important evidence that acute and chronic leukemia are varying degrees of
the same fundamental I?rocess.

SYMPTOMS AND PHYSICAL FINDINGS

The onset may be sudden or gradual. In some patients there are no signs
of illness until the sudden development of the disease. In others there are vari-
able periods of ill health usually extending over a few weeks preceding the more
severe manifestations. Preceding infectious states such as respiratory diseases,
tonsillitis, sore throat, influenza, and gastro-intestinal upsets may be he,ld responsi-
ble for the early and gradual development of symptoms. Patients can often date
their ill health from, some such disease.
The early signs may be those caused by ulcers and other infections of the
mouth, bleeding gums, benlOrrhages from other sources, extreme weakness, pallor
and anemia. In the early stages it may be confused with a large number of
infectious or hemorrhagic states.
In the fully developed disease the patient is acutely ill, with headache, pros-
tration, a septic type of fever and perhaps delirium and coma.
The hemorrhagic manifestations may predominate in the clinical picture witb
generalized purpura, bleeding from the gums, intractable nosebleed, and bleeding
39 2 THE LEUKEMIAS

from the bowel or uterus. Most patients show lesions of the mucous membranes
of the mouth and throat that range from redness and edema to ulcerations,
necrosis and gangrene.
There may be variable enlargement of the lymph glan$, usually of the
cervical group, or in some instances the spleen may be slightly enlarged .• How-
ever, no lymphadenopathy may be present. In the more prolonged cases there
is more chance for lymph gland enlargement. A common onset of a gradual type
in' children is that -of slowly developing cervical lymphadenopathy and a mild
febrile course over some weeks in which case there is suspected Hodgkin's dis-
ease, lymphosarcoma, infectious mononucleosis, tuberculosis, or other diseases
associated with lymphadenopathy.
There may be signs of heart failure, including palpitation, systolic hemic
murmurs, pulmonary congestion, ascites, edema of extremities, and enlarged
liver. There is tenderness over the sternum and other bones in some patients.
Symptoms may be observed that are referable to the gastro-intestinal tract, to
the bones and joints, or the nervous system. Lereboulet and Baize, discussing
the disease in France, stress the occurrence of the following symptoms: sudden
onset, chills, high fever, extreme lassitude, pallor, collapse, angina, and hemor-
rhages.
Among the nervous manifestations are meningeal irritation, facial paralysis,
convulsions, deafness, and cerebral hemorrhage. Cooke noted the following symp-
toms in order of frequency in his series of 50 children: asthenia with weakness
and pallor; dyspnea and cyanosis because of mediastinal enlargement; rheumatoid
pains; hemorrhages from nose, mouth, intestines, and urinary tract; cervical
adenopathy; varied abdominal pains; varying degrees of stomatitis; and neuro-
logic signs due to cerebral and spinal cell infiltrations.

HEMATOLOGIC FINDINGS
Because of the wide diversity of clinical findings and the confusion in dif-
ferentiation from other diseases, the findings in the blood are of the utmost im-
portance, especially from a standpoint of prognosis.
In the early stages there is very little anemia and the red cells may be near
the normal ligure. However, as the disease progresses they fall rapidly So that
counts of two to three millions per cu. mm. are not infrequent and they may be
as low as one million cells in the far advanced cases. The rate of red cell decrease
is quite rapid after it has begun and can be only temporarily checked by the use
of repeated transfusions.
'" In the early stages a study of the stained cells shows little variation from the
normal. If the course is prolonged there may develop varying degrees of anisocy-
tosis, poikilocytosis and polychromatophilia. Occasionally nucleated red cells
and megaloblasts are seen. The volume index is usually about normal. The
hemoglobin decrease is proportionate to the red cell deficiency and there is seldom
hyperchromia but nearly always varying degrees of hypochromia with a low
color index. In some cases the cells are macrocytic.
The blood platelets are nearly always reduced in number and in some in-
stances may be absent in stained smears. They ,are rarely above 50,000 per cu.
 THE ACUTE LEUKEMIAS 393
mm. but in an occasional instance may be near the normal figure. They seldom
increase at any time during the course of the illness but tend to progressively de-
crease in number. Their absence is responsible for the prolonged bleeding time,
the delayed clot retraction, and the resulting hemorrhagic disorders, so that there
always exists a thrombocytopenic purpuric syndrome.
Changes in the white cells are most important. The degree of immaturity of
leukocytes establishes the presence or ab~ence of the leukemic state, and the type
of predominating cell, the myeloblast, lymphoblast or monoblast determines the
type of acute leukemia. It is far more important to establish or rule out the
presence of leukemia than to establish the type, since from a practical standpoint,
the prognosis is the same, regardless of the type.
The leukocytes may range from only a few hundred cells per cu. mm. in the
aleukemic types to several hundred thousand in the leukocythemic forms. The
average is about 50,000 to 100,000 per cu. mm. When the count is high and the
majority of cells are definitely imn;ature the diagnosis can be made with cer-
tainty, but if it is within normal limits it may become exceedingly difficult. Also,
there are marked variations in the number of leukocytes at different times. We
have studied one patient whose total count was 75,000 on admission and four days
later was only 1000. A week later the count was up to 50,000 and on the day
before death two weeks later, the total count was only 600 cells per cu. mm. If
the patient had been studied for the first time in the leukopenic phase, it would
have been difficult to differentiate the condition from aplastiC- anemia, agranulocy-
tosis and other severe leukopenic states.
Regardless of the total number of cells the single most reliable criterion for
diagnosis is the presence of a SUfficient number of leukoblasts. In our experience
these are nearly always myeloblasts, which can be identified by the extremely
large cells, with deep staining round and oval nuclei, containing from three to
five nucleoli and the abundant dark cytoplasm (see plates for detailed descrip-
tions). Even though the total cell count is only a few hundred per cu. mm., if
these cells predominate, the diagnosis of a leukemic process can be made. These
cells are often incorrectly designated as large lymphocytes or monocytes by the
untrained laboratory worker.
Many acute leukemias are characterized by a predominance of small myelo-
blasts (micromyeloblast of Naegeli), that are no larger than small lymphocytes,
with purple nuclei, and a very thin rim of cytoplasm. Some of them appear to
consist only of nuclear structure with no cytoplasm. These cells, even in large
numbers, are often incorrectly designated as small lymphocytes and a diagnosis
of chronic lymphatic leukemia may be made incorrectly because of their presence.
A myeloblast may be unusually large (macromyeloblast), normal sized (normo-
myeloblast), or small (micromyeloblast), and the leukemic process characterized
by the presence of any of these. They may constitute as many as 99 per cent of
all of the circulating cells.
In some instances, particularly the aleukemic forms, there exists the "hiatus
leukemicus," in which there may be many myeloblasts and a considerable number
of fully segmented neutrophiles, but no intermediate forms. We agree with
394 THE LEUKEMIAS

N aegeJi that this is ahsolutely diagnostic of the leukemic state since we have
observed it many times.
The important question in these cases is whether or not certain cells can be
identified v;ith"llt <\Ile'i.ti"n "" bein~ bl""t {"tm'i., be<:.2.Il'i.e on thi.'i. may de~c"d the
diagnosis. This is a very difficult determination in many patients and the special
methods of staining offer little help unless older myeloid cells are present iIi suffi-
cient numbers to give a peroxidase positive reaction which will indicate that the
unidentified cell type is a myeloblast.
Myeloblasts react negatively to the peroxidase stains and also to that Imown
as the indophenol blue synthesis. They are best identified by tbeir characteristics
in the stained smear. If this cannot be done with certainty, it is then nec,essary
to make repeated and detailed studies of the blood at frequent intervals anti wait
for some change to occur that will enable the cells to be identified as blast types.
Some cases are characterized by predominating cells so immature that they
appear as primitive hematoblasts. Such instances have been called primitive cell
leukemia. By carrying out careful differential counts, the presence of an occa-
sional premyelocyte or myelocyte may be established, or a temporary outllOt of
these cells may be found, so that the unknoWn blast types can be identified "by
the company they keep," and found to be from the myeloid series and to be rnyelo-
blasts.
In summary, the hematologic findings that point to a diagnosis of a leukemic
process are: first, excessive numbers of leukoblasts, or predominating leukobla.ts
witb a normal, low, or high total white cell count; second, decreased numbers of
platelets; and third, varying degrees of anemia with low red cells and hemo-
globin.
The addition of transfused blood affects the hematologic picture very little,
exce~t to ~roduce a transient ·increase of red eel\,; and hemo~IQbi.n. We have
searched carefully for increased leukocytes two hours after a large transfusion
and are usually unable to detect any change in either the total number or the
various types of cells in tbe differential count.
In cases with low cell counts (the aleukemic types) the number of cells on the
stained smear may be so few that thorough morphologic study is impossihle. In
such instances we have found the concentration test to be very helpful (see ,ection
on technic).

DIAGNOSIS
The diagnosis of acute leukemia is based usually on a sudden or gradual
onset in a young person, weakness, pallor, sore throat, bleeding gums, cervical
lymphadenopathy, a febrile course, and purpura; and the presence in the hlood
of predominating numbers of leukoblasts.
It may be confused with various types of sepsis, purpura hemorrlJ.agica,
stomatitis, Vincent's infection, diphtheria, agranulocytosis, aplastic anemia, gland-
ular tuberculosis, Hodgkin's disease and any acute febrile illness. In many in-
stances the patient comes to a fatal termination without the correct diagnosis
being established and the true nature of the process is evident only on examination
 THE ACUTE LEUKEMIAS 395
of tissues removed at autopsy and even in these, there is little to aid in the exact
differentiation of the type.

PATHOLOGY
There is little difference in the pathological findings of the acute and chronic
forms except for the lymphadenopathy and splenomegaly in the latter. The two
major findings are extreme hyperplasia and proliferation of the hematopoietic tis-
sue in the bone marrow, spleen, and lymph glands. The marrow may show cellular
replacement in the shafts of the long bones crowding out the normal granulocytic
and erythrocytic centers. Organs and tissues are diffusely infiltrated with the
predominating cells, though tumor formations are rare. Other pathological
changes are those of secondary infectious states and of the various sequelae. In
gross examination at autopsy of the rapid fulminating cases, there is usually noth-
ing that would indicate the existence of a leukemic state, and this Can be estab-
lished only by histologic examination of the various tissues.

TREATMENT AND COURSE

It is questionable if a patient with this disease has ever recovered. All
writers agree that it is uniformly fatal. The course is rapid, progressively down-
ward, and the duration of life seldom exceeds three months.-
The treatment usually employed consists of repeated transfusions although
it is done with a sense of futility. Rosenthal recommends the use of radiation
very cautiously to enlarged glands, and the administration of Fowler's solution.
He believes that he has observed an occasional short remission. Most treat-
ment should be symptomatic with efforts directed to make the patient com-
fortable.
There is no more unfortunate diagnosis that can 'be made in medicine than
that of acute leukemia, especially since the patients are usually young people. It
should be made only after careful and thorough study, with extreme reluctance,
and only after every other possibility has been exhausted. When the diagnosis
has been established, it is a sentence to sure death, except that there is no
court to state the day, and no crime has been committed to merit the sentence.
 CHAPTER 31
MONOCYTIC LEUKEMIA
(Leukemic Reticuio-endotheliosis) (Monocytoid Myelogenous
Leukemia)
Monocytic leukemia is a progressively fatal disease characterized by wide-
spread hyperplasia of the tissue from which monocytes are derived, by increased
numbers of these cells in .the blood stream, and by their deposition in various
tissues and organs.
There can be no question but that there exists a type of leukemia that is
relatively acute or subacute, which is characterized by increased numbers of
monocytes in the blood. This type of leukemia was first described by Reschad and
Schilling in 1913 and much has heen .written about it in the last few years.
Recent discussions have centered about the academic question as to whether
the cells are derived from reticulo-endothelium or from the bone marrow. At
this time this question cannot be answered with certainty and it depends upon
the origin of the leukemic monocytes, which is an unsettled question.
Among those who have expressed their views are the group who believe
this to be a separate and third form of leukemia, including Schilling, Cl\)ugh,
Downey, Dameshek, Sydenstricker, Doan, and many others. On the other
hand Naegeli states that !lit is a temporary variant of myelogenous leukemia into
which it passes unless death intervenes." Among those who believe that the
monocytic leukemic cells arise in the bone marrow are Reich and Lawrence.
Bunting states that he always regarded the monocyte as a marrow cell and does
not believe that this represents a separate type of leukemia.
During the past few years we ha\'e studied 45, cases of this disease on file in
the Registry of Blood Diseases of the American Society of Clinical Pathologists,
and of this number there is no question but that 12 of them became definite
cases of myeloid leukemia before death. .
Then there are other patients who present large numbers of true mono-
cytes in the peripheral blood during life and the cellular infiltrations in the organs
reveal a predominance of granulocytes of all immature types.. These would
appear also to he instances of myeloid leukemia with a so-called monocytoid phase.
Wainwright and Duff studied carefully one patient who had \he hematologic
findings of monocytic leukemia but the tissues were infiltrated predominantly
with myelocytic cells. The authors refer to this as an "intermediate" form.
It is our opinion that there exists two types of this disease; one charac-
terized by the presence in the peripheral blood of typical adult monocytes, arising
from the reticulo-endothelium, and another type characterized by the presence
of atypical myeloblasts that are incorrectly designated as monocytes or mono-
blasts. Some refer to the latter as the monocytic phase of myeloblastic leukemia.
In an excellent review, Levine reports instances of both types of the disease.
396
 MONOCYTIC LEUKEMIA 397
Watkins and Hall at The Mayo Clinic reported 23 cases of the Naegeli type of
monocytic leukemia and six cases of the Schilling type of the disease, tbe former
being the myelogenous form, and the latter the reticulo-endothelial form. They
point out that there were no essential clinical differences and that treatment was
of no avail in either type.

INCIDENCE
In 45 cases in the Registry, collected from the United States, England and
Germany, the average age is 44, the oldest 79, and the youngest 5 years. Males
predominate in a ratio of two to one. There are no colored patients in the
series. Only a few more than 200 cases have been reported in the United States
(1941).

SYMPTOMS AND PHYSICAL FINDINGS

The initial symptoms and physical findings in this type of leukemia differ
little from those of other types except that the onset is sometimes relatively sud-
den, the course more progressive and rapid, the duration shorter, and the fatal out-
come comes more quickly. Therefore, it represents a more subacute type as
compared to the chronic myeloid and lymphatic types.
The onset of illness may be characterized by the development of a severe
anemia, by hemorrhagic disorders, by an acute febrile attack, sore throat, sepsis
of the oral tissues, or by symptoms referable to the teeth. In order of frequency
the following conditions were noted as the first signs of illness in forty-five
cases: Fever, sore throat, glandular enlargement, infection around teeth (gingi-
vitis), pain in joints, Joss of weight, purpuric manifestations; ear infection, and
jaundice. The disease was discovered in ten of these patients during other
medical treatment, including that for fractures, during pregnancy, and after
tonsillectomy.
Fever, sore throat, and variable glandular enlargement constitute the initial
picture in many patients. The onset may be acute, and the patient may be
treated for acute tonsillitis. Association with hemorrhagic disorders is frequent.
Examination reveals the patient usually to be acutely iII, with variable de-
grees of pallor because of anemia. There may be extensive purpura, bleeding
from the gums, diarrhea with bloody stools, or irregularities of menstruation.
In forty-five patients the following findings were noted in order of frequency:
Fever, pallor, purpura, glandular enlargement, stomatitis, enlarged liver, en-
larged:spleen, bleeding, and jaundice.
Forkner states that five of his six patients with acute monocytic leukemia
had enlargement ')f the spl~en. He further pointed out that all of them had
previously consulted a dentist in the early stages of illness for gingivitis. Mer-
cer also found that dental work was done in his two cases. This indicates
that the patients have the disease for some time before consulting a physician
and that disorders of the mouth and teeth are amo";,g the early manifestations.
Forkner has emphasized his belief that monocytic leukemia can be fairlv
well differentiated from the other types by the lesions of the mouth, which include
 PLATE XLI

MONOCYTIC LEUKEMIA (CHRONIC)*

o~
·-·
.
'i'....:,;~"

1 Monocytcs wIth C)toplaSID1C gnnules (mature icrms)

2. Non-granular monocyte.

Blood Fmdlngs: Differential:

Hemoglobin 9.6 gms. (Newcomer's method). Blasts , . 2%
RBC. 2,970,000 per c.mm. Segmented neutrophils . 2%
WBC 60,000 per c,mm Lymphocytes 5%
Platelets 240,000 per c.mm. Monocytes . .,9 1 %

Color IDeex . 09
Volume Inde::t 0,8
Erythrocytes; s11ght variation III SIze and shape, a slight microcytoSIS, and an occasional
normoblast.
* Drawn from a case on file in the Hematological Registry of the American Sodety of
Climcal PathologIsts
Plate XLI.
Plate XLII.
 PLATE XLII

MONOCYTIC LEUKEMIA (CHRONIC)

I. Non-granular monocytes (young forms).

2. Monocytes WIth faint cytoplasmic granules (mature forms),
3 Lymphocyte
Blood Fmdings:.. Differential:

Hemoglobin 92 gros. (Newcomer's method) Blasts "

RBC. 2.960,000 per c mm Myelocytes ,
WBC. 71,000 per c mm. Neutrophlls
Platelets Low Lymphocytes
Monucytes
Color Index 09 EosinophIl::;
Volume Index 0·9 Smudges

Peroxidase reaction showed 67% of mononuclear cel1s as positIve and 33% negative
Erythrocytes, slightly hypochromIc and microcytic, slight variation in me and shape, oc-
casional normoblast.
,. Drawn from a case on file in the Hema.tological Registry of the Ametican Society of
Clinical rathologists.
4 00 THE LEUKEMIAS

diffuse swelling of the mucous membrane, usually with ulceration and necrosis of
the soft tissues. He states that this seldom occurs in other leukemic forms.
Isaacs hag stated that monocytic leukemia is characterized by stomatitis, with
a relatively large liver and only a moderately enlarged spleen, whereas in myelog-
enous leukemia there is an extremely enlarged spleen but only a moderately
enlarged liver. I have noted marked degrees of stomatitis, gingivitis, ulceration
and necrosis, bleeding of the gums, swelling of the soft tissues of the gums in
instances of monocytic leukemia far more frequently than in the other forms.
Also, it seems that leukemic infiltrations of the skin which may be either diffuse Or
nodular, are seen more frequently in the monocytic forms than in the other typ~
of leukemia.

LABORATORY FINDINGS
The patients are usually anemic because of reduction of red cells which is
quite variable. The average red cell count is about two million per cu. m",.
and the hemoglobin reduced in proportion. There is little evidence of attempt.
at regeneration unless- the illness is prolonged and the process becomes relatively
chronic. The tendency is for the red cells to become progressively decreased
unless temporarily increased by transfusions.
The characteristic diagnostic picture involves the white cells which may
reach high levels or may vary widely in number with a tendency to increase
toward tbe end of tbe disease. Wbite cell counts in representative cases showed
the following fluctuations: From 5000 to 38,000; from 15,000 to 50,000; froln
13,000 to 70,000; from 600 to 3000; from 8000 to 170,000; from 1300 to
25,000; from I7 ,000 to 240,000. The white cells do not always increase con-
sistently but there may be periods of leukopenia with intervening periods of lell-
kocythemia.
The differential count reveals the type of leukemia, in that a considerable
proportion or a majority of the cells are usually immature with deep staining
round nuclei containing nucleoli, associated with more mature types that have
the cbaracteristics of adult monocytes. Some patients may have the adult mon,,-
cyte as the predominating cell, while others show the immature forms. The
latter types usually terminate more quickly. A few may have as the predomi-
nating cell a large, leukoblastic cell that appears as if it were derived from fixed
connective tissue (see plates for descriptions).
The per cent of monocytes has been variously reported as from 10 per cent
to 100 per cent.
The platelets are usually decreased in number, the bleeding time prolonged
and the clot retraction delayed. There exists then, as in other types of leukemill,
an associated thrombocytopenic hemorrhagic state.
Other laboratory findings are of little diagnostic significance and are tho~e
that accompany the development of complications. Cultures of blood during life
and at autopsy are usually sterile unless taken in the terminal stages after sell-
sis bas supervened.
 MONOCYTIC LEUKEMIA 401

PATHOLOGY
The pathologic findings are similar to those in other types of leukemia, the
major difference being that the type of infiltrating ceIl is usually the same as
that seen in the peripheral blood during Iiie. In some instances, however, the
circulating cells may be typical monocytes and the infiltrating cells be definitely
of the immature myeloid series. We have studied several such cases and that
of Wainwright and Duff is characteristic of this class.
It should be empbasized that it is quite possible to establish a diagnosis of a
leukemic process on the finding of infiltrating immature blood cells, but it is
extremely difficult to determine the type of leukemia on such findings, for the
reason that cells in the peripheral blood can be stained and studied in more
detail than those of fixed tissue stained with the usual hematoxylin and eosin
methods. This is especially true of these cells which are large, monocytic, un-
differentiated and blast-like in character. In other words, in a fixed tissue prepa-
ration it is questionable if myeloblasts, lymphoblasts and monoblasts can be
differentiated with any degree of certainty. Therefore, it is likely that if the
type of leukemia cannot be determined during life from hematologic criteria,
it is extremely unlikely that it can be determined after death from pathologic
criteria.
DIAGNOSIS
The diagnosis is based on the relatively sudden onset of fever, glandular
enlargement, sore throat, oral sepsis and purpuric manifestations, associated with
decreased red cells and platelets, with a predominating number of circulating
leukocytes of the monocytic series.
Those cases with high leukocyte counts are easily recognized but if the total
number of cells is only slightly increased, the condition has to be differentiated
from a large number of diseases characterized by lymphadenopathy, Sore throat
and monocytosis, or any combination of these. Those with low counts may be
confused with agranulocytosis, aplastic anemia, sepsis with bone marrow depres-
sion, or any disease characterized by leukopenia. In such cases careful ob-
servation of the changes in the patient and lin the blood will finally reveal the
true state. Some of those that are leukopenic finally become leukocythemic and
the leukemic character of the process becomes evident. In many the diagnosis
of leukemia is made only at autopsy by the finding of tissues and organs infiltrated
with diffuse cells or collections of cells.

TREATMENT AND PROGNOSIS

Practically all therapeutic measures have been employed that are used in
other types of chronic and acute leukemia. Radiation may be used to the en-
larged glands and to the spleen if it is enlarged. Arsenic preparations, liver ex-
tract, iron preparations, and many other agents have all proved to be equally
useless. Repeated transfusions may be given to support the patient and main-
tain life as long as possible. But with all of this, the outlook is hopeless, and
no patient has been known to recover.
 PLATE XLIII

SUBACUTE MONOCYTIC LEUKEMIA

(MONOCYTOID MYF.T.OGENOUS LRUKEMIA)

7 8 9

10 11 12
The cells in this plate were drawn from a case (If leukemia desIgnated as the monocytlC. type
based upon the prcdommatmg cells in the blood, samples of which are shown on this plate. These
cells presented characteristics of monocytes both in the stained smear and supra-vital reactions. ThIS
patient finally dIed with a blood picture and autopsy findings of typical myelogenous leukemia.
Cells No '1. 2, and 3 are smaller forms of monocytic cells showtng the characteristic foldmg, lobu~
tated, irregular nuclel with varIations In cytoplasmic color and granulation. Cells NO.4, .5, and 6
are larger forms of SImilar cells. Cell No, 4 shows one large nucleolus and two smaUer ones Cell
NO.5 5hows a !=lngle large nucleolus and cell No . ., is a heavily granulated monocytic cen with one
large nucleolus and one smaller one. Cell No. 8 shows some nuclear fragmentatIOn and a smgle
nUcleolus Cells NO.9, 10, Il, and 1.2 show further variations of the .predominating leukemic cells.
Plate XLIII.

"
I ..
,

-,

: ,~{A' I
,

-
Plate XLIV.
 PLATE XLIV

ACUTE MONO BLASTIC LEUKEMIA

1. Monob1asts.
2.Young mQnocytes. (These cell:; have the nuclear and cytoplasmic characteristics of mono~
blasts but the atypical type of g:tanulation suggests the older cell type. They appear to be
e:xamples of abnormal maturatioll in which the nucleus and cytoplasm fail to mature properly)
J Large cellular platelet.

Blood FlOdings: DIHerential:

Hemoglobm .• 10 gms, (Newtomer's method). Monoblasts, .
RBC.... 3,240,000 per c.rom. Immature monocytes
WBC. 52,850 per c mm. Neutrophils
Platelets Low Lymphocytes
Ruptured cells
Color Index . . 0·9
Volume Inde;t . . 0.8

Erythrocytes; slightly hypochromic and microcytic with variation in size 'and shape,
 THE LEUKEMIAS

The duration of illness after diagnosis has ranged from len days to Iwo
years with an average of 4S days, and the length of life depends largely upon
the acuteness or chronicity of the fundamental process rather than on the type
of treatment used. The more immature the predominating cell types the
shorter will be the duration and the more unfavorable the prognosis as to length
of life. In this respect it is similar to other types of leukemia.
 CHAPTER 32
ATYPICAL TYPES OF LEUKEMIA
ALEUKEMIC LEUKEMIA (ALEUKEMIC MYELOSIS) (HYPOCYTIC LEU-
KEMIA) (LEUKOPENIC LEUKEMIA)
The terminology of this state of leukemia is unfortunate. It should be recog-
nized as a part of the leukemic process, but because it usually offers a difficult
diagnostic problem, it deserves special but brief consideration.
Aleukemic leukemia is a stage in the leukemic process, characterized by
essentially the same clinical picture that is seen in the more typical forms, and
by the presence of normal or subnormal numbers of leukocytes.
" This condition may develop during the course of acute leukemia with typical
high leukocyte counts, or it may exist for considerable periods of time without
the count reaching figures ahove the normal level. It usually develops during the
course of an atypical leukemic process, and is often seen as a terminal stage of
acute leukemia. It occurs morc frequently in the acute types than in the chronic
forms4 It may arise spontaneously without cause or develop because of excessive
treatment with radiation or other agents that depress the leukocyte count.
The general process does not differ from the more typical leukemic states
except that there is the low number of leukocytes in the peripheral blood. The
pathologic changes in the bone marrow, spleen, lymph glands and in infiltrated
tissues are identical with those of other leukemias.
The symptoms and physical findings are usua11y identical with those seen
in acute leukemia and include the symptoms of anemia with pallor, fatigue,
dyspnea, and myocardial insufficiency; those of platelet deficiency, with hemor-
rhages and petichiae; those of cachexia; and those caused by the variable en-
largement of spleen and lymph glands.
The diagnosis is sometimes difficult to establish and it may be confused
with other leukopenic diseases such as. aplastic anemia and agranulocytosis but 1

it can be determined hy finding unquestionable immature cells in the blood in

substantial numbers.
We believe that the leukemic process may be either leukopenic or leuko-
cythemic, depending upon the activity of the spleen and other phagocytic tissues.
If the spleen is able to remove and destroy blast cells as fast as they are reo
leased into the blood· stream, then no cell increases will be evident in the blood
and the process designated as aleukemic. If the destructive action of the spleen
is unable to keep pace with increased marrow output, this will be reflected by
elevated leukocyte counts.
The appearance of low cell counts in a patient who has had high cell counts
is a bad omen, and is to be regarded with the same inte.rpretation as if there were
a marked shift to immaturity of the circulating leukemic cells. Although Naegeli
states that the duration of life is longer in the low count leukemias than in those
40 5
 THE LEUKEMIAS

with high counts, this applies only to those in which the predominating cells are
fairly mature, and if the patient lives for many years, then the original diagnosis
is open to question.
The treatment l course, prognosis and termination of aleukemic leukemia is
essentially the same as that in the acute leukemias.

PLASMA CELL LEUKEMIA

This is a rare form of atypical leukemia and at this time less than fifteen
cases have been reported. The most recent ones are those described by Reiter
and Freeman in 1937 and by Osgood and Hunter in '934, and by Patek and
Castle in 1936.
The process does not differ from the other types of leukemia except the re-
ported cell counts have not been so high and the white cells include variable
percentages of typical plasma cells.
A study of the cases shows that there can be no hard and fast line of dis-
tinction between plasma eel! leukemia in which the circulating blood contains
plasma cells with no tumor nodules, and plasma cell myeloma in which plasma
cells are not in the blood but are found-in multiple tumor nodules. In this the
situation- is analogous to chloroma and chloro-Ieukemia which are without doubt
closely related diseases and represent variations in the same fundamental process.
The blood is characterized by a considerable proportion of the white cells
having tire characteristics of plasma cells and their precursors, the plasmoblasts.
These cel1s are characterized by variation in size, an intense basophHic cyto-
plasm, and a definite perinuclear zone, with the typical eccentrically placed
nudeus containing chromatin material in radiating masses, giving the typical
cartwheel appearance. (See plate 9 for variations in plasma cells. Plate of
plasma cen leukemia drawn from tbe case of Osgood and Hunter.)
The important consideration in diagnosis is to recognize the plasma cells
and to bear in mind that they occur in the blood not only in leukemic states but
in such diseases as scarlet fever, measles, German measles, Hodgkin's disease,
generalized CMcinomatosis, rarely in tuberculosis, and occasionally in normal
blood.

CHLOROMA (MYELOID CHLOROMA) (CHLORO-MYELosrs) (CHLORO-

LEUKEMIA)
Chloroma is an atypical type of acute myeloid 'leukemia in which the im-
mature cells exhibit a tendency to infiltrate in green colored tumor masses rather
than in diffuse character. It is characterized by an acute fulminating course
similar to acute leukemia, and by the presence in the blood' of variable numbers
of immature myeloid cells.
This condition was formerly regarded as a separate disease entity of lym-
phatic origin, mainly because of the immaturity of the cell masses and their
tendency to tumor formation, but more recent studies have established the process
to be of myeloid character and characterized by the formation of a peculiar
green pigment in the circlllating cells, the blood plasma, and in the. tumor
mas~_

The clinical findings are identical with those of a~ute leukemia except a
 ATYPICAL TYPES OF LEUKEMIA

tendency for the tumors to be deposited in unusual locations, including the

periosteum, paranasal sinuses, the orbit, and the connective tissues of the skull,
spine, ribs and sacrum.
Among the outstanding clinical features are parosteal swellings, mainly in
the orbit; fadal, auditory and sciatic nerve paralysis; and tumor nodules be-
neath the skin, especially in the more acute forms.
There is often a striking protrusion of the eyeball with associated loss of
vision, and other symptoms referable to the cranial nerves.
The blood is characterized by the presence of large numbers of cells of the
immature myeloid series. The total leukocyte count may be extremely high
or as low as the normal leveL The blood plasma may be green tinged 'in rare
instances. Autopsy fmdings are characterized by the periosteal green tumors,
particularly about the ribs and sternum. The outlook is that of any patient with
acute myeloid leukemia.

MIXED CELL LEUKEMIA

This leukemic process is essentially the same as those previously described
except that the blood supposedly contains immature cells of both myeloid and
lymphoid types. There is question as to whether such a state actually exists, and
this depends upon the potentiality of the primitive blood cell. Certain cases
of leukemia may be lymphatic in type amI secondarily stimulate the marrow
to release immature myeloid cells. If the blood is studied at that time it may
appear that two fundamental cell types are involved. Also the converse may be
true, due to lymphoid hyperplasia because .of myeloid infiltrations. We do not
believe that there is any such disease entity as mixed cell leukemia.

ERYTHROLEUKEMIA
This is a term that bas been applied to those leukemic states, usually of the
myeloid type, in which the peripheral blood contains large numbers of immature
erythrocytes, including normoblasts, microblasts and megaloblasts. In rare in-
stances the red cell changes appear to be the most outstanding hematologic
finding. These changes are brought about by extreme irritation of erythropoietic
centers and in some instances represent a final effort of erythrocytic regeneration.
Such a blood picture might easily be confused with that seen in the acute phase
of polycythemia vera.

LEUKEMOID REACTIONS
In occasional instances there may occur transient periods in which the leuko-
cyte picture is characterized by large numbers of immature myeloid cells, to such
an extent that it resembles a leukemic process. Such blood pictures have been
observed in extreme sepsis, especially in children, the bone marrow crisis of
hemolytic jaundice and sickle cell anemia, syphilis, acute tuberculosis, poly-
cythemia vera, erythroblastic anemias of childhood, especially the von Jaksch and
Cooley types, infectious mononucleosis, pertussis, mediastinal tumors, osteo-
sclerosis t metastatic tumors in the bone marrow, and acute forms of Hodgkin's
disease.
Feldman and Stasney have produced "leukemoid" blood pictures in tubercu-
 PLATE XLV

PLASMA CELL LEUKEMIA'

1. Pla~ma cells.
2. Plasma cells with vacuoles in cytoplasm.
J. Nuclei with no cytoplasm.
4 MicrobJa~t Wlth basophilic stlpphng jn cytoplasm,
Blood Findiogs: Differential:
Hemoglobin .,' 6.34 gms. (Osgood-Raskin's method), Blasts, . 2%
R B.C. . . 1,920.000 per C nml. Juvenile neutrophils ., .,)"
W.E.C. ..... 341050 per c,mm, Segmented neutl'opbils . . .14%
PJatelets .. Low Plasma cells 52 %
Turk cells 10%
Color Index 1.13 Dbintegrated cells . 21%

Erythrocytes, anisocytosis, polychromatophilia. and an occasional normoblast.

*- Composit~ field from a case on file in the Hematological Registry of the American Society
of, Clmical Pathologhts.
Plate XLV.
Plale XLVI.
 PLATE XLVI

PRIMITIVE CELL LEUKEMIA*

~
g
L Primitive cells. (These cells do not :Ilt the picture of any classified cell They were the pre-
dommRte cell type In tbe blood of an atYPIcal fatal case of leukemia in :3 child They appear
to be YOllngt'r ('eJIs than leukobla"ts of any known type and we have called tbem pnDlltive
cells for lack of a better term)
2 Smudge
J. Lymphocyte

Blood Findings Differential

Hemoglobin 7 gms (Newcomer's method), PrJuutivc ceUs (?)
RBC ::,IOO,OOO per c mm, ScgItlented neutrophils
W.BC. ;120,000 per c mm. Lymphocytes
Platelets. 14,000 per c moo.
CoJor Index: .
Volume Index

Erythrocytes; normochromic. macrocytIC WIth marked variation in size and s.hape, occasional
nuckated erythrocyte. ~

* Drawn from a case on rue in the Hen:tQ.tologu:al RegtStry of the AmerIcan Society of Clintcal
Pathologists. I
410 THE LEUKEMIAS

lous rabbits by injecting them with tuberculin. In most of the animals the
total leukocyte count exceedeu 100,000 cells per cu. mm. with all types of im-
mature myeloid cells.

BIBLIOGRAPHY
THE LEUKEMIAS

BENNETT, J.
H.: "Two cases of disease and enlargement of the spleen in which death took place
from the presence of purulent matter in the blood" EdJn. Med. ami Surg Jour. 64, 413,
1845·
BERNARD, J.. "L'erythro-Ieucemique, experimentole provoquee par Ie gaudron." Sang 8, 28, j

]934·
BONNET, P.: "La retinite leucemique (teucemie myeloide),11 J. de mid. de Lyon., 14, Ifl9. 1933.
BUNGELER, W: "Die experimentelle Erzeugung von Leukamie, aleukiimischen Myelosen, Lymph-
adenosen und Lymphosarkom." Klin. Wchnschr., ll. 1982, 1932.
CASTLE, W. B., MEY.ER, O. 0., and C.w::w, W. B.: "Negative results of treatment of chrOlllC mye-
logenou!', leukemia as. a deficiency dls.eas.e." Proc. Soc. Exp. Biol. and .Med., 32. 660, 1935·
COOKE, J. V. ,"Acute leukemia in chIldren." Jour. Amer Med. Assn. 101, 432, 1933
CURSCHMAN~, H: "Uber familiare Leukamie." KIm. Wchnschr., 15. 18S, 1936.
DAMESHEK, W., BERLm, D. D, and BLUMGART, H. L.: "Complete ablation of the thyrOId gland in
a case of chronic lymphatic leukemia with hypermetabolism," New Eng. Jour. Mtd, 210,
723, I934·
DAVIS, C. L, and FlTz-HuCI:f, JR., T.: "Achlorhydria in the leukemias." Amer. Jour. Med. Sci.,
197, 763, I939.
DUORE, P., and BoncoMANo: "Leucemie aigue au cours de l'intoxication benzenique. Sur l'origine
toxiQue de certaines leucemies aigues et leur relatIon avec les anemies graves." J. de M ed. de
Ly07l, 9, 227, 1928. .
DoA.~, C. A..: "Cunent views on tbe origin and. maturation 01 cens 01 the blood," Jour. Lab.
and Clin. Med" 17, 887, 1932.
DOAN, C. A., and RlNEHART, H. L.: "The basophile granulocyte, basophilcytosis and myeloid leu-
. kemia. Dasophlle and mixed granule type." Amer. Jour CUn. Path. 11, I, 1941.
DOA.."i, C. A" and WISEMAN, B. K.: lIMonocyte, monocytosis and monocytic leukosis: clinical and
pathological study." Ann. Int. Med., 8, 383, 1934.
DOUB, H. P., and HARTMAN, F. W.: "Lymphocytic, myelocytic and monocytic neoplasms, roentgen
diagnosIS and treatment." Jour. Amer. Jled. Assn, 105, 942, 193.5.
ELLERMANN, V.: "A new strain of transmissible leukemia in fowls, (Strain H.)" Jour. ExP. lJed.,
33, 539, 1921.
FALCONER, E. H: "An instance of lymphatic leukemia following henzol poisomng." Amer. Jour.
Med Sd J 186, 353, 1933,
FELDMAN, W. H., and STASNEY, J: "Leukemoid response of tuberculoUS rabbits to administration
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FmlxNER, C. E' "Clinkai and pathologic diffe-rentiatinn of acute let.lkemi.as, with spedal yef!':I_
ence to acute monocytic leukemia." Arch. Int. Med. 53, I, 1934.
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FRIEDGOOD, H H,: "The effect of Lugol's solution on chronic lymphatic leukemia and its bearing
upon the pathogenesis of exophthalmic goiter" Amer Jour. Med SCt, 183, 515, r932
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FURTH, I., FERRIS, H. W., and RzZNlKOFF, P.: "Relation of leukemia of animals to leukemia of
man." Jour. Amer. Med. Assn> 105. 1824. 1935.
 ATYPICAL TYPES OF LEUKEMIA 4 II
GROAT, W. A" WYATT, T. C., ZlMM:t.:R, S. M., and FIELD, R. E: UAcute basophihc leukemia."
Arner. Jour. Med. Sa., 191. 457, 1936.
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4'2 THE LEUKEMIAS

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 CHAPTER 33
TREATMENT OF LEUKEMIA
By LLOYD F. !:RAVER, M.D.

The term "leukemia" includes a wide scope of disorders, including not only
the tissues that are normally responsible for formation of white blood cells but
also tissues which do not normally take part in cell production. In the leukemic
state they may do so, and in addition to this there are other tissues invaded by
abnormal white blood cells. Clinically, any part of the body whatsoever may be
affected by the leukemic process. In duration, leukemia may vary from a ful-
minating catastrophe, producing 'death within a few days, to an extremely chronic
affliction that is fairly tolerable for 15 years or more. To cope with the many
problems presented by leukemia in all of its aspects calls for all of the reSOUrce-
fulness of the clinician. Because of its varied manifestations and widespread
clinical symptoms it is obvious that its treatment cannot be standardized, but
each patient presents problems different from others.

CLINICAL AND PATHOLOGICAL TYPES OF LEUKEMIA

The following outline presents a fairly comprehensive classification of the

leukemic and allied disorders which serves as a satisfactory basis for discussion
of the various methods of treatment.

1. CHRONIC MYELOGENOUS LEUKEMIA

2. ACUTE MYELOGENOUS LEUKEMIA
3. CHRONIC LYMPHATIC LEUKEMIA
4. ACUTE LYMPHATIC LEUKEMIA
The above group includes the majority of cases that are currently recognized
and treated. However there are certain other varieties or phases that must be
considered even though they occur relatively infrequently.

5. MONOCYTIC LEUKEMIA
This is now regarded by many as a distinct entity derived from primitive
mesenchyme cells (Forkner) ; while by others it is regarded as a transitory phase
of myelogenous leukemia. Most of the cases are acute or subacute, and are
characterized by gingivitis or angina, by purpura and bleeding, together with
minimal or barely detectable enlargement of liver, spleen and lymph nodes. Some
chronic cases have been reported, but even so the duration of life is seldom Inore
than one year.

6. ERYTHROLEUKEMIA
In some cases the picture of polycythemia vera may be succeeded by a stage
with an unusual degree of splenomegaly, the development of anemia, and myelo-
413
 THE LEUKEMIAS

cytic leukemia. In other cases the picture of myelogenous leukemia may be

followed by a stage of polycythemia and in still other cases there may be a con-
current picture of myelogenous leukemia and polycythemia to the extent that
exact c1assification is difficult.

7. EOSINOPHILIC AND BASOPHILIC LEUKEMIAS

In rare cases the eosinophil count and in other cases the basophil count
may be so high as to justify the use of the appropriate one of the above terms.
Doan and Rinehart have described a case of leukemia in which the prenominating
cells showed a mixture of eosinophilic and basophilic granules which they call
myelogenous leukemia, mixed granule type.

8. POLYNUCLEAR LEUKEMIA
There are rare cases in which there arc the fundamental pathological changes
of chronic myelogenous leukemia but in which the peripheral blood shows a leuko-
cytosis, with an increase in mature polymorphonuclear neutrophilcs, and few or
no immature cells of the granulocyte series. In general these cases follow a rela-
tively benign course.

9. PLASMA CELL LEUKEMIA

These are cases in which there are such numbers of plasma cells. in the blood
with accompanying localized or generalized plasma cell myeloma or other forms
of plasmacytoma, that the term plasma cell leukemia may be applicable.

10. MEGAKARYOCYTIC LEUKEMIA (Piastrinemia of Italian Authors)

Megakaryocytes and blood platelets may be found in large numbers in the
peripheral blood in some cases, particularly in myelogenous lellkemia, but whether
such a condition as true megakaryocytic or megakaryoblastic leukemia or aleu-
kemia occurs, seems doubtful.

I J. LYMPHATIC LEUKOSARCOMA
With a lymphosarcoma of mediastinum, thymus, or other site, a lymphocytic
leukemia may be found at the same time or may develop later, sometimes very
rapidly, particularly in younger subjects. It is probably not justifiable to Jay the
blame for sucb an evolution on radiation therapy, as sucb a sequence of events
may occur in the natural course of the disease.

12. CHJ~OROMA
In this process, regularly described in text books, but seldom seen in practice,
tbere are greenish myeloblastic tumors, particularly in tbe bones of the skull, in
young subjects. It may best be regarded as a variant of a myelogenous~ form of
leukosarcoma.

13. SUBLEUKEMIA
In this group may be placed the cases of so-called aleukemic 'leukemia, and
low-grade leukemia, in which there are the clinical and pathological features of
 TREATMENT OF LEUKEMIA

leukemia but in which the peripheral blood shows a low total white cell count-
even a leukopenia in some cases-and relatively few abnormal forms. An aleu-
kemic myelogenous leukemia without an enlarged spleen, or an aleukemic
lymphatic leukemia without enlarged lymph nodes may present considerable
difficulties for diagnosis.

I4. LEUKEMOID STATES

In various conditions such as tuberculosis, metastatic carcinoma in bones,
pertussis, various septic conditions, and poisoning by various chemicals, (re-
cently sulfanilamide and allied chemicals) a leukemoid process may arise that
resembles leukemia. In this borderline zone occur the apparent recoveries from
leukemia, and it is usual to say that if recovery occurs the disease could not
have been leukemia. However, there would seem to be room for speCUlation as
to whether leukemia may not in its early stages be a reversible process, alld in
some of the cases it is most difficult to decide wbether to call the process true
leukemia.

METHODS AVAILABLE FOR TREATlvIENT

All methods of treatment of leukemia up to the present time may be con-

sidered only palliative, as there are exceedingly few if any reports of cures or
recovery that may be accepted.
Tbe methods available for treatment of the leukemic process may be divided
into:
I. those comprising a direct attack upon the leukemic tissues, as follows:
A. The various types of irradiation by means of x-rays, radium and
radioactive isotopes or other radioactive substances.
B. Arsenic and benzol.
II. those comprising adjuvants or measures more distinctly desigoed to re-
lieve various symptoms.
IRRADIATION ]\'iETHODS FOR LEUKEMIA

EXTERNAL IRRADIATION
This is tbe most generally available and most commonly employed method
of all.
a. Local x-ray therapy, that is, treatment delivered by an x-ray beam to local
parts of the body, as to cervical nodes or mediastinum. There is a wide range of
apparatus available for this method. Beginning with the least penetrating and
progressing to the more penetrating forms of x-ray therapy, there are the following
types.
1. Grenz rays, or x-rays generated at very low voltages, and suitable only
for treatment of skin complications.
2. Low voltage so-called "contact therapy" such as furnished by the Phillips
or Chaoul apparatus, using voltages of from 50,000 to 80,000, lightly filtered and
with bigh intensity. The scope of this modality· in leukemia is limited t~ the
 THE LEUKEMIAS

treatment of very superficial small lesions, which can by such apparatus be given
tbe desired dose quickly and conveniently. Neither of the two preceding types
of radiation has come into wide use for leukemia.
3.. Ordinary low-voltage x-rays, generated at about 100,000 volts, used unfil-
tered for very superficial lesions, or with from 1 to 4 mm. of aluminum filter, to
screen out the longer, less penetrating rays, and thus give a type of radiation
sufficiently penetrating to be suitable for lesions of some little thickness on, in, or
close heneath the skin.
4. Intermediate voltage x-rays, generated at 140,000 to 160,000 volts, filtered
by 3 to 6 mm. of aluminum or the equivalent. With the higher filtration this type
of energy may be useful even for some of the internal lesions of such a radio-
sensitive process as leukemia. Some clinicians prefer to use this type of roentgen
therapy at first even for deep~seated lesions, saving more penetrating radiation for
later, more refractory stages.
s. High voltage x-rays, generated C()mmonly at 200,000 Yolts, but in some
apparatus generated at 250,000 to 400,000 Yolts, and with filtration of from 0.5 to
2 mm. of copper or its equivalent. This type gives adequate penetration to
deliver a dose to deep parts tbat is quite satisfactory for treatment of leukemia.
It is commonly employed at target-skin distances of 35 to 50 cm. and the depth
dose may be further increased by increasing the target-skin distance to 70, 100,
or 150 em.
6. Super-voltage x-rays, generated at 700,000 to 1,000,000 volts, and pos-
sibly in the near future at even higher voltages, appear to have as their main
advantage the delivery of a greater depth dose in proportion to the dose affecting
tbe skin, and thus the skin can be mOle effectively spared. However, such power-
ful rays are probably entirely superfluous in the treatment of leukemic lesions,
even at the greatest depths.
b. Segmental x-ray therapy or large-field irradiation, usually employing in-
termediale voltage or high voltage apparatus, the distance and port factors being
so arranged that large segments of the body, as the entire chest or entire abdomen
may be irradiated in one sitting. Another use of the term segmental therapy
indicates the practice of some radiologists of treating leukemias and other lympho-
matoid processes by irradiating much of the surface of the hody, usually excluding
the head and the extremities, by dividing the surface into a certain convenient
number of fields, and systematically irradiating in turn one to a few of these
ports at a time until all have been treated.
It is but a step from this method to another method which has excited con-
siderable interest during the past decade, namely
c. Total irradiation (total teleroentgenotherapy; spray therapy). This is a·
method of simultaneously irradiating the entire body, or as used by some, of
irradiating half the body at a time or all the body exclusive of the head and
distal parts of the extremities. The usual technic is to use an intermediate or
a high voltage x-ray tube at a sufficiently great distance, (, to 2.5 meters) so that
the x-ray beam may encompass in one field as much of the body as the radiologist
wishes to treat, and to give doses of seldom more than 25 r at one sitting.
In the Heublein method of irradiating the entire body the target skin dis-
tance is greater (344 cm. in the present unit at Memorial Hospital; 540 and 730
 TREATMENT OF LEUKEMIA

cm. in the former nnit) and the intensity is reduced by appropriate adjustments
of milliamperes and filtration to only '.s rlhour (slightly less than , rlhour in
the former unit). With this low intensity it is possible to give the treatment
continuously for days at a time, as the average patient will receive about 18 to 20
hours of treatment in each 24 hours, therefore a dose of 24 to 30 r. Therefore the
unit is built between two adjacent hospital rooms, permitting the patients to rest
and sleep comfortably in bed while the treatment is given.
d. Local Radium Therapy. Formerly local radium applicators of small
radium content were used considerably by some radiologists in the treatment of
leukemia. This form of treatment proved to be fairly efficacious in producing
regressions of local masses, such as enlarged lymph nodes or an enlarged spleen.
It was a tedious method, however, and involved an undesirable amount of ex-
posure of the operator to gamma rays. For example, in order to treat an enlarged
spleen the applicator had to be repeatedly shifted from one small area to another
over the spleen surface. With the advent of larger radium or radon installations
of the bomb or pack type containing 2 to 4 Gm. of the element or the equivalent
thereof in radon, larger fields up to IS or 20 cm. or more in diameter could be
irradiated. Such apparatus may he used much like an x-ray apparatus for
efficient external irradiation of local fields. The longer exposure times for
treatments equivalent to usual x-ray doses, and the relative lack of sharp defini-
tion of the edges of the beam and the general unwieldiness of the apparatus have
combined largely to displace radium homb or pack treatments in favor of treat-
ment by modern x-ray installations.
However, in certain conditions the very fact that the beam of gamma rays
from a radium pack fans out much more widely than the beam Of x-rays from an
x-ray tube is of advantage. For example, a patient who is rather ill and at the
same time presents a widespread mass of leukemic tissue which one would prefer
to treat all together, such as an extensive intra-abdominal collection of nodes,
may to advantage be treated by centering the radium pack simply at one spot
over the center 01 the mass and letting the divergence of the rays include most
of the area at one time. In other words, at times the pack may rather efficiently
be used for a form of limited spray therapy. Yet there is little evidence for any
superiority of such a method over wide-field irradiation by x-rays.

INTRACAVITARY IRRADIATION
Rarely, in the treatment of leukemia there may be an indication for some
form of intracavitary irradiation. In some locations such as the mouth or vagina,
irradiation by x-rays may be most efficaciously directed to the lesion to be
treated through a metal cylinder of suitable caliber. In other locations such as
the nasopharynx or rectum, a radon-filled tube screened by suitable thickness
of platinum or other metal may be brought into close apposition to the lesion for
a time computed to give the desired dose.

INTERSTITIAL IRRADIATION
It is practically never necessary or desirable to treat a leukemic tumor by
implantation of gold radon-bearing seeds or by the insertion of radon- or radium-
containing needles, for leukemic processes are so radiosensitive and diffuse that
 THE LEUKEMIAS

they respond better to the more homogeneous irradiation effected from without,
than to the spotty irradiation produced by introduction within the tumor of
multiple point sources.

TIIORIUM, ETC.
Attempts have been made to treat leukemia by introducing within the body
various disintegration products of thorium, such as mesotborium, radiothorium,
thorium-x, and thorium emanation, as well as colloidal thorium dioxide (thoro-
trast). While definite favorable effects have been obtained with these substances,
there appears to be no evidence that they are in any way superior to the much
more readily available x-ray or radium therapy, and they have the hazard of
cumulative toxic effects of retained radioactive substances, similar to those seen
in the painters of luminous dials, who unwittingly ingested mesothorium and
radium salts.

RADIOACTIVE ISOTOPES
Possibly of epochal importance in the treatment of leukemia is the introduc-
tion of use of the radioactive isotope of phospharus by Lawrence and his co-
workers. By bombarding red phosphorus with high speed deuterons (nuclei of
heavy hydrogen) in the cyclotron, radioactive phosphorus is obtained. Con-
verted to a neutral solution of sodium phosphate and given by mouth in doses
estimated by experiments on monkeys and mice ta be less than one-tenth of the
lethal dose, calculated on a basis of weight, radioactive phosphorus shows greater
deposition in bone marrow and bone and probably greater deposition in leukemic
tissues} according to Lawrence, than in other tissues, and emits only beta rays,
having a penetration of only about 4 mm. in tissue. Furthermore the "half-life"
of radioactive phosphorus is only two weeks. By measurement with an electro-
scope or Geiger counter the amount of the radioactive phosphorus in the excre-
tions can be determined, and these measurements, together with a knowledge of
its rate of decay, make possible a fairly accurate estimation of the level of radio-
active phosphorus retained in the body at anyone time. It is even possible
roughly to estimate the dose on the basis of roentgen units (r) of whole body
irradiation. It is estimated by Marinelli that r millicurie of radioactive phos-
phorus retained in the body of an adult of average size for one day corresponds
to over 0.5 r of whole body irradiation.
Thus there is available a method whereby a leukemic patient may take by
mQuth at interval:=, (}of a few day:=, a :=,imple medicament, containing a radioactive
material which apparently is picked up rather selectively in those organs and
tissues where radiation is most needed, which emits there a localized radiation of
low penetrating power, and which has a rate of decay sufficiently slow to permit
convenient transportation and use before its value is lost, and yet not so slow as to
carry a threat of unduly prolonged radiation effects.
On these merits, radioactive phosphorus wordd appear to have a fair chance
to supplant other agencies of irradiation in the treatment of leukemia.
That radioactive phosphorus is capable of producing marked remission in
leukemia has been shown by Lawrence and is attested by the course of the one
 TREATMENT OF LEUKEMIA 4 19

case for which we have been able to obtain a sufficient supply of the material for
fairly adequate treatment. This patient, a man of 4' years, was first seen late in
January 1940, at which time he presented well-marked evidence of chronic
myelogenous leukemia. His spleen extended 4 em. to the right of the midline and
to the level of the iliac crest. His white cell count was 310,400 with 27 per cent

FlO. 3.-Pt. C. MeA. Graph showing response of a case of chronic myelogenous leukemia to oral
administration of ramo-acti,,"e isotope of phosphorus.

myelocytes, and his red cell count was 3.4 millions. At irregular intervals of from
2 to 40 days extending to early May, '940, he received radioactive phosphorus by
mouth in doses varying from 1.18 to 4.75 millicuries, the last dose being given on
May 7. The patient received no other form of therapy except hospital care and
oral hygiene. His condition improved generally; his appetite became unusually
hearty; his spleen sl~wly shrank, and his blood count improved at first slowly, and
finally in a dramatic fashion, so that in late May '940 his white cell count had
fallen to 29,600 with 4 per cent myelocytes, then to '4,000 with 5 per cent myelo-
cytes, while his red cell count had risen to 4.3 million, and the hemoglobiri to 91
per cent. On July 9, 1940 his hemoglobin was 97 per cent, tbe red cell count 4.84
million and tbe white cell count 15,900, with no myelocytes. The course in this
case is illustrated by the graph sbown in Fig. 3. _
It is of interest in comparison with total teleroentgenotherapy to note that
the dose of radioactive phosphorus in this case is computed to be equivalent to
175 to 200 r of whole body irradiation, spread over a period of 3' months.
We are informed by Dr. Lawrence tbat he now has 10 patients with leukemia
who, following treatment by radioactive phosphorus, show blood smears and
counts free from signs of leukemia for variable periods, one as long as three years.
420 THE LEUKEMIAS

ARSENIC AND BENZOL

ARSENIC
This ancient remedy for leukemia was resurrected and restudied by Forkner
about a decade ago, and he showed that hy relentlessly' pushing its administr"tion
to the point of toxicity, he could bring about striking remissions, particularlY in
chronic myelogenous leukemia. In general it produces responses best in those
cases which will also respond to x-ray therapy, but to some extent arsenic and
irradiation may complement each other, and one agent may produce some r"sult
after the other has appeared to faiL

BENZOL
Benzol has largely fallen into disrepute in the treatment of leukemia, because
of its dangerous toxicity. In our observation at 1I1emorial Hospital years ar,:o of
cases that had been treated elsewhere with benzol we were impressed with the
prolonged effects of the drug on the blood count, contraindicating x-ray theiapy.
However, there was failure of clinical betterment comparable to that seen fojlow-
ing effective irradiation.

ADJUVANT AND PALLIATIVE MEASURES

Transfusions. In general it may be said that transfusions are only of tril nsi -
tory benefit, at the most, in leukemia. Yet in many cases of acute leukemia and
acute and terminal exacerbations of chronic leukemia in which one fears tct use
irradiation to more than a minimal extent, if at all, one is left with hardly any
alternative to transfusion. Tn some cases it seems to postpone the lethal outcome.
In children repeated small transfusions seem to be preferable, and in ailults
m<lde.rate. am<lUtl.ts slJ.<:h as .,cc t<l 6cc <:<:. o{ ",hole hlood seem >;Ire{exahle. t<l l~r~r
amounts. However, it is not an uncommon experience in a leukemic subject to
find the red cell count lower instead of higher a few days after a transfusiotl has
been given.
I ron appears to be notably ineffective in general in causing hemoglobin re-
generation in leukemia.
Liver preparations are commonly employed but the effects are almost totally
unimpressive in leukemia.
Vitamins K and C seem largely ineffective in controlling the hemorrpagic
state in leukemia.
Rest is of great importance to the leukemic-rest of mind as well as of pody.
It is important to keep from the patient if possible a full realization 01 the
lethal nature of his dbease; and if he has been allowed to learn the diagnosis
and its significance it is helpful to minimize the serious possibilities for his
future and to allow him to believe that his is one of the atypical cases ,vhich
distinctly offers hope for restoration to health, though the road to travel m,W be
long and trying. Those who believe that honesty requires a complete revelation
to the patient of his affliction with leukemia and of its inevitable fatal termina-
tion have a very weak foundation for their position, save in unusual circumst£l.DceS
and witll patients of uncommon fortitude.
 TREATMENT OF LEUKEMIA 421

The patient should be spared all mental stresseo as far as possible. In two
instances I witnessed a rapidly fatal acute exacerbation of leukemia following
the death of a near relative of the patient.
Hygiene. The leukemic has poor resistance to infection. It is therefore par
ticularly important for him to avoid infection as far as possible by the practice
of good hygiene. Many of these patients are found to have dirty mout);S and
decayed teeth. Careful daily cleansing of the teeth with a soft brush and rinsing
the oral cavity using a mild soap or sodium perborate as a dentifrice, mouth
wash, and gargle are helpful in cleaning up an important source of sepsis. pental
work involving any trauma to the gums, pulp cavities or tooth sockets; tonsillec-
tomies, and intranasal or sinus operations should, in general, be avoided j if abso-
lutely necessary, undertaken only most carefully. Daily soap baths are doubtless
an aid in tending to prevent tbe occurrence of some of the pustular skin rashes so
prone to occur in leukemia, especially in the exacerbations of the lymphatic type.
The leukemic should observe extra precautions against re~piratory infections, by
oral hygiene, by avoiding crowds, overheating, fatigue, excessive smoking, chill-
ing, and exposure to others having such infections.
Ileliotherapy. The benefits of natural sunshine in moderation are often
fairly noticeable, and have been emphasized by Naegeli. In some cases natural
or artificial heliotherapy seems to tend to check the hemorrhagic tendency.

TREATMENT OF COMMON FORMS

EXTERNAL RADIATION

CHRONIC LYMPHATIC LEUKEMIA

TTIlica.Uy this is found as a disease o{ middle nr late li{e, ",resentin'l. a ",atient
with a rather uniform symmetrical generalized enlargement of superficial lymph
nodes, including those of the neck, axillae, groins and femoral triangles. Many
cases have extension of lymphadenopathy to somewhat unusual sites, as to pre-
and postauricular, occipital, submaxillary and submental, suprascapular, post-
axillary, lateral mammary regions, to the inner aspect of the upper arm in chain
fashion down to the epitrochlear group, and occasionally single nodes on the
lateral chest or abdominal wall may be found. The liver and spleen may be
enlarged to several finger breadths below the costal border, and there may be a
readily palpable central abdominal mass, made up usually of retroperitoneal
nodes. Usually a chest film shows only some accentuation of the hilar sMdows,
with or without minimal mediastinal widening, while rarely the lung parenchyma
may reveal nodular or diffuse infiltration, or the pleura may be infiltrated and
effusion may result. There are all gradations between this full blown pictufe and
the case which presents no palpable lymph node enlargement, and there are
likewise all gradations between the typical blood count of chronic lymphatic
leukemia, with hundreds of thousands of white cells in each cubic millimeter of
blood, mostly small lymphocytes, and the cases that have an aleukemic or sub-
leukemic count. Cases no doubt occur in which there is both an aleukemic blood
picture and an absence of palpable adenopathy and of enlargement of spleen and
liver. Such patients naturally present confusing diagnostic and therapeutic prob-
 THE LEUKEMIAS

lems. Some cases may simulate chronic myelogenous leukemia, by having huge
spleens, and little or no palpable peripheral lymphadenopathy (the splcnomegalic
type of lymphatic leukemia).
In the irradiation treatment of chronic lymphatic leukemia the general prin-
ciple is to attack the foci of lymphocytic infiltration. In the typical case with its
universal lymphadenopathy and relatively minor enlargement of spleen and liver
it has seemed logical to irradiate mainly the lymph nodc fields-tbe internal as
well as the external. In the splenic type, with a big spleen anil relatively minor
lymph nOde enlargement the first attack would logically seem hest directed to
the spleen. The more atypical cases call for even more individualization, thus
for direction of irradiation to whatever foci seem to be most threatening to the
patient's health.
In the program of irradiation of lymph node areas in the typical case, pre-
senting a rather uniform symmetrical lymph node enlargement one useful methorl
is to begin by treating the external nodes only. This serves as a test 01 the radio-
sensitivity of the nodes in the given case, and of the patient's tolerance to irra·
diation, as well as 01 the effect to be expected on the blood count. In such a cycle
appropriate ports are mapped on each side of the neck, each axilla and each groin
and femoral triangle, and each such port is usually treated with a single dose 01
from lOa to 300 r, using roentgen rays generated at from 140 to 250 kv. and fil-
tered correspondingly through from 4 mm. of aluminum to 1.5 mm. of copper.
One port is treated at a time and intervals between treatments may be from
one to three days. These factors are varied according to the general state of the
patient, the bulkiness of the lymph node masses, and the type of x-ray apparatus
most readily availahle. After a period of three to five weeks or more such a test
cycle may be followed by a considerable regression of tbe nodes, both those that
were directly irradiated as well as others, and there may be a marked favorable
effect on the blood count, along with a distinct improvement in the patient's gen-
eral stale. Anemia may largely disappear, the total white cell count, if it was
high, may drop to nearly normal, and lhe differential count may show a reversal
of the polynuclear :Iymphocyte ratio to nearly normal.
If such a degree of improvement occurs, the object of treatment, palliation,
has been well accomplished temporarily, and it seems good judgment to wait
and watch for some definite reason for resuming treatment. Some cases may
be able to get along very well for several to many months, after such a prelimi-
nary cycle. (Fig. 4)
Unless some special feature indicates otherwise, usually the next course of
irradiation is directed to the main internal lymph node areas, that is to the
mediastinal and prevertebra! retroperitoneal nodes. By irradiating a central
trunk zone about 10 cm. wide, divided into upper, middle and lower thirds an-
teriorly and posteriorly, these deep lying nodes can be effectively treated, prefer-
ably with roentgen rays that are generated by 200 to 250 kv., with filtration cor-
respondingly of from 0.5 to I.5 mm. of copper. As in the first test cycle, each
port receives a single dOSe of from lOa to 300 r, and the treatment intervals are
from one to three days, or one may go around twice or more with suialler doses, 50
 TREATMENT OF LEUKEMIA 423
to 'So r, at longer or shorter intervals, all depending on the clinical estimation
of the patient's ability to stand the treatment, based this time in part on observa-
tion of his response to the first cycle.

FIG. -4 -Pt. H. H. Graph of white-cell count over an eight-year period in a case of chronic
lymphatic leukemIa. Arrows indicate courses of roentgen therapy. Note that after five years the
need for treatment arose mUle frequently, and the 1cukocytOSI:3 reached lugher levels. Pallent 5ur-
vived about two years after last date indicated on the graph.

Subsequent courses of irradiation may be repetitions of these cycles to ex-

ternal or int!,rnal nodes, or both; or particular fields such as liver or spleen,
abdominal masses, or iliac, pelvic or inguinal nodes may require special attention.
As in other forms of leukemia each case must be individualized, and there-
fore the above description of a tecbnic applicable to some more typical cases
sbould not be taken to mean that all cases sbould be treated in this way.
Total body irradiation by the Henblein prolonged continuous method or by
intermittent exposures or by a segmental or large field technic may he very effica-
cious in some cases. (See Figs. 5, 6, 7, 8.) In fact the treatment of selected
cases may be begun and largely carried on by such a method, reserving focal
irradiation as a supplement to be used only if needed to care for especially promi-
nent or threatening local masses or infiltrations. In the rare cases in which en-
largement of lymph nodes, liver and spleen or other gross foci of leukemic
infiltration are minimal or absent, and in the occasional case with extensively
distributed skin infiltrations, one would naturally consider total irradiation as
a logical procedure. In the Heublein method doses of from 25 to 100 r to the
entire body ar~' commonly used.
The attack upon chronic lymphatic leukemia by irradiation is, then, in sum-
mary, based on the principle of best practicable palliation by means of directing
the treatment to foci of lymphatic infiltration in lymph node areas or elsewhere,
seeking always to keep the dosage within the bounds of what the patient can
stand and keeping in mind that after all it is palliation and not cure that can
be accomplished.
The degree of palliation may be very striking in some cases, moderate in
424 THe LEUKeMIAS

many and minimal Or absent in some. About nine or ten per cent will show a
fit.e year survival; most of the cases terminating within three years, while a few
will last 10 to 12 years or longer,

75,000 case J.V.

Lymphatic
Leukemia

R.B.C.
2,976,000
3,428,000
4,096,000

FIO . ., -Pt. J. V. Graph shO'Ws rise of red blood cells and marked decrease of white blood celh
following total irradiation. (2S0/0 SED;;.:;: z87 r; 10% SED == 75 r).·

CHRONIC MYELOGENOUS LEUKEMIA

The typical case of chronic myelogenous leukemia has a large spleen, which
may occupy almost the entire front of the abdominal cavity, a more or less
enlarged liver, and no palpable enlargement of lymph nodes. When nodes are
palpable in a cas'l, of myelogenous leukemia the prognosis is particularly bad.
While various technics of irradiation have been suggested it seems that in
general such typical cases are best treated initially by irradiation directed to the
spleen.
The spleen may be treated by x-rays through one or more large ports an-
teriorly and posteriorly, using 140 to 250 kv" corresponding filtration of from
5 rom. of aluminum to I.S mm. of copper, individual doses of 25 to 100 r at inter-
vals of one to three days, depending on the rapidity with which it seems advisable
10 treat the given case, and a total dose in the first cycle of 300 to 400 r to each
port. For a patient who is ambulant and who presents a typical case, the first
cycle may he completed in 6 consecutive days of treatment, giving 3 X 100 r of
200 kv. x-ray irradiation at 50 cm. target-skin distance, to a single large port
over the spleen anteriorly and the same dose posteriorly_ If the radium pack
is used in such a case, about the same result is accomplished by giving 3 X 2000
mg. hours anteriorly and posteriorly at 10 em. radium-skin distance.
In a favorable case such a course of treatment, while it may be followed by
'an early transient increase in leukocytosis, will result in about a month in a
marked improvement. The spleen may shrink so that it cannot be felt; the liver,
if it is enlarged, may likewise recede; the white blood cell count may be restored
 F_[G.,5. Plo J. \. Chronic lymphatic leu- FIG. 6.--Pt. J. V. Twelve week:!) later and 10
kemia before treatment. Edge of liver and dnys follo,.... ing- :;ccond course of total body irradia-
spleen outlined. \Vhile blood cells 275,(X)(). tion in Heuble'n lmit. Received Ig7 r in I2 days
Small Iymp.hocytes 100 per cent. and two month::; later recein:,d 75 r in four days.
\Vhite cell count one month after ~e-cond treatment
was 21,600 ·with 97 per cent small lymphocytes.
(See opposite for Fig. 7)

Fw. 8,- Pi. R. H. Chronic lymphatic 5ublellkemia. Chan:...::t" in appearann.' of patient in 2H

day~. ~ccond photograph taken lb day!;. following completion of 187 r of total body irradiation in
HeubleIn unit. Dose delivered in nine days of continuous residence in Heublein un.it.
 TREATMENT OF LEUKEMIA

to SO nearly normal that a diagnosis of leukemia would be difficult; the red cell
count may become normal; and the patient may gain several pounds and feel
as well as ever. (See Fig. 9.)
Before ordering such a course of treatment, however, one must beware of
failing to recognize certain signs indicating that the case is more acute than it
might seem to be at first inspection. The presence of much fever, of well-marked
hemorrhagic tendency, of infection, of severe anemia (for example, a red cell
count of less than two million) or of enlarged lympb nodes, should lead to a recog-
nition of II more acute type of process and hence the need for greater caution in
treatment. There is danger in the overdosage of the more acutely ill cases. Even
sudden death has followed the unwise irradiation of unrecognized acute cases by
doses suitable for typical cbronic cases.
It is commonly stated, and accepted, that it is not necessary or even advis-
able to try to reduce tbe wbite cell count to normal and tbat a fairly stable level
of 20,000 to 40,000, or even IOO,OOO white cells is compatible with a state of well
being. Certain cases, however, have suggested to the writer that wben it is pos-
sible to bring the white count down to a normal level without having the reduc-
tion go on into leukopenia, there is likely to be a more nearly complete temporary
restoration to bealth and a tendency to a longer interval before the next cycle
of irradiation becomes necessary.
Frequent re-examination of the patient is necessary in order to determine
when further treatment is indicated. Intervals between such examinations
sbould seldom be longer tban three weeks. At these times a blood count is taken,
tbe symptoms are carefully surveyed, and the patient is thoroughly inspected for
signs of the disease, particular attention being paid to such points as infection,
hemorrhagic tendency, lymph node enlargement, (usually a bad omen in'myelog-
enous leukemia), pain or tenderness about bones and changes in size and con-
sistence of spleen and liver.
The period of regression varies from a few weeks to several months or even
a year or longer. As long as the patient is comparatively symptom-free, and
the blood count is not showing increased evidence of leukemia, nothing is to be
gained by further irradiation; in fact, ground may be lost by overenthusiastic
attempts at "maintenance therapy" in an effort to preserve indefinitely the at-
tained favorable state. Yet certain experimental efforts in tbis direction seem
justified, as it is quite conceivable tbat if one could find the proper dose to give
in such circumstances, a certain small dose, perhaps to large segments or to the
whole body at suitable intervals, might serve to preserve a favorable state for
a long time. At present however slIch attempts must be entirely empirical as
we have no measurable factors otber than the blood count or possibly the
metabolic rate upon wbicb to base tbe determination of proper doses and proper
intervals for such maintenance therapy. Chemical researcb in the future may
be able to furnish some estimation of metabolites tbat will be useful in this
connection.
As for definite signs of the need for further irradiation, the blood count
usuall;, gives the first warning. When tbe white cell count again begins to show
 THE LEUKEMIAS

a distinct upward trend and the number of myelocytes begins to increase, one
has evidence that relapse is occurring.
At this stage, if the spleen has enlarged again it may be treated by another
cycle of irradiation. However, not infrequently the blood count and recurrence
of symptoms indicate the need for further treatment while the spleen and liver
still remain reduced to normal or nearly normal size. It is then a question
whether the small spleen should be treated again, or whether treatment should
be directed to some other part, such as the bones, of to large body segments, or
to the whole body. Particularly in those cases in which pains about the bones
and joints are a prominent feature does irradiation directed to the bones seem
indicated.
In treating bones, the sternuIll, entire spine, and ends of the long bones are
selected as the areas to be irradiated, and each such area is usually given a single
dose of 100 to 300 r high voltage roentgen therapy.
Sooner or later, commonly after repeated cycles of irradiation have been
given to the spleen, it becomes refractory and no longer softens and shrinks fol-
lowing treatment. Some authors have reported not only satisfactory results
on the blood count and symptomatic improvement in such refractory cases fol-
lowing total irradiation"but even a shrinkage of such spleens. The Heublein
treatment has never shown us a shrinkage of refractory spleens in myelogenous
leukemia, but no doubt such a result is quite possible.
The results of irradiation of chronic myelogenous leukemia can be measured
only in terms of palliation and length of survival. In only a relatively small
percentage (about 6 per cent) of the cases is there a survival beyond 5 years
after treatment is begun. Statistics seem capable of showing only some months
of increase of average survival in treated cases as compared with untreated
cases. However, the very marked palliations and the endowment of the patients
with periods of health and economic usefulness which they would by no means
gain to anywhere near the same extent by spontaneous remissions, fully justify
irradiation for this disease. Averages cannot tell tbe whole, story and undoubt-
edly in many instances the remissions produced by irradiation have staved off
for many years lesions that would otherwise have produced a rapidly fatal
course.

ACUTE LEUKEMIA
The treatment of the acute leukemias by irradiation offers comparatively
little and therefore there is no need of separately discussing the treatment of the
various histological types. Whether the acute leukemia be myelogenou's, lymph-
ogenous or monocytic makes very little if any difference in course or in prognosis
in relation to irradiation therapy.
There are a few instances in which apparently an acute process :has been
converted into a subacute or more nearly chronic one. In general, however,
there is considerable risk of increasing the severity of the disease by irradiation.
Especially the hemorrhagic tendency may at times seem to .have been aggravated
by irradiation.
 TREATMENT OF LEUKE1IIA

Judicious employment of small doses may be of some benefit, however. In

children the severe pains and swellings about joints, simulating rheumatic fever J
may respond to small doses of x-ray therapy. We have at times used as little
as 5 r of high voltage x-ray therapy either to the whole body or to large segments
of the trunk in treating children with acute leukemia. The spongy bleeding
leukemic gums may respond to some extent to small doses (100 to 200 r), but
as a rule the response is fleeting and minimal. In the leukosarcomas, such as the
cases of acute leukemia in children with bulky mediastinal masses, the tumors
may regress rapidly following small doses of irradiation, and at the same time
there may be an alarming drop in the total white cell count, giving a striking
warning of the probable dangerous effect of larger doses. (See Figs. ro, II.)
Painstaking nursing care, induding oral hygiene, (Figs. 12, '3) together with
repeated sman transfusions, perhaps the administration of arsenic, and most cau-
tious use of irradiation when it seems safe, summarizes what we are able to do
for acute leukemia. Irradiation by means of ingested radioactive phosphorus
seems worthy of further trial, or perhap~ some other radioactive isotope may
prove to be of value.

TREATMENT OF SOME COMPLICATIONS

THE HEMORRHAGIC TENDENCY

This is one of the most dreaded complications of leukemia. As a rule it is
accompanied by a low platelet count. When a low platelet count and bleeding
are present, great caution must be used in giving irradiation, for fear of increas-
ing the bleeding and of being forced to stand helplessly by and watch the patient
die. A severe hemorrhagic pictnre is always a sign that the end is not far off, but
a mild bleeding tendency in a case of chronic leukemia may disappear at least
temporarily following treatment of the leukemic deposits by irradiation. Helio-
therapy may in some cases seem to tenet to lessen the bleeding. Large doses
of calcium and increased dietary vitamin intake, especially of C, may also seem
to have a little favorable effect on the tendency to bleed, but an established
,evere hemorrhagic state is uncontrollable as far as we know today, despite the
availability of vitamins C and K.
Transfusions are employed when there has been marked bleeding, but their
replacement of lost blood is usually only very transitory, as usually the bleeding'
continues unabated.

INFECTIONS
These must be treated conservatively, avoiding surgical incisions and trauma
whenever possible. The dirty mouths and gums presented by many leukemics
can be cleaned up considerably by the frequent use of a mild soap as a denti-
frice and mouth wash. Only the softest of tooth brushes should be used. and if
the gums are very spongy a cotton swab is preferable. The extraction of teeth
or intranasal or sinus operations may be very hazardous in a leukemic patient
and may seem to initiate a downhill course.
 THE LnU'KEMIAS

It is an old observation, however, that in some leukemics an infection may

cause a transient reversal of the blood count towards normal and a remission of
symptoms and other signs of the disease.
We have recently treated a pneumococcus pneumonia in a child with acute
lymphatic leukemia, by giving full doses of sulfapyridine in the face of a white
cell count of only 900, and observed recovery from the pneumonia and no apparent
ill effects from the drug.

PREGNANCY IN LEUKEMITA
If early pregnancy is dhcovered in a leukemic the question of therapeutic
abortion always arises and must be seriously considered. However J if the leu-
kemic process is well advanced, or acute, there is likely to be a rapidly fatal
termination for both mother and child anyway, and therefore nothing would
be gained hy producing an ahortion either surgically or by irradiation. In such
instances it is probably best to be content to attempt merely to do whatever is
possible in a palliative way by irradiation, arsenic or other means and to let the
pregnancy alone.
Pregnancy occurring early in the course of a chronic leukemia may be able
to go to term without marked ill effect on mother or child. The child may be
normal and is very unlikely to show any signs of disturbed blood formation.
If irradiation is indicated it may be given to the mother with no particular
consideration for the complication of pregnancy, except for the important fact
that if the child lives and if it has received exposure to radiation during gesta-
tion, it may show some abnormality, such as one of the monstrosities or micro~
cephalic idiocy. Therefore, as suggested by Forkner, treatment of the mother's
leukemia by arsenic rather than by irradiation during the period of gestation
should be seriously considered in those cases of leukemia complicated hy preg-
nancy in which there is likely to he a viable fetus.
As leukemic women become pregnant only rather rarely, routine sterilization
does not seem to be in order.
No rule can be laid down for the treatment of gravid leukemic women. Each
case should be judged on its own merits, by consultation of internist, obstetrician
and -radiologist.

HERPES ZOSTER
Herpes zoster is a fairly frequent complication in leukemia, as in the other
lymphomatoid diseases. A self-limited disease, it frequently needs no special
treatment. Painting the area of vesicular eruption with collodion, or applying
a I per cent cocaine ointment may give sufficient palliation to tide the patient
over until the process subsides. If the lesions on the skin are unusually deep,
painful or necrotic, constant warm wet soaks in saline or magnesium snlphate
solution may afford considerable relief and prevent the formation of adherent
dry crusts retaining purulent secretions. Irradiation seems at times to hasten
the devolution of herpes zoster. A dose of 300 to 500 r of 200 kv. x-ray therapy
at SO cm. target skin distance with 0.5 mm. copper filtration may be given to the
 FIG. lO.-PL H. L. B. Th.h'diastinal tumor in ~Kute lymphatic leukemia in a. child.
F1C. 1l.~Pt. H. L. B. M:1rk~d rcgr(!ssion of mediastinal tumor lollowing ~maH doses o[ roentgen
therapy_ Also sharp decrease in white cells from 1501000 to 1500. Death after Lince months.

l-';1G. ]_2.-Pt. C, Z. Noma. in acule lymphatic leukemia in a chilJ..

}·lG. 13.-1J t. C. Z. Se\-'ere progression of noma in one week. Death one d3j' Jatcl".
 TREATMENT OF LEUKEMIA

appropriate segment of the nerve roots, and laO to 200 r of lightly filtered low
voltage x-ray therapy to, the areas affected by tbe rasb. Injections of pituitrin
and Vitamin B-r have been recommended.
Occasionally, in addition to the zonal herpes, few or many scattered vesicles
appear or generalized herpes. I bave never been able to find a history of con-
tact with a case of varicella in such instances.

GROSS LESIONS OF BONES

Such gross lesions as would be detectable roentgenographically are consid~
erably less common in leukemia than in Hodgkin's disease and lymphosarcoma.
Patients with bone lesions appear to 11ave no different c::our~e otllerwise, although
Copeland and the writer have noted a tendency for the cases of lymphatic leu~
kemia with bone lesions to be of the subleukemic type.
Regarding the bone lesions as simply further sites of leukemic infiltration
no special statements need be made regarding their treatment. Moderate
doses of high voltage x-ray therapy will usualJy suffice to cause relief from
symptoms, regression of tumors and sometimes bony~ repairs.

PRIAPISM
It seems that the empbasis and importance of priapism as a symptom of
leukemia has been exaggerated, particularly in textbooks. Its cause is not en-
tirely clear. No doubt some of the cases are due to stru;is of leukocytes and
thrombosis in the corpora cavcrnosa, or to leukemic infiltrations. Surgical in~
tervention is not the first measure to apply. The treatment of the leukemia is
the first consideration and several writers have reported satisfactory results by
treatment of the entire disease process.

BIBLIOGRAPHY

CKAVER, L. P.: "Clinical manifestations and trea.tment of leukemia." Amer. Jour. Cancer, 26, 1':14,
1935 .
CRAVJ!:R, L. F.: "Irradiation in the lymphomatoid diseases." Bull. N. Y. A(:ad. Merl., 1a. 442, 1939.
CRAVER, L. F." «Lymphomas, leucemias, and allied disorders in children." Jour. Pediatr., 15, 332,
r93f)·
CRAVSR, L. F.~ BRAUND, R. R., and TYLER, H. Y.: "Lesions of the lungs in thc lymphomatoid dis-
eases." Radiology (to be pUblished).
CRAVER, L. F, and COPELAND, M. M.: "Changes of the bones in the leukemias." Arch. Surg. 30.
639, 1935,
CRAVER, L. F., and l'tlACCoMB, W. S .. "Lymphatic leukemia with thymic enlargement: A brief Ie·
view of the bterature wjth case reports," Atner Jour. Cancer, 16. 277. 1932.
CRAVER, L. F., and MACCOMB, W. S.: "Heublein's method of continuous irradiation of the entire
body for gcner:l1.lZed neoplasms." Amer. Jour. Roentgenol and Radium Ther, 32, 654. 1934.
CRAVER, L. F.) and HAAGENSEN, C. D.: "A note. on the occurrence of herpes zoster in Hodgkin's dis·
ease,lyrnphosarcoma, and the leukemias." Amer. Jour Canc~rJ 16, 502, 1932.
DOAN, 1\. A, and RINER ART, H. L.: IjThe ha~phi.1 granulotyt~, basophi!cytosis, and myeloid
leukemia, basophil and Imixed ~ranule' tYPe3; An experimental, clinical and pathological ~tudy
wlth the report of a new syndrome" Amer. JOllr. elln. Path, II, 1, 1:941.
FO~][N:t.R. C. E.: "Leukemia and allied disorders." New York, Macmillan l 193~t
43 0 THE LEUKEMIAS

f{Ol"l'MAN, W. J., and CRAVER, L. F.: "Chronic myelogenous leukemia, value or irradiation and its
effect on the duration of life." Jour. Amer. Med Assn" 97, 836, 193:r,
LAWRENCE, J. R.o SCO"l"T, K. G, and TUTTLE, L. W.: j'Studies on leukemia with the aid of r.ldio~
active pbosphoru3." New Internat. Cbn, 3, 33, "1939.
LAWRENCE, J. II.. "Nuclear physics and therapy. Prehminary report on a ne," method for the
treatment of leukemia and polycythemia." Radiology. 35, 51, 1940.
MAJm{:t:;LLI, L. D., and KLNN"'l, J. M.' I'ersonal communication.
 SECTION SIX
MECHANISM AND CLASSIFICATION OF
HEMORRHAG IC DISEASES
ESSENTIAL THROMBOCYTOPENIC PURPURA
HEMOPHILIA
NON-THROMBOPENIC PURPURA
VITAMIN K
 SECTION SIX
HEMORRHAGIC DISEASES

CHAPTER 34
MECHANISM AND CLASSIFICATION OF THE HEMOR-
RHAGIC DISEASES
COAGULATION OF THE BLOOD

For a proper knowledge of the various factors that may he responsihle for
hemorrhages and for a thorough understanding of the underlying reasons why
certain hemorrhagic states are present, it is desirable that the formation of the
normal blood clot be discussed. This is especially import~nt in the classification
of the purpuric diseases and, therefore, for the use of treatment on an intelligent
basis.
The following discussion of normal coagulation is based on the theory eluci-
dated by Howell. The following are the components necessary' for the formation
of a normal blood clot, with other factors that may play a role in the process.
r. Fibrinogen is a substance that is found in the normal blood plasma. It
is probably formed in the liver, and released into the blood stream in
sufficient quantity to maintain the normal percentage of four-tenths of
one per cent. It is not demonstrable, has 110t been isolated, and is the pre-
cursor of fibrin strands that are seen in the normal clot.
2. Prothrombin, the inactive precursor of thrombin, exists in the blood as a
globulin structure, and is derived to some extent from normal blood plate-
lets. It is thought to be held in an inactive state and rendered inert in
the circulating blood by the presence of a substance known as antithrombin
(heparin).
3. Antithrombin (heparin) is formed in the liver, exists there in large
quantities, is released into the blood in sufficient amount to prevent the
activation of prothrombin.
4. Calcium salts are present in the whole blood to the extent of about 10
mg. per 100 cc. of blood. Their function seems to be the release of cal-
cium ions to unite with prothrombin and form the thrombin.
5. Thromboplastin (cephalin) (thrombokinase) is not present in' the blood
plasma. It is derived to some extent from the platelets after clotting has
begun, but largely from tissue juices that escape from ,incised or trauma-
ti~d tissue. This substance initiates the clotting process.
4 ••
434 HEMORRHAGIC DISEASES

6. The blood platelets (thrombocytes) are present in normal blood to the

extent of 300,000 to 500,000 per cu. mm. and in addition to releasing small
amounts of thromboplastin, they play their most important role in
causing a retraction of the clot from the wound edges, giving strength
and firmness to the' clot, as a final stage of the process of coagulation.
Platelets are responsible for the ability of the clot to retract. They play
a dual role, therefore, in the process of coagulation; they not only initiate
the process but complete it.
Normal coagulation is initiated by the escape of thromboplastin (cephalin).
This substance may arise from tissue juices in the case of extravascular bleeding
or from platelets in the case of intravascular clotting. Thromboplastin then unites
with antithrombin, which, ill turn, releases or activates the prothrombin. This
substance is activated by calcium ions to form thrombin. Thrombin in turn unites
with fibrinogen to form strands of fibrin, which is the first physical and micro-
scopic evidence of the coagulation process. Some of these suhstances in blood
clotting may act as enzymes; for example, Eagle has shown that a very small
amount of thromboplastin is sufficient for the activation of prothrombin to throm-
bin and even a hundred times that amount fails to increase the amount of
thrombin formed but only accelerates its formation. Furthermore, I mg. of a
crude preparation of tbrombin may convert several thousand milligrams of
fibrinogen to fibrin. This cannot be explained on a basis of chemical comhination
and quantitative relationship. It is probable tberefore, that thrombin itself is
a proteolytic enzyme.
The strands of fibrin, which are sticky, adhesive and interlacing, then serve
as a barrier in which large numbers of red cells, leukocytes, and platelets become
enmeshed, the entire mass constituting the blood clot. At this stage, although
the formation of the clot is complete and coagulation has occurred, bleeding does
not stop. The next major step is the retraction of the clot which gives the
necessary strength and firmness to effect the cessation of hemorrhage. This
function is carried out by the platelets. Therefore, in diseases characterized by
low or absent platelets, clot retraction does not occur. In such cases, the coagu-
lation or clotting is complete and the coagulation time is normal, but the bleeding
time is prolonged. An example of this is the mechanism seen in idiopathic
thrombocytopenia. ,
The process of clot formation and retraction may be illustrated by the fol-
lowing diagram.
Thromboplastin (cephalin} + antithrombin ~ The release of prothrombin.
Prothrombin + calcium ~ Thrombin.
Thrombin + fibrinogen ~ Fibrin.
Fibrin + the cellular elements ~ The completed clot.
The completed clot + platelets ~ The retracted dot with cessation of bleeding.
It can be seen at once that the complexity of the clotting mechanism and the
incrimination of so many substances have been responsible for the use of the large
 COAGULATION OF BLOOD 435
number of agents tbat are claimed to be of hemostatic value. Furthermore, on
tbeoretical grounds, a diminution of any of these substances would be responsible
for various hemorrhagic states and any of the following deficiencies could be re-
sponsible for prolonged bleeding:
r. Deficiency of fibrinogen. Since tbis is formed in tbe liver it is possible
tbat a disease characterized by marked destruction of liver tissue would
result in inadequate amounts for release to the blood stream. Example:
Atrophic cirrhosis; Banti's disease.
2. Deficiency of calcium. This is rarely seen, and it is doubtful if any hemor-
rhage ever occurs because of a calcium deficiency.
3. Deficiency of prothrombin (or Vitamin K). This has been stated to be
the missing factor in melena neonatorum (Whipple). See chapter on
Vitamin K.
4. Deficiency of thromboplastin. This is quite rare and it is questionable if
it plays any part in the prolonged bleeding if there is available any tissue
juice. The amount of thromboplastin from platelets is probably important
in hemophilia.
s. Deficiency of blood platelets. This is by far the most common cause of
hemorrhages, mainly because of failure of the clot to retract. It is seen in
thrombocytopenic purpura (purpura hemorrhagica), aplastic anemia, and
aleukemic leukemia.
6. Marked delay in the time of the entire process even though the quantita-
tive relationships of tbe components are normal. Eagle believes' that the
mechanism of hemophilia is the slow formation of thrombin.
7. Excessive amounts of antithrombin (heparin). Therefore tbe amounts
of thromboplastin would not be adequate to neutralize tbe antithrombin,
leaving some of it still retaining its union witb prothrombin, preventing
the latter from activation.
Since many concepts of blood clotting still remain somewhat speculative in
spite of the brilliant work of Howell, it seems probable that as knowledge in-
creases the above concept of clotting will be modified and changed in the light of
discovery of new facts. -

,CLASSIFICATION

From a study of the factors involved in normal blood clotting, it is obvious

that bleeding may occur because of a deficiency of either one of two large classes
of factors in the 'process. First, there may be a deficiency of one of the factors
enumerated above in the formation of tbe clot; or secondly, there may be a
deficiency of platelets with failure of the clot to retract. Also it has been
definitely shown tbat some types of hemorrhage occur because of an inherent
weakness of the capillary bed, tbis being known as increased capillary fragility,
decreased capillary resistance, or permeability of the capillaries, anQ in such in-
43 6 HEMORRHAGIC DISEASES

stances there is no defect of any kind in the clotting mechanism. Therefore,

all hemorrhagic states can be divided into three large classes, as follows:
I. Those due to decreased numbers of platelets. (No clot retraction.)
2. Those due to a failure of the clot to form. (Deficiency of one of the fac·
tors in formation of the clot.)
3. Those due to weakness of the capillaries.
The above basic classification can be slightly amplified by inclusion of some
of the various factors that may be deficient, including fundamental reasons why
platelets may be decreased. This would result in the following classification:
JExcessive destruction
Decreased numbers
I. Dysfunction of platelets 1Inadequate formation
Altered function
Deficiency in fibrinogen
2. Failure of the clot to form Deficiency in calciupl
Deficient prothrombin (or vitamin K)
Nutritional
3. Capillary weakness Infectious
Toxic (chemical)
Allergic
Finally, if one prefers to include in a classification the various sources of
these deficiencies and ennmerate the possible factors that may bring these about,
it can be amplified further almost to the point of becoming too complex for
practical use.
Splenic destruction
Ovarian deficiency
Allergic (drugs) <as sedormid)
Aplastic anemia
r
Decreased numbers Leukemia
Septicemia
Arsenic, benzene, radium
I. Dysfunction of platelets X-ray
Bone marrow dysfunction
Megakaryocytic aplasia
Altered function

Vitamin K deficiency
Liver diseases
Atrophic cirrhosis
Deficient fibrinogen Banti's disease
or prothrombin Chloroform poisoning
Phosphorus poisoning
2. Failure of clot to form Extreme fa tty degeneration
Deficient calcium Overwhelming sepsi~
Deficient thromboplastin
Excesive antithrombin
Hemophilia (Mechanism unknown)
 COAGULATION OF BLOOD 437
AllergiC (focal infection; Henoch's; Schonlein's)

!
3. Capillary weakness Nutri~ional (vit:'min C ~eficiency; protein deficiency)
InfectIons (sepSIS of varIOUS types)
Toxic (drugs, quinine, potassium iodide, etc.)

Although it is well to consider the possible sources of the hemorrhagic states,

when these factors are included in a classification, it is impossible to classify with
accuracy. Many of the conditions named in -the above table, although causa-
tive of hemorrhages, may not bring it about as indicated but through some other
mechanism. Therefore for practical usage and simplicity, we believe the second
classification is adequate in the light of present knowledge.
From a standpoint of the 'blood findings in .the purpuric diseases, they may be
classified as (I) thrombocytopenic, and (2) non-thrombocytopenic. The first class
includes all of those that are characterized by decreased numbers of platelets
(thrombocytes) in the circulating blood, and are referred to as instances of
thrombocytopenic purpura.
Without doubt the most important fact to be determined in the study of any
purpuric disease is to ascertain whether or not the platelets are decreased or at the
normal figure. As pointed out before, if they are decreased, the exact nature of
the disturbance in the clotting mechanism can at once be determined. This has
considerable practical importance since it is necessary to determine whether or
not the patient has the idiopathic type which often can be relieved with splenec-
tomy or whether the platelets are decreased because of inadequate formation in
the bone marrow.
If the platelets are decreased there will be a normal coagulation time of
capillary and venous blood as determined by the usual methods, but the bleeding
time is prolonged because of the failure of the clot to retract. Repeated studies
are often necessary to determine whether or not the bleeding is one of platelet
deficiency since many thrombocytopenic diseases have periods of remission and
relapse 'and the time when the sample of hlood is taken may coincide with a
period of improvement, even though hemorrhages have occurred only compara-
tively recently.
In every case of hemorrhage, there should be carried out platelet counts,
coagulation time, bleeding time, clot retraction time, and in those instances
where the platelet count is normal, the prothrombin clotting time and the calcium
time should be determined. Also other routine blood studies should be done.
If the condition is characterized by thrombocytopenia, the next question that
arises is the cause for this platelet decrease and it is probable that this comes
about from one of two reasons; either there is excessive destruction of platelets in
the reticula-endothelium, or inadequate output from the bone marrow. The de-
termination of the underlying pathogenesis in such cases is quite important, since
platelet destruction by the spleen can be corrected by the removal of that organ
and inadequate marrow output offers little promise for correction.
There seems to be general agreement that platelets are derived from the
giant megakaryocytes in the marrow, that they are released in sufficient number
 HEMORRHAGIC DISEASES

to maintain the normal numbers in the blood, and that they are finally destroyed
in the reticulo-endothelium, mainly that of the spleen_ There is no certain
method to determine whether or not the spleen or the hone marrow is at fault in
platelet deficiencies. A study of the sternal marrow in such cases has offered
very little information. ...
We have advocated a close stndy of the other cellular constituents of the
blood to determine whether or not the marrow is dysfunctional, this including
careful studies of red and white cells. If the white cells are low in number and
if there exists a granulocytic deficiency in absolute numbers, this is evidence that
the platelet insufficiency has its origin in inadequate marrow output, rather than
from excessive destruction in the spleen. If the leukocyte count is normal or
increased and there is no other evidence that marrow insufficiency is present, the
chances are that the thrombocytopenia is due to excessive splenic destruction and
the condition can then be classified as idiopathic thrombocytopenia. The im-
portance of prolonged and repeated studies to determine these factors should be
emphasized. .
There seems to be a "marrow threshold" for the formation and delivery of
platelets. In an occasional person the megakaryocytic tissue of the marrow may
be barely able to supply the normal number of platelets, and if splenic destruc-
tion becomes only slightly in excess this is at once reflected in decreased numbers
in the blood, followed by the hemorrhagic syndrome_ The fact that the removal
of spleens, which are normal grossly and histologically, is followed by permanent
remission of the disease is indication that there has been removed a sufficient
amount of platelet destroying tissue to enable the marrow to maintain the normal
number in the blood.
If no platelet deficiency can be demonstrated the purpuric states are then
due either to a defective clotting mechanism or to capillary weakness. Since there
is only one outstanding disease that is characterized by prolonged bleeding due
to defective clotting formation, the most likely diagnosis is hemophilia, if the
characteristic family history can be obtained. If the platelets are normal and
there is no prolongation of coagulation time, then the disease can be classifIed
as one of capillary weakness. In these cases there are no positive blood findings
except the prolonged bleeding time, whicb in some instances is not present, even
though the patient is spotted with purpura and shows the positive tourniquet
capillary resistance test. The tourniquet test is positive in this type of purpura
as well as those characterized by a low number of platelets.

BIBLIOGRAPHY
COAGULATION OF BLOOD

EAGLE, H.: "Studies on blood coagulation' The role of prothrombin and of platelets in the forma.~
tion of thrombin." Jour. Gen. Physiol., 18, 531, 1935.
EAGLE, H: ICStudies on blood coagulation: The formation of fibrin from thrombin and fibrinogen."
Jour. Gen Physiol., 18, 547, 1935.
EACLE, H.: "Studies on blood coagulation: The nature of the clotting deficiency in hemophilia."
Jour. Gen. Physiol .., 18, 813, 1935.
 COAGULATION OF BLOOD 439
EAGLE, H "Present status of the blood coagula.tion problem. A s),mposium on the bIeed." Univ.
of Wisconsin Press. 1939, p. 242,
HOWELL, W H.: "Theories of blood coagulation." Physiol Rev., 15. 435, 1935.
METTlER, g, R, and PURVIANCE, K.. "The hemorrhagic states." four. A mer. Aled. Ass1l, 168,
8" 1931.
 CHAPTER 35
ESSENTIAL THROMBOCYTOPENIC PURPURA
(Idiopathic Thrombocytopenia) (Purpura Hemorrhagica)
(Werlhof's Disease) .
Essential thrombocytopenic purpura was described by Werlhof in 1735. In
1910 Duke demonstrated clearly that the helllorrhages resulted from the decrease
in platelets. About 25 years ago there were two schools of thought concerning
the pathogenesis of the disease, one of which was headed by Kaznelson who
believed that tbe platelets were destroyed in the spleen in excessive numbers, and
the other by Frank, who believed that the bone marrow produced an insufficient
number. To demonstrate the truth of his concept Kaznelson removed the spleen
from a patient and ten years later reported that the patient was cured. Splenec-
tomy, therefore, has been done only during the last 25 years.
This disease is one of unknown cause, characterized by variable hemorrhages
from skin and mucous membranes, markedly decreased platelets in the circu-
lating blood, prolonged bleeding time, a normal coagulation rate, -failure of the
clot to retract, and a course of remissions and relapses, with eventual cure in
some patients either by medical or surgical methods.

INCIDENCE
The disease occurs in all parts of the world and seems to have no signifi-
cant geographic, racial, seasonal, or occupational incidence. We have observed
many cases in both negroes and whites, with no differences in the hematologic
or clinical manifestations. It is predominantly a disease of young people, since
the average age is 19 years in a series of over 200 cases we have collected from
the literature and it is quite rare in an old person. This age distribution is ac-
counted for to some extent by the probability that the more severe cases termi·
nate fatally in early life and some are cured by adequate treatment. A few, nc
doubt, have permanent spontaneous remissions. It seems to be more comnlO.
in females than in males, the ratio being about four to one. The disease ~eems t(
be quite rare in pregnant women, since Townsend was able to find only 52 case;
in the literature.

ETIOLOGY
The etiology remains unknown in that the cause of the platelet defiCienC)
has not been established. For that matter, there is not general agreement that tht
platelet deficiency is entirely causative, and such factors as increased capillar)
permeability and glandular dysfunction have been suspected. The opinion ha!
been advanced that the platelet deficiency is secondary and not the primary
process, on the basis that they may be decreased by deposition at the sites of
hemorrhage. Nygaard' has presented evidence that there exists a defect in
coagulation in addition to the platelet deficiency. Bemelrnans has stressed 1he
440
 ESSENTIAL THROMBOCYTOPENIC PURPURA 44'
streptococcus as a possible etiologic agent and states that the infection occurs
after wounds, undernourishment, fatigue, chilling, alcoholism, old age, and in
those with an hereditary predisposition. The evidence, however, is not con-
vincing. Based on experiments on rats, Anagostu believes that vitamin A has
some influence on the formation of platelets, since he observed marked platelet
reduction in rats with avitaminosis A.
The one consistent finding in thrombocytopenic purpura is the decreased
number of platelets. In view of their marked elevation after splenectomy, fol-
lowed by remission of clinical symptoms, it seems well established that this is
the primary defect. Whether or not the deficiency is due to increased splenic
destruction .or faulty marrow output is open to question. Storti has found con-
sistent platelet increases after ligation of the splenic artery in lower animals.
However, Holloway and Blackford found no essential differences in the platelet
counts of splenic arteries and veins in normal dogs. Lawrence and KnutH studied
the bone marrow of six patients and noted no deviation from the normal in
four of these, but it' should be borpe in mind that morphologic alteration may
not occur yet the megakaryocytes may be deficient in a functional manner.
In '932, MacCarty stated: "Twenty spleens were removed surgically at the
Mayo Clinic because of hemorrhagic purpura. I have not been able to distinguish
this type of spleen from the normal. spleen." Thus, careful studies have demon-
strated no morphologic alteration in either bone. marrow or spleen. It can be
concluded then that the deficiency in thrombocytopenic purpura is decreased
platelets and the cause of this is unknown.
Recent interest has centered upon the possible presence of a substance in the
spleen that is capable of destroying blood platelets in excessive numbers. Tro-
land and Lee prepared acetone extracts from spleens removed from patients with
thrombocytopenic purpura and after injection of these extracts into rabbits there
developed a marked thrombocytopenia, whereas similar extracts of normal and
other spleens did not reduce the blood platelets. Hobson and Witt have con-
firmed these observations. However, Pohle and Meyer have been unable to
demonstrate a platelet reducing substance from acetone extracts of spleens from
these patients. The most recent report is that of Rose and Boyer ('941) who
prepared acetone extracts from two spleens from thrombocytopenic purpura and
two other spleens from control patients. Upon injection into animals they state
that there was a marked decrease in the number of platelets. They consider
this as confirmatory of the work of Troland and Lee. At this time however the
entire question is in a controversial state.

SYMPTOMS AND PHYSICAL FINDINGS

The clinical findings are quite variable because the disease may occur in
both chronic and acute forms, and because of the tendency to relapses and re-
missions. The patient usually consults the physician, however, only when in
relapse, but the chronic types are far more frequent than the acute forms.
The patient usually gives a history of previous and perhaps repeated epi-
sodes of bleeding, which may have developed without apparent cause or may
have followed some infectious disease or operative procedure. The onset of
442 HEMORRHAGIC DISEASES

the attack is usually abrupt and characterized by anyone of the following: nose-
bleed, post-operative bleeding" prolonged menstruation, hematuria, or even a
cerebral hemorrhage, with varying degrees of purpura. Some have only the
purpura, others only the excessive menstruation, others repeated nosebleed, or
various combinations may be seen, Most pa tients give a history of a tendency
to easy bruising after slight trauma,
The patient may show a distinct pallor because of anemia or if there has
been but little loss of blood no anemia will exist and the skin color will appear
normaL Examination may reveal a generalized distribution of purpuric spots
ranging from pin head sized petechiae to large blue blotches varying in color
from a dull red to yellow, purple and blue, depending on the age of the extravasa-
tion of blood. There are no other physical findings of significance, The spleen
is not enlarged.

LABORATORY FINDINGS
The red cells are usually reduced in number, this depending upon the amount
of blood lost in previous hemorrhages or they may be entirely normal. The hemo-
globin is lower in proportion so that the type of anemia is hypochromic. The
leukocytes are at the normal level, or there may be a slight to moderate leukocy-
tosis with a shift toward immaturity of granulocytes. Especially is this true in
those patients who have lost considerable blood. The leukocyte count is an im-
portant finding, since there is a leukopenia in bone marrow insufficiency such as
that seen in aplastic anemia and aleukemic leukemia, while the count is normal
or above in thrombocytopenic purpura.
The platelets are markedly reduced in number and may be as low as two or
three thousand per cu. mm" but they are not always markedly decreased. We
have studied patients in whom the platelets varied betwen 100,000 and 200,000
per cu. mm. The platelets may be decreased and there may be no bleeding. Usu-
ally the decrease reaches 50,000 per cu. mm. before the beginning of hemorrhages
although this varies in different patients. After splenectomy the platelets may
or may not be elevated, in spite of the fact that a clinical cure is effected.
The coagulation time is normal; the bleeding time from capillary puncture
is prolonged; the dot fails to show retraction either in a watch crystal or test
tube; and the tourniquet test results in development of petechial spots on the
forearm. It should be pointed out that the capillary resistance test is positive
in those patients in whom there are either decreased platelets or alterations in the
capillary walls.
The platelets seen in the stained smear are quite variable in size and often
are large and cellular in type (see Plate No. 13).

DIAGNOSIS
Before making a diagnosis of thrombocytopenic purpura the blood studies
should be repeated many times. The consistency of the altered hematologic
ftndings over a long period and the continuous or intermittent periods of bleed-
ing, finally establish the diagnosis. There may be confusion with other diseases
characterized by thrombocytopenia, these including aplastic anemia, aleukemic
 ESSENTIAL THROMBOCYTOPENIC PURPURA 443

leukemia, acute leukemia, infectiou~ diseases as typhoid fever, miliary tubercu-

losis, cerebrospinal meningitis, and the acute exanthems of childhood. Also it
may be confused with the various types of allergic purpura following ingestion
of food or drugs to which the patient is sensitive. It is very important to rule
out all possible agents that may cause the platelet deficiency. Late in 1939
Falconer and Schumacher at the University of California reported a case of
thrombocytopenia caused by the ingestion of sedormid and collected 42 similar
cases from the literature. This widely used analgesic agent is capable of pro-
ducing the most serious depression of the platelets, and no doubt its use should
be abandoned for that reason. Falconer and associates have reported neoars-
phenamine as causative in seven patients with marked thrombocytopenia. Rus-
sell and Page have reported two cases from the administration of sulfapyridine.
I have observed one instance of marked platelet depression that apparently was
caused by the administration of sulfathiazole. Pohle has reported the platelets
low just hefore menstruation.
In any bleeding in a male :hemophilia should be considered, and this can be
established by finding the defect in the clotting mechanism and a normal number
of platelets. Also the hemorrhagic states of capillary fragility must be considered
in which the number of platelets is normal.

TREATMENT
There has been much written about the treatment of, thrombocytopenic
purpura and there is considerable disagreement concerning it. In general, these
conflicting opinions have developed over the relative merits of medical or surgical
treatment, the latter including splenectomy, and the former including mainly re-
peated transfusions of blood, removal of foci o(infection, and possibly radiation to
the spleen.
General treatment should include adequate bed rest, a high caloric diet, good
nursing and symptomatic therapy as indicated_ There is no question but that
this regime alone will induce a spontaneous remission in a considerable number
of patients. In addition to the above, repeated transfusions can be employed,
every three or four days, giving from 200 to 300 cc. of whole blood each time.
At the same time there should be a careful search for foci of infection and these
eradicated if possible. After this type of treatment nearly all patients with the
disease will recover from the acute phase of bleeding and become entirely free
from symptoms but in most instances this is only temporary. A considerable per-
centage of them will eventually return in another bleeding episode, and the same
line of medical treatment will have to be employed again. After this has hap-
pened several times the question arises as to whether or not it might have been
more satisfactory to remove the spleen during the first attack.
Jones and Tocantins observed 53 patients and of these I I recovered spontane-
ously and remained well, two recovered after removal of foci, and 24 recovered
after repeated transfusions. In only one was splenectomy employed. They
properly emphasize the fact that no treatment is of much value in the acute
fulminating type. .
Practically all patients that we have observed recovered from the acute
444 HEMORRHAGIC DISEASES

attacks with medical measures, especially with repeated transfusions, but nearly
all of them returned eventually with a recurrence of bleeding. It seems, therefore,
that the major fact to be determined in each patient is to find out whether he can
remain well and free from bleeding without splenectomy. If recurrences are too
frequent and bleeding too severe, then splenectomy is advisable.
Eliason and Ferguson reviewed 108 cases. of splenectomy from the litera·
ture and reported that all hut nve of these were cured or improved aiter splenec.
tomy, but in many of these the follow·up study was not long enough. Whipple
reviewed 81 cases after splenectomy and found 34 recurrences and 45 cured, this
indicating benefit in only about 60 per cent of the group. Spence found 22 re·
currences in one hundred cases. Also it must be considered that there is a natural
tendency to report only patients in which there has been clinical success. From
the available figures it would be a conservative estimate that 50 per cent of
patients are cured by splenectomy.
Splenectomy should not be attempted in the patient who has the disease in
the acute form with severe hemorrhages, nor sbould it be done in those who
are in a bleeding episode even though the course has been chronic. The spleen
should be removed only after the clinical course has become chronic, the blood
values r:estored by transfusion, and when there is no evidence of hemorrhages ..
We have observed several patients who were admitted to the hospital during the
acute phase. Remissions were brought about with transfusions and the patients
prepared for splenectomy, but the improvement was so marked that they did
not want to undergo the operation.
Radiation of the spleen was suggested by Stephan in 1925, but Pa!,coast and
his associates state that it has little value. Jones and Smith found it to be
ineffective in nine cases. This procedure probably has not had a sufficient trial
because the internist has good results from transfusions and the surgeons from
splenectomy. Wiseman and his associates at Ohio State University state that
radiation and ultraviolet rays have not been useful in the treatment of thrombo·
cytopenic purpura and stress the poi~t that splenectomy is the only satisfactory
treatment. On the basis of animal experiments Storti believes that ligation of
the splenic artery would be advisable, and especially in those instances where
the spleen cannot be removed without grave risk. Mettier and associates used
radiation to the spleen in five patients and found incr~ased platelets 24 to 48
hours later with improvement in clinical symptoms. They used 200 Roentgen
units daily or every other day until an average of 2600 units had been given.
After splenectomy there is marked improvement in, the state of the blood and
in the clinical symptoms. The bleeding ceases immedi~tely, purpuric spots
disappear, the bleeding time returns to normal, the clot retraction is good and
the tourniquet test is negative. The platelets rise within 24 hours in some in.
stances to the normal level and we have observed it to reach over one million
four days after operation. It slowly falls, however, a.nd may remain at the normal
level or may recede to the same level as before operation, but even so, there is' no
further evidence of hemorrhages.
In summarizing the treatment that should be employed in this disease" the
nrst point of emphasis is the absolute necessity for correct diagnosis. When this
 ESSENTIAL THROMBOCYTOPENIC PURPURA 445

is established, the patient should receive the benefit of a thorough trial with medi-
cal measures, including repeated small transfusions, bed rest, a high caloric diet,
good nursing, symptomatic therapy, and removal of foci of infection if possible.
In nearly all patients a remission will be induced, unless the disease is of the acute
fulminating type. After recovery the patient should be observed for a long period
of time to note the frequency and severity of hemorrhages. If these continue,
radiation or splenectomy should be employed if it becomes obvious that the
patient ,cannot live in good health and retain his spleen.
We have not observed a recurrence after splenectomy in any patient at the
Emory University Hospital in nine years (23 patients). We have observed re-
currences after adequate medical treatment had effected temporary remissions,
and we have seen only three patients in this group that have been free from
symptoms for as long as two years.
It seems probable that failures after splenectomy in some instances are be-
cause of incorrect diagnosis. We have studied one patient with subacute myelog-
enous leukemia which apparently developed shortly after a splenectomy for a
supposed thrombocytopenic purpura. This patient had leukemia from the be-
ginning, as later blood studies verified. Also in the Hematological Registry are
records of three patients who had been splenectomized for thrombocytopenic pur-
pura and who died later with typical myelogenous leukemia. These are instances
of incorrect diagnoses. Therefore, the most important part of treatment in this
disease is to know what is heing treated. It is quite possible that tbe statistical
results of splenectomy would be much better if there were assurance that the
operation is done only on patients with thrombocytopenic purpura. .
There is little evidence to indicate that the recently developed cevitamic
acid (vitamin C) is of value in the treatment of thrombocytopenic purpura. Also
the value of snake venom has not been proved.

'BIBLIOGRAPHY
ESSENTIAL THROMBOCYTOPENIC PURPUM
ANAGNESTU, J : "ExperimentcI1e untersuchungen iIbcr den einfiuss von vitamin A auf die throm·
bocyten und das knochenmark." Klin. Wchnschr, 18, 1277. 1939.
BEMELMANS, E.: "Criuque of the problem of purpura." IIaematologica, 16, 369. 1935.
DESAUSSURE, H W, and TOWNSEND, E W.: uPurpura hcmorrhagica in pregnancy." Amer. Jour.
Obst and Gynec., 29, 597, 1935.
DUZE, W. W. ClThe pathogenCSls of purpura bemorrbagica with especial reference to the part
played by blood-plalelets.1l Arch. Int . ..Med., 10, 445. 1912.
ELIASON, E L. t and FEROUSO~, L. K' "Splenectomy in purpura hcmorrhagica." Ann Surg, 96,
801,193 2,
FALCONER, E. H, EpSTEIN, N. N, and MnLS, E. So' "Purpura hacmorrhagica due to the arsphcna-
mines," Arch Tnt Med, 66,319,1940.
FALCO:NEIl., E. H, and SnUMACHER, Ie: "Purpura hemorrbacica caused by ingestion of sedormid
(allylisoprop)]acety]carbamide). Experimental observations and report of C.1"C." Arch. Int.
Med, 65, 122, 1940.
FRANK, R: "Die essentielIe thrombopenie {konstitutionel1e purpura; pseudo-hamophilie)." Berl.
klin. Wchnschr, 52, 454, 1915.
HoasON, F C. G., and WrrTS, L. J: "Platelet reducin~ extracts of the spleen II Brit. },fed. Jour"
1, so, 1940
 HEMORRlJAC1C DISEASES

HOLLOWAY, J K., and BLACKFORD, L M.: "Comparison of the blood platelet count in splenic,
arterial, and venous blood." Amer. Jour. Med. Sci, 168, 723. 1924.
1I
JONES, H. W., and S.MITB:1 R. M.: "Splenic irradiation in the treatment of purpura hemorrhagIca.
Ann. Int. Med.1' 11, 13l:I, 193&.
JONES, H. W., and TOCANTlNS, L.: "The treatment of purpura hemorrhagica." Jour. Amer. Med.
Assn, 100, 83, 1933.
KAZNEI.SON, P.: "Verschwinden der hlimorrhagischen diathese bei einem fa.lle von c55enticller
thrombopenie (Frank) nach milzexstirpation; splenogene thrombolytische purpura." Wien.
kIm. Wchnsch,., 29, 1451, 1916.
KRACKE, R. R.: "The effect of splenectomy in the purpuric diseases." South. Sutgeon, 2, 203,
1933·
LAWBWCE, ]. S, and KNU'l'f'I, R. E: "Bone marrow in idiopathic thrombotJemc purpura." Amer.
Jour. JIed. Sci ~ 188, 37, 1934.
l\!ACCA&TY, \V. C.' "Surgically removed spleens. Study III. Cytology and clinical significance."
Proc. Stoff .Meet., Mayo Clinic,. 7. 187, 1932.
METTlER, S. R, and SroNE., R. S.: 'JThe -effect a{ rcentgen ray irradiation on platelet produ'Ction
in patlcnts with essential thrombocytopenic purpura haemorrhagica." Amer. Jou,.. Med. Sci.
191, 794, 1936.
NYGAARD, K. K.: "Coagulability of blood plasma and cbanges in number of platelets in thrombo-
cytopenic purpura." Proc. Staff Meet, Mayo CUn., 9, 492, 1934.
PANCOAST, H. K I PENDERGRASS, E. p" and FITZHUGII, T.: "The present status of the roentgen treat-
ment of purpura bemorrbagica by irradiation of the spJeen." A mer. Jour. Roentgenol., 13,
5S8, 1925
PaRLE, F. J 'f and MEYER, O. 0.: "Inability to demonstrat(' a platelet reducing substance in the
acetone extract of spleen frorn patients with idiopathic thrombocytopenic purpura." Jour, CUn.
Invest, 18, 5.37, 1939.
ROSE, H., JR., and BoYEB, L. B.: "Thromhocytopen: A confirmatory report." JOfty. CUn. If'vest.,
20, 81, I94I.
RUSSELL, H. K., and PAGE, R. C.: "Thrombocytopenic purpura due to sulfapyridine." Amer. Jour.
Afed. Sci, 200, 495, 1940.
SPENCE, A. \V.: I'The results of splenectomy for purpura hemorrhagica.." Brit. Jour. Surg., 15,
466, 1928.
STEPHAN, R.: "Retikulo-endothelialer zelJapparat und blutgerinnung." Munchen. med. Wchnschr.,
67. 309, 1920.
S'IORTI, E: 4'Ligation of the !:plenic artery and splenectomy." Haema.tologic.a, 15. 107,1934.
TROLAND, C. E., anti LEE, F. C.: uThrombocytopen. A preliminary report on a platelet reducing
substance as seen in thrombocytopenic purpura." Jour. Arner. Med. Assoc., 11, 221, 1938.
WERLnOF, P. G: 'IMorbus maculosus hemonhagicus." OPera Omnia. Chat, 48, 748, 1775.
WnIPPLE, A. 0: J'Splenectomy as a therapeutic measure in thrombocytopenic purpura bemor-
rhagica." SUf'g. Gyn. and Obst., 42. 3:19, 19'16.
WISSMAN, B. K., DOAN, C. A, and WnsON, S. J.: uPresent status of thrombocytopenic purpura
with special reference to diagnosis and treatment." Jour. Amer. Med. Assoc., 115, 8, 1940.
 CHAPTER 36
HEMOPHILIA
Hemophilia is an hereditary disease, characterized by the transmission of a
sex linked recessive lIIendeli'ln defect th'l( results in delayed coagulation of the
blood, repeated hemorrhages throughout life, occurring only in males, and trans-
mitted by females and affected males.
This disease has been recognized for centuries and was referred to in religious
writings of the second century In 1820, Nasse elucidated his concept of trans-
mission of the disease and pointed out that it was manifested only in males and
transmitted mainly by females who were not victims. The clinical features Were
described in detail by Otto in 1803. In '9", Bullock and Fildes, by an extensive
review of the reported cases, and their own thorough investigations, verified
the hereditary transmission of the disease. Therefore, very little has heen dis-
covered about hemophilia except repeated verification of its transmissable char-
acter. :l10re recently, however, other contributions have heen made that promise
to throw light on the nature of the defect and the pathogenesis of hemophilia.
These include the observations of Birch in 1931, when she pointed out the possi-
bility that the disease manifests itself in the male because he may be deficient
in some substance that is present in the female. She reasoned that this must
be one of the ovarian or estrogenic substances. This concept has led to the clini-
cal trial of a large number of such agents in recent years.
1\[ore recent contributions'include that of Eley, Green, and lIIcKhann, who
reasoned that a substance in the placenta might accelerate the process of coagu-
lation, and they have reported that the administration of placental extracts in their
patients has been followed hy considerable success. This concept was developed
because of the well recognized infrequency of bleeding in young infants who later
become fully developed hemophiliacs.

HEREDITY
The inheritable constitutional defect in hemophilia is generally conceded
to he transmitter! as a recessive, sex linked character. A heterozygous female,
while showing no evidence of the disease, transmits the recessive gene to half of
her offspring and the dominant gene to the other, half, regardless of sex. The
daughters by normal father receiving the recessive gene will be like the mother,
that is, heterozygous and carriers. Since the rest of the daughters, while norrnal,
are imlistinguishable from the carriers, it is inadvisable for any of them to
propagate. The sons receiving the recessive gene will be bleeders since a sex
linked character has no allelomorph in the male. Therefore, those sons who do
not exhibit the disease cannot transmit it.
N aegeJi states that all hleeders are exclusively men, that there is no record
of a genuine instance of hemophilic bleeding in a female, and that a Walnan
447
 HEMORRHAGIC DISEASES

has never bled to death from hemophilia. He gives the possibilities of trans-
mission, as shown in the following outline:
1. Marriage of a normal man and a female carrier. This results in one
half of the sons as bleeders, and one half of the daughters as carriers,
with no way of telling which daughters are carriers.
2. Marriage of a bleeder (male) and a healthy woman. All sons show
no evidence of the disease and are not bleeders. The disease stops with
them and they do not transmit it. All daughters are carriers, and can
transmit to half of their sons and daughters. The male hemophiliac,
. therefore, transmits the defect to all of his daughters and none of his sons.
3. Marriage of a male bleeder and a female carrier. This is very rare,
but verified instances are on record, particularly among some European
royal bouses. This results in half of the sons being bleeders, the other
half free from the disease, and one half of the daughters carriers and the
other half bleeders. This is the only theoretical instance where a female
should show bleeding in this disease. Naegeli doubts that it has ever
occurred.
The probable reason why the disease does not occur in the female when it should
according to the Mendelian law, is the development of the double defect to the
point of a "lethal stage" and such embryos probably would not develop in utero
(Macklin) .
The correctness of these inherited characteristics has been amply verified by
detailed studies of many hemophilic families by many investigators, including
those carefully studied by Bullock and Fildes, the Pickett family reported by
Mills, the Hawkins-Cooper family reported by Birch, and many others.
Birch studied the genetics of 20 families of hemophiliacs, and noted that
both hemophiliacs and transmitters seem to have families above the average in
size; that most children of hemophiliacs were females, in a ratio of two to one,
and therefore, transmitted the disease. Birch concluded that "nature seems to
have provided that this disease shall go on forever, because the transmitters of
hemophilia have more sons than daughters, while persons with hemophilia bave
more daughters than sons."

THE NATURE OF THE DEFECT

It was shown by Wright in 1893, that the blood of hemophiliacs was charac-
terized by a marked delay in the time of coagulation, and this is recognized as
the fundamental defect by practically all observers. The coagulation time may
vary from 15 or 20 minutes to many hours, depending upon the amount of de-
ficiency. Naegeli took the blood of a 12 year old typical hemophiliac and noted
complete coagulation of blood from venesection in an ordinary glass dish at
the end of three hours, and in a paraffined dish at the end of five hours. The clot,
when formed, is normal in chemical content and in morphology. Once formed,
it retracts in a normal way. Therefore, the primary defect is apparently one of
the factors concerned in the process of coagulation. Conceivably it may be a
deficiency in anyone of the five factors producing coagulation, according to the
 HEMOPHILIA 449

Howell theory. Much work has been done by various investigators to determine
the exact nature of the deficiency. This has included that of Howell and of Addis,
who demonstrated the presence of prothrombin in normal amounts; that of
Minot and Lee who demonstrated that the thrombin content is normal; that of
Addis and of Feissly who showed that fibrinogen is normal in amount and func-
tion; and that of Hess who found normal values for calcium. Naegeli states
that among the various theories proposed for the nature of the defect are de-
ficient formation of tbrombin, decreased thrombokinase from blood platelets
(Sahli, Fonio), deficient prothrombin in the plasma, increase in antithrombin,
and delayed release of the factors initiating clotting (Hartmann, Opitz). From
the following discussion it seems that some of these theories are at least partly
correct.
It appears then that all of the theoretical constituents concerned in tha clot-
ting of blood are normal in amount, but the process is a deficiency of function
of one of these. Minot and Lee produced evidence that the platelets are deficient
in function, by adding the platelets of hemophiliac blood to normal blood plasma,
this resulting in a delayed coagulation time, and if normal platelets were added
to hemophiliac blood plasma the coagulation time approached normal. In view
of the fact that Howell has shown that it is necessary for the platelets to ag-
glutinate and undergo disintegration at the beginning of coagulation, releasing
thromboplastin, it would appear that the defect in hemophilia is a functional de-
ficiency in blood platelets. Current opinion is in general agreement that this
defect of thromboplastin from platelets, results in a prolonged period of conversion
of prothrombin into thrombin, with the resulting delay in coagulation. Therefore,
the defect seems to be an "excessive stability" of platelets in that they are re-
sistant to the process of normal disintegration and release of thromboplastin.
Brinkhous has also demonstrated that the defect in hemophilia appears to be a
slow conversion of prothrombin into thrombin and found that he could correct
this by adding minute quantities of tissue thromboplastin to hemophilic blood.
He reports that the addition of only I mg. of crude thromboplastin prepared
from lung extract corrects the deficiency in 100 cc. of hemophilic blood.
If the defect is a deficiency of thromboplastin from platelets, the question
arises as to why this is not provided by the tissue juice of incised wounds since it
has been shown by Gressot that the thromboplastin content of hemophilic tissue
juice is normal. This has been explained by the fact that tissue juice is present
only at the periphery of a wound, and clotting occurs only in that area and not in
the center.
However, some doubt has arisen recently on the validity of the above
concept. Patek and Stetson, in a series of well, controlled experiments with
normal and hemophilic blood, have, shown that hemophilic platelets function as
efficiently as those of normal blood;and that the addition of platelet-free normal
blood plasma reduces the coagulation time of hemophilic blood to within normal
limits. This substance in the plasma, as yet unidentified, is stable at icebox tem-
perature and potent in high dilutions either in vivo or in vitro. This appears to be
one of the most important recent contributions in this disease.
Pohle and Taylor state that the above substance is a plasma globulin fraction
45 0 HEMORRHAGIC D,SEASES

which is prepared by isoelectric precipitation at a pH of 6.0 from citrated cell.

free normal human plasma. It possesses marked clot accelerating properties for
hemophilic blood, both in vivo and in vitro. It is called globulin substance.
Patek and Taylor have prepared globulin substance from animal plasma and
showed that it possessed clot promoting properties for hemophilic blood. They
have used it as a local hemostatic agent. It is ineffective when given by mouth,
but acts as a local hemostatic agent. Only 750 mg. of final product is obtained
from roo cc. of plasma. The intravenous injection of this substance in saline
suspension produces a marked shortening in coagulation time in the hemophiliac.
Lozner. Kark and Taylor removed the prothrombin and fibrinogen from normal
human plasma that had been passed through a Berkefeld filter and the remain·
ing substance markedly reduced the coagUlation time of the hemophiliac in vitro
and in vivo. Injection of this material every six hours in hemophiliacs maintains
their coagulation time at a much lower level.

INCIDENCE
The disease apparently occurs in all parts of the world and is not confined to
any particular race of people. It is mainly a disease of young people since it has
been noted that there is a marked decrease in severity after the age of puberty.
Furthermore, because of the mortality rate in the young patients, those that live
to old age are comparatively rare. Naegeli cites the following ages of death in a
series of 42 patients:
Under 10 years ................ IS patients
From 10 to 20 years .... .. .... 13
From 20 to 30 years. .. ......... 8
All ages over 30. ................ 3

SYMPTOMS AND PHYSICAL FINDINGS

The outstanding symptoms are those of repeated and severe hemorrhages,
wbich begin in early childhood, altbough they are comparatively rare in infancy
throughout the first year. Tbe hemorrhages may be eitber spontaneous, as
seen in severe cases, or initiated by some type of trauma. They increase in
,everity to the age of puberty and usually decrease in severity and frequency
after that period. The bleeding may be continuous over a long period of time
or there may be periods of freedom from the bleeding which may extend for
months or even years in rare instances. There is no method whereby it may
be determined at what time or to what extent hemorrhages will occur in any
patient. One episode of bleeding may be very slight and another severe and
prolonged.
Bleeding may be initiated by the slightest trauma, such as friction on the
gums witb a toothbrush, accidentally biting the tongue, a very slight cut on any
part of the mucous membranes, a torn frenulum, the extraction of a tooth, the
spontaneous loss of a tooth, circumcision, tonsillectomy, and many other minor
traumatic incidents. Any of these may result in prolonged hemorrhage which
is impossible to suppress. Bleeding into the body cavities is unusual but spon·
taneous hemorrhages may occur in the muscles, muscle planes, connective tissue,
and joint cavities.
 HEMOPHILIA 45 1
Those into the joints are especially common and are seen so consistently
that some question the diagnosis of hemophilia unless this type of hemorrhage has
occurred. Kahn states that this is the most frequent type of hemorrhage and
that it occurs spontaneously in the following joints in order of frequency: knee,
ankle, elbow, and hip. The knee is by far the most common joint involved. Joint
bleeding is characterized by a sudden pain, marked swelling, diffuse redness, no
local temperature, and finally a varicolored skin over the joint. Absorption
takes place slowly and the pain may be severe. There is marked limitation of
motion, temporary invalidism, and the process is followed finally by varying de-
grees of bone destruction and deformity.
The external evidences of anemia, such as pallor, depend upon the duration
and extent of hemorrhages. Other than the hemorrhages there are no physical
findings that are characteristic of the disease, and in intervals between attacks
the patient may appear normal in every respect.

HEMATOLOGIC FINDINGS
These are characterized by prolongation of the coagulation time and pro-
thrombin time with other blood findings normal. According to Naegeli the bleed-
ing time is normal, the platelets are normal in number and in morphology, the
fibrinogen is abundant, the calcium is normal, the red and white cells are normal
in number and types, and the vascular resistance unimpaired as determined by
the tourniquet test.
The coagulation time may vary from a few minutes over the normal to sev-
eral hours. It can be determined in several ways. We have found the capillary
tube method, test tube method, watch crystal method, and large receptacle
method equally s;ttisfactory. Small amounts of blood are taken either from the
vein or by finger puncture and the coagulation time noted in capillary tubes.
There is considerable variation of coagulation time in the same patient at differ-
ent times.
The bleeding time is normal when blood is taken by finger puncture_ In
this instance there is sufficient admixture with tissue juice to initiate the clotting
process. Therefore, the bleeding time is normal by laboratory methods but pro-
longed by clinical standards. We use Duke's method for bleeding time.
The red cells and hemoglobin may show the changes of hypochromic anemia if
blood loss has been severe and a neutrophilic leukocytosis is not unusual. The
prothrombin time is prolonged but. the amount of prothrombin is normal.
The blood platelets are not reduced and they appear normal on the stained
smear. The clot retraction time is normal. -

PATHOLOGY
There are no pathologic findings of importance in this disease. Custer and
Krumbhaar have studied the bone marrow removed from three patients at au-
topsy, and found that the hematopoietic tissues showed normal regenerative
ability, which in two instances was erythroblastic and in a third leukoblastic.
They also noted a marked increase of megakaryoblasts and megakaryocytes in
the marrow> this indicating a possible relationship to the hemophilic process.
45 2 HEMORRHAGIC DISEASES

DIAGNOSIS
The diagnosis of hemophilia is based on repeated' attacks of prolonged and
intractable bleeding in a male from the time of childhood with a family history
indicating the hereditary transmission of the disease, and the finding of pro-
longed coagulation and prothrombin times in the blood. It is practically the only
disease that is characterized by such a defect. The finding of decreased platelets,
normal coagulation, prolonged bleeding time, and prolonged clot retractiorl is
sufficient to establish a hemorrhagic syndrome of some other type and exclude
hemophilia. Again it bears emphasis that decreased numbers of platelets elimi-
nate hemophilia and establish the process as thrombocytopenic purpura of the
essential or secondary types. With careful hematologic studies there should be no
confusion of this disease with other hemorrhagic disorders.

TREATMENT
The treatment is of two types: first, prophylactic; and second, that directed
to stop bleeding.
It is important that patients and their relatives be carefully instructed con-
cerning the genetic relationships and the factors involved in the transmission of
the disease. Macklin summarizes this as follows:
r. "The only persons in a hemophilic family who can marry with impunity
are the unaffected males and their descendants.
2. "All daughters of hemophilic males are carriers, and should not repro-
duce. Since, in a family in which' hemophilia is known to exist, it is
impossible to tell which women are carriers and which are free from the
latent defect until they reproduce, it is advisable for all women in such a
family to refrain from having children. Although this excludes some nor-
mal women from reproducing, it is the only safe rille to insure the elimi-
nation of the defect."
The above relationships may be further elucidated as follows:
I. The following members of hemophilic families may propagate without
fear of transmitting the disease:
(a) The unaffected males.
(b) The offspring, male and female, of unaffected males.
2. The following should not propagate:
(a) The affected males (those with the disease).
(b) Daughters of affected males.
(c) Females, whose mother, maternal grandmother, maternal great-
grandmother, etc., stand any genetic possibility of having been
carriers.
3. In fact, any female who is a member of a hemophilic family, should re-
frain from propagating unless her relationship to the hemophiliac can be
traced through an unaffected male.
In giving advice to these families, it should be brought out that it is not
necessary that they be denied the privilege of marriage, provided that the neces-
 HEMOPHILIA 453
sary steps are taken to prevent the possibility of propagation. This may be
effected either through sexual continence or by sterilization of the affected party.
Of course, it is apparent that the possible female carrier can be sterilized
either by radiation or by surgical methods. The sterilization of the hemophiliac
himself offers a more difficult problem, because of the dangers of hemorrhage in
operative procedures and the questionable results of radiation. However, in
light of recent knowledge, many of these can be obviated by thorough preopera-
tive preparation in the form of transfusions and the intramuscular injection of
whole blood, and doing the operation during a period of relative quiescence.
In the light of present knowledge it appears that the only hope of eliminating
this tragic disease is through such eugenic measures.
Other prophylactic treatment should include advice to the patient concern-
ing the nature of his illness and he should be cautioned against exposure to pos-
sible trauma. He should follow a sedentary and somewhat sheltered life, if
possible. If surgical procedures become necessary, these should be preceded by
transfusions, and Mills stresses the importance of the production of a state of
hypersensitivity to a foreign animal protein.
Mills sensitizes the patient by injecting him intramuscularly with three or
four cubic centimeters of sheep or hen serum, after first determining by intra-
dermal injection that the patient is not already sensitive. After two' weeks a drop
is injected intradermally and the presence of a definite urticarial wheal indicates
sensitivity. At that time the coagulation time of the blood is inarkedly shortened.
Isaacs and associates have pointed out that the treatment of hemophilia has
included a wide variety of substances including whole blood, citrated blood, human
plasma, human and animal serum, defibrinated blood, various hemostatic prepara-
tions, fibrinogen, cephalin in suspension, calcium chloride, sodium chloride, pro-
tein shock, liver and its products, whole ovary, ovarian extracts, and special diets.
It is questionable if the use of any of these products has been attended with any
considerable success. Furthermore, since in severe hemorrhagic states it is cus-
tomary to use many of these simultaneously and because of the fact that bleeding
Ceases of its own accord in some patients, it is very difficult to evaluate the results
of therapy.
The treatment of the patient during the stage of. bleeding should include
absolute rest, fixation of involved joints, the use of opiates if necessary, and trans-
fusions. Also the hemorrhages shonld be controlled if possible by the application
of absorbent cotton soaked in normal blood serum. These can be applied to the
bleeding surfa~es and compression should be used. The appiication of a 1 :1000
solution of epinephrine may be helpful. Minot and Lee believe that transfusion
of whole blood is the'most valuahle agent that" can be used and showed that the
coagulation time is shortened after its use, but that the effect was not pro-
longed over three days.
The local application of dilute snake venom has heen suggested by Peck
and his associates. Eley and associates have reported good results from the in-
jection and oral administration of placental extracts"with decrease of the coagu-
lation time and suppression of hemorrhages.
In recent years much interest has been stimulated by the work of Birch '
454 HEMORRHAGIC DISEASES

who repoIted good results using various preparations of ovarian substance. Re-
sults concerning this have heen conflicting. Stetson and associates found no clini-
calor hematologic effects after the use of ovarian and estrogenic substances by
mouth and by injection. Brown and Albright have also reported negative results
Novak st,ates that the use of these preparations (estrogenic) has been disap-
pointing.
Among agents recently recommended for shortening coagulation time is
oxalic acid. In 1939, Steinberg and Brown found that oxalic acid would reduce
the coagulation time of normal rabbits. Schumann tried it in a number of
hemorrhagic states and reported that it appeared to be of value in some of them.
Page and his associates gave oxalic acid to three hemophiliacs and reported the
coagulation time down to normal in two to three days. Van Buren at Emory
University has tried oxalic acid in four hemophiliacs, but with no effect on either
the clinical or hematologic findings.
The fact that Brinkhous has found that I mg. of tissue thromboplastin will
correct the clotting deficiency in roo cc. of hemophilic blood, and tbat th~ Har-
vard group has found so many characteristics of the effective globulin suhstance,
means that the outlook for the hemophiliac is by no means hopeless. At tbis
time (1941) repeated transfusions comprise the most dependahle treatment for
hemopbilia.
PROGNOSIS
The immediate prognosis in a hemophiliac who is bleeding is always poor.
Most patients die before the age of puberty, and if they attain manhood, as the
years go by their chances of long life become better. They are usually well
between attacks of bleeding. Many live the life of prolonged invalidism, barely
recovering from the disabling results of one attack before another sets in. The
mental outlook is poor since they live in constant fear of a recurrence of bleeding.

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HEMOPHILIA
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thrombin" Amer. Jour. Med. Sci., 198, 509, 1939.
BULLOCB, \V., and FnDES, P.: "Hemophilia.1! London University, Francis Galton Laboratory for
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CHEW, W. B, STETSON, R. P., SMITH, G. V. S., and SYIm, O. W.: "Estrogenic, luteal) and ganado.
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recent hterature." Arch. Int. Med., 57, :n86, 1936.
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prothrombin." Jour. CUn. Invest., 18, 603, 1939.
MILLS, C. A.: "Hemophiha." Jour. Lab. and elm. Med., 17, 932, :1932.
MINOT, G. R., and LEE, R I.: "The blood platelets in hemophilia." Arch. Int. Med., 17. 474,
1916
MACKLIN, M. T: "Heredity in hemophilia." Amer. Jour. Med. Sci., 175, 218, 1928.
NASSE, C. F.' e'Von einer erbIichen neigung zu todtIichen blutungen." Arch. f. Med. Erfahr .• 1,
385. 1820
NOVAK, E.: "The therapeutic use of estrogenic substances." Jour. Amer. Med. Assn., 104, 1815,
1935.
NAEGELI, 0.: Blutkrankhetten und blutdiagnostik, 5th ed., J. Springer, Berlin, 1931.
ORDWAY, T, and GORHAM, L. W.: Oxford Monographs, IX, Supp. to 1931. Oxford University
Press, New York, 1930
OTTO, J. C.: "An account of a hemorrhagic dlsposition existing in certain families." Med. Reposi_
tory. 6, I, 1803.
PAGE, R. C., RUSSELL, H. K., and ROSENTHAL, R. L.: "Effect of oxalic acid intravenously on blo()d~
coagulation time in three -hemophlliacs" Ann. Int. lIed., 14, 78, 1940.
PATEX, A, ]., JR, and STETSON, R. P "Hemophiha: Abnormal coagulatIon of blood and its rela-
tion to blood platelets" Jour. CUn. Invest., 15, 531, 1936.
PEcx, S. M., and ROSENTHAL, N.: "Effect of moccasin snake venom (Ancistrodon pjsdvorus) in
hemorrhagic:: condItions >I Jour. Amer. }.[ed. Assn., 104, 1066, 1935.
POHLE, F. J., and TAYLOR, F. H. L.: "The use of a globulin substance derived from beef plasma
as a local hemostatic in hemophilia." Jour. CUn. Invest, 17, 677, 1938.
QUICK, A. J., STANLEy-BROWN, M., and BANCROFT, F. W.: UStudy of coagulation defect in hemo-
philia a.nd jaundice." A.mer. hu,,.. .dIed. Sd., 190, sor, Tl}3S.
SCHUMANN, E. A. "Newer concepts of blood coagulation and the control of hemorrhage." Amer.
Jour. Obst. and Gynec., 38, 1002, 1939.
STEINBERG, 'S, and BROWN, W. R: "A new concept regarding the mechanism of clotting and tbe
control of bemorrhage." Amer. Jour. Physiol., 76, 638, 1939
STETSON} R. P., FORKNER, C. E., CImW, W. R, and RIeB, M. L.: "Negative effect of prolonzed
administration of ovarian substances in hemophilia." Jour. Amer Med. Assn, 102. 1122,
1934
WRIGHT, A. E.: "On a method for determining the condition of blood coagulability for clinical a.nd
experimental purposes, and on the effect of the administration of calcium salts in haemophUia
and actual or threatened hemorrhage." Brit. Med. Jour, 2, :t:z.3, 1893.
 CHAPTER 37
NON-THROMBOPENIC PURPURA
(Purpura of Capillary Weakness)
The term, non-thrombopenic purpura, although not specific, is used to desig-
nate that large group of hemorrhagic states that are thought to be: first, allergic
in type; second, those associated with chronic infectious states, and third, those
of avitaminosis, all of which are characterized by capillary dysfunction rather
than by cellular alterations. Since the causes of these hemorrhagic states appear
to be multiple, and the entire group poorly classified and ill-defined, tbey are
grouped together for convenience rather than for accuracy.
This group of purpuric disorders includes many syndromes that have been
given separate names in medical literature, and includes many varied manifesta-
tions that have masqueraded under a variety of different disease entities. Thus,
in the group are such diverse conditions as Henoch's purpura, Schonlein's purpura
(purpura rheumatica), erythema simplex, erythema multiforme, erythema multi-
forme bullosum and vesiculosum, erythema (nodosa, pigmentosa, papulatum,
pustulosa), urticaria, and angio-neurotic edema. It can be seen that the con-
fusion in terminology has come about largely because of the varied symptoms
and manifestations of the disease, depending upon whether the abdominal symp-
toms, or the joint symptoms, or the skin lesions predominate. The entire group
is characterized by symptoms referable to three divisions of the body, namely,
those of the joints, those of the abdomen, and those of the skin. .
It was pointed out by Osler that this group of varied manifestations were
closely related and he noted the similarity of the purpuric areas to the so-called
allergic phenomena including urticarial wheals and areas of angio-neurotic edema.
It seems generally accepted that the basic change is an unexplained weak-
ness of the capillary system, which may he general or local. This state has been
designated as capillary weakness, hemorrhagic capillary toxicosis, weakened
capillary resistance, and increased capillary fragility. The fundamental defect
seems to he an altered state of the capillary wall which is only temporary but
recurrent in most instances, in which there is escape of either whole blood or
serum from the vascular system, this occurring mainly in and beneath the skin.
The cause of this' weakness has not been determined. Several theories have been
proposed to explain it. Among these are first, the possible action of histamine
like products wbich may cause dilatation and increased permeability of the
capillary wall; second, the same factors as those producing the allergic state
whicb may be histamine like substances; third, changes in the sympathetic nuclei
(Castex) ; fourth, action of bacterial toxins; and fifth, vitamin C deficiency, in-
cluding tbe types seen in scurvy and so-called sub-clinical scurvy (Wolbach and
Howe).
456
 NON-THROMBOPENIC PURPURA 457
SYMPTOMS AND PHYSICAL FINDINGS
The onset of allergic (anaphylactoid) purpura is variable and the hemor-
rhagic manifestations may be preceded by signs of infection, such as fever, gen-
eralized muscular pain, headache, loss of appetite and general malaise. In others
there are no such early symptoms. Some patients are studied for a considerable
time in an effort to establish the presence of some infectious disease. Also joint
pains and tenderness may be early symptoms, usually of the knees and ankles,
and may shift from one joint to another. In Henoch's purpura the onset may
be preceded by symptoms of tbe gastrointestinal tract including nausea, vomiting,
diarrhoea, and generalized and shifting colic-like abdominal pains. Such patients
may be subjected to surgical procedures because of simulation of an acute surgi-
cal condition.
The hemorrhages, which usually follow and occasionally coincide with the
above symptoms, are varied, and may range from large intramuscular and sub-
cutaneous collections of blood to the smallest petechiae on the lower extremities.
There may be submucous bleeding, bleeding from the gums, nosebleed, and varied
purpuric spots, which range in size from one millimeter in diameter to large dis-
colored areas. Some are confluent and others are discrete. Most of them ap-
pear on the lower extremities, and then the arms, trunk, and face, in the order
named. Some are cliaracterized by increased purpuric areas toward the feet,
indicating the possible role of mechanical pressure in the capillary be~ of those
areas.
The purpuric spots vary in color from the red of recent hemorrhage to hlue,
purple, and yellow, in older ones. There may be a mixture of varied colors in
those of some days duration and the color of the spots gives some indication as to
the age of the hemorrhages. There may be areas of only simple erythema, or some
may be raised in papular form, others vesicular and filled with fluid, and others
pustular containing pus, especially the older ones second~rily infected. There
may be a predominance of diffuse subcutaneous fluid or urticarial wheals. The
lesions are symmetrical in some patients. Itching is not common, as it is in the
purpura of obstructive jaundice.
The degree of pallor depends on the presence of anemia which is not marked
in most patients. The joints may be swollen, red and tender (Schiinlein type) or
there may be abdominal rigidity and tenderness (Henoch type).
Many patients with the milder forms of the disease have periods of good
health for weeks and months with intervening episodes of purpuric spots which
cause little discomfort. Tbese fade away and the process is repeated at irregular
intervals. These mild types may be caused by chronic infections, such as infected
teeth and diseased tonsils, inadequate diet and possible vitamin deficiencies.
Other patients may develop the hemorrhages severely and quickly after the in-
gestion of some food or drug to which they are sensitive. Various drugs have
been incriminated in the production of acute purpura in tbe occasional person that
is presumably sensitive to their action. Such drugs have included acetanilid.
phenacetine, quinine, phenolphthalein, ergot, iodides, atropine, salicylates, chloral
hydrate, arsphenamine, and others. The purpura, seen in acute infections are
45 8 HEMORRHAGIC DISEASES

varied in type and extent. In rare instances the first manifestation may be a cere-
bral hemorrhage, or hemorrhage into the kidneys, which mayor may not he as,o-
ciated with purpura. We have studied one patient who tlied from acute nephritis
and uremia because of hemorrhages into the kidneys, which was associated with
the typical purpuric manifestation of capillary weakness.

HEMATOLOGIC FINDINGS
The red cells and hemoglobin are usually normal, unless marked external
hemorrhage has occurred, which is rare. Neutrophilic leukocytosis is common,
probahly because of absorption of products of hemorrhage and other tissue de-
struction. The bleeding, coagulation, and clot retraction times are normal.
The tourniquet test for capillary resistance is practically always positive. The
platelets are not affected, although there are many reports of moderately decreased
platelets in some of the,e syndromes. It should be emphasized however, that
anaphylactoid purpura is a non-thrombocytopenic type. The urine and stools
may contain variable amounts of blood, depending upon wbether hemorrhages
have occurred in the kidneys or in the intestinal tract.

DIAGNOSIS
The diagnosis is based on the occurrence of purpura and hemorrhages, usually
in a young person, and the disease is characterized hy a normal number of plate-
lets, normal coagulation time, normal clot retraction and a positive tourniquet
test.
It is important that this general class of purpuric states be differentiated
from th~ other two major "bleeding diseases, namely, hemophilia, and essential
thrombocytopenia. The following outline gives the salient hematologic features
of these and other diseases with which they may be confused.
Coagulation Bleeding Clot Tourniqutl
Disease lV.B.C. PlalcJcts T,me Time ReJraction rest
Essential Throm~ Normal or Decreased Normal Prolonged Prolonged Po')itive
bocytopenic increased
Purpura.
Hemophilia Normal or Normal Delayed Normal Normal Negative
increased
Anaphylactoid Nonnalor Normal Normal Normal Normal Positive
Purpura increased
Aplast1c Anemia. Decreased Decreased Normal Prolonged Prolonged -Poti.trve
Aleukemic Leukemia. Decrea:,ed, Decreased Normal Prolonged Prolonged Positive
immature
cells

TREATMENT
In general, the treatment of this class of purpuric states has been unsatis-
factory. This is because Qf tbe multiplicity of etiologic factors and the difficulty
in many instances of establishing causative agents.
Treatment should include a thorough study to determine whether or not the
bleeding results from an allergic state, and careful search should be made for sub-
stances to which the patient may he sensitive, either by contact or ingestion, and
this may be determined only hy careful history and skin testing. If there is a sen-
sitivity to certain foods, the diet should be arranged to exclude these, or if there is
 NON-THROMBOPENIC PURPURA 459
to
a sensitivity drugs, these should be stopped immediately. We have seen several
patients recover from an episode of bleeding, by being placed on a protein free
diet and with cessation of all medication. The acutely developing purpuras with
evidence of the allergic state in the form of wheals, will most likely respond to
this type of therapy.
In the patients with prolonged bleeding of a chronic type or those with re-
current attacks, a careful search should be made for foci of infection, and these
eradicated if possible. Occasion.ally the extra-ction of a dead and infected tooth
may be all that is necessary to clear up the disease.
Treatment should include general measures and symptomatic treatment as
indicated. For relief in the acute phase the administration of o.s to I.O cc. of
1 :1000 solution of adrenalin chloride is useful. If joints are involved they should
be placed at rest and elevated. Opiates may be necessary to control abdominal
pain, only when there is assurance that there exists no acute surgical abdominal
complication.
Recently advocated preparations that are claimed to be useful in the control
of liemorrhages and that are said to he curative in some instances, are prepara-
tions of snake venom, snake antivenin, injections and oral administration of
vitamin C (cevitamic acid), and the use of ergot preparations, mainly ergotamine
tartrate. We have used all of these and will discuss them under separate headings.
The use of dilute snake venom was suggested by Peck and Rosenthal. These
authors have recommended the repeated intramuscular injections of a 1:3000
dilution of moccasin snake venom, the injections being given in gradually increas-
ing doses over a period of several weeks. They report that the results have heen
excellent in the toxic, allergic, and endocrinal types of purpura, hut that its value
is questionable in the thrombocytopenic types. It has also been our experience
that it is a valuable agent in this class of purpuras but of no service in the
thrombocytopenic forms. We usually begin with o.s cc. of the I :3000 dilution,
and give injections twice weekly, to a maximum of I cc. per dose, for a series of
ten doses. In our opinion there is developed a type of immunity against the anti-
coagulant factor in snake venom. We have had little success with antivenin in tbe
acute phase of hemorrhages. If reactions occur, the patient should be desensitized,
and the dose decreased.
The use of ergot preparations and ergotamine tartrate in particular, has been
generally disappointing and seems to affect the course of the disease very little.
Cotti has reported rather remarkable success in the intravenous injection of
cevitamic acid ,in cases of thrombocytopenic and non-thrombopenic purpuras. He
gives lao to 200 mg. daily, intravenously, for three or four days iq acute cases,
and believes that it serves to correct the weakness of the capillary walls. The
value of this treatment in the scorbutic syndromes (scurvy and sub-clinical
scurvy) is generally recognized. In those instances where bleeding is the result
of vitamin C dietary inadequacy, this treatment is of much value. Engelkes used
150 mg. daily (Cebion, l\ferck) by intravenous injection with good results.
Wright and Lilienfeld have found it of service in cases of scurvy and sub-clinical
scurvy but of no value in other hemorrhagic states. Stephens and Hawley have
treated four patients with hemophilia and two patients with thrombocytopenic
 HEMORRHAGIC D,SEASES

purpura with large doses of orange juice, with no effect on the hemorrhagic mani-
festations or on the blood findings. They suggest that favorable therapeutic re-,
suits may be expected only in those patients in Whom capillary bleeding is asso-_
ciated with some degree of vitamin deficiency. Adequate cevitamic acid can be
given by mouth in orange juice (200 cc. orange juice contains 100 mg. cevitarriic
acid), but it is possible that some patients may not be able to properly utilize that
given by mouth so that injections may be indicated.
To summarize the treatment of non-thrombopenic purpura, it should include:
(I) A careful study to determine the causative agent; (2) Omission of the
offending drug or food: (3) Removal of foci of infection; (4) Use of snake venom
in the chronic types; (5) In d,etary deficiencies the use of cevitamic acid by
mouth in the form of orange juice, and by injection in acute cases; (6) Symp-
tomatic therapy, including adrenalin if necessary and opiates for control of pain
as indicated.
Transfusio~ and various hemostatic agents are of little value.

COURSE AND PROGNOSIS

The outlook for life is good, and fatalities seldom occur unless there is bleed-
ing into vital structures such as the kidneys or brain. There may be spontaneous
remissions and the type of treatment may be falsely credited with this. Recur-
rences are frequent and as the patient grows older the disease is lessened in
severity. Remissions in some are apparently permanent.

OTHER NON, THROMBOPENIC PURPURAS

The various forms of non-thrombopenic purpura that have been described
under a variety of names are probably a part of the group discussed above. These
include; purpura simplex, a mild type restricted to the skin, with no demonstrable
cause; purpura fulminans, a rapidly developing fatal purpura of children, purpura
senilis, seen in elderly and poorly nourished people; mechanical purpura, brought
about by violent muscular activity, the ,purpura of vicarious menstruation; and
the atypical hereditary forms that are rarely seen.
Davis in London has described r r families in which there was great frequency
of ecchymotic hemorrhages occurring chiefly in the female members. In eight
of these there was a history of purpura for successive generations. It was char-
acterized by a positive tourniquet test, but all of the constituents of the blood
appeared to be normal, including bleeding and coagulation times. The authors
refer to this as a hereditary familial purpura simplex.
There has also been described a condition known as hereditary fibrfnopenia.
Schonholzer reports two such cases characterized by absence of fibrinogen in the
plasma and consequently a prolonged coagulation of the blood.
Lozner and associates have described a patient with a non-thrombopenic
purpura, in which there was proved to be an anticoagulant circulating in the
plasma, the nature of which was not known. Fowler has reported two cases of
patients with prolonged bleeding time with hereditary features, which he ,desig-
nated as hereditary pseudo-hemophilia. Rubin .has reported instances of pro-
longed bleeding time in four infants from three to five weeks old, which he
 NON-J:HROMBOPENIC PURPURA

attributed to an allergy to cows' milk. Fox and Enquer have stated that the pur-
pura associated with scarlet fever is on an allergic basis with sensitivity to a
protein agent.

HEMORRHAGIC DISEASE OF THE NEWBORN

This is a non-thrombopenic purpura (morbus hemorrhagica neonatorum)

(purpura of the newborn) that is usually restricted to bleeding beneath the skin,
and if there is associated gastro-intestinal hemorrhages, it is usually called melena
neonatorum, and if bleeding is from the cord, it is known as omphalorrhagia.
It is said to occur once in every hundred births, is not hereditary, has no
relation to nutrition, and occurs equally in both sexes. The cause in many of them
seems to be a deficiency of 'Vitamin K. See p. 466. It appears spontaneously
between the second and tenth day after birth and hemorrhages may be in the skin
in the form of purpura, from the cord, from mucous membranes, from the in-
testinal tract, or they may be large and internal into a body cavity or the brain.
Symptoms depend upon the site and extent of the bleeding. Tbe cbild may show,
therefore, a generalized purpura, tarry stools, or localizing signs of cerebral hemor-
rhage, or even collapse and shock from loss of large amounts of blood internally.
The bleeding is continuous and if there is spontaneous cessation of bleeding it
does not recur.
The blood findings depend upon the extent of blood loss. There may be a
moderate leukocytosis. The platelets are normal but the coagulation and bleeding
times are prolonged. Whipple has found a deficiency of prothrombin.
Royster has suggested the prophylactic use of whole blood subcutaneously in
infants whose coagulation time is over ten minutes. For control of bleeding, the
subcutaneous injection of 20 to 30 cc. of whole blood every six hours has been
recommended and then subsequent daily transfusions ('S cC. of blood per pound
of body weight), until bleeding stop~. One transfusion is usually sufficient.
The prognosis is good and most infants recover (75-80 per cent) with trans-
fusions. Some recover spontaneously.. Death comes quickly (two to three days)
in those who do not respond to treatment. Therefore, prompt recognition of the
disease and early treatment are essential.

BIBLIOGRAPHY
NON-THROMBOPENIC PURPURAS

CASl'EX, M. R' j'La pathogenie due pUrpura hemorrhagique." La Presse Med., 32, 27'], 1924
COT'.l'I, L.: "Influence of vi!amine C on the hemorrhagic diatheses." Haematologica, IS, 923, 1935.
DAVIS, E.: "Hereditary familial purpura simplex." Lancet, 2, rno, 1939,
ENGELXES, H.; "Treatment of hemQrrhagic disorders with vitamin C." Lancet, 2, u85, Dec. 7,
1935.
FoWLb., W. M.o' "Hereditary pseudohemophilia." Amer. Jour. Med. Sci., 193, 191, 1937.
Fox, M. I" ~nd ENQUER, N: "A consideration of the phenomenon of purpura following .scarlet
fever." Amer. Jour. Jled. Sci., 196, 32I, I938.
HENOCH', E.: "Ueber eine eigcnthumlicbe form von purpura." Berl. kUn. Wthns,h,., 11, 64t, 1874.
 HEMORRHAGIC DISEASES

KUGELMASS, I. N., and TRITSCH, J. E.: uPrenatal prevention of potential hemorrhagic disease of the
newborn." Jour. Amtr. Med. Assn., 92, 531, 1929
LaZNER, E. L., JOLLIFFE, L. S., and TAYI.OR, F. H. L.: "Hemorrhagic diathesis with prolonged coagu-
lation tune associated with a circulating anticoagulant" A mer. Jour. Med. Sci., 199, 3IS,
194°·
OSLER, \V.: I'On the visceral manifestations of the erythema group of skm diseases" Amer. Jour .
.Me-a Sci., 127, I, 1904.
PECK, S. F., and ROSENTHAL, N.' "Effect of moccasin snake venom (Ancistrodon Piscivorus) in
hemorrhagic conditions." Jour. Amer. l.[ed Assn, 104, 1066, 1935.
ROYSTER, L. T.: "Hemorrhagic disease of the newborn." Va. lIed. Month, 51, 693, 1925.
RUBIN, M. 1.: "Allergic intestinal bleeding in the newborn; A clinical syndrome." Amer. Jour. Med.
Sci., 200, 385, 1940.
SCHONHOLZER, G: "Die hercditlire fibrinogenopenie." Deutsches Arch Klin. Med, 184, 496, 1939.
SCIIONLEIN, J. L.: "Allgemeine und specielle p3.thologie und therapie." Zweiter Tei!, IV Ed. (St.
Gallen, 1839), 42.
STEPHENS, D. J., and HAWLEY, E. E.: "The relationship of vitamin C to the hemorrhagic diatheses."
Jour. Lab. and Clin Meti., 22, 173, 1936.
\VHIPPLE,G. H.: "Hemorrhagic disease: Antithrombin and prothrombin factors" Arch. Tnt. Med.,
12, 637, I913.
WOLBACH, S. B, and HOWE, P. R: "Intercellular substances in experimental scorbutus." Arch.
Path. and Lab. Med, 1, I, 1926.
WRIGHT, I. S, and LILIENFEI.D, A.: uPharmacologic and therapeutic properties of crystalline vita-
inine C (ce\itamic acid)." Arch. Int. Med, 57, 241, 1936.
 CHAPTER 38
VITAMIN K
In '930, Hemik Dam, a biochemist at the University of Copenbagen, re-
ported that certain deficiencies in the diet produced in chicks a disease resembling
scurvy. Essentially it was a fat free diet and the disease was characterized by
the appearance of spontaneous hemorrhages, associated with a prolonged clotting
time and markedly decreased plasma prothrombin. Also in '930, Horvath noted
that the coagulation time in chicks was accelerated when they were fed a diet of
sprouted soy beans.
In 1931, McFarlane and his associates noted a similar hemorrhagic disease
in chicks, in a study of the fat soluble vitamins. In 1931 also Roderick had
noted that a hemorrhagic disease of cattle, long known to veterinarians as "sweet
clover disease," developed in the animals that ate spoiled clover, and that the
condition was corrected by feeding with properly cured, unspoiled clover. He
found further that (he cattle showed no deficiency of coagulation factors except
prothrombin.
From the above apparently unrelated findings by investigators working in
different fields came two outstanding facts: first, that the "hemorrhagic disease
of chicks was produced by a fat free diet and that the same disease in cattle
was caused by spoiled clover, and that both diseases were characterized by a low
prothrombin content of the blood. In 1934, Dam and Schonheyder showed that
the factor was a fat soluble, non-sterol fraction and named it vitamin K from
the Danish word "Koagulation."

DISTRIBUTION

Vitamin K is found mainly in green leafy vegetable~ but dried alfalfa has
been the source of the vitamin used by nearly all investigators engaged in this
work. In general the vitamin is richer in the top part.> of plants, such as carrots
in which the leaves are rich in the vitamin and the root contains very little.
Furthermore, it is more abundant in leaves that are sprouted in the light than
those. sprouted in the dark. Inner leaves of cabbage have only one-fourth the
activity of the outer leaves. Other good sources of the vitamin are spinach,
cabbage, kale, cauliflower, nettle, and chestnut leaves; also tomatoes, hemp
seed, sea weed, and soy bean oil. It is also found in hog liver fat, egg yolks,
unbulled rye and rice, and in sun flowers.
Vitamin K is also found in a considerable number of bacteria. It was noted
by Almquist that if ~ertain food stuffs became spoiled by bacterial action that
these developed a marked anti-hemorrhagic activity. Using alfalfa as a standard
with a potency of one, he found that B. subtiiis had a potency of 38, B. proteus
IS, Bacterium typhosum 3, ~Mycobacterjum tuberculosis II, and Sarcina lutea 20.
Molds, yeast, and fungi apparently contain none of the dtamin. This led to
4 63
 HEMORRHAGIC DISEASES

the preparation of vitamin K by allowing bacteria to act on wet fish meal.

Furthermore, the fact that the vitamin could be formed b)' the bacteria of the
intestinal tract of chicks gave a satisfactory explanation' for some cases of
spontaneous recovery from the state of vitamin deficiency in some of the young
chicks. The vitamin has also been found in certain tissues, particularly egg
yolk. The livers of young chicks contain very little but the livers of dogs and
hogs contain a considerable amount.
The biological measurement of vitamin K is based upon its capacity to
accelerate blood clotting. Nearly all workers have devised a different method
of assay and designed different units, this having led to much confusion in at-
tempted comparisons of potency. Fortunately most of these units are already
obsolete because of the rapid progress in identification of the vitamin and the
relatively pure synthetic compounds that are now available. (See section on
technic for methods.)

PHYSIOLOGY AND MECHANISM OF ACTION

It was shown by Dam and his associates that the impaired clotting power in
chick blood was because of a reduced level of prothrombin. Furthermore, it was
found that certain animals would have prolonged blood coagulation and low pro-
thrombin levels when bile did not reach the intestinal tract, through creation
of a biliary fistula. Greaves observed that the low prothrombin of bile fistula
rats could be corrected by oral administration of vitamin K, but not efficiently
unless the vitamin was given with bile or bile salts. Quick, at Marquette Univer-
sity, as well as Smith and his associates at the University of Iowa, working with
bile fistula dogs, obtained essentially the same results.
It has long been known that administration of bile would tend to correct
bleeding tendencies that are frequently associated with human obstructive jaun-
dice but the mechanism had never been explained before. Furthermore, bile
contains no vitamin K itself. The bile component concerned in the absorption
and transport of the fat soluble vitamin K was sbown to be choleic acid. When
vitamin K plus choleic acid was given orally, it corrected the prothrombin de-
ficiencies in bile fistula rats. Smith and his associates were able to overcome
the bleeding in obstructive jaundice by giving crude vitamin K with bile or
bile salts. Later, the intramuscular use of vitamin K concentrate dissolved in
oil came into use. Peritoneal injections in fistula rats have been found to be
effective. More recently, however, there are water soluble substitutes which can
be given orally, intramuscularly, or intravenously. They are absorbed directly
from the intestine regardless of whether or not bile is present.
If vitamin K is given by mouth, since it is a fat soluble vitamin, bile is neces-
sary as a carrier acroSS the intestinal mucosa. The mechanism of this is not
known but some believe that it is absorbed by the jejunum and not by the
stomach or colon; however, it is the colon that contains the bacteria which are
able to synthesize it in some cases and it is possible that some of it may be
absorbed there. The body obtains it either from ingested foods or by the putre-
factive action of the int~stinal flora on the foods. Then with the aid of the bile
 VITAMIN K
it is absorbed, carried to the liver, there changed into prothrombin itself, and
then released into the blood.
Vitamin K and prothrombin are quite different. The material from food is
an unsaturated hydrocarbon, while prothrombin from human blood plasma is a
pseudoglobulin. The fi~st is fat soluble, the other is water soluble. The first is
stable to physical agents, the other is unstable. The vitamin is -destroyed by
oxidizing agents and prothrombin is unaffected. The vitamin does not have
clotting activity whereas prothrombin has a powerful clotting activity. The
vitamin comes from the diet and the prothrombin probably comes from the liver
and perhaps other parts of the reticula-endothelial system. If the liver is re-
moved from dogs, there is a marked depletion of prothrombin, this indicating
that the organ is the seat of its formation. Andrus and associates removed the
livers from six dogs, and noted a prothrombin decrease of 45 per cent in one hour,
and 95 per cent in ten hours. Furthermore, if the liver is injured by operative
manipulations such as prolonged simple massage, there is a decrease in the
prothrombin level and treatment with vitamin K does not correct it, this indi-
cating that the damaged liver is unable to convert the vitamin into prothrombin.
Coagulation depends to a large extent on the quantity of prothrombin in the
blood, and the period of time is retarded in direct proportion to the deficiency of
prothrombin. A healthy adult has 50 per cent whereas a newborn child has only
one-fourth that much. If the prothrombin is rednced to less than 10 or ~D per
cent of normal there is a decided prolongation of coagulation time and the pro-
duction of hemorrhage. In normal infants during the first few days after birth
there may be a decided prolongation of coagulation time because of a moderate
vitamin K deficiency. It usually disappears within a week. Kato tested 100
mature and 75 premature infants and found that the prothrombin time was pro-
longed. Perhaps the introduction of harmless bacteria into the inte,tinal tract
initiates its formation and corrects the deficiency.

CONDITIONS IN WHICH VITAMIN K DEFICIENCY MAY OCCUR

From the preceding discussion it can be seen that -the vitamin normally enters
the body in the foodstuffs by way of the mouth; secondly, that it needs the proper
biliary content to act as a vehicle for its transportation to the liver; and thirdly,
that it needs a normally functioning liver to effect the conversion to prothrombin
Certain disease conditions, therefore, would finally result in the depletion of pro-
thrombin in the blood: first, an inadequate supply of the vitamin by mouth;
secondly, inadequate absorption of the vitamin from the intestinal tract; thirdly,
inadequate metabolism of the vitamin in the formation of prothrombin. Accord-
ing to Clark and associates there are four basic factors necessary for adequate
prothrombin formation, these heing (I) a diet containing ample vitamin K; (2)
presence of bile in the intestinal tract; (3) a normal absorptive intestinal sur-
face; and (4) a liver capable of effecting the conversion into prothrombin. Based
upon this the following diseases or deficiencies may interfere with the process- of
prothrombin formation.
 HEMORRHAGIC DISEASES

1. Dietary deficiency.
2. Deficiency of bile.
a. Obstructive jaundice from stone of common duct or carcinoma of head of
pancreas.
b. Biliary fistula.
3. Impaired intestinal absorptive surface.
a. Various tumors, malignant and benign.
b. Ulcerative colitis.
c. Intestinal polyposis.
d. Intestinal obstruction.
e. Short-circuiting operations.
f. Enterostomy tubes.
4. Damage to the liver.
a. Possibly obstructive jaundice.
b. Acute yellow atrophy.
c. Cirrhosis of varying types.
d. Poisoning with chloroform, phosphorus, carbon tetrachloride, etc.
e. Sweet clover disease of cattle.
f. Partial or complete hepatectomy.
g. Banti's disease.
h. Multiple or single abscesses.
i. Metastatic carcinolna.
5. Hemorrhagic disease of the newborn.
a. Inadequate prothrombin in mother.
b. Deficiency of storage in the fetus.
c, Icterus gravis neonatorum.
d. Congenital hydrops, possibly.

HEMORRHAGIC DISEASE OF THE NEWBORN

This is a type of alimentary vitamin K deficiency in the first few days of
life. The recovery may be spontaneous because of beginning bacterial action in
the intestinal tract after the ingestion of food. However, Tocantins observed
a full term infant whose plasma prothrombin was less than r per cent of the
normal; on the 11th day the prothrombin time became normal; the infant died,
however, from a complete obstruction caused hy duodenal atresia. In this situa-
tion the prothrombin level came to normal even though there was complete
obstruction with no food entering the intestinal tract. There was no contact
with bile and no synthesis of vitamin K by bacterial action in the intestine.
This would indicate that liver damage was responsible for the condition.
Treatment with vitamin K orally effects a prompt recovery. Kugelmass
states that in treating hemorrhagic disease of the newborn, therapy should not
be limited to a precursor of prothrombin, but that prothrombin itself should be
given in the form of one or more transfusions. When blood is given there is
absolute assurance that prothrombin is being given, but with the administration
of vitamin K there is no assurance that it will be converted into prothrombin.
Furthermore, the deficiency of prothrombin in the infant can be prevented by
giving vitamin K to the mother before delivery. It is possible that treatment
by injection of whole blood into these babies provides a sufficient amount of
 V,TAMIN K

prothrombin to tide the infant· over until bacterial action becomes established
in the intestinal tract.

OBSTRUCTIVE JAUNDICE
This situation is produced by obstruction to outflow of bile into the intestinal
tract, in such conditions as biliary fistula, blocking of the common duct by
carcinoma, stones, and inflammation. Even though the vitamin K intake is
adequate there will not be the mechanism to transport it across the intestinal
barrier. In such instances, of course, the water soluble vitamin can be given by
mouth or by injections ~nd will be effective regardless of the presence of bile.

HEMOItItHAGIC DISEASE OF LIVER DAMAGE

This can be produced hy trauma to the liver at operation and even by mas-
sage of the liver. There may he a hypoprothrombinemia after liver injury,
chloroform poisoning, p,imary liver disease, acute yellow atrophy, various forms
of hepatitis, cirrhosis, metastatic carcinoma, etc. The administration of vitamin
K by any route will not correct this situation since there must be a normal liver
to change it into prothrombin. It is true, however, that liver damage has to
be quite extensive in many instances before the function of prothrombin for-
mation is lost.

HEMOItItHAGIC DISEASES FROM OTHER CAUSES

These include conditions in which intestinal absorption is hindered and
therefore the prothrombin level falls. This includes sprue, hiliary fistula, .ulcera-
tive colitis and other abnormalities of the intestinal surface characterized by
deficiency of absorption. Such conditions are treated quite successfully with
vitamin K preparations if given in the injectable form.

PREPARATIONS OF VITAMIN K
The mechanism of vitamin K action is obscure since there is nothing known
about its conversion into prothrombin except that it occurs in the liver. Vitamin
K is not toxic in the ordinary sense and high doses can be given without evidence
of toxicity. Studies on the lethal dosage of vitamin K and two of its substitutes,
phthiocol and methylnaphthoquinone, have shown toxic effects of the two sub-
stitutes at levels a great deal higher than the normal therapeutic dose. None of
the preparations has shown toxic effects in ordinary clinical usage.
Up to 1933 it was necessary to obtain vitamin K either from alialfa or from
putrefied fish meal. The product from the former is now known as vitamin K,
and that from the latter as vitamin K,. In 1933, however, a group at Yale Uni-
versity isolated phthiocol from tubercle bacilli, and because of its close chemical
relation to the quinones, a group of investigators in California tried it as an
anti-hemorrhagic substitute for vitamin K, for which it was found to be very
effective. Therefore phthiocol was the first synthetic compound that was dis-
covered that has anti-hemorrhagic properties. Since then, other more potent
synthetic compounds have been produced, all of which are quinones. The most
active one of the group is 2-methyl-1,4-naphthoquinone of which 1 mg. daily is
 HEMORRHAGIC DISEASES

an adequate clinical dose. ,This is the product that is water soluble and can be
administered by practically any route and that has the widest clinical application
today.
Bacharach and Chance found that 2-methyl-IA-naphthoquinone was fully as
active in chicks as the natural vitamin. When administered in olive oil to fuur
patients with ohstructive jaundice it was effective.
Kark and Souter found that the water soluble derivative of, 2-methyl-Ik
naphthoquinone is very effective by either intravenous or intramuscular adminis-
tration. It is a sodium salt readily soluble in water, each cc. containing one
Jllilligram of the drug. They treated ,8 patients with ulcerative colitis, stricture
of the common bile duct, carcinoma of the ampulla of Vater, and hemorrhagic
iisease of the newborn and all responded with a rapid rise in blood prothrombin.
rwelve patients with hypoprothrombinemia presented clinical evidence of he-
patic disease and did not respond to intensive administration of either. Therefore,
the patient with a damaged liver cannot synthesize prothrombin from any of
the synthetic or natural vitamin K preparations.
The optimal dosage is one mg. daily which is usually a synthetic preparation
in oil and injected intramuscularly. If the water soluble preparation is used the
same dose can be given intravenously in an isotonic saline concentration. It is
water soluble at room temperature to the extent of 0.1 mg. per cc. and can be
incorporated in other solutions for intravenous use.

BIBLIOGRAPHY

I. ALMQUIST, H. J.: "Purification of the antihemorrhagic vitamin." Jour. BioI. Chem, 114, 241,
1936.
2. ALMQUIST, H. J.: "Vitamin K." Physiol. R~., 21, 194, 1941.
3 ANDRUS, W DLW, and LORD, J. W, JR.: "Correction of prothrombin deficiencies." Jour.
Am-er. Mea. Assoc., 114, ~336, 1940.
4. A..""l"DRUS, W DEW., LORD, J. W., JR, and MOORE, R. A: "Effect of hepatectomy on plasma
prothrombin and utilization of vitamin K." Surgery, 6, 899, 1939.
CLARK, R L, JR, DIXON, C. F, BUTT, H. R., and SNELL, A. M.: uDeficiency of prothrombin
assocIated with various intestmal dtsorders: Its treatment with the antihemorrhagic vitamin
(vitamin K)." Proc. StaB Meet. Mayo Clin., 14, 407, 1939.
6. DAM, H' Cholesterinstoffwechsel in hUhnereiern und huhnchen." Biochem. Ztschr., 215 f 475,
19 2 9.
- - ; "Uber die cholesterinsynthese im tierkorper." Biochem. Ztschr., 220, 158, 1930.
7. DaISY, E. A., BINKLEY, S. B., THAYER,S A., and McKEE, R. \V.: "Vitamin K." Science, 91,
58, 1940.
8. GRAV!:S, J. 0: "The nature of the factor which is concerned in loss of blood coagulability of
bile fistula and jaundiced rats." Amer. Jour. Physiol J 125, 423, 1939
9. HORVATH, A. A' "Change in hen's blood produced by a diet of sprouted soybeans." Amer.
Jour Physiol, 94, 65, r930
10. KARX, R., and SOUTJ:R, A. W.: "Synthetic vitamin K in treatment of hypoprothrombinemia"
Lancet, 1, II49, 1940.
II. KATO, K, and PONCH!:R, H. G: "The prothrombin in the blood of the newborn mature and
immature infants as determined by micro prothrombin test." Jour. Amer. Med. Assoc,
114, 749, I940'
Il. KUGELMASS, I. N.: "Vitamin K in hemorrhagic diseases of infants and children." Amer. Jour.
CUn. Path., 10, 673, r940.
 VITAMIN K
tJ. McFARLAND, W. D, GRAHAM, W. R, and RIC'.IIARm;ON, F.' "The fat soluble vltamm require~
ments of the chick. I. The vitamin A and vitamin D content of tisb, meat and meat
meal." Biochem. Jour, 25, 358, 1931.
14 MACFIE, J. M , BACIIARACR, A. L, and CRANCE, M. R. A.: "The vitamin K activity of 2-roetbyl-
1-4-naphthoquinone and its cbrucal use in obstructive jaundice." Brit. Med. Jour" 2, 1220t
1939·
IS. QUICK, A. J.: ('On the action of heparin and its relation to thromboplastin." Arn~r. Jour.
Physiol., 115, 317, 1936.
16 RODERICK, L M. CIA problem in the coagulation of the blood. Sweet clover disease of cattle."
Amer. Jour. Physiol, 96, 413, 1931.
11. SMITH, H. P .• WARNER, E. D, and BRINXH01]S, K M: "Prothrombin deficiency and the bleed-
ing tendency in liver injury (chloroform intoxication)" Jour. Exper Med., 66, ,801, 1937.
18. SNELL, A. M.: "Vitamin K.: Its properties, distribution and clinic.'ll importance." Jour. Amer.
Med. A.ssoc., 112, 1457, 1939.
19 ToCAm'INS, L. M.· "Probable mechanism of the physiologic hypoprothrombinemia of the new-
born." Amer. Jour. Dis. Children. 59. IO~4. 1940.
 SECTION SEVEN
INFECTIOUS MONONUCLEOSIS
POLYC YTHEMIA VERA
THE BONE MARROW
MALARIA
HODGKIN'S DISEASE: HISTOPLASMOSIS
BLOOD GROUPS AND BLOOD TRANSFUSION
THE BLOOD PICTURE OF NORMAL
. LABORATORY' ANIMALS
 SECTION SEVEN
MISCELLANEOUS

CHAPTER 39
INFECTIOUS MONONUCLEOSIS

(Benign Lymphadenosis) (Glandular Fever)

HISTORY

Because of the rarity of the monocytic response in contrast to the neutro-

philic response to an acute infectious process, infectious mononucleosis has at-
tracted attention at intermittent periods for many years. The first record of
the disease was in 1885 when Filatow, in Russia, described an idiopathic lym-
phadenopathy in children. In 1889, Pfeiffer established the existence o'i tbe dis-
ease as a clinical entity and gave it the title of glandular fever. 'He described it
as an acute infectious disease, principally of children, characterized by pyrexia
and rapid enlargement of the cervical lymph glands. Although his clinical descrip-
tion was quite thorough, he failed to describe any blood changes. Subsequent
observers agreed that the disease was infectious and reported a number of epi-
demics, the most important being the epidemic in Ohio reported by West in 1896
involving 96 children, This was the first record of cases in America. Later, in
1908, Terflinger reported ISO cases occurring in adults, thus showing for the first
time that this disease, though considered one of childhood, may affect adults of
any age group.
In 1909, Burns stated that the small monoilUclear elements of the blood'seem
to be the ones chiefly increased and he noted the lymphocytes to be increasea i as
high as 01 per cent in his cases. 'I
Reports were_ scarce in the literature until '920, when a wi,despread interest
was stimulated-by the report of Sprunt and Evans, who observed several cases in
adults presenting symptoms of an acute infection, a moderate enlargement of the
lymph nodes and of the spleen, and a mononuclear leukocytosis. They called this
SUpposedly new disease entity "infectious mononucleosis." Tidy and Morley,
in 1921, studied an epidemic with careful blood examinations and came to the
conclusion that Pfeiffer's glandular fever and infectious mononucleosis were
identical.
Following this, many reports have appeared under many different titles in-
473
474 l\!ISCELLANEOUS

cluding, "Acute Benign Lymphoblastosis," "Acute Benign Leukemia," "Acute

Lymphadenosis," "Monocytic Angina," and "Lymphocytosis of Infection."
The two terms "glandular fever" and "infectious mononucleosis" have per·
sisted. Downey and l\1cKinlay emphasize the occurrence in adults, the absence
of epidemicity, and the relatively severe throat symptoms in infectious mono-
nucleosis which, they believe, were lacking in the original glandular fever. How-
ever, in the years 1928 and 1930, the disease reached almost epidemic propor-
tions and has been carefully studied by observers over the world. There has
been noted in this same period, both in children and in adults, the predominance
of glandular swelling in some and of severe throat manifestations in others, but
underlying them all, the characteristic mononuclear blood picture. On these
grounds, it seems likely that the condition reported earlier as an epidemic disease
of children and that reported later as a sporadic disease in adults, are the same
clinical entity.
The most recent and valuable contribution was that of Paul and Bunnell,
in 1932, when they reported the occurrence of a high titer of heterophile
antibodies in the serum of patients suffering from the disease. The heterophile
antibodies were determined by the presence of agglutinins and hemolysins for the
red blood cells of sheep, and since they occurred in considerably higher titer in
infectious mononucleosis than in any other disease, the heterophile antibody test
was introduced as a diagnostic procedure. Conclusive statistics have been com-
piled attesting to the value of this finding. At the present time, there is some
doubt as to whether the antibodies are of the true heterophile type but their pres-
ence in a high titer seems specific for infectious mononucleosis and the test, with
modifications, is valuable in differential diagnosis. (See section on technic for
details of procedure.)

ETIOLOGY
Infectious mononucleosis has been reported from many parts of, the world
illcluding North America, Europe, A,!stralia, and the Orient. The only reports
from the tropics are a small epidemic from British Guiana and two cases from
Manila. There are no reports, so far as we know, of the disease in negroes and
it is questionable whether or not this race is exempt.
This disease is more prevalent in children and young adults, especially in tlle
epidemic type, although no age is immune. Infants up to the age of six months
seem to be immune, though one case is recorded at four months. Males seem
to develop the disease more readily than females. There appears to be no seasonal
or occupational incidence of significance.
The disease occurs in both epidemic and sporadic form and the degree of
infectivity seems to vary with the different outbreaks. Single cases ,will often
occur in a family and usually no other member becomes infected, but when
cases develop in a school or nursery, a large number of children may develop
the disease. Sporadic cases usually occur among adults, while epidemics occur
among children. Droplet infection is supposed to be the method of transmission
since the disease spreads from one child's bed to another and fairly close contact
seems necessary. The incubation period varies between five and fifteen days.
 INFECTIOUS MONONUCLEOSIS 475
Various bacteria have been reported as possible causative agents, but the eti.
ologic agent of this disease is unknown. There have been two main views regard·
ing the cause of the response of the blood in infectious mononucleosis. According
to the first view, a special infective agent gives rise to the characteristic Iymphocy.
tic response. According to the second, the lymphatic reaction is probably due to
an unusual constitutional disposition on tbe part of the patient reacting to an
ordinary acute infection; in other words, the type of response, and not the
stimulus, is the unusual feature. In weighing the evidence, we find that it over-
whelmingly favors the theory of a peculiar infective agent. Sprunt and Evans
summarize the following points upholding this view. First, the disease occurs in
definite epidemics in which a large proportion of those exposed may become ill.
Second, a person who has suffered from infectious mononucleosis may Shortly
afterwards or even during the disease exhibit a typical polymorphonuclear leuko-
cytosis in reaction to another infection. Third, it seems illogical to assume a con-
stitutional inferiority of the protective mechanism in a disease from which a
patient uniformly and promptly recovers. Fourth, there are other specific infec-
tious diseases associated with an actual lymphocytosis of the blood, as for ex-
ample, Whooping cough and rubeola.
In attempting to discover this infective agent, many bacteria have been
suspected, {ncluding a variety of organisms found in the oral and throat lesions,
such as various streptococci, diphtheroid organisms, influenza bacilli, avirulent
diphtheria bacilli, and the spirilla and fusiform bacilli of Vincent. 'Studies on
these organisms have revealed nothing significant; therefore, they may be con·
sidered as secondary invaders. The high incidence of Vincent's infection has led
some to believe that these bacteria are perhaps associated with the etiological
agent.
Blood cultures as well as cultures from the excised lymph nodes are usually
sterile although adenopathy would indicate that the essential lesion and focus
of growth of- the infective agent is in the lymph ";odes. It is believed by most
observers that the agent gains entrance to the body by way of the tonsils or upper
respiratory tract, and others suggest the gum margins as the portal of entry. It is
possible that the infectious agent enters the mesenteric glands by way of the
intestinal tract and travels up the thoracic duct to the cervical glands.
Many attempts have been made to produce the disease in laboratory animals
with varying success. A condition simulating infectious mononucleosis in man
has been produced in rabbits in three different instances. In 1926, Murry, Webb,
and Swann found that Bacterium monocytogenes was the etiologic agent for a
disease of ~abbits characterized by pronounced monocytosis; in 1929, Nyfeldt
isolated an organism called Bacterium monocytogenes hominis from the blood
and with this organism succeeded in producing the cellular blood picture of in-
fectious mononucleosis in rabbits: the following year, Bland produced a disease
closely resembling infectious mononucleosis in a rabbit by the injection of citrated
blood from a patient with the disease and reported a protozoon of the genus
Toxoplasma to be the etiological agent. .Hunt has described the characteristic
blood changes in the guinea pig following injection of pharyngeal exudate from
a patient with a severe case of Vincent's angina, and has reported further that
 MISCELLANEOUS

infectious mononucleosis developed in persons who had heen in contact with the
experimental guinea pigs. However, these experiments, important as they seem,
have not been adequately confirmed.
The discovery of heterophile antibodies in the serum of patients with in-
fectious mononucleosis has opened a new avenue for investigation of etiology.
Since the antibodies are characteristic of this disease, Bailey and Raffel believe
that the disease represents a specific response to a definite infective factor, either
intrinsic or extrinsic, rather than a reaction to a non-specific stimulation. Fwm
their experiments, they conclude that if the causative agent is found in the nose
or throat it is not culturahle and the organisms cultured from this region fail to
absorh the antibodies from the serum. Negative hlood cultures of infectious
mononucleosis patients, however, when injected into rabbits, will produce in the
serum a high hemolytic titer for sheep cells. Evidence is accumulating to indi-
cate that the cause of infectious mononucleosis may be a virus. Wising in
Stockholm injected monkeys with an emulsion of a lymph gland removed froll!
a patient suffering from the disease. He was unable to culture bacteria from this
material under either aerobic or anaerobic conditions. The monkeys' developed
clinical symptoms that resembled those of infectious mononucleosis and one of
the assistants who pricked his finger with a knife on which there was some of
the material, developed the disease.
Van den Bergh and Liessens have reproduced the disease in monkeys hy
the injection of blood from a patient who had the disease, the blood having been
passed through a Berkefeld filter, and cultured before injection. The animals
developed a positive heterophile antibody test. They feel that they have suc-
ceeded in transmitting the disease with a'virus and determined that the virus was
resistant to low temperatures for as long as 30 to 40 days.
Parker at Emory University has attempted to reproduce the disease in human
volunteers by spraying their throats with the Berkefelded nasal and throat wash-
ings from a patient ill with the disease but these experiments were unsuc,cessful.

SYMPTOMS AND PHYSICAL FINDINGS

The onset of the disease is gradual, averaging abont six days. The patient
complains of fatigue, dull headache, general malaise, followed by fever, sore
throat, and lymphadenopathy. The cervical, axillary, and inguinal glands usu-
ally become enlarged and there is tenderness in these regions. In some instances,
the patient has joint pains, chills and sweating.
Since there is such wide variation in these symptoms, it seems advisable to
classify the disease into four distinct types at the onset, as Canuteson has done'
after studying a series of forty-five cases;

(I) Those with sore throat, headaChe, and malaise, simulating an acute upper
respiratory infection.
(2) Those with swollen glands as the only complaint.
(3) Those with a slow insidious onset over a period of two weeks or longer
with complaints of fatigue, loss of weight, an occasional morning sore
 INFECTIOUS MONONUCLEOSIS 477
throat, subacute nasal infection, mild night sweats, insomnia, and some-
times, gastro-intestinal upsets.
(4) Those with a sudden onset with chills, fever, prostration, and few abnor-
mal physical findings.
Isaacs and his associates summarize the symptoms and physical findings as
follows: "Headache, general malaise, sore throat, tenderness of the glands, back-
ache, chilliness, anorexia, coryza, sweating, weakness, cough, dizziness, sore
bleeding gums, nausea, stiff neck, epistaxis, stomatitis, abdominal pain, rash,
photophobia and conjun~tivitis. The physical signs usually encountered are fever,
enlargement of the spleen, enlargement of the tonsils, membranous angina and a
maculopapular rash. The temperature usually varies between 101 0 and 105 0 F.
Enlargement of the glands is present in 100 per cent of the cases and tenderness
in 76 per cent. Suppuration of the glands has seldom been observed." Because
of the various clinical types, there is considerable variation in symptoms and
physical findings.
From a study of 50 cases in university students, McKinley gives a picture of
the clinical aspects of the disease. He states that the generalized enlargement of
the lymphatic glands is the outstanding and constant clinical feature. The
adenopathy usually begins and reaches its maximum in the cervical triangles,
with the involvement of the left nodes frequently predominating. The posterior
cervical lymphatic chains are constantly enlarged, -the extent of the adenopathy
being out of proportion to the intensity of the lesion in the throat. The deep
cervical nodes are massed in some cases under the sternomastoid muscle about
its middle aud somewhat below the angle of the jaw. The other individual nodes
usually reach a maximum of 3 em. in length and cause no appreciable pain. Sup-
puration occurs very rarely. The posterior auricular nodes usually are not en-
larged, but the axillary and inguinal nodes are enlarged rather constantly. The
epitrochlear nodes are frequently palpable.
Throat infection is present in about 78 per cent of cases. Frequently there
is marked infection of the fauces with swelling of the lymphoid tissue, the so-
called granular pharyngitis; less commonly· it is a membranous angina, indis-
tinguishable in appearance from tbat of diphtheria, and occasionally, follicular
tonsillitis or pharyngitis exists. The spleen is palpable in about 40 per cent of
the cases, its margin soft and its size never extremely large.
The infectious features vary but fever is almost always present with malaise,
headache, and in some cases, sweating and chills. The temperature ranges from
0
100 to 1030 F. and may last from three to four weeks. The pulse rate is propor-
tional to the temperature.
Other findings occurring more rarely are rashes, varying in type in different
patients and conjunctivitis of a dry catarrhal type. Also, jaundice is sometimes
seen. This condition may be caused by the enlargement of the nodes which
occlude the common duct.
As a rule, no complications occur and the disease runs a course of twenty
to thirty days. In rare instances, nephritis, retropharyngeal abscess, or otitis may
develop, but usually the patient recovers with no lasting ill effects.
 PLATE XLVII

INFECTIOUS MONONUCLEOSIS
(BENIGN LYMPHADENOSIS)

I. Large lymphocytes f .~-,. -, . . ":-

2. Lymphocytes with atYPical lobulation of~nuc1eus.
,3.Lymphocyte WIth vacuoles In cytopbsm,"
4 Small lymphocyte
5 Heavlly granulated monocyte
6, Neutrophil.

Blood Findmgs: Differential:

Hemoglobm 16 gms. (Ne'Ycomer's method) Neutropbils • 20%
RBC. . 5,210,000 per C.mm. Lymphocytes
38,000 per c,mm.
70%
W.BC. Monocytes •••• 100/0
Platelets . 390,000 per c mm.
Erythrocytes are normal.
Heterophil Antibody Test -Agglutination positive in dilution of I I to I: :128
Plate XL VII.
PI.!e XLVIII.
 MISCELLANEOUS

HEMATOLOGIC FINDINGS
In nearly every patient, the diagnosis can be made by the characteristic blood
picture. The red blood cells and the hemoglobin are not affected in cases of short
duration which is an important differential finding from a leukemic process. If
convalescence is prolonged, a slight secondary anemia may develop and persist
for several months.
The thrombocytes, as a rule, are unaffected. In rare instances, they may be
decreased and a hemorrhagic tendency may develop. However, some complicat'ing
factor is probably responsible for this rather than the disease itself.
The total white blood count is neither constant nor typical in infectious mono·
nucleosis. Most of the patients develop a leukopenia sometime during the course
of the disease, often with a leukocyte count as low as 3000 to 4000 cells per cu.
mm. Some observers believe that the onset is ushered in by a leukopenia. I
have observed this in a large number of cases and in rare instances the leukocyte
count may reach extremely low levels. Foard and Butt in their study of 42 cases
found that leukopenia was present in five of them in the early stages of the
disease. During the period of enlargement and subsidence of tbe lymph nodes,
the total count increases, possibly going as high as 20,000 or above. We have
studied one patient whose maximum white cell count was 50,000 cells per cu. mm.
About the third week after the onset the count usually returns to normal and
remains there during convalescence unless a relapse occurs.
The differentiaL cell count is the striking feature qf the blood picture. It is
constant and typical in the marked shift toward an increase in mononuclear cells
at the expense of the neutrophils. The mononuclear cells include both monocytes
and lymphocytes. The monocytes are present in normal numbers or are slightly
increased, while the lymphocytes show a relative and absolute increase of 50
per cent or above sometime during the disease. The lymphocytosis appears to
be higher in patients with high temperatures and higher in sporadic than in epi-
demic cases. The abnormal blood picture may persist for months after the
initial attack.
The lymphocyte of infectious mononucleosis is not the lymphocyte of normal
peripheral blood. It is an atypical cell which exhibits different characteristics in
various patients. Downey and McKinlay, in 1923, described thoroughly the
finer cytology of the abnormal lymphocyte. Because of the variation in the
atypical lymphocytes in different patients, they find it satisfactory to divide
the patients roughly into three groups, depending on the type of lymphocyte
which predominates in the blood picture, thus proposing a hematologic classifica-
tion. The following description of the three cell types is essentially that given
by Downey and McKinlay in 1923.
The cells of Type I appear most frequently. These cells are not excessively
large when compared with normal lymphocytes, but they are all atypical, patho-
logic forms. Many show a marked lobulation of the nucleus with chromatin in
the form of a coarse network of heavy strands and masses which are not sharply
separated from the parachromatin. This feature gives the nucleus a cloudy ap·
pearance which is characteristic of this type. In some cells, there is a slight con-
 INFECTIOUS MONONUCLEOSIS

densation of the chromatin with corresponding separation from the parachromatin,

resulting in the formation of a more definite network. These cells are highly
differentiated, mature, and functionally active. Their state of differentiation is
determined by the inner structure of the nucleus, indicating mature lymphocytes,
while the age of the cells and their functional activity are expressed by the lobula-
tion of the nucleus, the vacuolization of the cytoplasm, the large amount of
hyaloplasm, and the azure granules. The nuclei of the larger cells are frequently
placed eccentrically and may nearly fill the cell. The cytoplasm in this pre-
dominating type of cell is quite basophilic. The spongioplasm appears as dark
blue or slate blue, finely granular or flaky, material with a pale y~llow back-
ground of hyaloplasm and is so distributed that it gives the cytoplasm a vacu-
olated, foamy, or mottled appearance. One or more azurophil granules may b~
embedded in'the hyaloplasm. We consider this cell, described above, to be the
predominant characteristic cell of the disease and in our opinion, it can be found
in all cases at varying times (see Plate 47).
In the cells of Type II, the nucleus is somewhat similar to that of a plasma
cell. The chromatin strands are very coarse and there are several dense;rounded,
Dr angular masses of chromatin among them. They have more of a ~ashed ap-
pearance and the blocking of the chromatin is not so pronounced as in the plasma
cell. The cytoplasm has fewer vacuoles and its spongioplasm has a smoother
appearance which does not give the foamy, spongy, and stippled effect of cells
in Type I. Generally, the cytoplasm is less basophilic, the cell body wider, and
the nucleus more rarely lobulated than in cells of Type I.
The cells of Type III show more leukemic features than any of the others.
Tb'ey are rather large cells with vacuolated cytoplasm and some are quite baso-
philic. The nuclei appear immature, with a fine, siet·elike arrangement of the
chromatin. In the larger cells, there is, as a rule, relatively more hyaloplasm,
but the distribution of the spongioplasm remains about the same. Po?sibly o?e
per cent of the lymphoid cells contain a single large vacuole with an azurophil
rod located ;"ithin an identation of the nucleus. Azurophil rods ,.lie "features
which have been reported in many cases of acute leukemia. Some of the cells
seen in tlie more acute and fulminating forms of this disease may show alarming
degrees of immaturity. Bowcock has reported mitotic leukoblasts in the periph-
eral blood in infectious mononucleosis.
There has been a diversity of opinions concerning the derivation of the
atypical mononuclear cells. The point at issue has been whether they are of lymph-
oid origin or belong to the monocytic series. American authorities from the be-
ginning have believed the abnormal cell to be of lymphoid origin, and more re-
cently the European observers have come to agree. Besides the study of the actual
morphology of the cell, the peroxidase test on cells from infectious mononucleosis
is negative, thus giving a lymphocytic rather than a monocytic reaction. When a
lymph gland, considered by most hematologists as the site of manufacture of lym-
phocytes, is examined microscopically, the endothelium of lymph sinuses is usually
swollen and many free and fixed large mononuclear cells are seen in the substance
of the gland (McLean).
Controversy has also arisen as to the maturity of the atypi~al lymphocyte.
 PLATE XLIX

INFECTIOUS MONONUCLEOSIS
(LARGE CELL TYPE)

(() C)
2 3

(9 () -
4 5 6

7
OJ G 8
,

/?\
\f:__/
10
V 11
a 12
These cells were drawn from a Wright stain film from a case of infectious mononucleosis at the
height of the disease at which time the leukocyte count was 14,000 with SIxty per cent of the circulat-
ing cells being the type shown on this plate. Note the great variation in the morphological character-
istics of the ceIls. The nuclei vary markedly in size and shape. Cell No. I shows an irregular nucleus
with a smgle nucleolus and sky blue cytoplasm wIth a color accentuation at the peripl!ery of the cell
resulting in a pate peri-nuclear zone. This type of cell, as well as Nos. 3, 9, la, and II, could prob-
ably be classified as the plasma cell type frequently seen in infectious mononucleosis Cell No. :2 is
sinnlar but smaller and resembles a lymphocyte. Cell NO.4 could be mistaken for a neutrophihc band
form. Note that cells No. I, 5, and U, show small, oval structures resembling nucleoli. Bowcock
and others have reported mitotic figures in cells of infectious mononucleosis.
Plate XLIX.
Plate L.
 PLATE L

INFECTIOUS MONONUCLEOSIS

S 6 1

8 10

11
COeD
Ii?- 13

FIes 1-6, PathologlC "Ieucocytoid"·rnature lymphocytes from case of Type I. Climcal picture
01 tbi& patient was almost simllar to that of acute leucemia, but blood contained no immature cells.
FIG 7. Characteristic cell of Type II. Nuclei of these cells frequently resemble those of plasma
cells derived from lymphocytes. Cytoplasm IS not so basophilic and not so vacuolated as in Type I.
FIGS 8-II. Cells from Type III. In general cells resemble those of Type I, but some of them
show leucemic features, such as azurophlle rod in the large vacuole of Figure II. narrow-bodied
cells wlth indented nuclei (not illustrated). and cells with nuclei which are more or less immature.
ie., with dIffuse sieve-hke arrangement of chromatin and nucleoli (Figs 8 and 9)·
FIGS I2~I4. Immature cells from case of acute lymphatic leucemia. These are included to
facilitate comparison of mfectIous mononucleosis wlth acute lymphatic leucemia Total count and
lymphocyte percentage in thIS case were similar to counts in Case I (Figs 1-6). After Downey and
McKinlay, I923.
(From Handbook of HematoloGY1 Hal Downey, editor. Paul B. Hoeber, Inc, New York, I938.)
 MISCELLANEOUS

Downey states that the cells are mature and very rarely an immature cell is seen.
Others believe that the cells are immature because of their size and their com-
plete divorcement from the mature lymphocyte of normal blood. Stuart and his
co-workers uphold the latter view and report the finding of binucleated forms in
the peripheral blood.
Osgood has described a new feature of the lymphocytes of infectious mono-
nucleosis consisting of nuclear fenestrations, usually in the mature small lympho-
cytes. The fenestrated nucleus appears at first glance to be a nucleus containing
multiple nucleoli, but careful study shows that, in Wright's stained smears,
there are actually multiple holes, piercing the nucleus in various directions.
Though found in relatively small proportions, these nucleus fenestrations con-
stitute a definite diagnostic sign since they have been found in many of the cases
of infectious mononucleosis carefully studied for their presence.

DIAGNOSIS
The variation in modes of onset and the persistence of the enlarged glands
are the factors chiefly responsible for difficulty in diagnosis from a clinical stand-
point. Baldridge and his associates give an adequate account of the conditions
offering difficulty in differential diagnosis, as follows:
Cases with the septic type of onset must be differentiated from pyogenlc
septicemia, acute leukemia, acute Hodgkin's disease, miliary tuberculosis, influ-
enza, typhoid fever, dengue fever, epidemic thyroiditis, and tularemia. ~The
cases associated with fever and membranous angina may sometimes be confused
with diphtheria, scarlet fever, Vincent's angina, or follicular tonsillitis. Cases in
which there is only fever, tender glands, and a granular throat with moderate
constitutional symptoms may be confused with paranasal sinus disease or even
mumps. If abdominal pain and tenderness are the chief symptoms, appendicitis
may be suspected. The instances in which the lymphadenopathy persists and is
the constant feature, should be differentiated from other conditions in which
lymph nodes are enlarged, such as the malignant lymphomas, including Hodgkin's
disease, leukemia, lymphosarcoma and tuberculous adenitis. Finally, if the
onset is insidious and few symptoms are noted, the diagnosis may be missed
entirely and the disease classified as "idiopathic adenltis." Marshall has re-
ported four cases of infectious mononucleosis and emphasized the va~iety of
clinical symptoms that may be seen in the disease. Each of the four simulated
a different disease. One was characterized by a predominance of cerebral symp-
toms; another simulated lymphatic leukemia; there was one with all evidence
of a simple follicular tonsillitis and a fourth had weakness as the outstanding
symptom. Because of tbe variability of the clinical picture, the disease is espe-
cially important from the standpoint of differential diagnosis.
Much of the confusion resulting from the variable clinical symptoms can
be clarified by a study of the blood picture. It must he noted first, that few
infections produce an absolute lymphocytosis as is seen in this disease. Malaria,
typhoid fever, pertussis, malta fever, tuberculosis, and syphilis ar~ some of the
most common conditions producing a lymphocytosis, but the degree of increase
is usually much less than in infectious mononucleosis. Furthermore, the atypical
 INFECTIOUS MONONUCLEOSIS

lymphocyte is specific for infectious mononucleosis and should act as a definite

diagnostic criterion fQr the disease. During the winter of 1940 I studied a large
number of cases among University students in the Atlanta area, and in many of
these the diagnosis was quite difficult, mainly because of the presence of an
epidemic of wbat was called German measles. The two diseases appeared to be
very similar in clinical manifestations and in hematologic findings. German
measles is usually ushered in with leukopenia, sore throat, a faint macular rash,
and cervical lymphadenopathy. Frequently there are plasma cells in the blood
quite similar to those seen in infectious mononucleosis. In a few instances I
obtained positive heterophil reactions in cases that presented the clinical picture
of German measles, according to the judgment of experienced clinicians.
In many instances infectious mononucleosis bears its closest resemblance
to leukemia, both clinically and hematologically. The differential diagnosis is
of utmost importance here in order to predict the outcome since in infectious
mononucleosis nearly all patients recover and in leukemia the prognosis is
fatal. During the early stages of lymphatic leukemia, the fever, signs of an
upper respiratory infection, and the enlarged lymph glands resemhle closely the
symptoms of infectious mononucleosis. After a short while, however, the leukemic
patient becomes progressively worse with increasing fever, anemia and hemor-
rhages, while the patient with infectious mononucleosis remains the same or be-
gins to recover. The degree of lymphocytosis is usually more marked in lymphatic
leukemia but a low count is not enough to justify its exclusion. By careful com-
parison of the types of cells that are present, a correct diagnosis can usually be
made. Difficulty results from confusing the large basophilic lymphocytes of .in-
fectious mononucleosis with the immature lymphoblasts of acute lymphatic leu-
kemia. In lymphatic leukemia the lymphocyte shows definite evidence of im-
maturity as evidenced by the changes in the nucleus, the delicate, fine net of
regular chromatin which appears finely granular, and the presence of nucleoli
(see Plate 40). The atypical cell of infectious mononucleosis-is' more mature as
shown by the indented or lobulated nucleus, the coarse and definite nuclear
pattern, the absence of nucleoli, the vacuolation, and the relatively large amount
of cytoplasm. Another diagnostic feature is found in biopsy material from lymph
nodes; the hyperplasia of the lymphocytes is not so extreme or uniform in cases
of infectious mononucleosis as in cases of lymphatic leukemia. When there is
difficulty in differentiation from acute leukemia, the prognosis should not be
attempted. This can be more fully appreciated by the clinician who has assured
a group of relatives that the patient will recover, and within a few days or weeks
sees a fatal outcome from leukemia. The converse is not nearly so unfortunate,
but equally as embarrassing.

THE HETEROPHILE ANTIBODY TEST

In recent years, the heterophile antibody test has been introduced and has
proved to be the most valuable diagnostic procedure. In I9Il, Forssman observed
the presence of antibodies in the form of lysins and agglutinins for sheep erythro-
cytes in the blood of rabbits injected with various tissues of the cat, horse, and
guinea-pig. These are known as "heterophile" or "Forssman" antibodies. Studies
 MISCELLANEOUS

on normal human blood serum show the presence of sheep cell agglutinins and
hemolysins in a titer rarely exceeding I :8. In 1929, Davidsohn reported an in-
crease in titer up to 1 :64 in patients treated with horse serum or suffering with
serum sickness, an observation subsequently corroborated by others. In 1932
Paul and Bunnell reported the occurrence of higher titers in four patients with
infectious mononucleosis. During the next year, Bunnell ran tests for heterophile
antibodies in over 2,000 patients representing 76 clinical entities, including lS
additional cases of infectious mononucleosis. None of the 2,000 cases showed
a titer higher than 1 :32, while the antibody titer of serum from cases of infectious
mononucleosis ranged from 1 :64 to 1 :4096. Since that time various workers have
reported similar results and the diagnostic value of the test has become increas-
ingly evident. The presence of a definitely positive beterophil antibody test is
practically absolute assurance that the patient is suffering from infectious mono-
nucleosis. However, in rare instances there may be a false positive test as
illustrated by the case reported by Kent, which was a classic case of high cell
count, typical myelogenous leukemia. Nevertheless, the heterophil antibody
test was positive in a dilution of 1 :4096. Strauss has reported a technic for
heterophil antibody tests in which after the serial dilutions are made and (h\'-
sheep cells added, they are centrifuged at high speed immediately and readings
made. We have found this test to be quite reliable and satisfactory. For technic,
see page 662.
The titer varies in intensity, depending on the severity of the case. Any
dilution above 1 :64 is considered diagnostic if there is no history of serum sick-
ness. The titer reaches its beight, in most cases, a few days after the disease de-
velops and tends to parallel roughly the number of the atypical lymphocytes.
The titer seems to be higher when the temperature is high, but statistics on this
point are not conclusive. The high titer usually remains for a number of weeks,
then gradually recedes and becomes negative in about two to six months.
During the past few years, questions have arisen concerning the nature of
the sheep cell antibodies in this disease, that is, whether they are specifically
heteropbilic. At present, it appears that there exists a hitherto unknown class
of antigens which are thermostable, insoluble in alcohol, but not activated by
exposure to that reagent. This type of antigen produces antibodies with proper-
ties which are partly heterophilic and partly isophilic. The antibodies of in-
fectious mononucleosis belong to this class. Experiments hy Bailey and Raffel
have shown that the antibodies of infectious mononucleosis have the power to
hemolyze and agglutinate not only sheep red cells, but also to hemolyze and to
some extent agglutinate ox red cells, a typical isophilic antigen. These immune
bodies are not, however, isophilic since they are absorbed from the serum by
boiled ox and sheep red cells. So far as is known in~ diseas~s, this type of anti-
body is limited to infectious mononucleosis, and for this important reason its
presence serves as a diagnostic test.
In performing the heterophile antibody test with sheep erythrocytes, some
confusion results when the serum shows a low titer of normal heterophile anti-
bodies or when the serum of patients with serum sickness causes the titer to
be elevated. For this reason, Stuart and his co-workers have recently devised an
 INFECTIOUS lIIONONUCLEOSIS

adsorption test whereby differentiation can be made They found that the sheep
cell agglutinins in normal serum are absorbed by guinea pig kidney, but not by
beef cells; those in the serum of patients with infectious mononucleosis by beef
cells but not by guinea pig kidney; and those in the serum of persons with serum
sickness, by both guinea pig kidney and beef cells. By using these adsorption
tests, the exact type of antibody present can be determined. In most cases, a
correct diagnosis of infectious mononucleosis can be made from the clinical,
hematologic, and serologic picture, but in doubtful cases, in which the titer is
low, or in which serum sickness complicates the picture, the adsorption test is
valuable. For an excellent summary consult Davidsohn.*
I have observed at least three instances in which the serologic tests ,for
syphilis were positive during the course of infectious mononucleosis. This seems
to occur at a time when the heterophile antibodies are at their peak. A number
of such instances have been reported in the literature. Saphir has reported one
such case and there is another reported by Fowler and Tidrick. In their case
there was not only a positive Wassermann reaction but also a generalized
icterus. It is very important that these false positive serologic reactions be care-
fully and properly interpreted to avoid the possibility of lab~ling an innocent
person as a victim of syphilis.

PROGNOSIS
The prognosis is favorable. Complications are rare, and fatalities have been
so infrequent that they are never expected. A correct diagnosis early in the dis-
ease is important since a favorable prognosis can be given as soon as the diagnosis
is determined. The course and duration of the disease cannot be predicted with
certainty. Relapses or reCUrrences are not infrequent; there may be an enlarge-
ment of the glands on the side opposite to that originally affected or a retnrn of
swelling on the same side and such recurrences cause a slow convalescence.

TREATMENT
There is no specific treatment for infectious mononucleosis. In the more
acute stages symptomatic treatment is all that is necessary. If the cervical
glands are painful, hot or cold compresses may be used, or local ultraviolet radia-
tion may be effective when the lymphoid masses are large and soft, with peri-
glandular edema.
When convalescence is unusually prolonged or the patient suffers from sev-
eral relapses, a blood transfusion may contribute to the recovery. Blood from a
donor who has recovered from infectious mononucleosis may prove even more
effective. During an ordinary convalescence, it is advisable to administer iron
preparations to prevent development of an anemia which might result from the
disease. Proper nourishment and rest, however, are probably as effective in the
cure as any s"etific treatment used.
Infectious mononucleosis should be regarded as an infectious disease and
patients should be isolated. Sporadic cases are rarely dangerous, but care should
be taken here, also, since epidemics must originate from such cases. During epi-
• DAVlDSOHN, I.: Jour. Amer. Med. Assoc., 108, 289, ]937.
 MISCELLANEOUS

demics, patients should be isolated for approximately fourteen days and their
dishes and utensils sterilized as for any infectious disease.

BIBLIOGRAPHY

BULEY, G. H" and RAFFEL, S.: "Hemolytic antibowes for sheep and ox erythrocytes in mfettiou!
mononucleosis," Jour. Clin Invest, 14, 228, 1935.
DALDRIDGE, C. \V" ROHNER, F. I'I and HANSMANN, G. R.o "Glandular fever (infectJOus rtlono-
nuc1f'osis)." Arch. lnt Med, 38, 413, 1:926.
BLAND, J 0 \V.: "Glandular Fever: Experimental investigation," Lancet, 2, 521, Sept., I93C.
BOWCOCK, H.: "Mitotic leukoblasts in peripheral blood in infectIouS monoDuc1eo,,>is," Amer. Jour,
Med. Sci., 198,384. 1939. ;- _._o.,_ _ _ .

BUNNELL, W. W.: "Diagnostic test for mfeclious mononuc1e()S.~" A mer. Jour. Med. Sd.} 186,
346, 1933. ~
BURNS, J. E: "Glandular fever." Arch Int. Med, 4, lIS, 1909.
CANUTESON, R. I.: "Infectious mononucleosis (glandular fever) with report of cases." Jour. Kan .
.Med. Soc J 34, 2IZ, 1933.
DAVIDsoa~, I.: UHeteropbile antibodies in serum sickness." J()ur. Immuno!" 16, 259, 19'9
DAVlDSOBN, I : <lSerologic diagnosis of mfectious mononucleosis." Jour. Arner M ed. Assoc., 108, 289,
1937.
DOWNEY, H, and McKmLEY, C. A.' "Acute lyruphad<>nosis compared With acute lymphatic
leukemia." Arch. Int. Med., 32, 82, 1923.
DOWNEY, H.: "Infectious m()llODUcleosis. Part II. Hematologic studIes." J A.M.A., 105, 764,
1935.
FOORD, A. G, and Bun, E. M.: uThe laboratory diagnosis of infectious mononucleosis." Amer.
Jour. Clin Path., 91 448, 1939.
FOWLER, W. M., and TrnRICK, R. T.: "Unusual manifestations of infectious mononucleosis" 4mer
Jour. GUn. Path., 10, 548, 1940.
GORHAM, L. Vi., SMITH, D. T., and HUNT, H. D.' "The experimental reproductIon of the blood
picture of infectious mononucleosis in the guinea pig." Jour GUn. Invest., 7, 504, 1929.
ISAACS, R" STURGIS, C. C, BETHELL, F. H., and GOLDHAMER, S, M.: "Blood. a reVIeW of the
recent literature." Arch. ltd Med, 57, n86, 1936 .
.KENT, C, F.: "FaJse pOSItive Paul-Bunnell (heterophile) reachon?" Amer. Jour. CUn. Path, 10,
57 6, 1940.
MARSHALL, E. A.: "Infectious mononUcleosis." Arner Jour. Clin Path" 9, 298, 1939.
McKI?-.""LEY, C. A.' "Clmical aspects of infectious mononucleosis." Jour. Amer. Med Assn., 105,
761, 1935.
McLEAN, J. A.: "Supravital staining of large mononuclear cells in infectious mononucleos~ and
the acute leukaemias." Aled. Jour. Austral, 2, 734, Nov. 23, 1929.
MURRAY, E. G. D., WEBB, R. A, and SWANN, M. B. R.: "Disease of rabbits charactenzed by large
mononuclear leukocytes." Jour. Path. and Bact., 29, 407, 1926.
NYFELDT, A.: '~Etiologie de la mononucleose infectieuse." Compt, Rend. Soc. de BioI., 101, 590,
19 2 9.
OSGOOD, E. E ~ "Fenestration of nuclei of lymphocytes." Proc. Soc. Exp. B101. and Med., 33,
:uS, 1935
PAUL, J R, and BUNNELL, W. W.: lIThe presence of heterophile antibodies in infectious Dlono-
nucleosis." Amer. Jour. Med. Sd" 183, 90, 1932.
SAPHIR, W.: "The Wassermann reaction in infectious mononucleosis." Amer. Jour. Clin. Path,
9, 306, 1939.
SPRUNT, T. P: "Infectious mononucleosis (glandular fever)." Long Island Med. Jour, 24. 201, 1930.
SPRUNT, '1'. P., and EVANS, F. A: "Mononuclear leukocytosis in reaction to acute infections." BuU.
Johns Hopkins Hasp, 31. 410, 1920.
STRAUSS, R: "Simple slide and tube tests for infectious mononucleosis." Amer, Jour. CUn. Path,
6, 546, 1936.
 INFECTIOUS lIIONONUCLEOSIS

STUART, C A" GRIFFIN, A. M, FULTON, M.~ and A::WERSON, E G' "Nature of the antibodies for
sheep cells in infectious mononucleosis" Proe. Soc. Exp. Biol and Med, 34, 209. J936.
TuFLrnGER, F W.: "Epidemic of glandular fever." Jour. Amer. AleJ. Assn, 50, 765, 190B.
TroY, H. L., and MORLEY, E. B.: "Glandular fever." Brit. Med. Jour., 1,452, March 26, 1921.
VAN DEN BERGH, L., and LIESSEN'S, P: "Transmission della mononucleose infectieuse humanine au
Macacus rhesus. Resistance du virus aux basses temperatures." Compt. Rend. Soc. BioI., 132,
90, 1939·
WES1', J. P.: "An epidemic of glandular fever.'1 Arch Pediat., 13, 889. 1896.
WI5ING. P. J.: "Some experiments with lymph gland material from cases of infectious mODO~
nucleosis." Acta Med. Scandinav' 98, 328, 193Q.
J
 CHAPTER 40
POLYCYTHEMIA VERA

(Erythremia) (Splenomegalic Polycythemia) (Osler-Vaquez Disease)

Polycythemia vera is a progressive and ultimately fatal disease of unknown
etiology, which is characterized by excessive numhers of red blood cells, marked
splenomegaly, increased blood volume, and the symptoms resulting from these
altered states.
It was first described and given the name of polycythemia in ,892 by Vaquez
but attracted little attention until '903 when Osler described nine cases and
stressed the marked cyanosis that he observed in all of his patients.

ETIOLOGY
Polycythemia is a disease of middle and late life. It occurs with equal fre-
quency in men and women. We can find no record .of its occurrence in an infant
or young child. The disease occurs rarely in negroes. Reznikoff and his asso-
ciiites investigated the racial incidence of the disease in six large hospitals of the
United States and found that nearly one half of '34 patients with the disease were
European Jews. This incidence being so similar to that seen in the arterial dis-
ease, thromboangiitis obliterans, led these workers to investigate. carefully the
pathologic changes in the arterial system of the bone marrow, results of which
are discussed below.
There is some question as to the familial character of the disease, but more
than one case has been observed in a single family. Engelking has described five
cases in one family, occurring through three generations and other familial tend-
encies have been reported.
Among the older concepts of possible etiological factors have been first, that
the red cells live a longer span of life than normal because the normal destructive
processes are inhibited by some unknown agent, and secondly, that there is per-
sistent and sustained excessive erythropoiesis. The correctness of the last named
concept is indicated by the large number of red cells, the presence of immature
forms, the reticulocytosis, the coexisting leukocytosis with immature cells, the
enlarged spleen, the conversion of normally inactive marrow into functional types
and the relative increase of erythroid tissues in the hematopoietic sites. There is
little question that the basis of the disease is marked hyperplasia of erythro-
poietic tissues but the reason for this remains unknown.
The similarity of the blood picture in some cases to a leukemic process, the
splenomegaly, and the apparent change into an actual myeloid leukemia in an
occasional patient, has given rise to the concept that the entire process is neo-
plastic in character and that the same forces are at work in producing this dis-
ease as those producing myeloid leukemia. Minot and Buckman have reported
such an instance and Winter described a patient with myeloid leukemia that
490
 POLYCYTHEMIA VERA 49 1
eventuated into polycythemia vera. In an Qccasional patient whose leukocyte
count may be as high as 50,000 cells per cu. mm. with many myelocytes, the dif-
ferentiation is sometimes difficult to establish, and such instances have been called
erythroleukemia.
A possible cause of the excessive erythropoiesis that has received much atten-
tion in recent years is the presence of the hematopoietic principle in excessive
amonnts, perhaps elaborated in the stomach. l\Iorris suggested this as a result of
his demonstration of the so-called hematopoietic principle, "Addisin," in normal
gastric juice. Briggs and Oerting have treated patients by repeated daily gastric
lavage over a long period of time and report questionable success. This has also
been done by Kraemer and Asher. Singer has reported a patient with polycy-
themia who had normal blood values after gastric resection. Minot and Castle
state that serious consideration need not be given to the possibility that increased
gastric activity can produce polycythemia and base their statement on the failure
of the hematopoietic principle in liver and stomach preparations to raise the blood
levels in normal persons.
There is general agreement that erythropoiesis is controlled to a considerable
extent by the degree of oxygen tension in the bone marrow (see section on factors
influencing erythropoiesis) and, as Minot states, "the eventnal level of the red
blood cells is controlled by the oxygen tension in the bone marrow." This con-
cept lends importance to the work of Reznikoff and his associates who studied the
bone marrow in seven patients. In all cases they found an lncreased thickening
of the capillary arterial wall with fibrosis, and in six of these they noted a marked
subintimal and adventitial fibrosis of the subarteriolar capillaries, arterioles and
arteries. Thus, this process apparently resulted in narrowed lumina of marrow
vessels, decreased oxygenation, lowered oxygen tension and therefore, increased
erythropoiesis. In view of these findings polycythemia would appear to be the
thromboangiitis obliterans of the bone marrow instead of the lower extremities
as seen in Buerger's disease.
Beyne and associates in France have stressed the role of anoxemia as the
causative process, due to pulmonary fibrosis. If this be true, it would seem to be
a part of the general process of vascular fibrosis in the bone marrow as described
above.

SYMPTOMS AND PHYSICAL FINDINGS

The onset is insidious and the symptoms develop slowly hut progressively
with varying degrees of remission at irregular intervals. The first symptoms are
weakness and fatigue which may be accompanied by the development of a peculiar
:luskiness due to cyanosis, manifested chiefly on the face, lips and other mucous
membranes. It varies in intensity and is aggravated by cold weather. In other
~atients the first symptoms may be headache, dizziness and various types of
paresthesia. These were predominant in Adams' series. In others there may
ievelop a hemorrhagic syndrome. There may be irritability, emotional upsets,
periods of depression, inability to concentrate on work, with loss of memory in
m occasional patient. Reznikoff points out that the average patient is depressed
'49 2 MISCELLANEOUS

and seems to have a rather sluggish mentality but some of them show a combi·
nation of mental activity and irritability, which may create difficult social situa·
tions: Surgical procedures should not be attempted on these patients unless it is
absolutely necessary. Sensory disturbances include numbness and tingling of the
feet, roaring in the ears, visual disturbances, and there is often a marked sensi-
tivity to cold.
The abdominal and gastric disttlrbances are mainly referable to the spleno-
megaly and may include epigastric pain, digestive upsets. with excessive gas, and
nausea and vomiting. Various pains are noted chiefly over the bones and are
thought to be caused by the marrow hyperplasia and the generalized osteosclerosis.
Due to the frequency of hemorrhages, the patient may bleed in the gastro-
intestinal tract resulting in' tarry stools, or hematemesis. Wilbur and Ochsner
have commented on the frequency of peptic ulcer in polycythemia (8 per cent).
The patients rarely develop purpuric spots, but in women there are periods of
excessive and irregular menstrual' bleeding.
Because of the tendency to intravascular thrombosis the first sign inay be'
blockage of a cerebral vessel, or a cerebral hemorrhage. The excessive blood vol.
ume may be responsible for cardiac symptoms including palpitation, dyspnea,
and precordial oppression.
On examination the patient is usually apathetic and listless, and oftentimes
seems unconcerned over his condition. The dusky color of cyanosis is striking
and especially noticeable as irregular red and dark red splotches of color with
blueness of the lips. The fingers and nails are blue tinged.
Variable enlargement of the spleen is found in practically all cases. Occa·
sionally it cannot be palpated. The splenic border is usually smooth and not
tender. Klemperer states that the spleen is characterized by hyperemia and in·
farcts with engorgement of the sinuses and increased cellularity of the red pulp.
The pulp consists almost entirely of large cells with abundant cytoplasm and
clear nuclei with only a few red and white cells between. There is no evidence
of increased erythrophagocytosis and no increase in iron pigment.
Brown and Giffin reported enlargement of the liver in over one-half of their
cases. The blood pressure mayor may not be elevated. If markedly elevated,
the disease is then sometimes designated as Gaisbock's disease or "polycythemia
hypertonica." The cause of the hypertension is questionable since it may develop
secondary to the increased peripheral resistance due to the fibrosis of arterioles
and capillaries.
In summary, the diagnosis is based on the slowly developing weakness, the
peculiar color of cyanosis, the splenomegaly, and the most important finding,
the excessive number of circulating red cells.

LABORATORY FINDINGS
The blood findings are characterized by the high number of erythrocytes
with a hemoglobin content that is also much above normal but seldom increased
to the extent of the red cells. Therefore, in the usual patient the color index
is slightly below one. The red cells usually range between seven and ten million
per cu. mm. Naegeli records an instance of a red cell count of twenty million and
 POLYCYTHEMIA VERA 493
a hemoglobin percentage of two hundred and forty in another case. The red
cells should number at least seven million per cu. mm. before a diagnosis can be
established with certainty. There seems to exist a belief among many physicians
that six million cells per cu. mm. is adequate evidence for the diagnosis but it
should be borne in mind that this figure represents the normal level for some
people. Furthermore, in patients with high red cell and hemoglobin values, all
possible causes of compensatory erythrocytosis should be eliminated hefore reach-
ing a diagnosis of polycythemia vera, because of its serious prognostic import.
In fully developed cases the leukocyte count may be normal, moderately
increased, or markedly increased. In the cases that we have studied, the leuko-
cyte count is invariably increased and averages about 20,000 cells per cu. mm.
n may reach extremely high levels as shown by the cases recorded by Naegeli
(50,000) (9I,000) (87,000) (II4,OOO). In these instances the differentiation from
myeloid leukemia is difficult. The differential cell count usually reveals a marked
shift toward immaturity of granulocytes with the total number increased; for
example, one of our cases showed a total leukocyte count of 30,000 with 96 per
cent granulocytes, including myelocytes 8 per cent, juvenile types 12 per cent,
band forms 22 per cent, and segmented types 52 per cent. It seems probable
that the leukocytic stimulation represents merely an intensely active erythro-
cytic marrow in which the granulopoietic tissue is involved because of con-
tiguity. An occasional myeloblast may be found; eosinophils are usually in-
creased in total number, and the increase in basophils is sometimes a striking
phenomenon.
The blood platelets are usually increased and may reach high levels in the
peripheral blood and an occasional megakaryocyte may be seen. The red cells
in the stained smear may show some 'variation in size and shape, with varying
degrees of polychromatophilia and an occasional cell with stippling. Normoblasts
are not infrequent and reticulocytosis of varying degree is the rule. The red cells
are not macrocytic but tend toward microcytosis, because although the cells pack
to 60 or 70 per cent of the total blood volume, the volume index is usually below
one. l\lany of the cells show a central pallor, particularly if the color index is
low.
The total blood volume is increased from 50 to 100 per cent above the normal
(Rowntree, et al.), and the erythrocyte volume is increased to about twice normal
in high counts. Grossly the blood is dark red, very sticky and even viscous. The
viscosity may be several times the normal figure, and this may be apparent even
when sticking the finger and collecting a sample in a pipette. This increased
viscosity is present because of the increased cellular content and the plasma prob-
ably does not contribute to it. The viscosity of the blood may be from five to
eight times the normal and the specific gravity varies between 1.075 and 1.080.
The sedimentation rate is slow, and there is little disturbance in the bleeding
and coagulation times. There is no evidence of excessive cell destruction since
the icterus index and serum color are quite normal and the serum may even be
clear and watery in appearance.
The urine may show casts and small amounts of albumin, which may be due
494 MISCELLANEOUS

to the increased output of nuclear material as suggested by Isaacs, or to the pres-

ence of nephritis' on a basis of generalized arteriosclerosis.

TREATMENT
Treatment should be directed toward the reduction in the number of the
circulating red cells, and if effective, should result in restoration of normal red
cell volume, decreased total blood volume, decreased viscosity, lessened tendency
to thrombosis, some reduction in the size of the spleen, and a general sense of
well being and improvement.
In general, treatment has included radiation to the long bones, and to the
spleen, repeated bleedings by venesection, repeated gastric lavage, and chemical
methods including the administration of benzene, phenylhydrazine, and acetyl-
phenylhydrazine. ,
In recent years there has been a renewal of emphasis on the value of vene-
section. Removal of 500 cc. of blood once weekly can be continued for a long
time. It is said that venesection produces very little bone-marrow stimulation.
The removal of large quantities of blood at frequent intervals not only relieves
the patient of the symptoms caused hy increased viscosity and overloading of
the vascular system, but it also produces a mild degree of microcytic anemia
because the red cells have become smaller and even though there is an increased
number, the total cell volume, of course, would be less. By the process of bleed-
ing, the red cells should be kept below 50 per cent of the total blood volume.
In case cellular hypochromia develops, it is my.,opinion that such patients should
have iron medication at that time. Others believe that iron should be withheld,
but I have noted no ill effects from its administration during the time when the
color index is below one. The improvement after accidental bleeding and spon-
taneous hemorrhages has long been noted. The use of gastric lavage has not been
attended with striking success. Splenectomy has been done but apparently did
not alter the course of the disease. According to Naegeli, patients, have received
intensive treatment with Fowler's solution, oxygen inhalations, vegetarian diet,
thyroid tablets, thorium X, benzol, and other agents, but proof of their value is
lacking.
Modern treatment resolves itself between a choice of two methods, ihat is,
the use of phenylhydrazine or bleeding, or both. This drug, whiclr was intro-
duced in 19,8, has been used with much success. Its action is to destroy the red
blood cells, including those in the peripheral blood and the more immature types
in the erythropoietic centers. Giffin and Conner have attested to its value and
recommend the administration of I.S to 3.5 Gm. by mouth during it period of. a
week to ten days, with the daily dose not to exceed 0.3 Gm. (5 gr.), and preferably
it should be lower. At the end of a week or ten days it should be discontinued,
and its effect observed by repeated and careful blood studies.
After a course of treatment there will appear evidences of excessive blood
destruction, including increased bilirubin in the blood serum with high icterus
index, with dark colored urine and a slow reduction in the number of red cells
and hemoglobin. The drug seems to exert a prolonged or cumulative effect so
that it is important to check the blood findings for some weeks afterwards. Its
 POLYCYTHEMIA VERA 495
dangers include possible damage to the liver with acute fatal hepatitis, prolonged
action on the marrow to the point of aplasia, and accentuation of the tendency
toward thrombosis. After the reaction of the patient has been evaluated a main-
tenance dose can he established. A weekly dose of 100 to 200 mg. may be suf-
ficient to maintain the red cells at the normal level. Naegeli states that he
never gives over 0.3 Gm. (5 gr.) at weekly intervals, and prefers not to use it at
all. Bodansky has suggested the use of acetylphenylhydrazine because of its lack
of toxicity.
Among the first to report good results from radiation treatment were Milani
in 1929 and Pack and Craver in 1930. Recently Sgalitzer has reported the use
of radiation to the whole body in 34 patients with fairly good results, producing
remissions varying from one and a half to five years, but apparently not produc-
ing permanent remission in any case. He advocates the application of 25 roentgen
units for 20 minutes to the whole body for six successive days this being fol-
lowed by a weekly interval of rest with the course repeated until the red cell
count falls to the normal, carefully noting the decrease of leukocytes, and stop-
ping the treatment when they' reach 3000 cells per cu. mm.
Naegeli recommends the use of radiation and gives the following as his
technic. A systematic radiation of all bones using the skin erythema dose filtered
with 0.5 mm. zinc plus I mm. aluminum. On the first day both legs; on the
second day both thighs; on the third day both forearms; on the fourth day both
upper arms; then a rest of three weeks with careful checking of the blood picture;
then one day for the scapula; one day for the sternum; one day for the' pelvis ;
and one day for the vertebral column. He believes radiation of the spleen to be
useless. Various other methods have been reported in the treatment of poly-
cythemia vera. These include the use of liver extract, lipocaic, choline hydro-
chloride, ascorbic acid, and others, but all of these agents seem to be without
effect. Stenstrom and his associates at the University of Minnesota tried radia-
tion to the pyloric and Brunner's gland area of the stomach but failed to observe
beneficial effects.
Hunter has used the "spray therapy" of Sgalitzer, described above, in two
patients with excellent results. Langer has reported good results with radiation.
It would appear that radiation offers better results than the use of phenyl-
hydrazine, because of its better control and accuracy of measurement, freedom
from cumulative effects, and lack of damage to the liver. In instances where
such therapy cannot be obtained phenylhydrazine and bleeding are to be recom-
mended. The most important feature in any plan of treatment is careful check-
ing of the blood picture to guard against excessive destruction of hematopoietic
tissue and circulating cells.

PROGNOSIS
Polycythemia still remains an incurable disease. It is slowly progressive
with intermittent remissions, and if under proper treatment the remissions are
more prolonged. The average duration of life after diagnosis is reported as six
to eight years (Clough) with some patients living much longer. The prognosis
should always be guarded since complications are common and the patient may
 MISCELLANEOUS

die from one of these which include excessive hemorrhages, various thrombotic
processes, intercurrent infections, nephritis, and cirrhosis of the liver.

SYMPTOMATIC AND COMPENSATORY ERYTHROCYTOSIS

The term erythrocytosis refers to an increase in the number of circulating

red cells from any cause, and is analogous to leukocytosis of white cells. It is
to be distinguished from polcythemia or erythremia which is a disease entity,
analogous to the leukemia of white cells.
Erythrocytosis may be transient, lasting only a few hours, as seen in instances
of marked dehydration in high fevers and following excessive loss of fluids. In
such cases the red cell courit returns to normal after the plasma volume of the
blood is restored by the administration of fluids. Red cell values may reach
seven million cells per cu. mm. under such conditions. In this connection, it
should be emphasized that if a patient is being treated for anemia by transfusions
or other therapy, the red cell level may rise considerably because of dehydration
and concentration of blood if acute infection is present, and after correction of the
dehydration there may appear to be a decrease in the number of circulating cells,
where actually there is an absolute increase.
Physiologic erythrocytosis may quickly develop and remain sustained when
the normal person lives in a high altitude. Nearly all standard figures for normal
erythrocyte values are taken at sea level. Naegeli points out that the average is
8,000,000 cellsjn the Andes, over 8,000,000 cells in the lowlands of the Himalayan
mountains, and in the highest regions of Tibet the count ranges between 7.6 and
7.9 millions.
In instances of compensatory physiologic erythrocytosis there is an actual
stimulation of the erythropoietic tissue probably because of decreased oxygen
tension. The possible role of increased exposure to rays of the sun has been sug-
gested as a cause but tbis has been disproved by Meyer and Pick who found
the same erythrocytosis at high altitude in persons not exposed to sunlight.
The cell increases begin quickly, within 24 hours increasing one half million
or more, this being due probably to released cells from the splenic pulp. How-
ever, the bone marrow becomes stimulated to increased output within a few days
and soon accustoms .itself to a new and higher level of production. On return
from high altitudes the count rapidly falls to the normal level and the blood
attains normal values in a few weeks. During the period of erythrocytosis there
is no hematologic or clinical evidence of increased blood destruction~ The
hemoglobin content remains parallel to the red cell values, and the color index is
usually at the normal level. This is not the case in the compensatory erythrocy-
tosis of impaired pulmonary and cardiac function. During the period of erythro-
cytosis there usually is no evidence of increased marrow oulput except a slight
reticulocytosis and nucleated forms are absent. According to Naegeli short bal-
loon trips into high altitudes are accompanied by erythrocytosis, which disappears
quickly on'return to sea level. This is probably an instance of redistribution of
red cells.
It appears that any cause for the interference of adequate oxygen supply to
 POLYCYTHEMIA VERA 497
the tissues will result in erythrocytosis. Thus, marked red cell increases are seen
in various pulmonary diseases characterized by restriction of the alveolar gas
exchange capacity. It may develop, therefore, in massive atelectasis, wide-
spread pulmonary fibrosis, far advanced pulmonary tuberculosis, stenosis of the
hronchial tree, and in the so-called Ayerza's disease or sclerosis of the pulmonary
artery. The degree of erythrocytosis depends upon the amount of pulmonary
<lamage, and the red cell count may range between five and ten millions per cu.
om. The hemoglobin is usually lower in proportion, with a color index below
me. There is a slight and susiained reticulocytosis since the average life of the
circulating red cell is no longer than normal.
Similar degrees of· erythrocytosis are seen consistently in patients with any
ype of prolonged myocardial insufficiency, and especially in congenital heart
lisease. This finding is frequently seen in children since congenital heart dis-
,ase becomes evident in early childhood. The patients show varying degrees of
;yanosis, a dark dusky mottled color, marked dyspnea on exertion with the
;haracteristic findings of the heart lesions which vary with the type of pathology
Jresent.
In the erythrocytosis of impaired oxygenation of tissues, it is important to
:arry out careful blood studies with particular reference to the degree of red cell
ise, the amount of hemoglobin, and the estimation of the color index. We have
recently studied a patient with an erythrocyte count of nine million cells per
cu. mm., and hemoglobin of 70 per cent. The color index, therefore, was less
than one half, or each red cell was carrying only one half of its normal content
of hemoglobin. This patient, who had congenital heart disease,. was treated
with large doses of iron (ferric ammonium citrate), with a gradual reduction of
the red cells to six million per cu. mm. and increase of hemoglobin to 105 per
cent, with marked improvement of symptoms. This resulted in a marked decrease
in blood viscosity and relieved a damaged heart of an excessive burden of forcing
ed cells through the circulation that were functioning at less than fifty per cent
fficiency. Before such therapy can be employed it is essential that the diagnosis
e correct, for the administration of iron. would do little to relieve the increased
ematopoiesis of true polycythemia. Reznikoff points out that an increased red
ell count may be seen in persons who are nervous, high strung, and suffering
,om an anxiety state. It is also seen in hyperthyroidism and individuals who
ave heen taking thyroid medication. Polycythemia vera can be pro\fuced in
nimals by feeding cobalt salts.
Erythrocytosis is seen in those conditions where there are chemical changes
1 the hemoglobin of the red cells which render them incapable of transporting
,e normal amounts of oxygen. Such conditions include the development of
,ethemoglobinemia due to replacement of the normal oxyhemoglobin with
,ethemoglobin. This may occur with various drug and chemical poisons, in-
luding benzene, nitrobeneze, aniline, arsenic, phosphorus, acetanilid, and anti-
yrine. The condition may be either acute or chronic. Severe methemoglobinemia
may develop suddenly as a resul t of overdosage or ingestion of some of the
above named substances. \Ve have observed it on two occasions after operative
 MISCELLANEOUS

procedures, following which the patient received large quantities of acetanilid

and amidopyrine, respectively. In such instances, although there is a marked
methemoglobinemia which can be demonstrated by spectroscopic methods, the
erythrocytosis requires some few days to develop, after an initial period of
reticulocytosis. In the cases of chronic methemoglobinemia, there is a slowly
developing tendency to a dusky color which may not be apparent to the patient
but is oftentimes noticed by friends. Another important cause of acutely de-
veloping erythrocytosis is the condition known as hemoconcentration, usually
seen in surgical shock. This is caused by loss of fluid from the vascular system,
resulting in concentration of red cells. Since the patient does not show clinical
evidence of shock until the process has become quite severe, Moon stresses the
importance of repeated red cell counts in patients who are likely to develop the
condition, and states that impending shock can be detected by the gradually
increasing red cell count.

BIBLIOGRAPHY
POLYCYTHEMIA VERA

ADAMS, L. J : "Polycythemia vera. With special reference to nervous manifestations. Analysis of

nine case::;." Canad. Med. Ass. Jour I 32, 128, 1935.
BEYNE, J" BWET, L., and STRUMIA, M. V.: "On the mechanism of polycythemia originating in
the pulmonary system n Compt. rend. Soc. de BioI., 118. lI77, 1935.
BODANSKY, M.: "Effect of compounds related to hydrazine in producing anhydremia and experi-
mental anemia." Jour Pharm and Exp Ther.,. 23, 121, 1924 .
BRIGGS, J. F" and OERTlNG, H,' "Influence of gastnc lavage on familial and non-familial ery-
thremia. (With administration of erythremic gastric juice in two cases of perniciOUS anemia.)!!
,Minn. Med., 18, 499, 1935.
BROWN, G. E .• and GIFFIN, H. Z.. "Vascular cbanges in cases of polycythemia vera." Amer. Jour.
J/ed. SCi" 171, 157, 1926
CARPENTER, G.: "Failure to control polycythemia rubra vera with lipocaic and choline" Amer.
lour. Med., Sci, 200: 462, 1940.
CLOUGH, P W.: IIDiscases of the blood" Harper. Med Monograph. Harper and Brothers, New
York, 1929.
ENCELKING, E: uUebcr fanuliire potyzytbamie und die dabei beobachteten augenveranderungen."
Klin. M onatsb. /. Augenh., 64, 645, 1920.
GIFFIN1 H. Z.o and COm."ER 1 H. M: "The untoward effects of treatment by phenylhydrazine
hydrochloride." Jour. Amer. lIfed Assn., 92, 1505, 1929.
HARROP, G. A, JR.: UPolycyihemia." Meaicine, 7~ 1.91,1925.
HAY, J, and EVANS, W. H: l'Acute eosinophtlic leukemia and eosinophilic erythroleukemia."
Quart. Jottr. Med, 22, 167, 1929.
HIRSCH, E. F: "Genera1i2ed osteosclerosis with chronic polycythemia vera" Arch. Path., 19,
9I, 1<)35.
HUNTE~, F. T: "Spray x-ray therapy in polycythemia vera and in erythroblastic anemia." New
Eng. Jour-. JIed., 214, 1123, 1936
ISAACS, R.' "Pathologic physiology of polycythemia vera." Arch. [nt Ued, 31, 289, 192.3
KANDEL, E V.o and LERoy, G. V "Note on the lack of bemoregulatory effect ,of ascorb1(: acid on
patients with polycythemia vera." Amer. Jour. Med. Sct, 196, 392, 1938.
KLEMPERE~, P.: liThe pathologic anatomy of splenomegaly." Amer. Jour. CUn. Path., 6, 99,
1936.
KRAEMER, M, and ASHER, M.: IIAssociation of erythremia and duodenal ulcer. (Therapy by
stomach lavage)" Amer. Jour. Med. Sa., 191, 234. I936.
 POLYCYTHEMIA VERA 499
LANCER, H.: '~Roentgen therapy in hyperplastic blood dyscrasias j new technique for myeloid and
lymphatic leukemia, polycythemia rubra vera and Hodgkin's dIsease," Amer. Jour. Roentg,
34, 2I4, 1935.
MAJOR, R. H.: liThe effect of liver extract upon polycythemia vera." Jour. Lab. and elin. Med ..
24, 65, 1938.•
MILANI, G.: URoentgen treatment of Vaquez's disease." Jour. Ame,.. Med. Assn., 93, :1.205-1 ]9 2 9.
MINOT, G. R., and C.4.sru, W. B.: I'Year book of general medidne/' 412. Year Book Publishers,
Chicago, ~9J5.
MINOT, G. R., and BUCKMAN, T. E.: UErythremia (polycythemia rubra vera)." Amer. Jour J/ed.
Sd, 16, 469, 1923_
MORRIS, R. S, 8crnT.F, L., FOVLGER, J. H" RICH, M. L., and SHER!!AN, J. E: "Treatment of
pernicious anemia. Effect of single injection of concenbated gastric juice (Addison) " JOU1.
Amer. M ed. Assn, 100, 171, 1933.
NAtGELI, 0 ; "Blood diseases and blood diagnosis," 568-583. J. Springer, Berlin, 1931.
OSLER, W.O.: uChronic cyanosis WIth polycythemia and enlarged <;plccn. A new clinical entity."
Amer. Jour. Med. Sci., 126, 187, 1903.
PACK, G. T, and CRAVER, L. F.: "Radiation therapy of polycythemia vera" Amer. lotlr. Mcd.
Sci., 180, 609, 1930.
REZNIXO:Fl", P., FOOT, N. C., and BETmA, J. M.: "Etiologic and pathologic factors in polycythemia
vera." Amer. Jour. Med. Sci, 189, 753. 1935.
ROWNTREE, L G, DROWN, G. E, and ROTH, G M: uThe volume of the blood and plasma in
health and disease." W. B. Saunders Co , Philadelphia, 1:929.
SGALITZER, :M . "Total irradiation with roentgen rays in polycythemia." Wien. Klin. Wchnschr.,
48, 6)5, 1935.
SmGER, K.: Ills there a gastric polycythemia?" Klin. W chnschr., 14, 751, 1935.
STENSTROM, K.. \V., Hou.ocx, P. H., and WATSON, C. J' ('Negative results of irradiation therliPY of
the pylorus and Brunner's gland area in patients WJth polycythemia vera." Amer. Jour Med.
Sd, 199, 646, 1940.
VAQUEZ, H.: "Concerning a &pecial form of cyanosis a.ccompanied by an e~cessive and persistent
form of polycythemia." BuU. Med. Par, 6, 849, 1892.
WILBUR, D. L I and OcrrSNER, II. C.: "Association of polycythemia vera and peptic ulcer." Proc
Staff Meet. Mayo elin., 16, 166, 1935.
 CHAPTER 41
THE BONE MARROW
By R. P. CUSTER, M.D.

It is not enough that the modern hematologist be able to identify cells of

the peripheral blood. "A single examination of the blood is only one view of a
passing cavalcade which is constantly receiving reinforcements and constantly
losing members" (Haden, 1939Y: The vital importance of the bone marrow as
the major organ of blood formation must be appreciated and its normal and
abnormal functions understood to permit intelligent interpretation of the blood
picture in health and disease. The bone marrow is the largest organ in the body
and, with the exception of the blood proper, the tissue is undoubtedly the most
unstable.
During the past fifty years an enormous amount of research has been directed
toward the hemopoietic tissues througb autopsy, biop~y and experimental studies.
Although much has been learned, many problems remain unsolved and there is
great need for further investigation. "Out of the earlier analyses there emerged
five fundamental questions peculiar to and inberent in hemopoietic tissue, which
have formed the focus for all the more recent physiologic studies of the phe-
nomena involving the blood cells: (r) the nature and significance of cell origins;
(2) the factors essential to cell maturation; (3) the forces governing cell distribu-
tion; (4) the conditions influencing cell destruction; and (5) the functional
specificity and interrelationships of each cell type. New, sound therapeutic pro-
cedures have already been provided for clinical medicine as a result of these
modern studies, based upon the experimental method" (Doan, 1938).

TERMINOLOGY AND CELL MORPHOLOGY

Some of the existing confusion in hematology can be attributed to the varied

use of certain terms with reference to given cell types. For example, many
authors apply "megaloblast" to the primitive blood cells of early embryonal life,
thereafter only to the nucleated red cells in the relapse phase of pernicious anemia.
The term is also used by others for cells more primitive than those usually desig-
nated as early erythroblasts that are found in actively regenerating marrows.
However, for those who disagree with the broad use of the name "megaloblast,"
it is suggested that "proerythroblast" be substituted throughout this chapter
except with reference to pernicious anemia. (A full discussion of the latter
reference is maue by Jones, 1938.)
Again, the dualists use "myeloblast" to denote the progenitor of all formed
elements of the blood except the lymphocyte, whereas the other schools apply
the term to the parent cell of the granulocytes and it is in this latter sense that
it appears here.
500
 THE BONJO: MARROW 501
or
It is highly desirable to work toward uniform terminology, but at present
it is more important to understand the connotation of terms as used by a given
author. For this reason the following scheme of blood formation is included to
illustrate the nomenclature in this chapter:

FIG !4.-Scbeme of hematopoiesis, to illustrate nomenclature.

Detailed cytologic description is given elsewhere, but brief comment on the

m<lre <lutstanding features <If the various cell types seems desirable Itere, as well.

UNDIFFERENTIATED CELLS
These can be classed as fixed and free, the former being elements of the
reticula-endothelial system, the latter probably immediate derivatives therefrom.
The reticulum cells lie in the interstices of the marrow as an inconspicuous mesh-
work supported by fine argyrophilic fibrils; the abundant, lightly basophilic cyto-
plasm may be vacuolated or mottled and the nucleus presents a fine chromatin
structure containing several indistinct nucleoli. The endothelial elements are
usually seen as flattened cells forming the walls of the blood sinusoids; in an
active phase, as when phagocytic or engaging in erythropoiesis, they assume the
morphologic characters of reticulum cells. The free undifferentiated cells show
considerable variation in size; the amount and basophilia of the cytoplasm differ
and some contain a few azure granules while others do not. The perinuclear
membrane is delicate, the chromatin hazy and nucleoli fairly large and distinct.

THE ERYTHROCYTE SERIES (The Erythron)

The promegaloblast presents a narrow rim of deeply basophilic cytoplasm
surrounding a large nucleus witb indistinct membrane, the chromatin being
sparsely distributed in tiny clumps and the nucleoli indistinct; no az.ure granules
are present. In the megaloblast the granule-free cytoplasm is more homogeneous
and deeply staining, the chromatin skein coarser and the clumps larger, nucleoli
502 MISCELLANEOUS

being more sharply outlined. The early erythroblast shows an increase in the
amount of cytoplasm and still deeper basophilia. The nucleus becomes more
compact; the filaments are coarser and the chromatin clumps block-like in the
later stages. Nucleoli are lost at this stage. The intermediate erythroblast has
the typical cartwheel nucleus and the cytoplasm is polychromatic due to acquisi-
tion of hemoglobin whiclI gradually increases in amount so that in the late
erythroblastic stage the basophilic substance is largely replaced. The nucleus
gradually shrinks and in the normoblast it has become a homogeneous blue-black
mass whiclI is extruded. The reticulocyte is a little larger than the adult
erythrocyte; in the stained smear it may present slight diffuse or even punctate
basophilia which rapidly disappears as the mature stage is reaclIed.

THE GRANULOCYTE SERIES

The myeloblast, the parent cell of all three subdivisions, is a fairly large cell
with clear vesicular nucleus, thin sharp perinuclear membrane and prominent
nucleoli supported by extremely delicate threads of chromatin. The cytoplasm
varies considerably in amount and is usually lightly to moderately basophilic,
containing a few azure granules which increase in number with maturation. The
promyelocyte contains fairly large numbers of azure granules and a few specific
granules at first (neutrophil, eosinophil, or basophil) which gradually increase in
number concomitant with disappearance of tlIe azure granules, until the myelo-
cyte stage is reached when the full complement has been acquired. During these
stages the chromatin becomes coarser, the nucleoli are lost, and the nucleus is
smaller and more compact. From this point to complete maturation there is no
further cytoplasmic change. The nucleus becomes indented in the metamyelocyte
stage, then pyknotic, finally segmented.

THE THROMBOCYTE SERIES

The mega)mryoblast retains many of the characters of the primitive cell
and is distinguished by the somewhat larger nucleus and increase in bulk and
basophilia of the cytoplasm. In the promegakaryocyte stage one commonly notes
a folding of the nucleus which becomes rapidly more complex as to lobation
through multipolar mitotic division which concerns only the nucleus, the cyto-
plasm growing progressively larger and acquiring fine, 'dust-Iike, azurophilic
granules, the mature cell reaching a diameter of 20-40 microns. (It is possible
for megakaryocytes to reach functional maturity and remain mononuclear,
however.) The cell margin is ragged and platelets are thought to bud from the
edge and become detached. The' megakaryocytes subsequently undergo degen-
eration, as evidenced by nuclear pyknosis and cytoplasmic disintegration.

THE PLASMOCYTE SERIES

Under normal conditions few, if any, plasmocytes are formed in the bone
marrow. Their progenitor, the plasmoblast, presents a round nucleus with evenly
 THE BONE MARROW 50 3
distributed strands of coarse chromatin and one or more nucleoli; it lies centrally
or excentrically in a fairly abundant dark blue cytoplasm which acquires an
acidophilic perinuclear "Hof" as the cell matures. The nucleus shrinks, loses its
nucleolus and the chromatin forms radially arranged blocks, giving a cartwheel
appearance in the mature pl~smocyte.

THE LYMPHOCYTE SERIES

Lymphocytes are not usually present in the bone marrow in numbers greater
than can be accounted for by the circulating blood. Occasionally one finds a
circumscribed aggregation in the tissue proper and rarely an actual follicle with
so-called germinal center.

THE MONOCYTE-MACROPHAGE SERIES

The fixed undifferentiated cells of the general reticulum and sinusoidal endo-
thelium possess phagocytic powers which stamp them as fixed macrophages or
clasmatocytes. They have the ability to become detached, and the so-called wan-
dering macrophage bears essentially the same morphologic characters. Through
the medium of the free undifferentiated cell the monoblast may develop, a cell
with a moderate amount of cloudy basophilic cytoplasm and a spongy nucleus
with one or more sharply defined nucleoli. During maturation the nucleoli are
lost, the nucleus retains its same skein-like character and the cytoplasm a~sumes a
dusty blue-gray hue and contains few or many azurophil granules and vacuoles.
In tissue culture they have been seen to take on the appearances of the so-called
"epithelioid" cells which in turn may undergo hypertrophy and become wandering
macrophages.

ORIGIN AND DEVELOPMENT

The first suggestion of a marrow cavity begins bet\veen the third and fourth
month of intrauterine life, beginning in the center of the shafts of the long bones.
The cartilage cells become vacuolated, disintegrate, and are later resorbed, con-
comitant with development of the myeloid vascular tree. The blood vessels which
enter thi~ softened center arborize into a rich plexus of large sinusoids walled by
a single layer of endothelium; these extend toward the epiphyses as the cartilage
is resorbed. In addition, the mesenchymai'cells fill the interstices and are sup-
ported by a rich framework of reticular fihers. Hematopoietic tissue appears
during the fourth month and gradually assumes the burden of blood formation,
So that at birth there is little or no blood-forming tissue outside of the marrow.

STATUS AS AN ORGAN

The bo'!.e marrow is the largest organ in the body, its estimated volume
ranging from 67 to 91 cc. at birth and from '320 to 4'92 cc. in adults. Males
MISCELLANEOUS
 THE BONE l\1ARROW 505
have greater volume of marrow space than females and elderly individuals
greater than young adults.
In postnatal life there is a gradual receosion of hematopoietic tissue in the
bone marrow and replacement by fat, so that in the young adult one finds "red
marrow" almost exclusively in the vertebrae, sternum, ribs, os innominatum,
bones of the skull, clavicle, scapulae and to small degree in the proximal ends of
long bones (Fig. IS). Some investigators have stated that the marrow is solidly
cellular up to the age of 7 years, but I have observed a few fat cells in the marrow
of infants. This process is a physiological one and probably indicates the growth
of the marrow spaces beyond the need of the body for blood-forming tissue. Fat
here, however, is in an exceedingly labile state and, should stimulus to marrow
hyperplasia occur, gives way rapidly to the spreading blood-forming tissues; I
have seen under experimental conditions a transition from completely fatty to
solidly cellular marrow within two days.
Temperature is thought. to be a factor in hematopoiesis, a lowered tempera-
ture predisposing to involution and fat replacement_ For example, if the tip of
the tail of an albino rat is transplanted into the abdominal cavity, the marrow
of the implanted vertebrae will undergo hyperplasia while those which remain
outside of the body retain their fatty structure.

STRUCTURE

The supporting tissue of the bone marrow is formed by a delicate network

of reticular fibers to which the reticulum cells are intimately attached.
The circulation is peculiar in that the nutrient arterial branches almost im-
mediately form a vast sinusoidal network, the radicles having a considerably
larger potential caliber than capillaries elsewhere. The combined lumina are of
such volume that the marrow confmes make it impossible for all to be dilated at
the same time, so that a considerable portion of the vascular bed is reduced to
capillary size and is not open to the circulation at any given time, an important
factor in red blood cell formation.
The marrow is poor in nerves_ Glaser has described fibers accompanying
larger vessels, but ganglion cells have not been demonstrated. Sensory fibers
are definitely present, as demonstrated by the pain experienced when one enters
the marrow cavity during biopsy, for example.
The cytologic pattern of bone marrow varies considerably in normal indi-
viduals. The free cells occupy the spaces between fat vacuoles. Cells of the
granulocyte series furnish a more or less diffuse background and erythrocyte
formation tends to be focalized. Megakaryocytes are sparsely distributed and
may often be seen lying in close juxtaposition to sinusoids. The erythro-myeloid
(E-M) ratio varies from 1:2 to 1:6 "in healthy adults (some observers have
recorded it as high as I :xo) and these series together comprise about 90 to 95 per
cent of the total cells. The usual pattern is shown in the following differential
count:
506 MISCELLANEOUS

TABLE XXII
THE DIFFERENTIAL .COUNT
Cell types to be listed in performing a differential
count of bo-ne marrow are as follows (figures indicate
normal range in per cent):
Undifferentiated cells ................... .
],fyeloblasts .................. ........ 0.0 - I 2
Promyelocytes
Neutrophil " . . . .. . .. ,...... 0 5 - 9 0
Eosinophil ................ . , . O. I - :2 0
Basoplnl*..... ... ............ 0,0
Myelocytes
NeutrophiL.. . . . . . . . . .. ., .... ~o 0 -34 6
Eosinophil . . . . . . . ,..... ..... 0.3 - 2.0
Basophil·...................... 0 0 - 0.3
Metamyelocytes
Neutrophilt .................... 14.8 -33.0
Eosinophil ......... ,. .... 0.3 - 3.7
Basophil'" '" ... ..... ....... 0 0 - 0 3
Segmented forms
Neutrophil. .. .......... ..... 3.0 -17.4
EOSInophIl .................... 0 I - 1.0
Dasophil"'......... ... ... .... 0.0 - 1.0
Promegaloblasts.. ................... 0 0
11egaloblasts .. "., .. ,.,', ... , .. ,... 0,0
Erythroblasts ...................... 4.2 -18.2
NOfmoblasts ....................... 13 3 -20.0
Megakaryoblasts .. ................ .. 0 0 - 0.25
Megakaryocytes. . . . . . . . . . . . . . . . . . .. 0.25- 0 8
Reticulumcells ....................... 0.2 - 2.0
Endothelial cellJ........................ 0.1 - 0 6
Lymphocytes.......... ................ 0 0 - 1.8
Pla.smocytes.... . . . . . . . . . . . . . . . . . . . . . . . . 0 0 - 1 0
• Basophils ale not encountered in paraffin sections.
t MetamyelocytC$ include both juverule and stab
forms of the Schilling classification.

FORMATION AND DELIVERY OF BLOOD CELLS

ERYTHROCYTES
The demonstration by Doan tbat the vascular tree is a closed bed with only
a portion of the sinusoids open to tbe circulation at anyone time offers an
attractive explanation for the formation and delivery of the non-motile erythro-
cytes. In regenerating marrows after cellular depletion he was able to show that
erythropoiesis occurred in the closed capillaries through hyperplasia and differen-
tiation of the lining endothelium, passing successively through megaloblast,
erythroblast, normoblast, and reticulocyte stages. When maturation is complete,
one can presume that tbese erythropoietic capillaries becom~ open to the circula-
tion and the ripened cells are thus delivered. Only under conditions of mMked
stimulation, however, are the first two steps in tbis sequence in evidence; at the
normal rate of erythropoiesis red blood cells are thought to be supplied througb
division of intermediate and late erythroblasts and by enucleation of the normo-
blasts thus produced. Again, it is almost impossible to demonstrate the outlines
of these erythropoietic capillaries in a crowded marrow, due to the preponderance
of granulocytes.
 THE BONE MARROW

GRANULOCYTES
It is generally agreed that the granulocytes take origin in the main from
fixed reticulum cells, either .through the medium of a rounded-up, free stem cell
or sometimes as a stellate cell that has acquired specific granules in situ; occa-
sionally myelocytes are seen in an endothelial position. Here again stem cells
and myeloblasts are not in evidence in the normal marrow, proliferation of
promyelocytes and myelocytes apparently being adequate for maintenance of the
blood level. Maturation to segmented forms within the marrow is usually most
prominent in the vicinity of patent blood sinusoids which they enter by ameboid
movement.

PLATELETS
The derivation of megakaryocytes from reticulum cells through indistinctive
blast stages is readily demonstrable in active marrows. Serial sections show them
lying chiefly in close juxtaposition to sinusoids and occasionally one finds pseudo-
podal cytoplasmic intrusions into the v!lscular lumina, this being the probable
mechanism of platelet delivery. Megakaryocytes may arise from sinus endo-
thelium as well, seen usually in metaplastic formation in tlie spleen, liver and
lymph nodes (Custer, 1933).

MONOCYTES AND MACROPHAGES

Reticular and sinus endothelial cells can assume phagocytic quality in situ
(fixed macrophages, clasmatocytes) and may take free, amehoid form (wander-
ing macrophages, monocytes, "epithelioid" cells). The bone marrow shares the
formation of these cells with the other tissues of the body, notably the spleen,
liver and lymph nodes, thereby playing a part in blood destruction and the defense
mechanism. The motility of the free cells in this group permits entry into the
blood stream and transport to distant parts.

LYMPHOCYTES AND PLASMOCYTES

While these cell twes are relatively foreign to myeloid tissue, the potentiali-
ties for their formation exist, nonetheless, through differentiation of the lllulti-
potential stem cells. Their mere presence does not necessarily imply that they
were formed in the marrow, in that they are motile, but the finding of true
lymphoid follicles and the so:called reticular lymphoid and plasma cells does.

DESTRUCTION OF BLOOD CELLS

That the cell content of the blood may be maintained at a fairly constant
level, a balance between replacement and destruction must exist. The marrow
participates in the disposal of effete cells through the phagocytic activity of its
reticulo-endothelial elements which also playa role in the breakdown of liberated
hemoglobin and the storage of iron. Degenerated megakaryocytes may undergo
disintegration in situ; sometimes the shrunken nuclei find their way into the
sinusoids and are carried to the lungs where they lodge in the capillaries and
become autolyzed.
508 MISCELLANEOUS

MEANS OF STUDY BY BIOPSY

Marrow studies during life are possible through sternal biopsy and should
be practiced with greater frequency in those cases which do not permit diagnosis
from clinical features and careful examination of the peripheral blood. Apart
from the diagnostic aid the course of a given disease can he followed by repeated
biopsy and new light may be thrown on the nature of diseases of the hemato-
poietic system.
One must never forget that normal bone marrow varies markedly in its
composition (Custer, 1932; Schulten, 1936), certain pathologic marrows even
more so, and the recognition of all disorders of the blood-forming organs obviously
cannot depend on sternal biopsy. The diseases that present a specific marrow
picture upon which a definite diagnosis can be based are comparatively few.
Even though the marrow appearances may not be specific, however, an intelligent
correlation with the clinical aspects and blood picture greatly widens the pos-
sibilities of the procedure.

The several methods are safe if performed under strictly aseptic technic,
bearing in mind the danger of introducing infection into the myeloid cavity. If
care is given to the local anesthesia, the patients suffer little or no discomfort;
even by the trephine method there need be merely a twinge of pain as the marrow
space is entered. Idle curiosity should not prompt biopsy, however, when there
is nothing to be gained from the patient's point of view.

THE TREPHINE METHOD

Apart from the fact that it is an operating-room procedure, this is the method
of choice. A button of bone and attached marrow are removed from the ventral
plate of the sternum with a I cm. diameter trephine, fixed in Helly's fluid, decal-
cified in sodium citrate-formic acid solution and embedded in paraffin; 4 micron
sections from the deep surface of the button, stained by ]\fa~imow's a2Ure II-eosin
method, afford excellent preparations from which accurate disease diagnoses are
sometimes possible and non-specific changes are almost always important. After
removal of the bone-hutton, fragments of marrow are curetted from the margin
of the cavity, imprinted against and drawn gently across clean micro slides, and
stained by the May-Griinwald-Giemsa technic soon after drying; other slides
while wet can be fixed for one minute in absolute methyl alcohol and stained
later. Tissue cultures and supra-vital preparations on neutral-red-Janus-green
filmed slides may also be made; the last two procedures are especially useful
when cells in the peripheral blood are not clearly identified with Romanowsky
staining. Reticulocyte counts may be performed in the same fashion as with
blood smears.
It must be emphasized that dry imprints at least are to he coupled with the
sections, hecause intrinsic cellular detail is clearer, comparison with blood smears
is possible and a link between the peripheral blood and the marrow sections is
thus obtained.
 THE BONE MARROW 50 9

FIG 16-Showing position for injection of large-bore needle with stylet (Is-gauge avera!;"c).
The needle IS thrust through the locally anesthetized skin and fascia, then through the ventral plate
of the sternum into the myeloid caVlty.

THE ASPIRATION METHOD

This is the method most widely used because it is the simplest (Fig. 16). A
large-bore' needle with stylet (average IS gauge) is thrust through the locally
anesthetized skin and fascia, then through the ventral plate of the sternum into
the myeloid cavity; the stylet is withdrawn, a syringe attached, and bone marrow
admixed with blood is withdrawn. The needle should be withdrawn as soon as
blood is observed at the tip of the syringe, thus avoiding too great a dilution of
the marrow. Smears are prepared and stained after the fashion of a blood smear;
the residual material can be expelled into fIxative, centrifuged and sectioned, but
the preparations are not very good. Some hematologIsts prefer to omit the
stylet in the hope that tiny fragments of bone and marrow will enter the needle as
it passes into the cavity, to be recovered for section; this may merely clog the
needle with periosteum and cortical bone, however.
Comparative Value. The value of well-prepared histologic sections of marrow
in studying the hematopoietic pattern cannot be overestimated. Using the trephine
method one can determine with considerable assurance a state of hyperactivity,
bypoplasia or defective maturation in one or all of the cell series. These are
matters of some question in examination of aspirated marrow, in, that cells do not
retain their original relationship and probably not their relative proportions, and
the material withdrawn may not be representative. Focal lesions which may be
of diagnostic importance are almost sure to be missed, and aspirated marrow is
bound to be appreciably diluted with blood. Finally, cells are apt to be damaged
in preparing smears of the marrow-blood mixture, whereas they are well preserved
in dry imprints.
The aspiration method has the advantage of simplicity which permits many
repeated biopsies in the same individual, and is quite satisfactory in some in-
510 MISCELLANEOUS

stances, such as the detection of certain aleukemic leukoses and other conditions
characterized hy the presence of cells which are abnormal or foreign to the
marrow.

ABC
ll::~.
FIG. 17 -A, dotted figure shows direction for introducing cannula and trocar through skm
When this maneuver is completed, instrument is rotated in upright positIon B, cannula is pushed
down to rest on sternum. C, drill ha! penetrated sternal plate and tip i1'o within marroW' caVlty.
(From Favorite, 1940, Courtesy of the C. V. Mosby Company.)

THE DRILL METHOD

As a compromise method Favorite has devised a drill sheathed in a cannula
(Fig. 17). After the drill is introduced into the marrow cavity, it is rotated, thus
withdrawing rragments from which imprints and sections can be made. If the
cannula is retained in place, the drill can be reintroduced at different angles and
additional material obtained.

EXAMINATION OF BIOPSY MATERIAL

Many tecbnical difficulties confront tbe student of bone marrow cytology;
it is not easy to prepare legible sections and smears, particularly the former, and
disappointing results naturally lead to neglect of the tissue. The various technics
are outlined in Section Eight and it is up to the individual to select those best
adapted to his facilities. Considerable practice should be had on autopsy tissue
before biopsy is attempted.
Study of the sections should include observation of the general hematopoietic
pattern and differential counts. The first requires an estimation of percentage
cellularity versus fat; this is compared with the expected normal for the par-
ticular bone at the given age. Low-power view also gives evidence of normal or
abnormal grouping or dispersion of the various cell types as well as the presence
of focal lesions and tissue foreign to the marrow. The differential count of at
least 500 cells should be performed according to a definite plan; thus, using a
ISX ocular equipped with a net micrometer or parallel hair lines, cells in every
fourth field are allocated, avoiding fat cells, blood channels and bone trabeculae.
I prefer to move to another portion of the slide after counting ,four fields in
sequence; an additional 500 cells should be counted if there are marked discrepan-
cies suggested by a sharply changing count.
 THE BONE MARROW 5I l
TABLE XXIII
COM~ARISON OF COUNT OF FIRST AND SECOND 500 CELL GROUPS
Best Fit* Po ...es' Flit
First Second First Second
500 500 500 500
Granulocyte series:
Myeloblasts . .. .............. . ..... '75 ,85 25 JI
Pcomyclocytes (neutrophil). . . . . . .. . ... '4
Myelocytes (neutrophil). . . . . . . ...... ,
lZ 36
73 lIS
'4
Myeiocytes (eosinophil). . . .. " ...... 9 3
Metamyelocytes (all types) ............ 67 89
Segmenter.;; (all types). . ........... ,6, '00
- 19° - IQ9 -372 - 3 42
Erythrocyte series
Mcgalobla.sls. . ...... .. ....... . .. .. '9 ,6 2
Erythroblasts .. .... ....... .. 92 95 3' 60
N ormoblasts . .. ......... '0' 97 43 6,
- 21 3 - :208 75 - 123
Thrombocyte series.
Megakaryoblasts ......... . . . .. .... . . 0
Megakaryocytes .................. 2

'4
Reticula-endothelial cells:
Reticular cells }
Endothelial cells ........... .......... 50 47 18 '4
Wandering cells .... ............ " . 4 23 'S
54 S6 4' '9
Lymphocytes ............ .... ......... '3 '9
Plasmocytcs .... .,. ...... ...... ...... 9 4
3' '3
" Case of idiopathic agranulocytosis.
t Case of septic neutropenia.
(From Krumbhaar and Custer, 1935, courtesy of Lea and Febiger )

If one is interested in a sparsely distributed cell type, such as the mega-

karyocyte, the total number of cells per average field in the given marrow is esti-
mated; the particular cells in each field are counted until the approximate total
of all cells reaches 12-15,000, and the percentage then calculated.

MEANS OF STUDY AT AUTOPSY

Bone marrow specimens removed at autopsy are prepared in tbe same manner
as biopsy material. Selection of the material is important in that samples should
be taken from several bones; a thin wedge of vertebra, a narrow cross-cut through
the sternum and a pencil from mid-femur are the minimum for proper study.
"The inconsistency in the tissue itself should be sufficient indication for a careful
and uniform technic and a survey of more than one bone. On the contrary, how-
ever, except when performed by those workers carrying out special studies on the
bone marrow, the reverse is too often the case. For example, the tissue selected
for study by many pathologists is removed at random from anyone convenient
bone, perhaps the tibia on account of its availability. Often one notes in protocols
that the bone used is not even identified" (Custer, 1932).
5 '2 lvf,sCELLANEoUS

APPEARANCES IN ANEMIA

As result of an immediate demand for red blood cells there is primarily a

redistribution of tbose already in tbe vessels by reason of increased vasomotor
tonus, accelerated heart action and splenic contraction. This is followed by the
prompt response of the normoblast reserve in the bone marrow and the release
of reticulocytes and erythrocytes into the 'general circulation. If this is insufficient
to re-establish the normal level of the blood, the bone marrow undergoes hyper-
plasia, fat being rapidly resorbed and the new hematopoietic tissue taking its
place. The adequacy of the hone marrow to restore hlood may be judged by:
(I) the erythrocyte count; (2) the quantity of hemoglobin per cell; (3) the size
and shape of tbe cells (anisocytosis and poikilocytosis) ; (4) the degree of reticu-
locytosis; and (S) the presence of nucleated elements in the peripheral blood.
As anemia per se may result from extraneous factors or from bone marrow
insufficiency, or both, it is probably better to consider the marrow appearances
under the headings of hyperplasia, hypoplasia and displacement.

WITH HYPERPLASTIC MARROW

The bone marrow in hemorrhagic and hemolytic anemia undergoes hyper-
plasia in proportion to the demand for new red blood cells, there being an inver-
sion in the erythro-myeloid ratio with late erythroblasts and normoblasts pre-
dominating in the milder forms. Basophilic erythroblasts and still younger cells
of the series appear in progressively greater numbers as the severity of the anemia
increases. Although the relative proportions of granulocytes and megakaryocytes
are diminished, there is an actual increase in these cells per unit area, often re-
flected in the peripheral blpod by leukocytosis and a "shift to the left" in the neu-
trophils. Reticulocytosis is the usual indication of increased erythropoietic activ-
ity, but release of nucleated elements may mark the more excessive hyperplasia.
During a hemoclastic crisis of certain hemolytic anemias cells of the reticula,
endotbelial system are hyperplastic and contain whole or fragmented erythrocytes.
When iron deficiency exists, whetber from chronic blood loss, poor absorp-
tion, or inadequate intake, much the same marrow reaction is observed, but the
poverty of hemoglobin is apparent in the staining reaction of the usually hemo-
globiniferous cells. It is not uncommon in idiopathic hypochromic anemia for
normoblasts to appear in section as a compact nucleus surrounded by a colorless
halo.
Although the initial stages may not show medullary megaloblastosis (Lambin
and de Weerdt), the most striking alteration is seen in pernicious anemia in
relapse when megaloblasts and their progenitors dominate the field and more
mature forms are relatively sparse. Transitional stages between cells of the
reticulo-endothelial system and promegaloblasts are often demonstrable, either
directly or through tbe medium of the free, undifferentiated cell. Some authors
prefer to regard this as a completely different developmental sequence rather than
 1'1(,;-. IS.
;':ORli.\L STER'\: \1. B():\I': ),I .\IUI()'\

Undiffcrentiated ccll~.

).[n·lobl:t>it".. 0 ()
i~rom~'e!~'cyles (neutrophil) 9.0
(co"inophi.l), 0 0
:'I!yelocyLcs (neut rophil ) 3.+.(1
(l'osinophil) 2.0
.\lcUlmyclocyLl'S (nculrophilJ q ()
(eosinopl1 il l
~l·f.:!ml"nt("rs (neutrophil,. 2 f)
(L'oo:lIl0phil).

Total 63.S
l!rO!'lcgaloula,.:j ~ 0.0
~lcgal.obla,.;t." 0.0
Erythroblasb. [4.~
~orm{)bla.sLs.. i8 .2

Total . 33 0

.\ ltgakaryocyLt::i .
Reticular Cells ..
[':ndot]wli,lI C(·lls ..

FJG. 19.
PER:\lClOLI:-. ,\:\1',~1I,\ <S "[AGJ ~ OF RET •. \I'~I I

l'ndifierelltiated ccii,.;" 2. !
\1 \'cloblasts . o. ,"I
:Prumyc!ocytcs (neUlmpil il l. a.S
(l:o"inopitii). 0.0
~lycl(,cytl''' (nel.llrophil~ . ,.4
(cns.inophil ). 2 . .'-\
::\ lel<tmyeioc,')te:-; (ne ul mphil ~ . ...j., (,
(l"o~in()phill.
:-;('~tlll'nh:rs (neU lr4)l'hil ~.
(cosinn pll il). .1 2

Tutal 27·0

I'rumt:galoblasb. ~. ,'\
.:\[cgalobla;.:.Ls ;~S, (.
Eryth.rQhbl-:h . lJ ,S
;\1ormohlash. . 5 . ..J.

Tot~d (JO. (I

\h:gakar_\ ·uc~·tcs.. 0 :..;

J..1,diculat CcII:,.. 5.2

r':n(jothelial l'dl", .. -I- a
 FJ{~. 20.

PERNrCJ.oL;~ .\XE'H. \ (E.\IH .... · 1{.c~n:-::.ilO~)

Ul1di(fercntialeu (d!s.. 0.6

":\Tyc1Qhb'":t'!'- ... ' 1.0
Promydocytes (nelltropbil) . . I . (,
(co~ lnoJ)hil). 0 -+
~\Iydocylt."~ (neutrQphit} .')..0
(eosin0phill. 3. a
::\l~tamyeluc.rtl'~ (neutrophil I .1 oS
(eosinophk\) 2.b
Scgmenlers. (neutrophil) 8 . ..(-
(eo"inophiJ'1 r . ,"i

Total.. 28 (J

Prot'negalQbb,Sls .. 1 .0
Megaloblasts. ~. >
Erythr-obla:sts. 47··~
~Qrrnoula"h... 10 ~

Tolal. . 66.S
Megakaryocytes. o .S
Reticular c:dl~ I (j

Endothdial cdIs. r 6

FIG. 2].

P£R~"""ICIOt:'S .\XE"IA ( \ .. .-\n~ RI-:\nSSB.r;-..;- 1

Undifferl'lltlat~d cells. 0 -I-

~\ly(.·lobla~ts. I 4
Promyd<)Cyll.:':'. (h-c:utI'Oph~l'.. 6 _(~
(eosinophil).. o ,fJ
)-lyelo(.'ytc:"- (neut rophil ). . 15 .....
(~o,inophil). .0
)ldamydocytcs (neutrop,bil,i 9.
(eosinophil) .0
SegmcDteTs (!1cu~rop;,nl. ~
(coi'iil'lophill. 0 S

TotaL. . . <1-3 :-:

Prorncga1obl<l!'t~L 0 :..:;
]\.1")I.lo1>la'\" .<J
En'throhl;l~t~. 12 .J
Normohh~t:-:." 27 Z

TutaL 53·S
.Mq:!;akarroC}'l es. r 0

Reticular cells. 0 .•'-:

E:ndot_hdial cells o. 1:
 FIG. 22,
Ht ~lORlHL\GIC ,\XE~lL-\

L"nciiffcrL'nLiated cell::;. 1,:-\

1IydobLtsts. 0 :
Promydoq,tcs (neutrophil) . 2.-J.
(co:-;~nophil).... 0.0
\lydl)crtt:~ (nc.:utropbil) 5·-J.
(i.:IJ::iLnophil).. ( .6
l\lctamyclnc)'lcs (neutrophil). 8. 4
(cosinophill. 0.8
Scgmcnlers (neutrophil) 3.4
(eusinophil)

Total. 22-4
Prom('wllobla~ls. r .s
r-.lcgalohla,:ls. 0.6
EI),throhlasb. 3.3 (J
~ornl()bla~ts. 32.0

Total 63.0
:J.IL'~.\karyo'(,.'Yl{'~. . 1: l

R-l'licular ('dIs. 3
Enuolhelinl cells. -'.--l
 FIG. 23.
P_"-R.\SIT\(' \-..;,- ,n \
(XEC.\TOJ( . \ \ll,"UC.\"\l"~)

"Cndlffc.renlial\:tll..:db. 1 2

:.\lyelob]a~ts a .J
l'romydnt'ytt-... l)\.' UlN))hil i :-:;
t;o",illnpl,ill , 1
':\Ircloc~'l('~ neutrophil t () 0
(lw.. inllphil -' J
.\h·tanH'el"(T!t'S (nt:'tllrophil , (J a
- . (i:\):'oint)p~i\ :! ()
Segmenll'r'i (nl'lilrophil . .l ....
(t.'o~ inophi! j I (,

Total JJ 2
Promcgalohla!it~. 4 6
~[cgalobli.l~ls . 3 .2
ETythrobla~t~. 3' ~
)J"ormolJlil,,1 s .JJ .2

TOlal ()2.8
:.\It:gakliryocyle::; 0 4
Reticular cdl~ r 4
j~ndolhl_'lLll n-1b.

FIG. '4.
Ivrol'A'rlllC llYPoCIIRO\UC .\XJo:lllA

L' J}(li\tcrcl1liatl'u celis. :1 ()

'I\'l'lohlasls ., 0.:-1
'ProlJlyl'loc,yt(.'!oi . (nt:t1l~;J;I~il J" . I. is
(eosinophil), 0.0
_\(ydocylc~ (neutrophil i ,-1-
(eos>inophil 0 _~
.\I(·tilmYl'iI)("~·lt·~ (neutrophil () :)
l L'O",j nophil I .2
Sl·~nlL'ntt·r.;. (!lfutrophil l _ _;; I
(",'()~ inophil 0 .0

Total !i 2

Prornq.!,d(·bla . . t" .• -4
)I(.g .. dobla~t~ ~ S
Er\'lhmhl "I:-, .J; l -I-
\'(:rlll0hlaQ... !:) 2

Total OJ.S
\h:~;~k;\[yo\:y l~'S. 0 . .-\

Rctku lar cells. 5.0

i':ndothdial cclI!:.. :! 0
 FIG. 25.
_\PL\STIC ,\_;';L_\lL\

Undifferentiated cells, " 0,8

:,jl)'e!oblasts. 0 ()
Promyelocytes (neutrophil) 0 4.

••
(eosinophil'. 0.0
).rydoc)"l~s (neutrophil) 0."";
(eosinophil I. . .

•
0 !
~[clam.rdocytes (neutrophil}
(eosinophil" 0,2
SL't;mcnlcrs (neutrophil 1 0.0

-_ Total.
(eosinophil).. 0.0

2.6

•
• I
"Promcgaloblu!'ts.
Mc~aIQbla'6!;j.
Etythroblasts.
)l"ormoblasts ... ,
0.:2
O.-$-
45.2
30. l

• • Total
)1 ega.karyocytes. .
;6 . ..:
0 •J

Re:tlcuiar cells. . . I J .0

••
ErJodothe-JiaJ ccll~. f) 2

(:\Ioderatc inllu.\ 01 lymphocYles and pla:-,-

mocytc~)

Flc. 26.
S[Ch:LE CELl •. \XJo: . . IU

l'mLiffcrcntbted celb 2 ..

. . . h'cloblasts ... oX
'Promyelocyt('s (neuLrophm .. I ,~
( t:'o~inQphill 00
}Iydocytl::S (neutrophili .. 8 2
(t.'Osinophil) . o ,~
}.Ictamyclocytcs (neutrophill ro S
(eosinophil ) , 0 .. ~
Sl'gnH.!nlcr:-; (neutrophil) + ,;
(CQSinOfllll!) .. Q.2

ToLd. 2;
PrOlnegaiobla~t~.
1\[ega.lobla~l~ "
I. :1
2,4

,1
J."
ErythrolJrasts. 33 4
Xormobla:;t~. :24. 0

Total
)tep;akaryocytes.
Reticubr cdls
Endothc1i'll cdIs.
6J
0.8
4
4 0
.0

0
.
 FIG. 27,-:\:,!ranlllocyto~i:3, Hypcl'pla~ia of mydoblasts in the femoral manow with pra.cticall)
r.o maturation tc g;rflnular forms; a few lymphocyte~ a.nd plasmocytc-s arc admixed <X (90).
FlG. :!8,--A;.!ranulor~'to<:l~. Showiu;.!. normal furmation of erythrocyte progenitol"s and mega-
karyocyte!> (X 690),
FIG. 29.-Agranulocylosis. Hypopla.';ia of marrOW fo]lm"'ing: degeneration of m~'cloblasts.j the
lntennJ:rJgJcd normoblast:;; do nut :-:h.:{r·" I he degerwri:i(iurt (X ogo).
FIG . .3o.-Acute myelosis {acllte mye'og0nou::; leukemia). The marl'O"\\' space::;. arc almost COID-
pJeif'Jy replaced by myelnbhlF-!S anrl promyr]oryll's; mitotj[ ngur.r... art: numerou~ (X 550),
FlG. 3L-Chroi)jc mydo!)js (chronic myelogenous lclJkemja). The marro'!y is totaUy cellular
:IS result of marked hYJJcrpi<l.sia of aH of the celt series} especially the .c:ranuloc~.... tcs which shm,\'
maturation lo the segmented stagL"j O1r-gakaryoblasts and mega~N.ryocylcs are also prominent
(X 600).
FIG. 3:?~Chronic myelo~is (chronic myelogenous leukemia). Hcmopoies;s is largely intra-
I;ascular (and c~lr3-mcdul:ary); mCj.!aknryor:ylcfi arc ~lI1usually prominent and the p::oripheral blood
:;.ho\\'ed marked tbrombocytosis (X 400).
FIC. 33.-Acllte lymphadenosis (acute lymphatic kukcmia). Hemopoietic tissue is displaced
by J~'mphobra.sts -::hO"""inj!: rather .... esIcular nuclei and prominent l1uch::oli; the Hinfiltratlon" is often
1005("1' than this and the dC.[!:oChtrativC" chan~('::. in the marrO\\i more apparent eX 1500).
Fre, 34,-Chronic Irmph<ldeno~l.- (chronic Jymph.aHc Jeukem;a). Termjna} repla.cement of the
marrow by Iytnph(lcyle~ of r('lati\'ely nlaturr type (X 600)
F1G. J.5.-AruLr retku]o~b (.(lcute mon(]c.'·tic leukemia). Early ~ta2;~ 5howing proli[eration of
c('lIs. of the. reticuloendothelial s}"~tcm, mostly r~taj(Jihg their fi:...ed ti~suc po::;ition and rdation to
reticular fibrillae (X50o),
Frc. 36.-Acutc reticulosis (acute monocytic leukemia). Latc. slage (auLop5Y, sam~ case) ~ mos1
of the cells arc free and ha\·c 35sumcd lhr morphologic -characters of monobbsts and monocyte~ ,.
hemopoietic ti~;;;:u(' i~ c(Jomplclely rep\;.tced (X :mool
FICo. 37.-:\clltt. pla:-mocyto:-=b {a("Ull' p!CI..c:.nwL")·tic h~llk~l11ia). The m~dority of ('en~ an.' pla.c; mo-
hlaHs: fJuL cn()u)!n ha\'e matllrcd qlitici('nL!y l-O present the eccentric. cari-wheel nucleus to be dearly
identifi:;.blc as. pla~moc.YLes (X 600),

...
 THE BONE MARROW 513

a pathological deviation from the normal (Jones, '934, et a1.). Israels (1939)
presents the following scheme for comparison of the two series:

Hemocytoblast
I

7~~,
Normoblast A Megaloblast A
(basofhiliC) (basorhilic)
Normoblast B Megaloblast B
(polychrom.) (polychrom.)
I I
Normoblast C Megaloblast C
(orthochrom.) (orthochrom.)
and states that megaloblasts have no place in the development of normal blood
cells in extra-uterine life, that they appear only when the proper activity of the
liver principle is in abeyance. Lack of the hematopoietic principle affects
the neutrophil series as well and, while some of these cells mature norm,llly,
others pass through bizarre myelocyte stages to form the so-called "macro-
polycyte" with muitilobated nucleus. In some cases megakaryocytes are de-
creased in number and occasionally display a deeply basophilic agranular cyto-
plasm although the nucleus may be polymorphic.
The rapidity with which this state may change is remarkable after adrnin-
istration of the anti pernicious anemia principle; on the day following a single
injection of potent liver extract most of the cells are in a mid- or late-stage 01
maturation. Some observers believe that the megaloblasts undergo normal matura-
tion, while others state that they follow their abnormal developmental sequence
and that the normal erythron is established subsequently. The neutrophil series
also reverts to normal under adequate treatment. Similar but less rapid and
complete change is seen with spontaneous remission.
Megaloblastosis of the marrow with imperfect maturation is found in sprue
and pernicious anemia oj pregnancy, and a somewhat similar picture may be seen
in Cooley's anemia although the immaturity of the cells is less marked. In the
pseudo-pernicious anemia of gasttic carcinoma and Diphyllobothrium la tutn
infestation, in chronic liver disease, pellagra and other macrocytic. anemias there
may be scattered foci of megaloblasts but the more mature forms predomiltate .
The anemia of nephritis is usually associated with a normally cellular or
hyperplastic hone marrow of the normoblastic type; there is nearly alway? an
inversion of the erythro-myeloid ratio.
One generally ~sociates chronic exposure to benzol with anemia due to
bone marrow hypoplasia. Mallory and his associates (1939), however, observed
this in only six of 19 cases; three presented normal cellularity (with qualit:ltive
variations), three moderate and five marked hyperplasia. Activity was ,nore
 MISCELLANEOUS

marked on the part of the granulocytes, megakaryocytes and macrophages than

in the red cell series, and in the severe cases stem cells appeared in large numbers
and displayed numerous mitoses. Two cases developed an actual leukemic
status. Fibrosis of the marrow was a feature of some cases, with replacement of
fat rather than hemopoietic tissue.

WITH HYPOPLASTIC OR APLASTIC MARROW

Depression in hemopoietic activity may occur in one or more of the' various
cell series, usually all, with leukopenia and thrombocytopenia being as much a
feature of the peripheral blood picture as the anemia.
The so-called idiopathic aplastic anemia, in which, no etiologic agent is
demonstrable, presents a marrow which is more or less completely fatty and
largely devoid of hemopoietic elements. The paucity of granulocyte progenitors
is often more striking than erythrocyte, as shown in the differential count in
Fig. 25.; nucleated red blood cells are fou~d chiefly in the late erythroblast and
normoblast stages, the few more immature elements being observed in rare tiny
foci of regeneration. Cells of the reticulo-endothelial system (undifferentiated
mesenchyme) are shown to be increased in the count, but this is relative, Dot
absolute. Whether the marrow hypoplasia is due to lack of stimuli for differentia'
tion of these cells or to their inability to respond has not been determined. An
influx of lymphocytes and plasmocytes, perhaps a reaction to degeneration, is
usually noted in the marrow of aplastic anemia.
Bone marrow hypoplasia or aplasia secondary to known toxic influences,
such as overwhelming infections, irradiation, or chemical agents (usually an idio-
syncrasy) will display essentially the appearances already described. The varied
pictures of chronic benzol poisoning have been considered. In many cases the
marrow will regenerate very rapidly if the cause of the depression is determined
and removed.

WITH DISPLACED MARROW

Marrow spaces in the "myelophthistic" anemias are largely replaced by
fibrous tissue, bone or tumor, or by reason of unrestrained proliferation of one of
the hemopoietic series as in leukemia. The effect on the peripheral blood depends
on the ability of the other organs, notably the spleen, liver and lymph nodes, to
revert to their fetal blood-forming status. Usually there is a progressively in-
creasing appearance of distorted erythrocytes, a reticulocytosis and a later ap-
pearance of nucleated forms, associated with an outpouring of immature neutro-
phils, which Vaughan designated "leuko-erythroblastic anemia."

APPEARAN.cES IN HEMORRHAGIC DISEASES

In the several hemorrhagic diseases the formation of erythrocytes and leuko-

cytes is normal and these developmental series may undergo hyperplasia in the
usual fashion in response to anemia or infection. Megakaryocytes, however, may
or may not present numerical or structural alterations. Some cases of thrombo-
 THE BONE MARROW

cytopenic purpura will show a normal megakaryocyte count (Nickerson and

Sunderland, 1937), others exhibiting marked hyperplasia of these cells (Limarzi
and Schleicher, 1940) and metaplasia in spleen, liver and lymph nodes; still
others will show paucity of megakaryocytes which may be in a degenerated state.
Scarcity or absence of cytoplasmic granules has been described ("hyaline mega·
karyocytes"). There is an increase in the number of megakaryocytes in hemo·
philia (Custer and KruJl)bhaar, 1935).

TABLE XXIV
GROSS ESTIMATION OF THROMBOCYTE SERIES*
.... (:zoo consecutive.fields, averaging 60 cells each)
(expressed as number per 12,000 total cells and in per cent of the :12,000)
M egakaryo~ Normal Degen.
blasts megakaryocyles megakaryocyte! Tolal

No. % No. % No. % No. %

Hemophilia:
Case I -Sternum 1:2 0.10 o ,8 36 0.30 60 0 58
Vertebra r6 0 13 21 0 IS 35 0 29 7:2 0 60
Case 2,-SternuIl1.. ... . . .. .,. 7 0 06 6 0.05 32 0 27 45 0.38
Vertebra. ......... 8 0 07 :26 0.22 25 0.21 50 0 49
Case 3 -Sternum . 8 0 07 20 0.17 23 0 19 51 0 43
Femur. .. ........ 16 0.13 23 a 19 9 0 oS 48 0 40

Average 57:; 0.48

Non-hemophilic controls:
Symptomatic purpura ........ 0 05 10 008 IS 0 1:2' 31 026
Symptomatic purpura. .... •. 1: 0.01 :2 002 15 0 12 18 0.15
Polycythemia vera . . ... . 4 0 03 0'7 :20 017 44 Q. 37
Chtonic hemorrhage... ..... 4 0 03 9 0 08 13 0 II 26 0 :2:1
Arsphenamin neutropenia ... ,.. 4 0 03 16 0,13 7 006 27 0.23
Third degree burn. . . . . . 5 0 04 12 010 13 0 II 30 0 :25
Brain tumor . . . . . . . . . . . . . . . 0 02 8 007 21 0.18 31 0 26
Bronchopneumonia .. . ... 0 os 009 14 0 II 31 0 26

Average :29 7 0.25

• {From Custer and KrumbhaarJ 1935, courtesy 01 Lea and Febiger.)

APPEARANCES IN POLYCYTHEl\lIA

Polycythemia in general is associated with an extremely active marrow, the

hyperplasia being shared by all developmental series, most marked in the erythron.
In certain instances the granulocytic reaction is associated with the appeara1ice
of immature forms in the peripheral blood, in others this is true of the mega-
karyocytes, and it is sometimes difficult from study of both the marrow and the
blood picture to differentiate this condition from chronic myelosis (myelogenous
leukemia) in a polycythemic phase.
At least some cases of polycythemia vera present the additional feat).Ire of
thickened, occasionally thrombosed blood vessels. This has been regarded by
some observers as a possible etiologic factor in the disease, in that it may produce
a state of anoxemia of the marroW and thereby create a stimulus to erythro·
poiesis.
 PLATE LI

BONE MARROW'
(NORMAL: HYPEI{PLASTIC: APLASTrc)

o
Top. Normal Bone Marrow. Center: Hyperplastic Bone :Marrow wit}l Matu~
1.MHIoblast. ration arrest at myeloblastic level (from
2'Premyelocyte. patient WIth arute myeloblastic leukemia).
3 Neutroph!lic mye}ocytes. r Myeloblasts
4 EosinophilIc myelocyte 2. Myelobla&t in division.
5. Juvelllle neutrophils 3. Premyeloc)'tes
6. Band neutrophIl, 4. Myelocyte.
7. Segmented neutropbJ1. 5. Megal()blasts.
8. Lymphocyte 6. Macroblast
9. Monocyte. 7. Normoblasts.
10. Megakaryocyte.
n. Macroblast':.. Bottom Aplastic Bone 1:!.-1arrow (from 'Patient
12. Normoblasts. with aplastlc anemia).
13. Primitive free cell <.?).
r. Lymphocytes.
2'. PrImItive free cell (?).
* Drawn from l>erum spread. 3. Degeneratmg cells
Plate LI.
 THE BONE MARROW

APPEARANCES IN SIMPLE NEUTROCYTOSIS

The bulk of the hemopoietic tissue of the marrow is devoted normally to

replacement of the short-lived (3-5 days) neutrophilic leukocyte and it has
been estimated roughly that 5 to 10 billion mature neutrophils are delivered
to the circulation daily. Infection and certain non-bacterial toxemias furnish
stimuli for increased production and delivery of these cells, primarily through
rapid maturation of the myelocyte and metamyelocyte reserve, further by actual
hyperplasia of the myeloid tissue and appearance of more immature cells. If
the cell destruction proceeds apace or exceeds new formation, the leukocyte count
will remain normal or decrease, but the immature ceJls will be released. The
degree of bone marrow activity, therefore, is reflected in the peripheral blood by
the number of circulating neutrophils and/or the percentage of younger forms.

APPEARANCES IN NEUTROPENIA

Neutropenia of moderate degree is compatible with good health in certain

individuals, apart from a tendency toward fatigability, and, although the bone
marrow is apparently functioning at a lower level than usual, it is capable of
byperplasia and responds to infection by leukocytosis.
Severe grades of neutropenia or complete absence of these cells from the
peripheral blood can be conveniently catalogued according to the general state of
the bone marrow by reference to Table 25.
A specific pattern is found in idiopatkic agranulocytosis (Custer, 1935) viz.,
myeloblastic hyperplasia with arrest of maturation at the promyelocyte stage,
normal erythropoiesis and thrombopoiesis, and folliculoid aggregations of lym-
phocytes; some cases show degeneration with a relatively empty marrow. This
disease should probably be classed as a proliferative allergic state, with amido-
pyrine as one of the more important causal agents.
Septic neutropenia shows a very marked granulocytic hyperplasia with com-
plete maturation in this series; in typhoid fever, focal necrosis and reticulo-
endothelial hyperplasia are characteristic.
Aplasia, idiopathic or resulting from arsenic, benzol, irradiation, etc., presents
a fatty marrow in fatal cases; during the recovery period from a lesser effect of
the destructive agent, however, extreme hemopoietic activity, particularly granu-
locytic, is the rule.
The bone marrow pattern in the aleukemic leukoses will be described in the
following section_

APPEARANCES IN THE LEUKOSES (LEUKEMIAS)

The cytologic picture in the bone marrow in the various leukoses is usually
sufficiently specific to permit diagnosis, is essentially the same whether leukemic,
 l\hSCELLANEOUS

TABLE XXV
.ru.SENCE OR STRIKING DIMINUTION IN NUMBER OJ! NEUTROPHILS IN
THE BLOOD STREAM
I. With relatively ICfull" marrou', as result of.
(0) Severe t01emia. (usually bacterial), through primary stimulation of panulopoietlc tisSoue-,
then destruction of cells in situ or on entry into the circulating blood.
(b) The leukoses (leukemta.s), WZ.:
1. Aleukemic myelosis, through overproduction of gittnuJocytes that either do not l~\'e
the marrow or are destroyed on entering blood.
2. Lympha<lenosis, through replacement of granulopoietic tissue.
3. Reticulosis, through replacement of granulopoietic tissue.
(c) Jdjopathlc agranulocytosis (agranulocytosis 01 Schultz, agraowocytic angina, maligtlant
neutropenia); through defectlve maturation of royelobla'Sts (most cases show full mariow;
see II-e).
II. With relatively "empty" ma"ow, as result of:
(.a) Severe toxemia. (usually chemical), sometimes specific for neutrophils.
(b) Marrow exhaustion, through protracted anemia, toxemia or infection.
(c) Aplastic anemia. (idiopathic).
(d) Irradiation (Roentgen ra.y or radium).
(.t) IdiopAthic agranulocytosis (the occasional case).
(From Custer, 1935, courtesy of Lea. and Febiger.)

TAllLE XXVI
Dll!FERENTIAL MAlUlOW COUNTS (PER CENTS)
Azure II-Eosin. (1000 cells each.)
Agranulocytosis
Early MarkM.
Changes Changes Arsphen. Septic.
(Case 4) (Case 10) Neutropenia Neutrop;ma
Granulocyte series:
Myeloblasts. .. . . . . . . • . .. • •.•• 0.6 37·3 45·1 6.6
Promyelocytes.... .. .. ••. . .• 9. 0 2.6 95 82
Myelocytes (neutrophil) ...... . 34.6 0.' 1 • 29 •
~"';"\"":l\"'" ' ' ' ' ' ' 'l>'' ' > .. " ..
Myelocyte, (basophil) ........ .
~.<>
00
"','
00
"S
04 00
Metamydocytest (neutrophil) .•. '4 6 06 16.1:

E~~:\~ds~ri~~ (all types) ... . 3·0) 00 0.0 5·0

Megahblasts ................ . 0.0 3·5 os 1.0 0.'
Erythroblasts. •• .. .. . •..... '4 8 '9 2 65 ~4·4 9 ,
Normohlasts .... •.... • .... 18,2 ~9·9 108 10 J
Thrombocyte series:
MegakaIyoblasts ..•. " .•.•. 0.2 r 2 14 I.. 0.1
Megakaryocytes .............. . 0.8 1.4 2.1 I • o.b
Reticulo-endothelial cells:
Reticular forms. .............. 7·9 3 4 3. 8 .
Wanderingforms.............. 0.:1 1.:;1 1.3
Lymphocytes... ... .... ........ Co 0 4.2 11.6 02
Plasmocytes . . . . . . . . . . . . ,. .. 0 0 1.3 3.,5 0.0
(Dense folliculoid accumulatio-ns of lymphocytes were avoided in agranulocytosis ca.ses.)
• Case of b:a.in tumor (white female, a.ged '2) with norma1leukocyte count.
t 1\{etamyelocytes include all cells between myelocyte and segmented form for simpl.ifica.tion.
t Tbis figure is lower than usual.
(From Custer, 1935, COUl'tesy of Lea and Febiger.)

subleukemic or aleukemic, but differs considerably between acu,,, anu C.llru"'~

types. Doan (1938) has laid down four criteria of disordered maturation which
can well be applied to this group of diseases: "( I) hyperplasia and frequent
 THE BONE lIIARROW 51 9
heteroplasia; (2) immaturity in cell type; (3) asymmetry in cell division, and
(4) arrhythmia in cell delivery."
Evidence thus rar presented indicates that the unrestrained hyperplasia of
leukemia, at least in man, is best regarded as truly neoplastic; the apparent
relationship between chronic benzol poisoning and myelogenous leukemia fits well
with the carcinogenic properties of various coal-tar derivatives, and the incon-
stancy of this effect takes into consideration the possible hereditary factor. The
etiology of llukemia, however, is properly left ,,:S an open question for the
present.

MYELOSIS (MYELOGENOUS LEUKEMIA)

In the acute form the marrow becomes rapidly filled with myeloblasts which
do not usually mature beyond the myelocyte stage and ultimately replace the
normal hematopoietic tissue, sparsely scattered clumps of late erythroblasts and
normoblasts persisting, and feW" megakaryocytes usually remaining; rarely one
finds megakaryocytic hyperplasia in the marrow and thrombocytosis in the
peripheral blood.
Displacement of erythropoietic tissue is much less marked in chronic myelosis
and there is often actual hyperplasia. Maturation is rar more complete in the
granulocyte series than in the acute type, the majority of cells being found in
the myelocytic and later stages, although foci of myeloblasts are encountered
frequently. Megakaryocytes are often hyperplastic and may even dominate the
cytologic picture; the platelet count in such instances may reach I to 2 millions
per cu.mm., as in the case illustrated (Fig. 3') where the interstices of the marrow
were relatively empty and blood formation was confined largely to the sinusoids
and extra-medullary tissues_ The more usual picture is seen in Fig. 3 I; even
here, however, there is hyperplasia of megakaryoblasts and the platelet count
reached a million on one occasion. Anemia and thrombocytopenia generally
occur during the later stages of the disease, however, as the granulocytic elements
take the foreground.

LYIIIPHADENOSIS (LYMPHATIC LEUKEMIA)

The acute type is characterized by a rather marked degree of marrow de-
generation associated with a loose or sometimes dense "infiltration" (metaplasia?)
of Iymphoblasts and lymphocytes, themselves often somewhat degenerated.
In the chronic form, well preserved small lymphocytes tend to form folJicu-
loid aggregations, between which normal blood-forming tissue ,is found. It is for
this reason that aspiration biopsy in 'the aleukemic stage may fail to show the
true state of the marrow. These accumulations tend to become more confluent
in the later stages and "replacement anemia" gradually becomes more marked.

RETICULOSIS (MONOCYTIC LEUKEMIA)

Massive proliferation of cells of the reticulo-endothelial system character-
izes this disease; in the bone marrow this goes on to the ultimate exclusion of
5 20 M,SCELLANEOUS

normal myeloid tissue. Early they retain their fixed po~ition in relation to
reticular fibrillae (Fig. 35); later they become rounded up and free, often
phagocytosing erythrocytes and debris (Fig. 36). ;rhe individual cases may
display various degrees of differentiation, from mo,fubla<;ts hardly identifiable as
such to rather mature forms of monocytes or macrophages.

PLASMaCYTOSIS (PLASMA CELL LEUKEMIA)

There is more or less marked dissemination of plasma blasts and plasmocytes
throughout the myeloid cavities, readily recognizable from the characteristic
morphology of the more mature forms, although they may sometimes be con-
fused with intermediate erythroblasts. Differentiation from plasma cell myeloma
is based on the diffuse rather than focal proliferation and the absence of
oste01ysis.

MEGAKARYOCYTOSIS (MEGAKARYOCYTIC LEUKEMIA)

This is practically impossible to differentiate from cases of chronic myelosis
with extensive megakaryocytic proliferation (the "pan-myelosis" of some German
writers), if it exists as an entity at all (see Favre et aI., 1933). Boros and
Korenyi (1931) described a case of acute megakaryoblastic leukemia, but ad-
mitted that the cells were too undifferentiated to permit positive identification.

APPEARANCES IN STORAGE DISEASES

The so-called lipoid and non-lipoid histiocytoses (Niemann-Pick, Hand-

Schiller-Christian, Gaucher, etc.), being more or less generalized conditions, affect
the reticulo-endothelial elements of the bone marrow as well as the spleen and
other organs, and may produce local areas of myelophthisis and bone rarefaction.
The presence of extraordinarily large, mononucleated cells, with or without
Iipoidai material in their cytoplasm, is diagnostic according to the type.

TUMORS

PRIMARY TUMORS
Of the primary tumors, multiple myeloma is the commonest, nearly always
being of the plasmocyte variety; myelocytic and erythrobJastic types are rare.
They are characterized by focal tumor masses of the particu1ar cell type, y.>hich
produce rarefying lesions of the bones, the process occasionally being diffuse.
Chloroma presents the microscopic appearance of leukosis (usually myeloblastic),
differing grossly through formation of intra- and extra-medullary tumor masses
of grass green color which fades rapidly to dull gray on exposure to air. Reto-
thelial (reticulum cell) sarcoma is occasionally primary in bone and produces
a rarefying lesion which can be differentiated histologically from Ewing's tumor
 THE BONE MARROW 52 1
(endothelial myeloma) by the relatively large, pleomorphic, occasionally phago-
cytic cells.

SECONDAIt'( TUMORS
These are sometimes encountered in bone marrow biopsy, masquerading
clinically as a primary anemia.
 CHAPTER 42
MALARIA
By W. ELIZABETH GAMBllELL, PH.D.

Emory University, Ga.

Malaria is unquestionably the most wide spread and destructive parasitic

disease affecting man. In the United States each year from 2,500 to 4,500 deaths
are certified as due to malaria, despite the fact that the diagnosis is relatively
easy and specific drugs are available for treatment. This excessive malaria
mortality is indicative of other tremendous losses, for approximat.!'ly one thousand
cases occur for each registered death; consequently one million to four million
individuals are incapacitated annually by this disease. To the hematologist this
presents a cballenging problem, since tbe parasites are essentially hemocytozoic
in that stage of tbeir life cycle affecting man, and furthermore, tbe final
criterion of a correct diagnosis of the clinical disease rests on the demonstration
and identification of the parasite by blood examinations.

HISTORY

For centuries human heings, particularly those living in tropical and sub-
tropical climates have b~en victims of intermittent and continuous fevers. The
symptoms of chills and fever appeared so consistently among the inhabitants of
low marshy regions that an early correlation of disease with topography resulted,
hence the labels "marsh fever" or "marsh miasm" for these clinical manifestations.
Thus in the prebacterial era, the etiology of chills and fever was ascribed erro-
neously to the vapors, gases, or damp night winds arising from marshes, and
transition to the Italian term "malaria" literally meaning "bad air" was natural
and easy when this word was introduced into English medical literature by Mac-
cullock in 1827.
The cure for the disease was known long before the cause was recognized.
In Europe in 1640, the Countess of Chinchon returning to Spain from Peru,
introduced quina bark which had been used extensively in South America as a
specific for certain fevers. The cause of the fevers remained a mystery until 1880
when Laveran, a French army surgeon working in Algeria, discovered and de-
scribed a protozoan parasite in the red blood ceIls of patients with the disease.
His report came at an unfortunate period, for the medical world influenced by
bacterial discoveries was more inclined to accept the claims of Klebs and Tomassi
Crudelli for Bacillus malariae as the true etiologic agent. It was not until
r885 that Laveran's work was recognized and the term "malaria," restricted to
an infectious fever caused by a parasitic protozoon. Shortly after this, Golgi
(1885-86) described the asexual cycle of the quartan parasite and differentiated it
522
 MALARIA

from the one causing tertian malaria. He also recognized the significance of
"crescents" in the peripheral blood of patients as belonging to a third type of
malaria, and was the first to correlate the paroxysm with the rupture of cor-
puscles which liberated new broods of parasites into the blood. Marchiafava
and Celli named the causative agent of quartan malaria Plasmodium malariae in
188S, Grassi and Feletti called the causative agent of tertian malaria, Plasmodium
vivax in 1890, and Welch gave the name Plasmodium Jalciparum to the parasite
causing estivo-autumnal malaria in 1897. The term "malaria" really covers three
or more different clinical diseases in man and even more in other animals, each
type of the disease being caused by a "different species of the parasite.
The role of mosquitoes in transmitting the disease was worked out in bird
malaria with Culicine mosquitoes by Ross in 1898, at the instigation of Sir Pat-
rick Manson. In the same year Grassi, Bignami, and Bastianelli described the
sexual cycle of human malaria in Anopheline mosquitoes. They succeeded in
infecting three human volunteers by allowing infected mosquitoes to feed on
them. In 1900 Manson confirmed these findings by infecting two men in London
with mosquitoes brought from Italy. Previously, MacCallum (1897) had de-
scribed the process of exflagellation of gametocytes in avian malaria and in·
terpreted its significance. For a more detailed account of these discoveries,
extensive reports such as those of Thayer, Wenyon, and Boyd should be consulted.
In recent years two events have given a distinct impetus to the study of
this disease; first, Wagner Jauregg's introduction in 1917 of the therapeutic
application of malarial infections in cases of general paresis, thereby openfng
new fields for further studies on the biology of the parasites, and finally, the
synthesis by the German chemists of new drugs for the treatment of malaria.

CLASSIFICATION AND LIFE CYCLE OF THE MALARIA PLASMODIA

The organisms causing malaria belong to the phylum, Protozoa, class, Sporo-
zoa, order, Haemosporidia, family, Plasmodidae, and genus, Plasmodium, because
they are unicellular animals, with ,organs of food ingestion and locomotion absent,
forming spores at one stage of the life cycle, haemocytozoic and capahle of pro-
ducing pigment. Coatney and Young in reviewing the taxonomy of the human
malarias concluded that the controlled studies afforded by malaria therapy have
confirmed the evidence that there are several species of human plasmodia and
that these separate species are made up of races or strains. The latter, while
indistinguishable on morphological grounds, can be separated on the basis of
antigenic properties, effect of drugs, virulence and infectivity. Four species of
human malaria are known, Plasmodium vivax, causing the tertian type of fever,
Plasmodium malariae, causing the quartan type, Plasmodium falciparum, caus-
ing estivo-autumnal, subtertian or malignant fever, and Plasmodium ovale. Re-
cently considerable attention has been accorded Plasmodium ovale. A complete
description and discussion may be found in reports by Stephens, Craig, Giovan"
nola, Meleney, Yorke and Owen and James, Nicol and Shute. In view of the
wide distribution in nature of different species among other animals, it is quite
 FLATE LII

MALAjUA PARASITES
(Blood Pl'llelets on bottom row)

Plasmoaium fivax (.l'erttanJ

I. RIng forrn 25. Ring form
2. Ameboid form and Schuffner's dots in the 25. Double mfection of erythrocyte.
erythrQc} teo 27. Ring·form with two chromatin dots..
3. 4 and 5 Ameboid forms 28 , Triple infection of erythrocyte.
6, 7 and S. Schuonts. 29 and 30, M1Crogame.tocytes (Crescent).
9, 10 and I t. Segmenting stages. 31 and 32, Macrogametocytes (Crescent).
12. Liberated roerozOltes
13 and q. ]\:hcrogametocytes. Blood Platelets
IS and 10. Macrogametocytes.
3,3, 34, 35 and 36. Blood platelets sUperim-
Plasmodtuhl Malariae (Quartan) posed upon erythrocytes. Shown tn tlm.
11. Ring form plate because they are frequently confused
18, 19 and 20. Schizont); with malarIa parasites by the untrained
2I and n Segmentmg stages. laboratory worker.
:23. Mkrogametocyte.
'4- Macroganetocyte.
 Plate Lit

." •

•
•
 MALARlA

likely tba! tbere are more than four species affecting man, but the description in
this instance will be limited to the four generally accepted species.
Any description of the morphology of the parasites necessarily entails a brief
resume of the life cycle, which involves two hosts, the mosquito, the definitive host
where the sexual or sporogenous cycle takes place, and man, the intermediate
host where the asexual or schizogenous cycle takes place. In both hosts,
tremendous multiplication of the parasites is accomplished, so its chances of
perpetuation are great.

THE SEXUAL OR SPOROGENOUS (:YCLE IN THE MOSQUITO

The bite of an infected female Anopheline mosquito initiates the disease in

man. Certain previous developmental procedures in the mosquito have led
to its infectiousness, and since this is similar for aU four species of the parasites
affecting man, the events will be described in general. The sporogenous cycle
begins when the mosquito bites an infected person whose blood contains the
sexual forms of the parasite, known as gametocytes. The female form is called
the macrogametocyte and the male form the microgametocyte, and it is necessary
for the mosquito to ingest a sufficient number of each kind to become infected.
The gametocytes are carried to the stomach witb the blood meal, and there
the microgametocyte undergoes exflagellation, the process of extruding several
long fJIamentous, motile bodies 'known as microgametes. One 0) these fertilizes
the macrogamete which has developed by maturation of the macrogametocyte.
After fertilization this zygote becomes actively motile, therefore is called the
ookinete. This migrates through the stomach wall and in about three days comes
to rest under the elastic membrane surrounding the intestine. It quickly assumes
a spherical shape, develops a cyst wall and is known as an oocyst. Some stomachs
of mosquitoes contain hundreds of these oocysts. They grow very rapidly
and small spherical bodies (sporoblasts) form inside. In about five days
after a gradual enlargement, the cytoplasm undergoes vacuolization and
many, small, spherical, chromatinic bodies are formed within the sporoblast
from the division of the nucleus. A week after infection, a large number of
delicate filaments have developed within the oocyst. These are the sporozoites.
The oocysts eventually rupture and the sporozoites enter the body cavity of the
mosquito. Since they are actively motile, tbey soon migrate to the tubules of the
salivary glands ano are stored there.
The entire process requires about I4 days, and if the mosquito At the end
of this period bites another individual the sporozoites are carried with the saliva
into the wonnd, taken up by the blood and eventually infect the red cells of the
new host thereby beginning the second phase of its life cycle. The sporogenous
cycle ends and the schizogenous cycle begins with the entrance of the sporozoite
into the human bpdy.

THE ASEXUAL OR SCHIZOGENOUS CYCLE IN MAN

When the infected mosquito takes a second blood meal from a human being
the young malaria parasites, the sporozoites, enter the tissues and invade the
526 MISCELLANEOUS

erythrocytes. Here the slender sporozoite assumes a spherical or ring form

and begins growth within the cell. As the parasite grows it fills the cell, some-
times causing it to enlarge, and consumes the cell contents as food. The process
of growth takes from 48 to 72 hours depending on the species of Plasmodium.
During this time, the nucleus of the parasite divides until finally one ring form
has given rise to from 8 to 32 similar forms known as merozoites. These cause
the cell to rupture and the young parasites are free in the blood plasma. At
this stage some die or are phagocytosed by _the leukocytes. Only a few escape
destruction but these invade new cells and begin the asexual cycle again. The
rupture of tbe cells, the debris and toxic products of tbe parasite liberated at this
time of completed segmentation cause the chills and elevation of temperature.
Certain merozoites develop into sexual forms of the parasite, forming male, or
microgametocytes, and female, or macrogametocytes. These require a longer
time to mature than the asexual forms and do not multiply. They usually ap-
pear a few days later in the infection than the trophozoites and form only a small
percentage of the total number of parasites. Darling estimated tbat in estivo-
autumnal infection 12 gametocytes per cu. mm. of blood must be present before
the patient was infectious for mosquitoes. Mayne has infected mosquitoes from
patients with tertian malaria having one gametocyte to 650 leukocytes. The
presence of gametocytes in the peripheral blood constitutes one of the major
problems in the epidemiology of malaria.

EXO-ERYTHROCYTIC STAGES

For many years some malariologists have suspected- that other types of
development of the malarial parasites besides the usual schizogony in erythrocytes
migbt take place. Golgi (1893) observed parasites which did not appear degen-
erated in the phagocytic cells of the internal organs.-· Since he found all stages
of the schizogenous cycle within these cells he suggest~a 'that development might
occur here, thereby protecting the parasites against -dr_ug treatment. James
(1931) in studying the response to quinine in mosquito-induced malaria and
malaria produced by blood inoculation advanced the i4,ea ,that the former was
more resistant to drugs because sporozoites underwent, a ,special development in
connective tissue cells. Huff (1930) described a new','species of avian malaria
in which schizogony occurred in tbe cells of the interna!.organs. Huff and Bloom,
Raffaele, James and Tate, 'Volfson, Hegner and Wolfson, and others showed that
in certain species of avian malaria, schizogony occurs 'in other cells than erythro-
cytes, chiefly monoc~tes and the endotbelial cells of the spleen, liver, kidneys, and
capillaries of the brain, and that all asexual stages were found in all blood and
blood forming cells-the majority being in the cells of the erythrocytic series.
Tbe parasites which occur in other cells than erythrocytes are unpigmented and
are known now as exo-erythrocytic stages. The taxonomic significance of this
observation is apparent, but this is overshadowed by the great practical impor-
tance of this finding in the study of tbe chemotherapy of malari~.
In human malaria, very few studies have been made in regard to the exo-
erythrocytic cycle. Many observations of parasites in the endothelial cells and
 MALARIA

those of the lymphoid-macrophage system have probably been disregarded as

phagocytosis rather than considered as an exo-erythrocytic cycle. In bone
marrow smears from two patients with P. vivax infections induced by sporozoites
five days previously, Raffaele (1937) found one extracellular rosette and two
young trophozoites in a large tissue cell. Tarsitano and Lucrezi (193.9) reported
exo-erythrocytic schizonts in the bone marrow of another patient inoculated with
P. v;vax. Brug (, 940) reported three intra~ellular schizonts in an infection with
P. v;vax which he found in a smear from the bone marrow after S6 hours' search.
In infections with P. /alciparum only one case has been reported. Casini (1939)
found ma'1Y large inclusions in the endothelium of the brain capillaries in a fatal
case of estivo-autumnal malaria. For an excellent critical review of exo-erythro-
cytic schizogony the report of Porter and Huff ('940) should be consulted.

MORPHOLOGY OF THE PARASITES

PLASMonWM VrvAX
This parasite usually requires 48 hours to undergo schizogony. The first
stage in the cycle is the ring form having a diameter of ahout 3/'. The nucleus
is eccentrically placed and a thin border of cytoplasm surrounds a very con-
spicuous, centrally located vacuole, giving the parasite the appearance of a
signet ring. With the Romanowsky stains, the nucleus stains red and the
cytoplasm, a bluish purple color. No hemozoin or malaria ,pigment is present
in this first stage. The parasite enlarges in the process of growth and assumes
many shapes, since it is capable of very active ameboid motion, hence:-the name
vivax. After six hours most of the ring forms have becoine ameboid in· shape
and contain a slight amount of pigment within the increased cytoplasm. The
parasitized erythrocyte also changes, and appears paler, swollen, and granular
(Schiiffner's dots). These changes are of diagnostic significance, being peculiar
to P. vivax. Growth continues for 36 hours, then nuclear ·clr'mges take 'place.
The chromatin begins to divide, first into two nuclei, then four, and finally into
a number of nuclei ranging from, 2 to 24. At this time the parasite is quite large,
filling the erythrocyte, wh,ich has increased in. Qiazifeter to 10 or "I'-. The
hemozoin tends to aggregate in one area. Each ·of the newly formed nuclei
takes on a portion of the cytoplasm and appears as a small ring form. T,hese
are known as merozoites and form a cluster, which is often called the segmentiiig
form or "segmenter." At the end of 48 hours, the red cell, which has been re-
duced to 'a mere shell, ruptures and the merozoites are set free in the blo;d
plasma and enter other cells to repeat this cycle. At each rupture, or.segmenta-
tion, hemozoin is liberated and this is picked up by the leukocyte~ and ,fi'fed
macrophages. The large mononuclear cells are particularly active in phagocYJosis
and are usually increased in numbers during an infectiolJ.
During the course of the asexual cycle, some forms develop into gametocytes.
In infections with P. vivax these usually are not seen until several days after
the beginning of the initial attack though Boyd reported finding gametocytes at
the same time the first trophozoites were detected. They tend to reach a maxi-
mum number shortly before the termination of the infection.
5,8 M,SCELLANEOUS

Pregametocytes are hard to detect but are more compact than the asexual
forms. Development takes longer and usually complete maturity is not reached
untii after 96 hours of growth. When mature, the. gametocytes are sexually dis-
tinct. The male or microgametocyte possesses a hyaline cytoplasm which stains
a very pale blue. The nucleus is large and diffuse."''rhe female or macrogameto-
cyte is 12 to 141' in diameter, its cytoplasm stains an intense blue while the nucleus
is small and takes a deep red stain. Both forms have a large amount of the
yellow-brown pigment granules scattered throughout the cytoplasm, rather than
clumped, as it is in the schizont. The macrogametocytes are generally more
numerous than the microgametocytes.

PLASMODIUM MALARIAE
The schizogenous cycle of this parasite requires. 72 hours for completion,
and in its development is very similar to that of P. vivllx. The ring form is ap-
proximately the same size, but the cytoplasm is denser and stains more deeply.
This difference is not great enough to enable a distinction to be made on morpho-
logical grounds. P. mlllilriae grows slowly and is not actively ameboid, there-
fore the parasites are more regular in form when stained. One characteristic
feature of this parasite during the growth stage is its tendency to stretch out
across the erythrocyte in a band form. In young parasites the bands are narrow,
hut become broader as the parasite grows older. The infected red cell appears to
shrink rather than enlarge. .
The nucleus of the parasite divides to forlll eight or ten daughter nuclei and
these arrange themselves ahout the centrally located pig~tnt. When fully mature,
the merozoites arranged around the pigment make a rosette form, filling the
erythrocyte. The gametocytes are very similar to those of P. vivax, although they
are smaller in size and very scanty in number.

PLASMODIUM FALCIPARUM
Schizogony of this parasite lacks the pronounced synchronism that is char-
acteristic of the other two species, but apparently occurs over a period of 36
to 48 hours. The ring forms are much smaller in size being about 1.251' to 1.51'
in diameter. Multiple infections in one erythrocyte are common. The nucleus
often shows two chroma tin dots rather than a single mass, and these with the
small size serve as points of differentiation from P. vivax. The ring forms and
the crescent shaped gametocytes are usually the only forms found in the periph-
eral blood unless the patient is in a moribund state, for multiplication takes place
in the capillaries of the viscera and bone marrow. Only occasionally can a
segmenting form be found. Sudden showers of parasites in all stages of develop-
ment in the peripheral blood indicate a grave prognosis. Examination of bone
marrow, or blood aspirated from the spleen or liver, reveals the developing stages
of the parasite. The adult schizont is much smaller than t!l.e erythrocyte,
usually meas\,ring 4.5 to 51' in diameter. The merozoites vary in number from
8 to 24. The hemozoin is blackish brown and occurs in well defined blocks. The
infected cells show basophilic granules known as Maurer's dots.
 MALARIA ~29

TABLE XXVII
DIFFERENTIAL DIAGNOSIS OF PLASMODIA AFFECTING MAN
P. wtJax P. malariae P·falciparum P. ovale
I. Type of fever Tertian Quartan Quotidian or sub- Atypical tertian
tertian (estivo-
autumnal malig-
nant tertian)
2. Length of 48 hours 72 hours 24 to 48 hours. 48 bours
asexual cycle Irregular~
3. Morphology
of asexual
para-site
(a) Ring form Signet ring, irregu- Signet rings, regu- Small ring 1.25 to Signet ring wit~
!ar outline. 3 or lar outline. 3 in I.S in dtameter, large chromatm
4- in diameter, diameter. Indis- regular outline, dot. R.B.C.
multiple miee- tinguisbable from often two chro- contains
tion of R.B C. P.vitlax. matin dots, multi- SchllfIner's dc)t3·
occasional. pie infection of
R.B.C. common.
(b) Older Very irregular out- Band forms or Not seen in periph- Round a.nd ov~l
tropho-- lines~ pseudo- very regular era! blood. with vacuole in
zoites podia! processes. rings. Smaller early stages
Larger than than erythro- smaller than
erythrocytes. cytes. erythrocytes.
(c) Number of 12 to 24 6-10 8-20 6-10
mexozoites Av. - 18 to 20 Rosette form Usually 16 UsuallyS
(d) Hemozoin Yellowish-brown Yellowish-brown Blackish-brown Dark brown.
Ba.t;_illiform Large coarse Large coarse
grain. grains or irre~~
tar masses.
4. Gametocytes
(a) Micro- Spherical or oval Spherical or oval Plump crescent Similar to P.
gameto- shape. Light shape. Light or shape. Pinkish· malarial but
cytes blue cytoplaiiro. greenish-blue blue cytoplasm. found in oval
Diffuse nucleus. cytoplasm.. Dis- Diffuse nucleus. erythrocytes con·
Peripheral pig-
ment.
persed. nucleus.
Peripheral pig-
Pigment s.cat-
tered throughout ~~~o~~iifi-
Ul't'tl.\.. CY\'q>~"'ttl.
(b) Macro· Spherical or oval Same Long slender cres- Similar to P.
gameto- shape. Deep cent. Blue cyto- malariae but
cytes blue cytoplasm. plasm, compact found in oval
Compact nucleus, nucleus, centrally erythrocytes.
eccentrically placed. Pigment containing
placed. Pigment around nucleus. Schilffner's dot-s.
irregularly dis-
tributed.
s. Effect on eryth- Increased I n size, Not increased in Unaltered in size. Increased in s~e,
racytes pale, stippled size, may be Pale, sometime.s oval or irregular
with Schuffner's smaller. coarse granules in shape. Every
dots. (Maurer's dots). parasitized cell
showsSchliffner'.
dots.

The gametocytes are different from those of other species, assuming the
characteristic crescent shapes. The ends, however, are not pointed but rounded.
They are large, averaging from 9 to I41' in length and 2 to 31' in breadth. Often
the remnant of the red cell is seen stretched aoout the parasite. The microgameto-
cyte is broader than the macrogametocyte with a diffuse nucleus which occupies
most of the ccli and with hemozoin scattered in the cytoplasm. The macrogameto-
cyte is slender with a centrally placed nucleus having the hemozoin more con·
centrated about it.
53 0 MISCELLANEOUS

PLASMODIUM OVALE
This species is relatively new, having been described by Stephens in 1922
in an infectioa contracted in Africa. Giovannola ([935) considered it a modified
form of P. vivax after a long residence in the human host, but much experimental
data accumulated by James and his co·workers (1933) at the Horton Malaria
Therapy Center in England differentiate it from Plasmodium vivax. Schizogony
takes place every 48 hours. The ring forms have a large chromatin dot and the
infected erythrocytes containing these forms show numerous Schuffner's dots. As
the trophozoite matures, the infected erythrocyte assumes an oval shape and
sometimes acquires a ragged appearance around the edges. The Schliffner's dots
are pronounced and appear in every infected red cell. Often the cytoplasm .of
the cell stains so faintly that only the parasite and the Schiiffner's dots are
apparent. The erythrocyte is slightly enlarged but not so much as in infections
with P. vivax. As segmentation takes place the parasite fills about three-fourths
of the infected cell. From 6 to 12 merozoites are produced, the usual number
being 8.
The gametocytes resemble those of P. malariae, being approximately the same
size and shape, and staining in the same way. However, the presence of 'the
eosinophilic stippling in the erythrocyte often leads to confusion with the gameto-
cytes of P. vivax. The size of the parasite and the irregular shape of the red cell
differentiate P. ovale from P. vivax. In infections resulting from mosquito bites
the periodicity of the asexual cycle and the characteristic morphology of the
parasites are maintained.

CLINICAL SYMPTOMS

Malaria is characterized typically by chills and fever_These symptoms hold

such a close association with the disease that, in endemic areas, the laymen and
often the physician make a diagnosis of "a touch of malaria" for any and every
physical manifestation of these ,characteristics. Boyd called attention to features
which distinguish this disease from other commonly encountered communicable
diseases; first, that while malaria in its initial stages presents acute manifesta-
tions, the disease is characteristically chronic and the parasites persist in the
body for indeterminate periods, and second, that under the clinical term "malaria"
we are in reality confusing three diseases which possess certain individual char-
acteristics depending on the particular species of parasite causing the infection.
The ~most striking clinical features of the infection are fever, anemia, and
splenic enlargement. James reported tbe characteristic symptomatology as a
primary series of paroxysms of fever and anemia followed, after a period of
apyrexia, by a number of secondary series of similar attacks called ·relapses. He
called attention to the mild types of malaria which occur during years of normal
endemic incidence and the severe or malignant attacks which occur during epi-
demic years under unusual conditions, irrespective of the type of infection.
 MALARIA 53 1

INCUBATION PERIOD

The time between the bite of the infected mosquito and the appearanc(- of
clinical symptoms in the patient depends on the dose of sporozoites injected, the
particular strain of the parasite, and the general physical condition of the patient.
In experiments with induced malaria, James found that by mosquito bite the mean
period of incubation in tertian malaria was 14.II days and by blood inoculation
11.2 days. In estivo-autumnal malaria the incubation period is usually shorter,
and in quartan malaria longer. Some infections show a delay of several mor1ths
or a year in developing and are known as latent cases. Ross estimated that the
patient showed no symptoms of illness until the number of parasites had in-
creased to approximately 50 per cu. mm. of blood or one parasite to one hUll<jred
thousand erythrocytes. He designated this the pyretogenous limit. Low~ in
a recent numerical study found that in P. vivax infections a minimum of 500
parasites per cu. mm. of blood was usually necessary to cause fever, and it! P.
falciparum infections 600 to 1500 parasites per cu. mm. were necessary. For
several days before the disease is evident, the patient often shows signs of !len-
eral malaise characterized by headache, aching limbs, lassitude, a desire to yllWn
and stretch and sometimes vomiting. A sligbt rise in temperature generally ac-
companies these symptoms.

THE DEVELOPED INFECTION

A primary attack of tertian or quartan malaria consists of three well defined-

stages: first, the chill, rigor or cold stage; second, the hot stage; third, the
sweatitl\!; sta\!;e. These are (allowed hy a Vedod of av"reKia itl wmch th.e s"mQ-
toms abate and the patient feels comparatively well but after a definite interval
another paroxysm sets in and the cycle af events is repeated.
At the initiation of the cold stage, the patient feels chilly, and this sePsa-
tion of cold spreads over the body causing the teetb to chatter and the body to
shiver. This feeling of intense cold is entirely subjective as the temperature at
this time is usually several degrees above normal. The skin becomes blue, the
lips and nails are livid and the extremities cold. Vomiting frequently occurs.
Quite often this stage lasts only an hour but it may be prolonged for three hours.
The hot stage begins with alternating waves of warmt!) which gradually bring
comfort and relief from cold but soon change to intense febrile distress. The
skin becomes dry and hot, the pulse is rapid and full, the face flushed, and the
temperature rises, sometimes higher than 106 0 F. The patient complains Of a
severe throbbing headache and is often delirious. Vomiting also occurs at this
stage. In one or two hours these symptoms abate and the sweating stage begins.
The patient becomes covered with perspiration of a peculiar odor so profuse
that clothes and bed are saturated. A f_eeJing of languor and weakness repl~ces
the former discomfort and the temperature returns to normal. Very often the
patient .sleeps for an hour or two, and at the cessation of the sweating period (eel.
53 2 MISCELLANEOUS

well and goes about his usual duties. Herpes labialis commonly accompanies
malaria, especially the benign tertian infections. The interval between rigors
may last from one to three days depending on the type of infection.

ESTIVO-AUTUMNAL FEVER

From a clinical standpoint, this type of infection is more important than

tertian or quartan malaria. It more often terminates fatally and because of its
protean character is likely to be confused with other diseases such as typhoid fever,
bacterial septicemia and cerebral disturbances caused by other conditions. It
differs from the other two types of malaria in the following respects: the rigor is
not so pronounced and the fever is usually very irregular extending over a much
rpore prolonged period. Often sever~ headache, persistent vomiting, diarrhea and
hyperpyrexia accompany these stages. At any stage of the infection more per-
nicious characteristics may suddenly develop. Often the patient is comatose
before he receives medical attention. Several patients have been aomitted to
Emory University Hospital with the diagnosis of diabetic coma but blood studies
revealed that they were suffering from Ii comatosis caused by estivo-autumnal
malaria. Destruction of red blood cells is particularly marked in this infection.
Manson-Babr in a very detailed description classified the main clinical types as
follows:
Bilious remittent.
Characterized by bilious vomiting, gastric distress, saffron tint of skin
and sclera from increased serum bilirubin.
Pernicious.
Cerebral-caused by emboli of malarial parasites in cerebral capillaries
of the various nerve centers.
Hyperpyrexia!.
Temperature rises to IIO' to II2°" <\elirium, coma, followed by death
in a few hours.
Comatose.
Temperature 104' or less, coma which sometimes passes at sweating
stage, often ends in death from collapse.
Convulsive.
Epileptic or tetanic characteristics often seen in children.
Paretic.
May simulate cerebrospinal meningitis, delusional insanity, de-
mentia, acute alcoholism, apoplexy, paralyses complicated with
aplasia.
Algid.
Characterized by collapse, extreme coldness of the body surface with
elevated axillary and rectal temperature, and a tendency to fatal syncope.
Gastric form.
Acute catarrhal dyspeptic trouble, accompanied by incessant vomit-
ing (vomitus may contain blood). Severe epigastric distress, tender
retracted abdomen.
 MALARIA 533
Choleraic form.
Numerous loose stools with other symptoms accompanying great loss
of fluids·.
Dysenteric form.
Dysenteric symptoms often accompanied by hemorrhage from bowel.
Hemorrhagic form.
Hemorrhages may occur in any organ.
Syncopal form.
Usually occurs during profuse sweating stage leading to collapse
and syncope. Cardiac failures result from fatty degeneration of
myocardium.
Acute hemolytic anemia.
Rapidly developed anemia in fourth or fifth week of primary at-
tack. Great pallor of mucous membranes and conjunctiva, retinal
hemorrhages, cardiac distress and dyspnea, erythrocytes under
1,000,000 per cu. mm. of blood, extreme leukopenia, hemoglobin ten
per cent, microcytes and megaloblasts in peripheral blood.

TYPE OF FEVER AND DEVELOPMENT OF THE PARASITE

James distinguishes malaria from other forms of fever by two outstanding

characteristics: intermittency, by which is mearit the cessation of fever at inter-
vals during which the temperature is normal or "lightly subnormal; and peri-
odicity, "the habitual tendency of the fever to recur at stated intervals of time."
Ordinarily three types of fever may occur: Quotidian, daily at 24-hour inter"
vals; Tertian, every third day at 48-hour intervals; Quartan, every fourth day
at 72-hour intervals.
Boyd states that the first type is characteristic of initial infections caused by
any species of parasite, or that it may be produced by double infections in which
generations of parasites mature on different days. Manson'Bahr points out that
any of the three parasites may cause what was formerly known as remittent fever,
that three generations of P. malariae, maturing on three successive days will
cause the quotidian type of fever as well as two generations of P. vivax maturing
on different days. Mixed infections of two or more species may also occur and
must be borne in mind.
The character of the fever is definitely related to stages of growth of the
parasite. The paroxysm is initiated by the rupture of the parasitized erythrocytes
with the release of debris, cell fragments, products of the parasite, and the para-
sites themselves into the blood plasma. Abrami and Senevet regarded the malarial
paroxysm as being partly an anaphylactic process. They considered the symp-
toms manifested at this time as evidence of a hemoclastic shock characterized by
lowered arterial tension, leukopenia, alteration of the differential count, diminu-
tion in the number of red cells and changes in the coagulability of the blood.
Later work by Sinton, Orr and Ahmed agreed with these findings. When the
parasites are again within the erythrocytes and undergoing the process of growth,
the toxic effects are not felt; therefore, the temperature returns to normal until
534 MISCELLANEOUS

the next segmentation occurs. In searching for parasites in a stained blood film,
the clinician should bear in mind that the parasites will be larger anil more
obvious in the hours just preceding the chill, and that they-will be smaller (r'
lorms) and more numerous just after the chill.

THE BLOOD PICTURE IN MALARIA

The hematologic findings in malaria are twofold: first, the morphology of

the parasites themselves within the infected erythr09't!'~; and second, concomitant
changes in the cells and hemopoietic centers due to the pathologic effects of the
parasitic invasion.
The most direct result of acute primary infections is anemia, for every
erythrocyte inhabited by a malarial parasite is destroyed each time schizog~ny
takes place. Often the red cell count will fall below 2,000,000 per cu. mm. after
a week or two of the disease. Sometimes the drop in cell numbers is precipitd US ,
showing a decline of more than 1,000,000 per cu. mm. after each chill. this
anemia is out of proportion to the actual number of red cells parasitized. It nlay
be caused to some extent by intense erythrophagocytosis as well as to destrUC-
tion by parasites. Brown demonstrated experimentally in rabbits that malarial
pigment (haematin) acted as a hemolysin and caused capillary hemorrb;tge
by its action on the endothelial cells. The number of erythrocytes reache? a
low level and tends to stay there indefinitely, unless the patient receives treat-
ment. Chronic cases in malarious regions usually show some degree of anerrlia .
Molina and Gonzalez in a complete hematologic study of ,00 cases in Puerto
Rico, 83 per cent of which were chronic, and 17 per cent acute, found the degree
of anemia slight, the mean erythrocyte count being 3.95 millions and the
"'~"." t.~"'\;>~\\;>""'U, '">.:) c,,,,. ~'<>~ \"11>"'" \;><,. "."~"",, ~'A"'~",",--e'" 'D'j ~~ ~;\;>"'1> 'il""'''-
as follows: macrocytic anemia, 34 per cent; normocytic-'lnemia, 37 per cent;
simple microcytic, ,6 per cent, and hypochromic, '3 per ·cent. Only 35 per cent
showed the characteristic leUkopenia. The Schilling hemograms showed a de-
generative shift in the granular leukocytes, and changes in platelet and sedimerl ta·
tion rates were within the normal range.
~ The parasites are believed to have a predilection for young cells, especilllly
reticulocytes. Eaton, by utilizing supravital stains, and Jacobstal, with a com-
bination of Loeffler blue and Giemsa stains showed that the majority of the
parasitized cells were reticulocytes. Hegner found that vivax merozoites had a
special affinity for reticulocytes, but that falciparum and malariae parasites
invaded older cells. He ascribed the large size of the vivax-infected erythrocytes
to the fact that they were actually large reticulocytes rather than enlarged eryt~ro­
cytes. Kitchen found a greater percentage of reticulocytes infected thin mature
red cells throughout the course of two .vivax infections. Multiple infections
were more frequent in reticulocytes. He also observed that in three faldparum
cases the mature erythrocytes infected always exceeded the number of infected
reticulocytes. P. malariae was always found in both greater relative and absolute
numbers in mature erythrocytes.
 :MALARIA 535
The infected cells in tertian malaria are enlarged, and often filled with small
granule-s which take an acidophilic stain and are called Schliffner's dots. SchUff-
ner thought these granules were derived from the reticulum of the erythrocyte
and the recent work of Hingst offers additional evidence to support this concept.
In the other two types of malaria the infected cells are not enlarged, but the cells
in estivo-autumnal malaria may have large basophilic granules described first by
Stephens and Christopher and known as Maurer's dots.
Not only are the parasitized cells affected, but often the other red cells
undergo changes, and poikilocytosis, anisocytosis and basophilic stippling are
evident. In pronounced infections, the picture of primary pernicious anemia
may occur and nucleated red cells appear in the peripheral circulation.
The hemoglobin is also reduced in amount usually in direct proportion to
the erythropenia, which in turn depends on the severity and duration of the'
infection. This decrease is greater in eslivo-autumnal infections, the amount
of reduction varying from 10 to 40 per cent in a short time. Sometimes the dis-
ease terminates fatally so rapidly that the reduction is slight.
The numbers of leukocytes also decrease. Ross and Thomson were the first
to show that during paroxysms there is a leukocytosis but shortly afterward
(three to four hours) the total count averages 3,000 to 5,000 per cu. lI)m. These
results have been confirmed by many subsequent workers, but it must be borne
in mind that a leukocytosis does not rule out malaria. Bunker, in a study by
hourly leukocyte counts on 10 cases of general paresis treated with tertian
malaria, reported that the leukocytes were reduced during the paroxysm and in-
creased afterwards during the descent of the fever. The large mononuclear cells
show a relative increase, often comprising 'S or 20 per cent of the total white
cell number. Thoinson found that the percentage of large mononuclear leuko-
cytes gave a curve which is exactly the reverse of the temperature curve, i.e.,
higher during apyrexial periods and low when the temperature is at its peak.
The polymorphonuclear leukocytes in the peripheral blood and small capillaries
are engorged with pigment which they have phagocytosed. This is the hemozoin
derived from the hemoglobin by the action of the parasites, and set free in the
blood plasma when the erythrocyte.ruptures at the segmentation of the parasite.
The finding of pigment-containi.!l{1e~kocytes in the peripheral blood is regarded
by some as proof of the presence of malarial infection. Large phagocytic cells
of the reticula-endothelial system, the macrophages, are present in abundance
in the capillaries of the spleen and other viscera. These contain large amounts
of pigment and often entire erythro-parasite' complexes. Mulligan, in a study
of the reticulo-endothelial system in relation to malaria, found that cells from this
system were increased in the peripheral blood in acute and chronic infections.
He found that general leukopenia usually occurred but that clasmatocytes,'
monocytes and cells with a positive peroxidase reaction increased above the nor-
mal number.
The blood sugar is decreased. The serum-bilirubin estimated by Van den
Bergh's reaction shows a rise during paroxysms. Sinton found that wanges in
 MISCELLANEOUS

the surface tension and the refractive index of the serum occurred in tertian
malaria when the blood was collected at the time 01 the paroxysm.

PATHOLOGIC CHANGES

THE SPLEEN
Splenomegaly is one of the most frequent physical findings accompanying the
fever and anemia of malaria. In primary acute attacks, the splenic area becomes
tender and painlul and during the first paroxysm, an increase in the are11 QI
dulness is noted on percussion. At this time, the spleen itself is swollen, tender,
hyperemic, and usually salt in consistency although it may not be palpa.ble.
During the apyrexial period, enlargement subsides, but after repeated paroxysms
the decrease is not enough to counterbalance the increase, and finally a greatly
enlarged spleen results. In chronic cases which have had little or no treatment, it
often forms a hard tumor mass and can be detected easily by palpation in the
majority of these cases. The degree of enlargement depends on several factors
such as race, age, sex, and especially the number of attacks of malaria and the
type of infection. Greater enlargement occurs in tertian malaria than in qual'tan
and estiva-autumnal. Boyd states that pronounced splenomegaly in cases of the
latter infection usually indicates undiscovered infection with P. vivax also. C~ses
of spontaneous rupture of the spleen during acute infections of malaria are on
record.
The splenic index is used in epidemiologic studie)')ls a simple method of
estimating the degree of malaria in affected areas. A ,detailed discussion of the
various methods of obtaining this index has been presented by Boyd.
The spleen, because of its rich endowment with numerous phagocytic cells,
acts as a filter for parasitized and damaged red corpuscles. Usually parasites
at" 'ffi\)'" I)''i';''''
'0.\)"""''0.''\ '" \\\'" \\\'0." '" \\:,,, '1>""'1'\:'''''0.\ b\l)\)\t '"" a ,,,cent "",\m..-
sive study on experimental infections in monkeys, Taliaferro and Cannon' have
shown that the ratio of parasites to red cells in the spleen was often 20 times the
ratio in the peripheral blood. During the acute rise of the infection, the para-
sitized cells occurred in both the splenic cords and sinuses but at the crisis (be-
ginning of marked defense activity) the parasitized cells were pack~d in the J3il1-
roth cords and scarce in the sinuses, indicating that normal and infected cells
enter the cords from the artery, but only normal cells can pass into the venOUS
sinuses when the defpnse reactions have become active.

THE LIVER
The liver as well as the spleen becomes tender and enlarged during attacks
of malaria. This enlargement is caused by simple hepatic congestion, and is usu-
ally accompanied by hepatitis. In cases where repeated attacks of malaria have
occurred this congestion becomes permanent. Siderosis is usually present .. When
an excessive amount of free hemoglobin occurs through abnormal destruction of
erythrocytes by the malaria parasites, the liver attempts to take care of this by
increased secretion of bile. Excessive polychoJia frequently accompanies maladal
 MALARIA 537
infections and causes the pathologic effects which Manson-Bahr classifies as
"bilious symptoms," bilious vomiting and bilious diarrhea. He expresses the
opinion that the yellowness of the skin and sclera is caused by tinting of the tis-
sues with liberated hemoglobin rather than cholemia from bile absorption. The
Kupffer cells of the liver are very active phagocytes, hence engulf parasites and
pigment.

THE KIDNEYS
Quartan malaria is often accompanied by subacute nephritis. An inflamed
kidney initiates this process but acute nephritis is rarely associated with malaria,
the more common form being chronic interstitial nephritis. Craig states that
in most cases of tertian and quartan malaria the urine shows no important patho-
logic changes. Polyuria may occur when the temperature is declining, and the
chlorides usually increase. In estivo-autumnal infections the urine may show
definite pathologic changes, such as albumin, hyaline and granular casts, increased
urea, and increased specific gravity. These changes usually appear during the
paroxysm.

MORBID PATHOLOGY
Since malaria is typically a chronic disease, opportunities for studying the
pathologic changes taking place in the various organs during the course of infec-
tion are rare. Only the extreme or pernicious cases come to autopsy and these
present pathologic pictures of a greatly exaggerated nature. Cannon in discussing
the general pathologic effects of the disease aptly stated, "Human malaria is a
type of infectious disease in which the duration, severity and outcol11e are affected
by a number of variables, such as the species of the parasite, the variation in its
biologic properties, the length of infection, the age and race of the host, the
differing grades and kinds of resistance, the effects of relapse, the kinds and
amounts of therapy used and the concomitant influences of intercurrent and
terminal infections. It is apparent from a consideration of these facts that
malaria can no more develop according to a simple pathologic pattern than can
tuberculosis, syphilis or any other disease with a variable clinical course." The
disease is characterized by diminution in tbe actual blood volume therefore the
organs at autopsy show little general congestion except in early acute infections.
The most outstanding changes occur in the spleen, liver and bone marrow. The
spleen is generally enlarged and its surface presents a pigmented appearance, being
dark blue or jet black in color. The cut surface is often tarry, particularly in
chronic infections of long duration.
Microscopically, smears or sections show numerous red blood cells containing
parasites in different stages of development. The phagocytic cells of the reticulo-
endothelial system are greatly increased in numbers and contain a large amount
of hemozoin occurring in rods, blocks, and granules. This is sometimes free in
the venous sinuses. Often the macrophages contain entire erythro-parasite com-
plexes. Hemosiderin from the destroyed erythrocytes is present as a golden yel-
low pigment. The splenic sinuses are congested and distended witb blood, anei
 IIhsCELLANEoUS
thrombosis of the capillaries is evident. Usually the splenic-pulp shows local
areas of necrosis. Areas of hemorrhage may be observed. Taliaferro and Can-
non in their study on monkey malaria reported lymphoid hyperplasia evidenced by
increase in size of follicles and increase in the red pulp, accompanied by lymphor-
rhexis. As the infection progressed irregular islands of varying sized basophilic
cells appeared in the red pulp, especially around the trabeculae and trabecular
arteries. Except for this latter change involJing plasma cell basopbils, they felt
that the cellular changes in the spleen were concerned with the formation of
lymphocytes and reticular cells which, in turn, produced macrophages.
The liver is usually pigmented, therefore, of a dark green or olive' brown
color. The capillaries are distended with parasitized red cells and hemozoin,
and the active Kupffer cells are enlarged and swollen. The liver cells are some-
times atrophied, often show cloudy swelling and fatty degeneration. Hemosiderin
is present together with hemozoin.
The bone marrow, especially of the sternum and bodies of the vertebrae, IS
filled with parasites and hemozoin and therefo~' markedly congested. The
hemozoin is contained within the macrophage cells of the parenchyma away from
the blood vessels. Osgood, in studies of human marrow obtained by sternal pun~-.
tures on five cases of inoculation malaria, reported intact parasites as well as
partially digested forms in neutrophilic leukocytes.
The brain and nerve tissues show evidence of degenerative changes. In in-
fections with P. falciparum which have terminated with fatal coma, the capil-
laries and small arterioles of the pia mater and brain cortex are blocked with
erythrocytes containing parasites in all stages of development. Cen free para-
sites are present also, together with pigmented leukocytes, free pigment and
macrophages. The parasite infected corpuscles tend to adhere to the vessel walls,
resulting in complete occlusion and thrombosis. James stated that the malarial
symptoms referable to the nervous system are due in part to the mechanical effeds
of this blockage, and also, to the action of toxins which cause degeneration of
the nerve cells and endothelium of the vessels, thereby resulting in punctiform
hemorrhages. Focal degenerations or granulomata as a direct result of these
hemorrhages have been described by several workers. Manson-Bahr in sum-
marizing their description noted that they resemble tuhercles and are formed
from the conglomeration of glia cells around the center of degeneration.
Dudgeon and Clarke have found a diffuse fatty degeneration of the heart in
rapidly fatal cases of estivo-autumnal malaria. They considered these lesions
similar in all respects to those of acute diphtheritic 'toxemia. The cardiac capil-
laries were filled with parasites. This may explain the sudden deaths from
malaria simulating acute cardiac failure. The suprarenal glands often show
lesions chiefly from the reduction of fatty lipoids of the cortex. Dudgeon and
Clarke felt that the pathologic basis for t}ie great muscular weakness and low
blood pressure often noticed in eslivo-autumnal malaria was due to cerebral
changes rather than to the lesions in the suprarenals alone.
Other organs such as the lungs and alimentary canal often show phagocytosis
 MALARIA 539
of pigment by the macrophages and congestion of the vessels with parasitized
erythroc). tes.

BLACKWATER FEVER
(Malarial Hemoglobinuria) (Hemoglobinuric Fever)

The etiology of this disease has never been definitely settled but at the
present time it is considered a result of repeated attacks or continuous infection
with estivo-autumnal malaria. The chief characteristi<:s are a sudden acute hemol-
ysis of red blood corpuscles liberating large amounts of· hemoglobin in the blood
stream consequently producing hemoglobinuria. The incitant of this sudden
hemolysis is not known but evidently some change occurs in the resistance of the
erythrocyte. Thomson reported a greater occurrence of corpuscles with Maurer's
dots and deformities called "brassy bodies" in cases of subtertian malaria which
developed blackwater fever. Manson-Bahr is of the opinion that the only abso-
lutely certain fact is that 60 to 80 per cent of the erythrocytes are destroyed within
24 hours.
The onset is sudden and the average mortality is about 25 per cent, although
this varies in different regions. The general pathology is very similar to that of
fatal cases of estivo-autumnal malaria. Most pronounced changes are found in
the blood picture and in the kidneys. The erythrocyte count drops precipitously,
usually within 24 hours, and cases have been recorded in which the count was
500,000 cell? per cu. mm. Shadow cells are common in fresh preparations.
Malaria parasites are usually not demonstrable during the attack and patients
have been known to recover completely from malaria after an onset of black-
water fever. During recovery intense regeneration of red cells takes place and
many immature cells are present in the peripheral blood. The grayish-brown
kidneys are congested and usually enlarged. The tubules are usually blocked
with infarcts of hemoglobin, and with hyaline and cellular casts. The capillaries
contain hemozoin, but the collecting tubules are usually filled with hemofuscin.
Anemia and general debility sometimes follow an attack of blackwater fever, but
in many individuals recovery is quite rapid. This disease closely resembles
paroxysmal hemoglobinUria, and involves many factors which are yet unsolved.

TREATMENT OF MALARIA

Three drugs in various forms are now available for the treatment of malaria:
the cinchona alkaloids, plasmochin, and atabrine. Until 1924, the problem of
therapeutics was relatively simple, quinine or some other alkaloid derivative of
cincbona bark being regarded as a highly specific remedy. The results of its use
for three centuries as a malarial febrifuge justified this belief, and the only real
questions were which derivative to use and how much to administer. During the
last decade, the increase in knowledge concerning the parasites themselves, the
reaction of the infected individual, and the advent of synthetic remedies have
540 M ISCELLAN1<OUS

greatly complicated the issue from the standpoint of the clinician. The unsettled
world conditions have been reflected in the active searcb for new anti·malarial
drugs and it is quite possible that, as a result of this, new drugs will be synthe-
sized or found which will [lfove more effective than tbe ones now in use. Maier, in
1940, pointed out that two serious deficiencies common to tbe present drugs bave
impaired their effectiveness in controlling malaria as a world problem; they do
not exert a specinc prophylactic action against tbe sporozoite, and they can not
be depended upon to sterHize the infection.
For a comprehensive analysis of the present status. of the problem, two reo
cent critical reviews with extensive bibliographies are available, the Third General
Report of the Malaria Commission.of the League of Nations, and Sinton's subse-
quent detailed discussion of this report. ~
Many complex factors entcr into the choice of the best remedy. Williams
states, "Tbe response to anti-malaria therapy varies tremendously between dif-
ferent races, between individuals of the same race, is unequal between the species
of malarial plasmodia, and even varies between different strains of the same
species." With these factors in mind, the physician must make an individual
choice in the uS'l of the present available drugs, before intelligent treatment can
be instituted. This choice should be influenced by local conditions, that is;
whether the individual to be treated runs no risk of reinfection, or wbether be is
likely to be reinfected or superinfected shortly after the termination of treatment
(Sinton). In the nrst case the object of treatment should be a radical cure as
early as possible. In the latter case Sinton and the Commission botb suggest that
patients in highly endemic areas may ,benefit by developing a natural toler~nce
to the strains of parasites in their area of residence, hence a less strenuous course
of treatment is advocated. No drug in present use kills all the parasites in every
patient. No drug acts as a causal prophylactiC" for there is none available which
will destroy and prevent the development of sporozoites.

QUININE
Cinchona bark contains numerous alkaloids, some amorphous and some crys-
tallizable, which act specifically on the parasites of malaria. Quin~ne is more
generally used, althougb quinidine is equally effective and Dawson bas shown
that it may be substituted for quinine when individuals show a sensitivity to the
latter. Tbe Malaria Commission of tbe League of Nations has advocated a
standardized cinchona febrifuge (70 per cent of crystalline alkaloids of which
less than IS per cent, is quinine), under the name of "totaquina" for use where
price is a big consideration. Tbe standard treatment recommended by the Na·
tional Committee on Malaria in this country in 1919, and wbose efficacy was
confirmed by Bass is: 10 gr. of quinine sulphate in capsules by mouth three
times a day for three or four days followed by 10 gr. every night at bed time for
eight weeks. For infections without acute symptoms 10 gr. every night for eight
weeks without the first intensive treatment is advised. In children proportions
based on age are administered and the doses advocated are as follows:
 MALARIA 54 1
Less than [ year. . . . . . . . . . . . . . . . . . . . . . . . . . . 'y, gr.
I year ............................. "...... 1 gr.
:2 years ................................... 2 gr.
3 to 4 years .............................. " 3 gr.
5,6, 7 years ........ " " .. .. ... . . . . .. . ..... 4 gr.
8, 9, 10 years. . . . . . . . . . . . . . . .. . . . . . . . . . . . .. 6 gr.
II, 12,' 13, 14 years. . .. .. ...... . .. ... ...... 8 gr.
15 years. .................. •........ .... 10 gr.

Weight and size as well as age should be considered. Quinine sulphate is slowly
absorbed because of its relative insolubility (1.725). Its action, when admin·
istered orally, is greatly enhanced by clearing the digestive tract with an alkaline
purge prior to its administration. Recently, the efficiency of smaller amounts of
quinine and other cinchona derivatives given uver a shorter period of time has
been tested clinically by many workers with most gratifying results. Sanders, in
a report on the short course methods now in use, concluded from his own experi-
ence with 1,547 cases that quinidine is superior in controlling acute attacks and
preventing relapses. Craig in 1940 censured the "short term" treatment with
quinine and recommended that it be abandoned since in his opinion the aim of
the physician should he the elimination of the infection which can be accom-
plished only by the administration of this drug in adequate doses over a long
period of time.
Intravenous medication is necessary for patients who are in a comatose con-
dition or suffering from severe gastro-intestinal upsets. Ten grains of quinine
bihydrochloride dissolved in 10 cc. of sterile physiologic Nael should be admin-
istered slowly. The time required for the injection should take at least three
minutes. Often in severe cases of estivo-autumnal malaria, the quinine is added
to 250 ce. or 500 cc. of physiologic saline and 5 per 'cent glucose, and a-Bministered
slowly. Not more than three of these injections should be given during 24 hour
periods, although two are usually sufficient. Small doses (3 gr.) given at more
frequent intervals sometimes prove more effective than the larger doses given at
longer intervals. For children the size of the dose should be proportionate to
their age and size. In some cases intramuscular injections are used. The best
site for injections of this nature is the upper outer quadrant of the gluteal region.
Unfavorable local reactions, such as necrosis and damage to blood vessels, often
result from these injections and because absorption of the drug is slow and some-
what uncertain, this method should be nsed only when no other route is available.
Although quinine is an effective anti-malarial, it has many objectionable
features, such as its bitter taste, its tendency to cause tinnitus aurium, visual dis-
turbances, deafness, cardiac depression and gastric disturbances. Some individ-
uals show an idiosyncrasy to the drug. Dawson suggests that scratch tests on the
patient hefore administration of any drug will reveal whether or not he is sensi-
tive, and often quinidine or some of the newer synthetic remedies may be suh-
stituted for quinine.
The use of quinine for malaria in women who are pregnant has be~n seriously
questioned. Manson-Bahr feels that the drug is less harmful than the paroxysms
of the disease ("ague fits"). He advises the administration of 5 gr. of quinine
542 MISCELLANEOUS

daily during and several days after labor in regions where malaria is very prev-
alent !Is parturitio'n often brings on attacks or relapses of the disease.

PLASMOCHIN C'
Plasmochin, a quinoline derivative, was produced in' 1924 by Schuleman
and Memmi, at tbe Research Laboratory of Farbenindustrie, Elberfeld, Ger-
many. The chemical formula of this drug is N-diethyl amino-isopentyl, g-amino
6-methyl oxyquinolin made into a salt suitable for tablets. Tablets of 1/3 gr.
(0.02 Gm.) are dispensed and the dose recommended is·three tablets daily for
five days. This drug acts primarily on the gametocytes, and is of little use in
therapy against the asexual forms. Since this is useful in preventing infections of
mosquitoes, but of little value directly to the patient, it has been combined with
quinine in tablet form, known as plasmochin compound, or chinoplasmin (quino-
plasmine). Each tablet of plasmochin compound contains r(6 gr. (0.01 Gm.)
of plasmochin and 2 gr. (1.125 Gm.) of quinine sulphate. In chinoplasmin the
ratio of plasmochin to quinine is a 1:30, 0.01 Gm. plasmochin to 0.3 Gm. (40 gr.)
quinine. Three tablets each day for three to five days is the recommended dose
for adults. These compounds supposedly contain enough of each drug to rid
the patient of both asexual and sexual forms of the parasites.
Plasmochin causes the spleen to contract, thereby reducing its size. Often this
sudden contraction causes an acute spasm of pain in the left hypochondrium.
Cyanosis sometimes accompanies the administration of this drug.

ATABRINE
The same laboratory which synthesized plasmochin, on evaluation of that
drug, produced another as a specific for asexual forms of the parasite. This has
been advertised as the "five-day treatment" for malaria, and is sold under' the
trade name "Atabrine." It was synthesized in 1930 by Mietsch and Mauss, the
chemical formula being dihydrochlor-alkyl amino-alkyl amino-acridine. Tablets
of 0.1 Gm. (10 gr.) are dispensed and a dose of one tablet three times daily
for five days on a full stomach is recommenued by the company. This drug was
tested experimentally on birds by Kikuth and clinically by SioH. In the- past few
years numerous reports have accumulated regarding results of its use in the hands
of other workers. The consensus of opinion is tbat atabrine is a valuable addi-
tional weapon against malaria, that it exerts a peculiarly specific action on the
schizonts of all malarial parasites, but has no direct action on the gametocytes,
especially those of P. falciparum. It may eventually dispose of the gametocytes
by cutting off the source of supply. Walters and Manson-Bahr have shown that
it does not affect their power of exflagellation. The drug remains in the body
for extended' periods (average 26 days) after a course of treatment. Untoward
symptoms have been reported from the use of this drug, chiefly, a yellow tint to
the skin, and cerebral excitation. Molitor in a recent review of the synthetic
anti-malarial drugs concurred with the opinion that treatment should not be
repeated within an interval of less than eight weeks and that tbe total amount
given in anyone period of treatment should not exceed 2 Gm. (0.3 Gm.'per day).
Dawson, Gingrich and Hollar showed that atabrine, when administered in-
 MALARIA 543
travenously, caused untoward and even dangerous symptoms. Atabrine mus-
sonate, now sold as "atabrine for injection," is available for intravenous tberapy.
Adverse reports are accumulating in regard to the administration of atabrine
and plasmochin together as each drug tends to increase the toxicity of the other.
Patients in general, however, are favorably inclined towards atabrine as it appeals
to their taste, and brings speedy relief from the symptoms of the disease. The
increased use of atabrine since its introduction and the accumulation of favorable
results from its many clinical trials have proved that this is an effective anti-
malarial agent. Present results indicate that ali three specifics for malaria are
valuable, but none is a perfect remedy, and entirely without untoward effects on
some individuals.
BIBLIOGRAPHY

A:CRAMI, P., SENEVET, G: "Pathogenie d l'acces palustrej la crise bemoclasique initiale." Bull.
et mem Soc med d hop de Par I 43, ,3Td Ser, 530, 1919.
BASS, C. C LIThe standard treatment for malaria" New York 1J.fed Jour, 117, 740, 1923.
BoYD, M. F.' "Introduction to malariology." Harvard Uruversity Press, Cambridge, Mass. 1930
BoYD, M. F.: "On the schizogonous cycle of plasm()dzum vivax, Grassi and Felettl." Amer. Jour.
Trop ..Med ~ 15, 605. 1935.
BOYD, .M F, STRATMAN-'J'ImMAs, W. K., and MUENSCH, H.: "Tbe occurrence of gametocytes of
plasmtJdium 'jJlvax during the primary attack." Amer. Jour. Trop. Med'J 16, I33, I93-6.
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 CHAPTER 43
HODGKIN'S DISEASE: HISTOPLASMOSIS

HODGKIN'S DISEASE

This is a systemic disease of unknown etiology, characterized by a painless

progressive lymph gland enlargement, with a specific histological structure.
H,STORY

The disease was first described in 1832 by Thomas Hodgkin wbo was
pathologist at Guy's Hospital in London, and since then has borne his name. He
described seven cases of "Some Morbid Appearances of the Absorbent Glands
and Spleen," but only two of these would now be accepted as true Hodgkin's
disease. The other five are now known to have been tuberculosis or some form
of neoplasm. Wilks of Guy's Hospital, was the first to realize that Hodgkin had
described a new clinical entity and in 1856 he described more cases of the same
syndrome and again in 1865 he reported IS additional cases and suggested then
that the disease entity bear Hodgkin's name.
In the past the disease has been and still is known by numerous names.
Walhauser has listed over 50 terms that have been used. In 1864, Virchow
classified the disease as a lymphosarcoma because of the histological structure.
In current literature the disease is frequently referred to as lymphadenoma by
the British, and lymphogranuloma by the Germans. In this country, Mallory
and Means have maintained that it is a neoplastic process of .mesodermal origin
and have suggested the name lymphoblastoma, malignant lymphoma, or one of
the subtypes of lymphoma.
The establishment of a characteristic histological picture bas provided the
criteria necessary for diagnosis and separation of this condition from the other
lympho-reticulo-endothelial diseases_ This advance WlIS due largely to Sternberg
of Germany in r898, Andrews of England, and Dorothy Reed of this country in
'902. Dorothy Reed, at that time, on the basis of a study of eight cases of mronic
lymphadenopathy of the Hodgkin's variety, gave such an excellent description of
the giant cells that writers since that time have labeled them as Dorothy Reed
cells. Grenfield of London recognized these large cells as early as 1878 and
from that year on pointed them out to his students, while Goldman first observed
the numerous eosinophils in 1892.
ETIOLOGY

The etiology of Hodgkin's disease is unknown. Furthermore, the very nature

of the process remains sufficiently obscure to the extent tbat it is considered
inflammatory in origin by some and neoplastic by others. Those wbo have con-
sidered it inflammatory have suggested that it may be:
546
 HODGKIN'S DISEASE: HISTOPLASMOSIS 547
L An atypical form of tuberculosis.
2. A specific infective granuloma of unknown origin.
3. A vi rus disease.

AN ATYPICAL FORM OF TUBERCULOSIS

Many instances of tuberculosis associated with Hodgkin's disease have been,
noted. According to Zeigler approximately 20 per cent of the patients have
tuberculous lesions, and in 10 per cent the bacilli can be demonstrated or recov-
ered by animal inoculation. In 1898, Sternberg first advanced the concept that
the process may be tuberculous as he observed tuberculosis so frequently along
with typical Hodgkin's lesions. In the majority of cases of gland tuberculosis;
however, the organism can be demonstrated by staining or animal inoculation, so
that it is generally accepted that tuberculosis is a separate entity that frequently
accompanies Hodgkin's disease. On the other hand, it is well known that the
organism cannot always be demonstrated in proved tuberculous lymphoma.
Furthermore the histological picture of Hodgkin's is similar to that seen in the
diffuse hyperplastic form of tuberculosis in unsensitized individuals. L'Esperance
presented evidence that it is caused by an attenuated strain of avian tubercle
bacilli but this has not been adequately confirmed.

AN INFECTIOUS GRANULOMA OF UNKNOWN ETIOLOGY

If this is true the condition might be correctly termed Hodgkin's granuloma.
The pleomorphic and diversified cytological changes, necrosis, and subsequent
fIbrosis, indicate the presence of an infectious agent. On the other hand, infec-
tious granulomata seldom have the regularity in distribution that IS seen in
Hodgkin's, and the invariably fatal outcome.
The history of this disease is replete with various claims for specific etiological
agents. In 1913, Bunting and Yates isolated a large diphtheroid organism from
the lymph nodes and were able to reproduce lesions of the lymphoid organs closely
resembling those seen in Hodgkin's disease. However, the same organism was
later isolated from patients with leukemia, lymphosarcoma, tuberculosis, and
arthritis deformans. Other workers have attached etiological significance to
a wide variety of other bacteria but none of these claims has been substantiated.
In recent years Parsons and Poston at Duke University have isolated the Brucella
melitensis from the blood or lymph nodes of '4 consecutive cases of Hodgkin's
disease. Furthermore, they found the Brucella only once in a control group of
67 patients with other lymph node diseases such as lymphosarcoma, leukemia, and
tuberculous adenitis. Later (1940) a group from Duke University reported the
histological findings in Brucella infected guinea pigs. They did not claim to have
reproduced the lesions of Hodgkin's disease, nor even mentioned the disease in
their discussion but any pathologist could see that the lesions were very similar,
if not identical with those seen in human Hodgkin's disease. This work prom-
ises to be important but has not yet received confirmation.
A study of comparative pathology has been of little assistance in solving
the nature of Hodgkin's disease. Lymphosarcoma and leukemia are much more
 MISCELLANEOUS

widespread in the animal kingdom for only two cases of Hodgkin's disease hve
been reported in the hog and four in the dog. Feldman, in a study of 600 ca~es
of animal neoplasms, and Fox and Radcliffe, in a long autopsy experience at the
PbHadelphia Zoo, have not observed any cascs that could be classified as
Hodgkin's.
In studying tissue cultures from active cases of Hodgkin's disease, Le",is
bas obtained or produced some large hyperchromatic cells closely resembling the
Sternberg-Reed giant cells, which, from their reaction and habits of growth, more
nearly resemble inflammatory than neoplastic cells and do not resemble megakary.
ocytes.

A VIRUS DISEASE
The study of human virus diseases may finally prove the solution fo the
etiological problem. This phase of investigation has not yet reached sufficient
proportions to exclude viruses as possible causative agents. Schoen ohserved
intracellular bodies in mouse sarcoma cells after inoculation with a ,so-called
Hodgkin's virus.
The adherents to the concept that the disease is a neoplastic one are those
who believe it to he a primary neoplastic process and those who believe it to be
a transition between a chronic inflammatory process and a tumor. Mallory and
others have regarded the condition as a form of lymphoblastoma, a generic name
which has also been applied to lymphosarcoma and lymphatic leukemia. Wart)lin
claimed to have seen cases of Hodgkin's change into lymphosarcoma and lym-
phatic leukemia, but others have failed to observe this. The neoplastic theory' is
upheld mainly on the basis of the widespread infiltrative quality of the lesions and
on the invariably fatal outcome.
In 1937, Medlar, Hornbaker, and Ordway suggested that it is primarily a
disease of the bone mal'row with the giant cells arising from megakaryocytes,
thereby justifying the term megakaryoblastoma. They found cells that t}ley
interpreted to be megakaryocytes in the peripheral blood in 50 out of 56 cases
diagnosed as Hodgkin's disease by histological section. However, they observed
the same type of cell in other diseases and they apparently consider a megakary-
ocyte to be a cell with a single irregular lobulated nucleus that throws out sm,!I1
pseudopodia. This' description of the so-called megakaryocyte would, to m:lny
hematologists, describe the monocyte.
Symrners maintains that Hodgkin's disease holds an intermediate position
between infectious granuloma and true tumor, as seen in mycosis fungoides.
Ewing's concept of the transformation of some cases of Hodgkin's granuloma
into a sarcomatous process' is generally accepted and compatible with present
day knowledge of carcinogenesis. The neoplastic cells seem, to be of endothelial
origin, yet they lose their endothelial appearance and appear as large round cells
similar to those found in lymphosarcoma rather than endothelioma. Krumbh~ar,
in a study of one group consisting of 62 biopsies and 34 autopsies, has never
observed an undoubted Hodgkin's lesion that developed'into or was accompanied
by a true Hodgkin's sarcoma.
 HODGKIN'S DISEASE: HISTOPLASMOSIS 549

PATHOLOGY
Hodgkin's disease may be regarded as a primary involvement of the reticulo·
endothelial system in which the principal lesions occur in the lymph nodes, spleeh.
liver and bone marrow. All or only localized parts of the lymphoid tissue may be
involved. The process usually begins in-the cervical region and later becomes
generalized.
The lymph nodes are firm, large, and usually discrete until the late stages
when they lose their identity, develop an increase in fibrous tissue, and become
matted together. On cut section they are grey to pink in color, clear, moist, and
with or without areas of necrosis. Necrosis is a predominant feature in the
acute fulminating cases while fibrosis and lesser degrees of necrosis are present
in the more chronic cases. The invasion of the capsule and the matting together
of the glands are regarded by many as features suggesting malignancy but the
same tendency is present in other well·established granulomatous conditions.
The lungs are invaded in about 40 per cent of the cases of mediastinal Hodgkin's.
According to Moolten in about 10 per cent of the cases the disease apparently
starts in and spreads along the bronchial walls.
Involvement of the spleen is only a part of the generalized process. Primary
disease of the spleen without changes in otber parts of the body has been reported
but this is rare. The spleen shows a patchy instead of homogenous type of
necrosis with the outer and cut surfaces studded with yellow to white elastic
areas that may be several centimeters in diameter.
Lesions of the liver occur infrequently and are confined for the most part te>
the portal regions. Invasion of the bone marrow is found fairly often and when
extensive may account for the accompanying anemia. Osteolytic and osteo·
sclerotic changes are often combined, although the former are more common with
perforation of the cortex in occasional instances. The two processes may involve
the vertebrae resulting in pressure on the spinal cord or nerves but the inter-
vertebral discs are seldom invaded. In many instances it is difficult to separate
the condition from other chronic inflammatory processes. Single or multiple
nodular lesions that frequently ulcerate may occur in the skin and when studied
microscopically resemble Hodgkin's disease. Gastro-intestinal lesions are rare
and when present are difficult to differentiate from those of tuberculosis and
lymphosarcoma.
Microscopically the lesion of Hodgkin's disease has a pleomorphic cytology
in which large characteristic mononuclear and multinuclear giant cells of the
tumor type occur among lymphocytes, plasma cells, eosinophils, proliferating
endothelium, and varying amounts of fibrous tissue. These giant cells are
known as Sternberg, Dorothy Reed, or Sternberg-Reed cells and probably orig·
inate from the hyperplastic reticulo-endothelium. The earliest cytological change
is the appearance of new large pale cells with vesicular nudei which are similar
to and possibly the same as the chronic inflammatory "epithelioid" cell of tuber-
culosis. There may be an apparent increase in the lymphoid tissue during the
early stages of the disease hut this disappears and is replaced by the hyperplastic
reticulo·endothelium. Varying numbers of eosinophils are usually. present and
55 0 M ISCELLAN EOUS

frequently in large numbers. It is not known whether they are produced in the
lymph glands or in the bone marrow. Necrosis is sometimes extensive, par-
ticularly in the acute fulminating cases. In the more chronic cases fibrous tissue
changes, with less necrosis, gradually replace the more cellular elements.
HEMATOLOGICAL FINDINGS

The blo1ld has been extensively studied in Hodgkin's disease but there are
no changes that occur with sufficient consistency to be considered characteristic.
A progressive hypochromic anemia frequently develops in the chronic cases and
may be accentuated if the process involves the bone marrow. The total leukocyte
count has a tendency to be slightly increased in the earlier stages. However,
extremely high counts have been recorded, but normal white cells or even leuko-
penic counts are encountered. The leukocytosis, if present, is mainly a neutro-
philic one though the Jymphocytes and monocytes may also be increased. Bunt-
ing has stated that a poor prognosis is to be expected if the lymphocytes decrease,
as this indicates that the reticula-endothelial activity is crowding out the lymphoid
centers. Eosinophilia is occasionally observed and may be as high as 60 per cent,
but does not occur with sufficient consistency to be of diagnostic or prognostic
significance. Eosinophilia is seen more often in those cases that have an accom-
panying pruritus. An occasional case shows an extreme eosinophilia as the one
reported by Stewart (100,000 eosinophils per cu.mm.) and the one by Major
and Leger (150,000 eosinophils per cu.mm.).
Roth and Watkins have carefully studied the blood findings in 40 cases at
The Mayo Clinic and found no definite eosinophilia in any of them. They con-
cluded that there is a slight leukocytosis with a neutrophilic left shift in most
cases and that the extensiveness of the disease does not affect the blood findings
except to accentuate the anemia. They concluded that there is no specific change
in the leukocyte picture that is diagnostic of Hodgkin's disease.
Krumbhaar states that although there is no characteristic blood picture, the
blood studies present useful diagnostic evidence. Bunting has observed that the
platelets of the peripheral blood were increased in size and number.
Craver has stated that an increase in the serum phosphates may indicate
Hodgkin's disease with involvement of the bones. Studies of blood cholesterol,
Upase, fibrinogen, globulin, and basal metabolic rate have not been sufficiently
constant to be of diagnostic value.
THE GORDON TEST

In 1932, Gordon discovered that the intracerebral inoculation of a suspension

from a Hodgkin's node into guinea pigs or rabbits produced a characteristic
encephalitis which be attributed to a virus. The view that the agent responsible
for the reaction is a virus has not been substantiated but the reaction has become
accepted to be of some diagnostic value. The reaction can p~obably be considered
a nonspecific one, as a similar encephalitic syndrome can be produced by a
variable group of animal bloods and tissues when injected as a suspension into the
rahbit's brain.
Stei~er (194I) considers the encephalitis producing substance to be a non-
 HODGKIN'S D,SEASE: HISTOPLASMOSIS 55 1
living agent, probably enzymatic in nature. Of the cases reported in the litera-
ture as having a Gordon test done (310 cases) 76 per cent have been positive and
Steiner found the test positive in 76 per cent of his 21 cases. The test was uni-
formly negative in his control group of 40 cases of lymph node diseases which
had to be differentiated from Hodgkin's disease, this group including lympho-
sarcoma, the leukemias, tuberculous and non-specific lymphadenitis. Therefore,
if the histological examination of a lymph node does not provide a conclusive
diagnosis a positive Gordon reaction would be of diagnostic value in indicating
the probability of Hodgkin's disease and would give almost absolute assurance
that the patient does not have some of the other lymph gland disorders mentioned
above. Steiner concluded that "the test shows uncanny ability in differentiating
closely related types of lymphadenopathy." A negative reaction would make
the diagnosis of Hodgkin's less likely.
CLINICAL FEATURES

The disease is seen mainly in young people but cases have heen recorded of
infants less than one year old and in adults during the eighth decade. It is two
to three times more frequent in males than females and there are no racial or
geographical features of significance. The average duration of the disease was
formerly between two and three years after diagnosis, but with the use of radia-
tion therapy, the period of survival has been increased several years in many
instances. The acute fulminating types are often fatal within a period of six to
eight weeks.
The superficial lymph nodes, usually the cervical group, are the first to
become enlarged, this being followed by other groups. Symmers, from autopsy
observations, has found that the abdominal and t!lOracic nodes are involved in a
larger number of cases than the cervical group. X-rays are of considerable
value in establishing or confirming the presence of mediastinal involvement
before pressure symptoms occur. The spleen is enlarged in 60 to 70 per cent of
the cases, and in rare instances the condition may be confined entirely to the
spleen.
A periodic elevation of the temperature for several days with a gradual
return to normal for a period of ten days to two weeks is frequently seen and is
known as the Pel-Epstein type of fever.
Skin manifestations occur in 20 to 40 per cent of the cases, with pruritus
being the most common one and this may be present many months before
glandular enlargement takes place. In some instances it is generalized and in
others localized. The latter can be explained by irritation of the sensory root
nerve or ganglia by pressure or actual invasion by the pathological process.
Occasionally the itching may lead to excoriation, scarring, or be accompanied
by a pigmentation that suggests adrenal involvement. In rare cases nodular or
ulcerated infiltrations occur in the skin. These present a histological structure
typical of Hodgkin's granuloma and have been designated by some "'riters as
lymphogranulomatosis cutis.
Bone marrow lesions may dominate the clinical course and if extensive, are
accompanied by a progressive severe anemia. The lesions are destructive and
 55 2 MISCELLANEOUS

may even perforate the cortex. Collapse of the vertebrae is not uncommon but
fractures are rare. Loss of weight, pallor, and weakness are pronounced in the
late stages of tbe disease. The liver mayor may not be enlarged.
The disease process may begin in and remain localIzed to the mesenteric or
retroperitoneal nodes for a long time without palpable enlargement, thereby.mak-
ing the diagnosis difficult. There is frequently an elevated temperature, cachexia
. and mild anemia. Involvement of the gastro-intestinal tract is rare. Herpes
zoster is a frequent complication. The symptoms and physical signs vary widely
and depend chiefly on the mechanical pressure of the enlarged glands on the
cardio-vascular, biliary, gastro-intestinal, or respiratory systems. Pleural effusion
and ascites are not uncommon.

DIAGNOSIS

The differentiation of Hodgkin's disease from lymphosarcoma, reticulo-

endotheliosis, and reticulum cell sarcoma is best done by excision of a superficial
node and the demonstration of the pleomorphic histological picture of reticulo-
endothelial cell proliferation, in which are scattered a few large or small lym-
phocytes, varying numbers of eosinophils, fibrosis and necrosis, and tbe endothelial
giant cells of the Sternberg-Reed type. The leukemias, including aleukemic
leukemia, can be excluded by a thorough study of the peripheral blood. Lymph
nodes are found to vary in their histological appearance, especially in early c::tses.
One node may reveal nothing more than a chronic lymphadenitis but other nodes
from the same area may show a structure typical of Hodgkin's disease. The
Gordon test is a non-specific reaction which is more reliable in differential diag-
nosis of lymph node disease if it is accompanied by histologic examination.
TREATMENT AND PROGNOSIS

Tbe treatment o! Hodgkin's disease is best carried out by the radiologIst,

by giving large doses of x,ray therapy at properly spaced intervals to the areas
involved. The average survival period has been increased from one to three
years by radiation therapy. A few cases of Hodgkin's disease have been reported
that are still in good health after 20 to 25 years. Therefore, the outlook for some
patients is better with x-ray therapy and the prognosis of 100 per cent mort::tlity
can perhaps be modified.
Nevertheless, the prognosis remains unfavorable as more than half of the
cases will not survive the first year and more than 75 per cent die within three
years. The cases of localized glandular enlargement offer the best prognosis and
those with generalized involvement, the poorest. Repeated exacerbations of
severe fever, marked changes in the leukocyte count, poor results with radiation,
or a coincident tuberculosis, indicate a poor prognosis.
Wise and Poston (Duke University) have directed their therapeutic efforts
toward the Brucella infections that were present in their cases and'have reported
a decrease in the size of the lymph nodes following the administration of sulfanil-
amide or sulfapyridine. Further confirmation is necessary, as other workers have
not met with the same degree of success. Immune serum therapy, commercial
antiserum, or serum from persons having a high titre of agglutinins for the brucella
 HODGKIN'S DISEASE: HISTOPLASMOSIS SS3
organism, has not been found to be effective alone or in combination with sulfanil-
amide or its derivatives_

HISTOPLASMOSIS
(Reticula-Endothelial Cytomyco;';s)

Histoplasmosis was first described as a disease entity by Darling in 1906.

Until very recently tlris disease was considered quite rare, but Meleney in '940
reviewed tire thirty-two cases which have been reported and pointed out that the
apparent increase since 1938 'may be due to more general recognition. The
causative organism is a fungus, Histoplasma capsu/atum, belonging to the Crypto-
coccus group. Microscopically, the organism in man occurs as a yeast-like form,
closely resembling the Leishman-Donovan bodies. Darling at first thought the
parasite was a protozoon closely related to Leishmania but Rocha-Lima in 1912
pointed out its similarity to Cryptococcus farciminosus, the cause of epizootic
lymphangitis in horses, and concluded t~at it was in reality a yeast-like organism.
CLINICAL FEATURES

Clinically, the disease presents a wide variety of symptoms· and signs.

Meleney classified the cases in which the organism had been found into four
general groups: 1. Those in which the clinical picture corresponds to a systemic
febrile disease similar to kala-azar, with an enlarged liver and spleen, a septic
temperature curve, anemia and leukopenia; 2. Those in which lymph node enlarge-
ment predominates and which may simulate Hodgkin's disease, leukemia, lympho-
sarcoma, or aplastic anemia; 3. Those with pulmonary symptoms in which the
infection is complicated by or superimposed upon pulmonary or generalized tuber-
culosis; 4. Those in which the infection begins as a skin lesion which sometimes
develops into a generalized ulcerative skin condition and with purpura sometimes
occurring as a terminal condition.
PATHOLOGY
The macroscopic pathology is as variable as the clinical manifestations. In
most cases, one or more organs show gray or white nodules or extensive areas of
necrosis, and the lungs present abscess or tuberculous cavities. The pleura often
show evidence of a recent fibrino-purulent organizing process or fibrous adhesions.
Caseation of the adrenals may occur. meers in tire small or large intestine or both
have bee~ found, and nodules sometimes occur in the submucosa or deeper coats
of the intestine_
Microscopically, the lesions usually consist of an area of necrosis with a
central proliferation followed by complete loss of tissue and cellular structure
and surrounded by granulation tissue with large numbers of macrophages COn-
taining the parasites. The organisms are found mainly in tire organs of the body
which are rich in reticulo-endothelial cells. In severe systemic infections, they
may be found in large mononuclear cells in the pe_ripheral blood. The possibility
of histoplasmosis should be considered in any patient presenting symptoms of an
obscure disease of the blood-forming organs or lymphatic system, and in those
554 M,SCELLANEOUS

having an enlarged liver or spleen, particularly if there is any resemblance to

leishmaniasis, as this latter disease is not known to be endemic in the United
States.
Histoplasma capsulatum is a round or oval-shaped organism irom 3 to 5
micra in diameter and when stained with the usual blood stains such as Wright's
or Giemsa's, or with hematoxylin and eosin, consists of a round, crescentic or
vesicular central staining mass surrounded by a clear zone, and a refractile cap-
sule. In advanced systemic infections, the yeast-like organisms may be found in
either thin or thick smears of the peripheral blood. The latter should be de-
hemoglobinized as in malaria. They may be obtained by puncture from the
spleen, liver, Iymph.node or sternum, and may also be found in the huffy coat of
centrifuged, dtrated blood. Cultivation of the organism from any of the above
sites is sometimes possible and should be attempted. Cultures should he kept
for at least a month, at 37° C. and at room temperature.
At autopsy, smears from various organs should be examined and if no
organisms are found animal inoculations should he made from the tissues.
TREATMENT

No proved method of treatment is available. Meleney recommends trial of

antimony preparations such as potassium antimony tartarate, fouadin, and
neostam.

BIBLIOGRAPHY
HODGKIN'S DISEASE
L BUNTING, C. H.: uBlood platelets and megalokaryocytes in Hodgkin's disease." Bull. Johns
Hopkins Hosp.,. 22, II4, 19II.
- _ ' liThe blood picture in Hodgkin's disease." Second paper. BuU. Johns Hopkins HosP.
25. 173. 1914.
2. CRAVER, L. F.: "Local and general irradiation in Hodgkin's disease." Radiology, 31, 4Z, 1938.
3. EWING, J.: IINeopIastic diseases." Philadelphia, W. B. Saunders Co. 4th ed 1940.
4. FELDMAN, W. H: uNeoplasro of domesticated animals." PhIladelphia, W. B. Saunders Co J
193 2.
5 Fox, H.: "Remarks on the presentation of microscopical preparations made from some of the
original tissue described by Thomas Hodgkin, 1832/' Med~ Hut' J 8, '370, 19-26.
6. GOLDMAN, E. E . "Lympham, malignes." Zentralbl. Allg. Path. u. Path. Anat, 3, 665, 1892.
7. GORDON, M. H.: uRemarks on Hodgkin's disease." Brit. Med. Jour .• I. 641, 1933.
8-. GREENFmLD, W. S.: "Lymphadenoma with infiltration of the lungs and skin." Trans. Path Soc.
Lan, 27. 275, 1876.
9. HODGKIN, T.~ "On some morbid appearances of the absorbent glands and spleen." Med. Chlr.
Trans, 17,68,1832.
10. KD.'UM»HAAR, E B.~ "Pre5ent statu'S of Hodgkin's disease." Symposium on Blood. Univ. Wis-
consin Press. 1939 j P 148.
II. VESPER»l'CE, E. S.: "Experimental inoculation of chickens with Hodgkin's nodes." Jour. Im-
munal, 16. 37, 1929.
12. LEWIS, 'V. H.: "Normal and malignant celIs." SCIence, 81, 545, 1935.
13. MAJOR, R. H, and LEGER, L. H: uMarked eosinophilia in Hodgkin's disease." Jo-ur. Amer.
Med. Assoc., 112, 2601, 1939.
14 MEANS, J. H : "The symptomatology of lymphoma." Jour. Amer. Med. Assoc., 113, 646, 1939
 HODGKIN'S DISEASE: HISTOPLASMOSIS 555
IS. MEDLAR, E. AI., HORNDAKlm, J. H., and ORDWAY, W. H.: "An interpretation of the nature of
Hodgkin's disease." Folta Haematol, 57, 52, 1937.
16. R.E!:D, D. 1\.1.: liOn the pathologIcal changes in Hodgkin's disease with especial reference to it:!!
relation to tuberculosis." Johns Hopkins IIasp, Rept., 10, 133, 1902.
17. ROTH, G. M I and WATKINS, C. H.: "The leukocyte picture in Hodgkin's disease." Ann. Int.
1.Ied} 9, 1365. 1936.
]8, SCHOEN, R: "Evolution du virus Iymphogranulomateux dans les elements neoplasiques s,arco-
mateux." Compt. Rend. Soc. BIOI., 128, 135, 1938.
19. STEINER, P. E "Reliability and significance of the Gordon test in Hodgkin's disease." ftrth.
'- Path, 31, I, 1941.
20. STERNBERG, C.: "Ueber eine eigenartige unler dem bilde der pseudoleukamie verlaufende tt1ber-
culose des lymphatischen apparates." Ztschr. Heilk J 19, 21, 1898. '
ZI. STEWART, S. G.: "EOSinophilic hyperleukocytosis in HodgkIn's. disease with familial eosinolJhilic
diathesis." Arch. Int. Med J 44, 772, 1:929.
22. SYMMERS, D.: liThe chnical sigruficance of the pathological changes in Hodgkin's disease."
Amer Jour. Med. Sci J 167, IS7j 313. 1924.
23. TuRNER. J. C., JACKSON, H" JR •• and PARKER, F., JR.' '(Etiologic relation of eosinophil t(' tho
Gordon phenomenon in Hodgkin's disease." Amer. Jour. Med. Sci" 195, 27, 1938.
24. VAN RooYEN, C. E.: "A biological test in the diagnosis of Hodgkin's disease." Brit. Med.
Jour., 1, 644, 1933.
25, \VARTHIN, A. S.: "The genetic neoplastic relationships of Hodgkin's disease, aleukemic and leu-
kemic lymphoblastoma and mycosis fungoidcs,1I Ann. Surg, 93, '153, 19310.
26. WILKS, S.: "Cases of lardaceous disease" Guy's Hosp- Rep., 2:103, 1856, "Englargement elf the
lymphatic glands and spleen." Guy's Hasp. Rep., 11, 56, :1865.
z7. WISE, N. B., and POSTON, M. A : "The coexistence Qf brucella infection and Hodgkin's dis(lase."
Jout'. Amer. Jled. Assoc., 115, 1976, 1940.

HISTOPLASMOSIS

CLEMENS, H H., and BARNES, M L.: "Histoplasmosis of Darling. Report of a case." South. Med.
Jour" 33, II, 1940.
DARLING, S. T.; uA protozoon general infection producing pseudotubercles in the lungs and focal
necroses in the liver 1 spleen, and lymph nodes." Jour. Amet'. Med. Assoc I 46, 1283, 1906·
MELENEY, HENRY E: "Histoplasmosis (reticula-endothelial cytornycosis)' a review with mention
of thirteen unpublished cases." Amer. Jour. Trop. Med, 20, 603, 1940.
ROClU-LWA, H.: "Beitrag zur kennfnis der blasfomycosen. LymphangItis epizootica und Jtisto~
plasmosis." Zentmlbl. Bnkt. (Abt. 1 Orlg.), 67, 233, 1912. •
 CHAPTER 44
BLOOD GROUPS AND BLOOD TRANSFUSION
By FRANCIS P. PARKER, lif.D.
Emory University, Ga.

HISTORY

The prevalence of untoward reactions following the transfusion of hum,l!>

blood, during the early years of the use of this procedure, resulted in investigation
of variations in different bloods. The work of Landsteiner, De Castello and SturIi,
Jansky, and Moss showed that all bloods can be classified into four groups. Tbis
was determined by random cross matches of a large number of people. These
groups were shown to be based upon the distribution of two specific antigens in
the red cells and two antibodies in the serum. The former are called isoag-
glutinogens A and B and the latter isoagglutinins a and b. The isoagglutinins
are also designated by the Greek letters alpha and beta, and tbe terms anti· A
and anti·B. Other agglutinogens, namely M, N, P and Rh, may also be present
in the red cells but since no corresponding' agglutinins occur in the serum, these
have no bearing on the problem of transfusion. These bodies will be discussed
subsequently in relation to medicolegal applications.
To avoid confusion, the American Medical Association Committee on Medico-
legal Blood Grouping Tests has recommended that the term "group" be used
only when referring to classification according to agglutinogens A and B and that
the term "type" be used with relerence to agglutinogens M and N.

THE FOUR BLOOD GROUPS

The distribution of agglutinogens A and B and the corresponding agglu-

tinins has given rise to three classifications of the blood groups, which differ
from each other in terminology and all of which are in common use. These are
given in Table XXVIII.

T AIlLE XXVIII
Classmeation Groups Agglutinogen Agglutinin
Moss Jansky Landsteiner
1 IV AB A andB None
IT IT A A b
ill In B B a
IV r 0 None a and b

It is evident from the above table that in the use of the Moss and Juns]<y
55 6
 BLOOD GROUPS AND BLOOD TRANSFUSION 557
nomenclature there is liability of error due to the reversal of groups I and IV
unless the exact classification is always stated. In the Landsteiner classification
this danger is obviated by naming the groups according to their agglutinogen
content. This nomenclature is now recommended by the Health Committee of the
League of Nations and the American Association of Immunologists, and is the
one that is used in this discussion.

RACIAL AND GEOGRAPHIC

The factors responsible for the blood groups are not characteristic of any
particular race of people; however, examination of large numbers of individuals
of any given race will show a certain distribution of the groups. The studies of
L. and H. Hirszfeld on European races and those of Snyder, Tiber, Culpepper
and Ableson, and Sanford on the Caucasians of this country, along with some 600
other studies including practically every existing race, indicate that the frequency
of occurrence of the blood groups is a racial characteristic. The Hirszfelds also
believe that there is a geographic as well as a racial relationship, and point out
that in Europe, the incidence of agglutinogen A decreases from West to East
while agglutinogen B increases. In general, however, group 0 (43 per cent) and
group A (40 per cent) predominate, while group AB (10 per cent) and group B
(7 per cent) are present in a much smaller number of people. The percentages
represent only average approximations.

BLOOD GROUPS AT BIRTH

In reference to the establishment of the blood groups in early life, it has

been shown that the agglutinogens are usually demonstrable at birth and in one
instance have been demonstrated as early as the 37th day of foetal life. The ag-
glutinins, on the other hand, are frequently absent from the serum at birth (so
per cent) and when present, are identical with those of the mother. Furthermore,
many cases are on record in· which the agglutinins of the newhorn disappeared
shortly after hirth, to be replaced subsequently by different ones. This has led
to the assumption that the original agglutinins were carried over from the mater-
nal circulation. In any event, after birth, both agglutinogens and agglutinins
gradually increase in titer, reaching their normal concentration at about 20 years
of age.
After the hlood group of an individual becomes established, which is usually
hy the end of the first year of life, there is no evidence that it ever becomes altered.
There are numerous reports indicating this possibility in certain infectious dis-
eases, blood dyscrasias, particularly leukemia, after repeated transfusions, X-ray
therapy, and after the prolonged administration of certain drugs such as quinine
and opiates, but in no such case has the possihility of technical error been com-
pletely eliminated.
55 8 .lII,SCELLANEOUS

ANOMALIES IN BLOOD GROUPS

Among the various anomalies attributed to the blood groups, that of deficient
or defective groups is a theoretical possibility. A defective group is one lacking
in its normal agglutinogen or agglutinin content, a condition which occurs but
with extreme infrequency. In the cases reported only the agglutinins have been
involved.
A condition which occurs with much greater frequency is the presence of
sub-groups. This is an anomaly first described by von Dungern aud Hirszfeld,
in which a group B serum was absorbed with group A cells until agglutiMtion
no longer occurred. The serum was then shown to be able to agglutinate large
numbers of other group A cells. It was presumed that the original group A
blood contained aggl utinogens A different from those usually present. This work
was subsequently verified and at present agglutinogens At and A. are recognized
as occurring in connection with groups AB and A, giving rise to subgroups At,
A., AlB, and A.B. Corresponding agglutinins can be demonstrated in relation to
their respective groups. Thus agglutinins a, or a, may be present in the serum
from group B or group 0 bloods. In addition the serum of groups At and A,B
may contain agglutinin iI. and that of groups A, and A,B may show agglutinin
at. There is a question as to whether agglutinins at and a. differ from each
other qualitatively or only quantitatively; however, Landsteiner favors the for-
mer view. Agglutinins a, a, and a2 show considerable overlapping in their re-
actions with cells containing agglutinogen A, and A2. Agglutinin a reacts strongly
with A, cells and moderately with A. cells; agglutinin a, reacts moderately with
Al and weakly with A.; while agglutinin a2 reacts moderately with A2 cells and
strongly with group 0 cells. This ability of agglutinin a2 to clump group 0 cells
\w."!. \>~" 'L,tl!o'" "l>, ~"''''~'''L1!o \\\.,.t "l>2 " "I"''''\'·aA'''I!o\'j "',«~,~,,\ )'~m "1.
Agglutinogen Az is noted for its low level of reactivity, frequently being re-
sponsible for false grouping in which group A. is mistaken for group 0 and group
A2B for group B. However, with potent group B serum (anti-A) both aggluti-
nogen A and A2 react completely. A small per cent of group A bloods have
been shown to react feebly with such sera, indicating the possihility of a ~hird
type of agglutinogen A. This has been designated as agglutinogen A3 by Fischer
and Hahn and by Friedenreich, giving rise to two additional subgroups A, and
A,B: 'Viener has recently reported the occurrence of such a blood group in a
negress and her child and points out the even greater possibility here, of mis-
taking group A. for 0 and A,B for B. The importance of these reactions 01 the
subgroups in cross-matching hefore transfusion will be discussed under that
subject. '

HEMAGGLUTINATION

Henmgglutination, or clumping of the red cells, with subsequent hreakdown

and hemolysis, occurs as a result of the interaction of like agglutinogen and ag-
glutinin. For example, the mixture of A and a, or Band b produces this reaction,
 BLOOD GROUPS AND BLOOD TRANSFUSION 559
giving rise to certain interrelations among the blood groups. These are graphically
shown by the diagram from Todd and Sanford (Fig. 38) .

.._----~
Landsleiner B.
Moss lll.
Jansky Ill.

1
Landsieiner A.
Moss II.
lansky II.
+-----8
FIG 3R.-Interrelations Qf the blood groups.
Landsieiner O.
Moss IV.
lansky I.

On the basis of the conditions necessary for agglutination, it is seen that

the serum of any group will agglutinate the red cells of the group to which the
arrow points. Thus the serum of group AB will not agglutinate the red cells of
any other group nor will the red cells of group 0 be agglutinated by the serum
of other groups. This has resulted in the designation of group AB as the "uni-
versal recipient," and group 0 as the "uuiversal donor." 'Vorkers who condone
the use of the "universal donor" in transfusions feel that sufficient dilution of the
donor's serum and absorption of the incompatible agglutinins by the tissue and
blood cells of the recipient occur to make negligible any action of these ag-
glutinins. However, when the interrelations of the subgroups are considered, it
is seen that there is neither universal recipient nor donor for the serum of groups
AlB and Al may agglutinate the cells of group 0 as well as those of groups
A,B and A2 • Thus the indiscriminate administration of group 0 blood without
regard to the possible subgro:up of the recipient often eventuates in post-
transfusion reactions, many of which are cited by Davidsohn. The advisability of
this practice will be discussed at greater length under the subject of transfusions.
It will be noted then, that only when homologous blood groups are mixed,
is the reaction completely compatible. Therefore, it is important to determine
not only the group of a given blood, but also, because of the possible presence of
subgroups, that these homologous groups show no anomalous agglutination. The
former is ascertained by the process of blood grouping, and the latter by cross
matching (donor vs. recipient).
BloQd grouping is carried out by mixing the red cells of the individual to be
grouped with the serum of group A and of group B wbich contain agglutinin band
agglutinin a, respectively. (Some workers prefer a third preparation in which
group 0 serum containing agglutinins a and b is used. This is an added check
on the procedure but is not essential.) The resulting presence or absence of ag-
glutination will indicate the type of agglutinogen in the red cells. From this
560 :'1 IS CELLAN];oU S

setup, one of four possible reactions will result. These reactions, with their
interpretation, are shown in Table XXIX.

TABLE XXIX
Group Serum A.(b) Serum B.(a)
AB ............. . + +
A ................. . +
B ................. . +
0 ................. .
+ Indica.tes agglutination.
- Indicates no agglutination.

TECHNIC OF BLOOD GROUPING

The procedure for blood grouping has a number of applications: anthrOPO-

logical, medicolegal, in the field of genetics, but most frequently it is used clin-
ically for the selection of a donor for transfusion. In this connection it is not a
procedure of necessity, but rather one of convenience, particularly where pro-
fessional donors are available. Here the recipient may be grouped and a prOper
donor called, thus eliminating the necessity for many random cross matches.
Preparation of grouping sera can be carried out in the laboratory, but utore
often they are purchased. The sera used should be of high titer to eliminate the
possibility of a false negative reaction with red cells of low agglutinogen s<nsi-
tivity. Healthy group A and group B male adults who are neither very yaung
nor elderly, usually produce sera of satisfactory strength. This should further be
ascertained by the actual titration of varying dilutions of serum against a red
cell suspension of the proper group.
Collect blood by venipuncture under completely sterile precautions, and allow
it to clot. Loosen and separate the clot from the serum by centrifuging ot re-
frigeration for a period of time. Pipette off the serum and transfer to sterile
I cc. vials. Inactivate in a water bath at 560 C. for 30 minutes. Label and
store in a refrigerator. At refrigeration temperature potency is usually retained
for several months. Preservatives of various types are available and are recom-
mended by some workers.
Determination of titer of sera according to the technic recommended by
Wiener is as follows:

Group A Serum.
I. For each serum prepare five tubes labeled I through 5 as follows:
Tube I-Undiluted serum.
Tube 2-(Dilution I!1O) Add 0.1 cc. undiluted serum and 0.9 cc. physiolog-
ical salt solution. Mix.
Tube 3-(Dilution I :20) Transfer 0.5 cc. from tube 2 and add 0.5 cc. salt
solution. Mix.
Tube 4-(Dilution I!40) Transfer 0.5 cc. from tube 3 and add 0.5 cc. salt
solu tion and mix.
 BLOOD GROUPS AND BLOOD TRANSFUSION 561
Tube S-(Dilution 1:80) Transfer 0.5 cc. from tube 4 and add 0.5 cc. salt
solution and mix.
2. In a separate tube for each dilution, place one drop of serum mixture and one
drop of a suspension of group B cells.
3. Allow to stand for one hour and examine microscopically for agglutination.
The titer is indicated by the highest dilution in which clumping can be detected
with the naked eye. Only those sera giving r"!lctions at dilutions of I :20 or
higher should be used for grouping tests.
Group B Serum.
1. Prepare serum dilutions as above.
2. These dilutions should be tested:as given above, against known group A, and A,
cells.
3. If these are not available test against group A cells. In snch case agglutination
with dilutions of at least '''10 is necessary for satisfactory testing serum.

Preparation of red cell suspension should be done immediately before each

grouping. A few drops of blood are taken by finger puncture and mixed with
physiological salt solution to make a 2 to 5 per cent concentration. This may be
used as such or washed free of serum by centrifuging and resuspending in salt
solution. With experience, the proper cell concentration can be satisfactorily
estimated by the depth of color in the suspension. Using a serological test tube
(10 mm. inside diameter) it is possible to see but not to read printed type
through the suspension.
The cell suspension should be freshly prepared before each grouping and
should never be used when more than 24 hours old. 'Red cells washed free from
serum quickly lose their reactivity; clotted blood suspended in serum, however,
retains its reactivity as long as a week if kept at refrigeration temperature.
The procedure JOT blood grouping varies widely and may be performed satis-
factorily by anyone of a number of methods. For routine work we have used a
hanging drop preparation exclusively. A drop or loop of serum of group 11. is
placed un one coverslip, and one of group B on another. An equal amount of
cell suspension is mixed with each. The coverslips are rimmed with mineral
oil and placed over the concavities of a double concave slide. The preparation is
then examined at intervals with low. power magnification to determine the
presence or absence of agglutination. The setup should remain for at least 30
minutes before final reading. In the case of infants or young children, the mini-
mum time Should be an hour.
Agglutin;J.tion in tbe mixture is evidenced by the formation of irregUlar,
coarse clumps of cells which can usually be distinguished grossly_ These settle
oul rapidly leaving a clear fluid medium. Microscopically, the agglutinating cells
are seen to be attracted toward each other until clumping is accomplished. 'the
clumped cells become distorted, lose their individual identity and soon appear as
amorphous masses. Hemolysis mayor may not occur with agglutination. When
noted, it is due to specific hemolysins similar in type to, but not identical with,
the associated agglutinins present in the serum. These hemolysins are effective
against red cells of similar agglutinogen content, for example, the hemolysin of
 MISCELLANEOUS

group B serum would act upon red cells of groups AB and A while the serum of
group Ali could contain no hemolysin.
Two other slide preparations are in common use. One differs from the above
only in the substitution of a plain glass slide in place of the double concave slide.
This method has the disadvantage of accentuating rouleau formation. The other
method is a macroscopic preparation in which a drop of whole blood taken by
finger puncture is mixed on a slide with the typing sera. This is a quick method
but will not detect the finer degrees of agglutination capable of demonstration
by the microscopic technic.
Some workers, notably Landsteiner, recommend a test tube method as being
quite sensitive and better adapted to the examination of large numbers of bloods.
This method consists of mixing one drop each of the cell suspension, salt solu-
tion, and the testing sera, in a small test tube (7 mm. diameter). This is allowed
to stand for one hour, examined grossly for agglutination and checked micro-
scopically if necessary. This method has been modified by Schiff. To speed the
agglutination reaction he recommends centrifuging the tubes at 2000 revolutions
per minute for tbree minutes. They are then shaken in a rack until the packed
cells are resuspended. The presence of coarse clumps or a solid clot indicates
agglutination.

CROSS MATCHING

Even though grouping is used routinely it is still advisable to cross match

the prospective donor and recipient to eliminate any possibility of an untoward
reaction which might result from error in grouping or the undetected presence of
subgroups.
In performing a cross match either the slide. or the test tube method (see
technic for blood grouping) may be used. Blood is collected by venipuncture, a
portion used to prepare the cell suspension, and the remainder allC;JWed to clot for
separation of serum. Diggs has recently advocated the use of clotted blood alone
for cross matching and grouping. The cell suspension is prepared by transfer-
ring, with a wooden applicator, blood from the clot still suspended in serum.
Allowing the red cells to remain in contact with the serum preserves their ag-
glutinogen content and makes unnecessary further collection of. blood where re-
peated matchings must be performed or the use of several tubes and proper salt
solution at the time the blood is withdrawn. Oxalated blood has been shown
by Becton to be equally satisfactory for both Cross matching and grouping. Cell
suspension is prepareo, the blood centrifuged, and the supernatant plasma used
in place of serum.
In the preparation with the hanging drop method, a small drop or loop of
donor's serum is placed on one coverslip and with it is mixed an equal amount
of the recipient's cell suspension. Another coverslip is similarly prepared using
recipient's serum and donor's cells. These are mounted as descl ibed above on a
double concave slide and examined for agglutination, at intervals· from 30 min-
utes to an hour. Care must be exercised to label each end of the slide properly
and to be certain that the sera and cells are actually crossed.. The error of
 BLOOD GROUPS AND BLOOD TRANSFUSION

mixing donor's serum and cells on one end of the slide and recipient's serum
~nd cells on the other has occurred with the resulting possibility of a false nega-
tive reaction. If the test tube method is employed the amount of serum, cell
suspension, and salt solution, as well as the technical procedure, is identical with
that given under blood grouping. Regardless of the method used, the same errors
may occur here as with blood grouping.
With the widespread use of sulfanilamide and its derivatives, the assertion
has been made tbat these drugs interfere with cross matching. Kreinin has
performed studies on patients before and after they received sulfanilamide and
found that the drug per se does not disturb cross matchings. He concluded that
any inability to cross match was due to changes in the blood brought about by
the disease.

SOURCES OF ERROR

Anyone of the above methods is open to certain sources of error, giving rise
to false reactions. Wiener divides these roughly into two classes: false negative
and false positive reactions. Of these, false negative reactions are far more dan-
gerous since the failure to detect agglutination when it should occur may result
in transfusion of mismatched blood with subsequent serious or even fatal reac-
tion. False positive reactions are not liable to such grave consequences. How-
ever, they may cause great inconvenience in the search for a proper donor or
embarrassing error in grouping for medico-legal purposes. False negative reac-
tions may be due to anyone of the following causes.
Grouping Sera of Low Titer. This may be due eitber to an inberently low
content of agglutinin or to the gradual decrease of these bodies due to the age of
the serum. Bacterial contamination will also decrease the agglutinin content
but such growth can be detected grossly and the serum discarded.
Low Agglutinogen Reactivity. Agglutinogen A2 in the cells of groups A2 and
A2B, particularly in the latter, frequently will react so weakly that errors in
grouping occur. It is not uncommon to find that the bloods of group A, are
designated as group 0 and. those of group A2B as group E. Whenever repeated
cross matches involving thes~ groups show incompatibility, the bloods should
be regrouped. The use of grouping sera of high titer or grouping with unknown
serum and group A and group B cells will make such errors unlikely.
Hemolysis may occur so rapidly that agglutination is masked. This is par-
ticularly likely to occur where the test tube method is used but should be de-
tected easily by the clear red color of the supernatant fl uid.
Too great concentration of the cell suspension may result in the absorption
of agglutinins from the testing serum without production of agglutination.
Weak concentration of cell suspension may prevent agglutination by making
the distance between cells so great as to overcome the attractive force of the
reaction. Such occurrence would be seen only in slide preparations.
H asle, in that the preparation is allowed to stand an insufficient length of
time for agglutination to occur. This is of greatest importance when working
 MISCELLANEOUS

with bloods of infants and children in which the concentration of agglutinogens

and agglutinins is less than in adults. We have seen, in cross-matching an
infant with nine prospective donors, perfectly compatible preparations for 45
minutes, and then aggl utination occurred in eight of the nine during the next IS
minutes.
Carelessness 01 improper marking oj tubes may result in failure actually to
cross cells and serum in cross matching.
False positive re.1ctions may be due to a number of conditions.
Pseudoagglutination or rouleaux jormation is one of the most frequently
encountered. This characteristic of the red cells of some bloods was first de-
scribed by Shatlock, and consists of an arrangement of the cells in rows, as a
stack of coins. Groups of these rows radiating irregularly from a common center
often have the appearance of true agglutination. An experienced observer can
usually distinguish this reaction from agglutination by examination of the prep-
aration with high dry magnification for each cell retains its normal shape and
individual identity as contrasted with the amorphous character of the agglutinated
cell. Furthermore, agitation of the preparation will disperse the cells in rouleaux
formation while it accentuates the agglutination reaction. If the distinction is
still uncertain, a new preparation in which the serum is diluted 1:2 or I '3 with
physiological salt solution will not show rouleau. With the test tuhe method
such dilution is present and therefore rouleau is rarely seen in these prepara-
tions. This phenomenon has been attributed to abnormal protein concentration
of the serum.
Cold agglutinins occurring in sera have been described by Landsteiner and
Levine. These, if mixed with cell suspensions at temperatures between o· and
5· C., will produce agglutination. This reaction generally can be prevented by
performing all tests at room temperature. In addition this phenomenon may
result from the action of a type of specific agglutinin thought to be identical
with agglutinins a, and a2 noted in connection wit]l subagglutinogens.
-Bacterial contamination oj sera which have stood for long periods of lime
may also cause agglutination, so care must be taken to prevent this.
Autoagglutinins, non-specific in nature, are often present in diseases asso-
ciated with liver damage. Since these react best at low temperatures, they are
often considered types of cold agglutinins. Autoagglutinins may be of sufficient
strength to produce agglutination at room temperature in which event error of
grouping may result. Rarely does the reaction occur at body temperature al-
though we have seen a patient with such potent autoagglutinins that red cell
counts could hardly be done and it was impossible to obtain a compatible donor
for transfusion. This patient eventually died with a progressive hemolytic
anemia.
Panagglutination, a phenomenon described by Hubener, Thompsen, and
others, occurs with respect to bloods which have been allowed to sland for a
period of time. These cells may then be agglutinated by any serum added, even
that of the same person. This is due to growth of certain bacteria which produce
an enzyme capable of activating a latent agglutinogen present in red cells. Such
reaction may be avoided by using only fresh red cell suspensions.
 BLOOD GROUPS AND BLOOr> TRANSFUSION

Wharton's jelly, often present in blood collected from the umbilical cord,
may cause a clumping of cells resembling agglutination. This clumping will
disperse on standing, only to reappear with slight agitation of the preparation:
With the increasing use of placental blood for transfusions such reactions should
be borne in mind when grouping or cross matching.

OTHER AGGLUTINABLE BODIES IN RED CELLS: THE BLOOD TYI'ES

During the past few years agglutinable bodies other than agglutinogens
A(A A2 ) and B have been shown to be present in red cells. Agglutinogens M,
"
Nand P were demonstrated by Landsteiner and Levine and more recently
agglutinogen Rh by Landsteiner and Wiener. None of these agglutinogens are
associated with naturally occurring corresponding agglutinins, therefore they do
not enter into the problem of transfusion. Agglutinogen Rh may be an exception
in this respect since in certain instances the formation of agglutinins has been
induced. The significance of such occurrence is discussed in connection with
transfusion reactions.
Agglutinogens M or N or both occur in the red cells of all persons. They
are inherited as Mendelian dominants giving rise to bloods of types M, N,
and MN. It is recommended that the term "type" rather than group be used
in designating such bloods. Since no corresponding agglutinins occur in human
sera, these agglutinogens can be demonstrated only hy the use of immune sera
prepared by injecting cells of the proper type into animals, usually rabbits.
These agglutinogens are fully developed in the newhorn. They have been demon-
strated, according to Davidsohn and Rosenfeld, in embryos as early as one and
a half months and at 4 to 6 months they are of equal strength to that in the adult.
The M-N type does not change during life; however, it can be determined re-
liably only in the red cells. These types have their greatest application in
anthropological or medico-legal work and will be considered further under that
head.
Methods for the preparation of anti-M and anti-N testing sera have been
devised by both Wiener and Levine. These utilized the procedure of imm uniza-
tion of rabbits against cells of groups MO and NO by repeated injection of the
'proper cell suspension. The ~erum is collected, the non-specific agglutinins
removed by absorption and the M or N agglutinin content determined by titra-
tion. All animals injected do not respond with equally satisfactory agglutinin
production. Anti-N serum is the most difficult to prepare in adequate strength.
Davidsohn and Rosenfeld have compared critically the two methdds and for
detailed directions for serum preparation this work or Wiener's book on blood
groups (see bibliography) should be consulted.
Agglutinogen P, first described by Landsteiner and Levine, is probably identi-
cal with an agglutinogen designated as 0 by Furuhata. Anti-P agglutinins occur
naturally in the serum of a number of animals, particularly the pig, but have been
demonstrated in only one human. Agglutinogen P appears to be inherited as a
dominant. It occurs with gleater frequency in the negro than in the white race.
566 MISCELLANEOUS

The lack of corresponding agglutinins in human serum makes unnecessary its

consideration as a factor in blood transfusion, but its determination may be
valuable in medico-legal problems.
Agglutinogen Rh is the most recently demonstrated agglutinable factor in
red cells. If blood of a Rhesus monkey is repeatedly injected into a rabbit,
specific agglutinins are formed against these cells. This immune serum will
agglutinate the red cells of approximately 85 per cent of humans. These bloods
are designated as Rh +. Application to transfusion is indicated in that Rh -
persons have shown anti-Rh agglutinins in their serum following transfusions with
blood from Rh + donors. These agglutinins were not permanent but if a second
transfusion of Rh + blood were given within a short period of time, agglutina-
tion of the donor's cells occurred with resulting hemolytic reaction. Wiener and
Peters report three such cases and point out that such reactions also may occur
after the first transfusion of Rh+ blood into an Rh - woman who has recently
carried an Rh + fetus.

BLOOD TRANSFUSION

The procedure of transfusion, to which these methods have their greatest

application, is one of antiquity. Its history has been characterized by alternating
periods of enthusiasm and disfavor. The period since the World War has been
one of enthusiasm in which the procedure has had wide application to clinical
medicine. The prevalence of untoward reactions responsible for tile periods of
disrepute in past years, has heen curbed largely by the application of our knowl-
edge of the isoagglutination groups and by refinements in technical methods_
In the past, freshly drawn, whole blood was the sole agent used in transfusions.
At present this procedure also utilizes stored or preserved whole blood and
plasma which may be stored as such or in a desiccated form.

TRANSFUSION OF FRESH WHOLE BLOOD

The hroad consideration of this subject necessitates some attention to the
methods of transfusion, although details of the technic are not within the scope
of tbis ~iscussion. The melhods generally in use are divided into two types:
direct and indirect. Both have certain advantages and disadvantages; both
have their staunch advocates. . Of the direct types, the technics of Lindeman,
Scannell, Unger, Kimpton and Ilrown, and Rudder, are used and all consist of the
rapid transference of unaltered blood from donor to recipient usually by some
adaptation of the syringe principle. Advocates of these methods cite as ad-
vantages that blood may he given rapidly, with a minimum of manipulation
and extra-vascular contact, and in as nearly as possible a physiologically unal-
tered form. Certain disadvantages include, first, that it is necessary for donor
and recipient to be in close proximity to each other; and second, that these meth-
ods make use of certain mechanical apparatus requiring considerable training
and skill on the part of the operators. In respect to this latter contention, it is
true that when clotting occurs or "something goes wrong" it usually necessitates
 BLOOD GROUPS AND BLOOD TRANSFUSION

discontinuing the transfusion. However, in respect to skill of operators, re-

gardless of the method used, transfusion should never be attempted by inex-
perienced persons. The Lindeman or multiple syringe method even circumvents
mechanical complications since all of the apparatus required are syringes of 20
or 50 ce. capacity, needles, and normal salt solution.
The citrate method is the indirect type most widely used in this country. It
consists of the removal from the donor of the requisite amount of blood, which,
as it is removed, is mixed in a sterile receptacle with sufficient sodium citrate solu-
tion to make a final concentration of 0.25 to 0.30 per cent. At a subsequent time
this mixture is injected into the vein of the recipient. The chief advantage cited
for this method is its simplicity, since it requires no apparatus which is not
available even in tbe smallest hospital and if necessary can be performed without
assistance. It makes the meeting of donor and recipient unnecessary. It also
eliminates the factor of speed requisite in the direct methods, since the blood
may be given hours after its removal from the donor and, in addition, the actual
injection may proceed as slowly as is necessary. The disadvantages of the citrate
method include: first, ad<litional chance of bacterial contamination because of
the more cumbersome technic; second, the addition of a foreign body in the form
of citrate; and third, the effect on platelets of stirring and exposure to air. The
simplicity of the method is even cited as a disadvantage since it may place the
procedure of transfusion in the hands of unqualified persons.
The ultimate choice between the use of direct or indirect method should be
governed by an analysis of the per cent of post-transfusion reactions resulting
from large numbers of transfusions in diversified conditions. Here again there IS
no uniformity of reports. Lewisohn pointed out that, with the proper attention
to preparation of apparatus, and particularly solutions, the per cent of reactions
in the citrate method become negligible. This assertion has been substantiated
during the past four years by demonstration that the type of febrile reaction
so prevalent with the indirect method and which was attributed to citrate, is
actually due to the presence of so-called pyrogens in improperly prepared dis-
tilled water. Recognition of this has resulted in a definite shift to citrate technic.
Wiener and his co-workers show that, in 1936-37, 78.5 per cent of 1209 transfu-
sions were by direct methods, while, in '939, 78.0 per cent of 1213 transfusions
employed citrated blood. The incidence of all types of reactions in this latter
group was only 2.9 per cent. Fresh whole blood may be used for transfusions
in any condition in which such therapy is indicated. It is paJ ticularly adapted
to the needs of small hospitals having an average of less than three transfusions
dally. ~

TRANSFUSION OF STORED 'WHOLE BLOOD

The use of stored or preserved whole blood for transfusion,. was described
by Rous and Turner as early as 1916. The Russian workers revived the proce-
dure through their use of cadaver blood. Yudin describes the use of such blood
without anti-coagulant, in 924 transfusions with only 5 per cent of mild reactions.
In this country the revival took the form of the establishment of so-called "blood
5 68 MISCEI.I.ANEOUS

banks" in which citrated or preserved blood was stored at low temperature until
needed for transfusion. Withdrawals from the bank were covered by deposits
of an equal quantity of fresh blood and a balance of blood readily obtainable
was thus maintained.
This type of transfusion service is applicable only to the larger hospitals
where the daily number of transfusions given is sufficiently great to assure rapid
turn-over in the stock of blood and thus reduce to a mininlUm the amount of
blood which must he discarded as a result of prolonged storage. Furthermore,
the establishment of a blood bank should not be contemplated unless adequate
equipment and personnel are available for the proper conduct of the service.
Diggs lists the following items as essential:
l. The employment of a full-time technician with sufficient training to as-
sume charge of all operations_
2. Adequate refrigeration facilities. These should include one or more re-
frigerators of sufficient size to accommodate at least 60 flasks of blood.
There should be an automatic 24 hour recording thermometer and signal
lights to indicate dangerous temperature variations. These controls are
particularly important since either elevation of temperature or freezing
of the blood may produce hemolysis sufficient to necessitate the discarding
of all blood in storage.
3. Laboratory facilities to conduct the necessary grouping, cross matching
and serological procedures as well as bacteriological cultures and hemolysis
tests.
4. Facilities to take blood from donors, which activity, however, is usually
carried on by the house surgical staff.
5. Facilities for keeping adequate records not only on the status of blood
in storage but also on its ultimate disposition.
Methods for preservation and storage of whole blood are generally uniform in
all of the institutions where banks have been in operation for sufficient time to
evaluate the procedure properly. Strict aseptic technic must be observed
throughout all manipulations.
The blood is collected in 1000 cc. pyrex flasks containing 100 cc. of 2.5
per cent sodium citrate solution (Merck reagent grade) and stoppered with a
two-hole stopper with attached air filter and rubber tubing. Withdrawal of the
blood from the donor may be effected in several ways, by free flow from the
vein, suction applied to the collection flask or by the use of specially prepared
vacuum bottles (Baxter). When collection is completed, the blood remaining in
the tubing is placed in a test tube to be used for grouping, cross matching and
serological tests. The filled flask, with the tube of blood attached, is sealed
and placed immediately in the refrigerator at 2· to 6· C. Both are labeled with
the proper blood group and the date of collection. When a patient is to be
transfused, his blood is grouped and then croSs matched with the oldest flask
of the proper group. If not compatible, the next oldest is tried and so on until
a satisfactory match is effected.
 BLOOD GROUPS AND BLOOD TRANSFUSION

The blood is then tested for hemolysis. According to Diggs and Keith, the
routine inclusion of this procedure has resulted in a decreased incidence of
reactions following transfusion. Their procedure consists of removing 10 cc. of
mixed blood, usually after the whole quantity has been filtered through sterile
gauze and transferred to the infusion flask. This blood is centrifuged and the
supernatant plasma removed. If this is definitely red, the blood is unfit for use;
if the discoloration is questionable or definitely absent it can be used.
The administration of stored blood is by drip infusion at such rate that the
hansfusion of 500 ce. requires from 30 to 60 minutes. The blood should not
be warmed to body temperature since hemolysis may result. No deleterious effects
bave occurred as a result of its intravenous administration at refrigeration or
room temperature.
Routine bacteriologic culture of all flasks is no longer deemed advisable.
Examination of a single flask selected at random each day serves as adequate
check on technic. Significant contamination can be detected grossly and gen·
erally causes sufficient hemolysis to necessitate discarding the blood.
Much work has been done or is in progress concerning the changes which
take place in stored preserved blood. There is general agreement that the red
cells show increasing fragility proportional to the length of storage time. Roe
reports that this change is definite in 3 days and after 8 days the cells hemolize
in salt sol\ltion 80 per cent isotonic. Wiener and Schaefer have made similar
investigations by determining the survival time of the transfused cells and find
an average decrease in survival time of about 6 days for each day of storage.
Fresh transfused cells remain in the circulation approximately 120 days while
cells stored for 2 I days before transfusion, survive only 24 hours. They recom-
mend storage for only 7 days at most.
Leukocytes, particularly neutrophils of stored blood, rapidly decrease in
number, showing degeneration and beginning disintegration as early as 24 hours.
Phagocytic activity is lost and Kolmer feels that these cells in transfused blood
are incapable of function.
Platelets are affected even more quickly, showing clumping and degeneration
in 24 hours; they become scarce in 48 hours and are practically absent after
5 days. Beck, Henry and Rosenstein report an average decrease from 216,000
to 44,000 per cu. mm. in 2 days.
Active substances in solution in the plasma are somewhat more stable, com-
plement being preserved for IO to 2 I days and isoagglutinins showing no decrease
for IO days and only slight reductions occurring thereafter. Bactericidal ac-
tivity as tested against Staphylococcus aureus, Streptococcus haemolyticus, B.
coli and B. typhosus is retained for 7 days but rapidly deteriorates with longer
storage.
Prothrombin time on stored blood by both Quick's and Howell's methods
was found prolonged and the prothrombin content of such blood determined
by Reinhold, Valentine, and Ferguson, showed reduction- to 55 per cent
in 6 to 8 days. Quick confirms these findings and feels that stored blood
is inferior to fresh blood in the control of hemorrbage in jaundiced states.
57 0 MISCELLANEOUS

Such deterioration of blood elements is in a degree influenced by the

type of preserving fluid used. Kolmer and Howard critically· compared
the rate of degenerative change in bloods preserved with four different fluids;
sodium citrate solution, Moscow Institute of Hematology solution (M.l.H.),
modified Rous-Turner solution (R-T), and Maizels and Whitaker solution
(M-W). The latter two differ from the others mainly in that they contain car):JO-
hydrate either as dextrose or dextrin, and show definite superiority as a preserva.
tive of red cells against dehemoglobinization, fragility, and disintegration. Supe-
riority with respect to other elements was noted but to a lesser degree. DeGowin
and his co-workers report similar observations with respect to red cell preservation
and recommend the Rous-Turner blood-dextrose-citrate mixture as safe and practi-
cal for human transfusions.
Rous-Turner Blood·Dextrose·Citrate mixture.
Blood ......... ~.. . . . . . ... ................. 10 volumes
Aqueous dextrose solution 5.4 per cent ........... 13 volumes
Sodium citrate (dihydric) solution 3.2 per cent. . . .. 2 volumes
These changes constitute a definite limitation to the use of stored blqod for
transfusion. Kolmer and Howard, Fox, Wiener, and others feel tbat ·stored
dtrated blood is useful in states of acute hemorrhage and shock but if more Wan
3 days old should not be used in the treatment of anemias, infections, or hemor-
rbagic dyscrasias.

TRANSFUSION OF STORED PLACENTAL BLOOD

Placental blood is used as a source of supply ·for blood banks in a number
of institutions. The technic for collection as described by Goodall and his co-
workers and by Gwynn and Alsever is reported to be harmless to the mother
and does not interfere with the normal routine of delivery. Both the Moscow
Institute of Hematology solution and dextrose preservatives are employed and
the blood used for transfusion after storage for as long as 50 to I20 days. Heyl
has found the use of such blood satisfactory in emergency transfusions or in
those where an adult donor is not available but prefers adult blood for elective
transfusion.

TRANSFUSION OF BLOOD PLASMA

Blood plasma has been shown by many investigators to be equally or even
more effective than whole blood for replenishing the protein content or restoring
or sustaining the fluid volume of the blood stream. Thus in many conditions,
some of which requiring prompt treatment, available plasma may be given im-
mediately and the delays necessitated by the use of whole blood avoided. 'fhe
administration of plasma is a safe procedure 9ut in this respect distinct jon must
be made belween plasma and serum. Plasma is the fluid portion separated from
oxalated or citrated blood while serum· is that removed after coagulation has
occurred. Thus serum contains little or no fibrinogen, an alteration of importance
perhaps since the intravenous administration of serum is attended by a high
incidence of febrile reaction. This occurrence has led to the almost univefsal
 BLOOD GROUPS AND BLOOP TRANSFUSION 571
abandonment of serum in favor of plasma for transfusion. For greatest use-
fulness plasma should be prepared in advance and stored so that it is immediately
available when needed. It may be collected from fresh dtrated blood or salvaged
from "bank" blood on which the time limit for safe storage has expired. Separa-
tion of cells from fluid may be accomplished by either sedimentation or centrif·
ugation. The latter method results in a greater yield of plasma but necessitates
the use of a special centrifuge head to carry the large size flasks. Strict asepsis
must be observed throughout. Aspiration of the plasma from the sedimented
cells may be accomplished by water suction or by the use of a special vacuum
flask. The latter is most convenient and offers less hazard of contamination but
is somewhat m<)re expensive than the other more crude method. Some workers
prefer to add to the finished product sufficient merthiolate to give a final con-
centration of I :10,000. This completely inhibits bacteria!" growth and may be
given intravenously without reaction.
Elliot and his co-workers recommend observation of the following precau·
tions where plasma is being prepared from freshly collected blood.
I. Use anly fasting donors. Plasma from persons who have eaten shortly
hefore collection of blood is often milky in color. This lipemia does not
ban its use but gives a product of unsatisfactory appearance.
2. Store whole blood in the refrigerator for 24 hours before converting to
plasma. This minimizes the amount of precipitate which forms in clear
plasma after a few days of storage.
3. Avoid shaking whole b·lood before it is transfused to centrifuge flasks.
This curtails hemolysis and prevents disc01or!'.ti9n of the finished product.
4. Refrigerate centrifuged blood for I2 .hours before aspiration of plasma.
This allows sedimentation of the fevt:i,ed'<;ceI1s not removed by the centri·
fuge. .'
s. Dilute plasma with an equal.'~olume of physiologic sodium chloride solu-
tion, plain or with 5 per ce';i:!Jextrose added. This also helps to prevent
formation of undesirable color or precipitate on standing.
6. Add mertkiolate to a concentration of I :ro,ooo as a preservative. Plasma,
even without added preservative, is quite stable with the exception of
the complement and prothrombin content. It is best stored at refrigera-
tion temperature although samples kept at room temperature for as long
as '70 days did not deteriorate, develop toxic products or lose their
therapeutic effectiveness.
Preservation may also be accomplished by desiccation according to the
"lyophile" method of Flosdorf and Mudd or the "adtavac" process developed
by Hill and Pfeiffer. Both of these procedures involve rapid freezing and dehydra-
tion in vacuum. The apparatus used is somewhat complicated to build and
operate and the method therefore is hest suited to volume production. Plasma
so processed is stored as a dry powder or a porous cake and appears to be com-
pletely stable for an indefinite period of time. This preparation is readily
soluble in distilled water which is added to the desirable concentration im-
57 2 MISCELLANEOUS
mediately before injection. A more simple and economical method of dehydra-
tion has been devised by Hartman. This utilizes cellophane cylinders in which
plasma is rapidly desiccated by revolving in a current of warm air. The re-
SUlting product is stable and readily soluble in distilled water.
Plasma may be administered intravenously by drip infusion, in dilute form,
normal strength or concentrated as the occasion demands. Hill advocates a con-
centration of four times normal for most conditions. There is no necessity fqr
blood grouping and cross matching since sufficient adsorption and neutralization
of incompatible agglutinins occur to obviate danger of reaction. This is par-
ticularly true where pooled plasma is used, as shown by the work of Levinson and
Cranheim where the mixture or pooling of a number of plasmas of different
blood groups results in a suppression of the agglutinins. The titer of such a
mixture is far lower than the titer of the individual plasmas before pooling.
Plasma must be filtered before administration and a stainless steel 200
mesh filter as incorporated in the Baxter apparatus is recommended. If such
is not available, several layers of sterile gauze through which sterile saline has
been rUn serves the purpose adequately.

INDICATIONS
The greatest present danger in the application of transfusion lies in its
prevalent and frequently unwarranted use as a therapeutic agent in lhe treatment
of disease. Therefore, it is important to consider some of the indications for its
use. It is difficult to set forth definite indications because each patient presents °a
different problem which must be handled on the basis of its own merits. To facili-
tate discussion, the somewhat arbitrary division into surgical and medical indica-
tions is used. .
In the former group the condition of shock has long been looked upon as
best treated by immediate transfusion. In light of more recent work, a distinc-
tion must be made between shock with or without hemorrhage. Where blood loss
has been considerable it is generally agreed that the transfusion of whole blood
is the procedure of choice. Where hemorrhage is not a complicating factor, there
is considerahle difference of opinion. Many writers are of the same opinion as
Bock, that gum acacia in saline may be just as effective as blood and it has the
advantage of greater availability. Other workers feel that there is a certain
amount of danger associated with the administration of acacia and believe, as does
Haden, that no fluid is comparable to blood in its ability to supply\he-physiologic
neeas present in shock. A more impurtant application is in the prevention of
shock where a preoperative transfusion is far more effective than the same pro-
cedure instituted in postoperative shock. The therapeutic use of transfusion in
hemorrhage is generally conceded with the possible exception of the hemorrhage
of peptic ulcer. Here, it is contended by some that this procedure may cause
added bleeding by increasing the blood pressure. There is general agreement,
however, that in the presence of a lowered blood pressure, increase to normal by
transfusion will not produce added hemorrhage, and if the pressure is already
normal the administration of blood will not alter it. It has also been show[l
 BLOOD GROUPS AND BLOOD TRANSFUSION 573

that the clot formed in exsanguinated individuals is soft and jelly-like but after
the transfusion of whole blood it is firmer and better able to block efficiently the
bleeding point.
Transfusion as a supportive measure in general surgical conditions has a
broad and rather loose application which from necessity must be dependent upon
the judgment of the surgeon. In such conditions as operable malignancy, the
actual basis for indication is usually anemia and here a transfusion may enable
the patient to weather the storm of an extensive operative procedure.
In infections, particularly of the blood stream, it is debatable whether trans-
fusion has sufficient specific effect on the course of the di,ease to warrant its uni-
versal advocation. In the past, transfusion in septic states was considered of
value only when a sufficient degree of anemia existed but now there is increasing
evidence that such treatment enhances the ability of the body to combat infec-
tion. Slightly better results have been recorded following the use of immuno·
transfusion. Here, either a donor having a high ti ter of specific antibodies
against the infecting organism, or one in whom the antibody titer lias been
increased by the administration of autogenous vaccine, is used. Such donors
are so infrequently available that the procedure has not enjoyed wide application.
In medical conditions, anemia is by far the most frequently encountered indi-
cation for transfusion. Ultimate judgment as to fue validity of the indication
must rest, however, not only upon the degree of anemia but also upon ilie causa-
tive factor involved. Generally when the hemoglobin content is down to around
40 per cent and ilie red cell count reduced to 2.0 to 2.5 millions, transfusion in
conjunction with other therapy is justified on the basis of more rapid recovery.
The use of the procedure where the red cell count is 4.0 millions or above, even
though there is considerable hemoglobin deficiency, appears unnecessary and
even economically wasteful, since ,it has been shown that red cells in excess of
the normal number are rapidly destroyed. In such cases adequate iron fuerapy
is usually sufficient. In the treatment of such conditions in elderly persons, par·
ticularly where there is some degree of myocardial failure, the factor of over-
loading the circulation must be considered. In younger persons with normal
cardiac musculature, this factor may be largely discounted.
A certain small per cent of iron deficiency anemias do not respond satis-
factorily to adequate ;Con therapy until a transfusion is given. According to
Haden the beneficial effect is probably on the basis of stimulation of hematopoietic
centers.
In the treatment of pernicious anemia transfusion was once the only thera·
peutic meaSure available, but due to the efficacy and economy in the use of liver
preparations, it is now seldom used. Occasionally an individual in severe relapse,
showing signs of extreme anoxemia and circulatory failure, may be sustained only
by transfusion, until sufficient amounts of liver can be administered.
The hemorrhagic diseases such as hempphilia, purpura hemorrhagica, and
hemorrhagic disease of the newborn are all benefited in a greater. or less degree
by transfusion. Patek and Stetson have shown that transfusion materially de·
creases the coagUlation time of ilie blood of hemophiliacs. They point out that
574 MISCELLANEOUS

this effect )s transient and in persistent hemorrhage a series of small transfusions,

One every second day, may be necessary until healing is well advanced. In
purpura hemorrhagica, of ei ther the secondary or the idiopathic thrombocytopenic
types, diminution of bleeding usually follows single or multiple small transfusions.
In the idiopathic type this method of treatment is considered by some to be far
superior to splenectomy. The transfusion appears to be fo'lowed by a rise in the
number of platelets greater than can be accounted for in the volume of blood
added. In any event, the procedure is of greatest importance in the preoperative
preparation of the patient for splenectomy.
The associated hemorrhages of obstructive jaundice may also be improved by
transfusion. In such patients, due to the remarkable though transitory beneficial
effect it has on the bleeding tendency, it is clearly indicated as a preoperative
preparatory measure. In familial hemolytic icterus, remission may be produced
by transfusions but the possibility of hemolytic reaction must be kept in mind.
Pretransfusion alkalinization to urine pH of 7.0 at least, is a wise precaution.
Many other conditions are characterized by associated hemorrhage, as well
as by extreme degrees of anemia. In the leukemic states, both myeloid and
lymphatic, the use of transfusion is a debatable question. There is complete
agreement that this therapy is only palliative since the eventual outcome is
purely a question of time. The medical profession at large, quite commendably,
feels that the use of any therapeutic measure capable of prolonging the life of
the patient is justifiable. Others take the view that prolongation of life by such
measures in this disease only serves to increase the. suffering of the patient and
the financial burden of the family.
In aplastic anemia, even of the idiopathic type, which so frequently eventu-
ates fatally, transfusion is indicated for two reasons: first, since cells administered
bave practically the same span of life as those formed by the recipient, repeated
tr,\nsfusions may tide the patient over until the marrow regenerates spontane-
ously or can be stimulated; and secondly, there is the possibility advanced by
many workers that transfused blood acts as a stimulating agent. The same line
of reasoning may be applied to the treatment of agranulocytosis, for this disease,
in a sense, may be looked upon as an aplasia of tbe myeloid elements of the bone
marrow. The results with this type of therapy, on the whole, however, have
been discouraging.
Poisoning by such agents as carbon monoxide or substances producing ex-
treme degrees of methemoglobinemia may produce fatal anoxemia by inter-
ference with the oxygen carrying ability of the red ceIls. Transfusion in such
cases may supply sufficient functional cells to prevent death until the toxic agent
can be neutralized or eliminated.
The availability of stored whole blood and plasma in addition to fresh whole
blood for transfusions necessitates consideration of the type of preparation which
may be used most effectively. The constituent of blood actually needed is not
always the same, £0 that rational choice of the proper preparation depends
upon a knowledge of the functional requirements in the various conditions
 BLOOD GROUPS AND BLOOD TRANSFUSION 575
previously discussed. These are logically considered on the basis of physiologic
function.
One function of blood is the transportation of oxygen by the red cells. In
those conditions characterized by red cell or hemoglobin deficiency, correction
can be accomplished only through the medium of whole blood transfusion. More-
over, fresh whole blood is preferable. If stored blood is used, it should not be
more than three days old since the increased fragility of the red cells in older
specimens so decreases the life of the transfused cells that any benefits are
distinctly temporary. Obviously the transfusion of plasma in anemic states can
serve no useful purpose.
The restoration of the fluid volume of the circulating blood is another
important function of transfusion. This involves not only the replacement of
fluids but the prohlem of keeping such fluid within the confines of the vascular
system. This is largely a question of preserving or restoring the normal osmotic
relationships hetween vascular and tissue fluids. These pressure relationships
are dependent primarily upon the protein content of blood plasma and secondarily
upon the electrolyte content. Deficiency of protein whether by loss from the
body, insufficient intake or abnormal passage into the tissues is therefore the
factor of primary consideration in states showing abnormality of fluid volume
or balance. In shock, Whether primary or delayed, the basic condition is
one of disparity between the volume of the vascular system and the bulk of the
contained fluid. The capillaries become increasingly permeable to the fluid
element of blood resulting in a tremendous loss into the tissues. The administra-
tion of isotonic solutions will not correct the deficient vascular bulk unless the
normal osmotic pressure relationships are restored by the administration of
protein. Whole blood either fresh or stored may be used but since red cells are
frequently not needed, plasma is the medium of choice for transfusion.
The same basic condition obtains with burns and with acute hemorrhage, in
each of which the primary need is for fluids and protein. The need for cells in
acute hemorrhage is of secondary importance and if shock is successfully com-
bated these will usually he replaced satisfactorily by the hematopoietic tissues.
The lack of need for red cells is even more striking in burned persons where loss
of fluid produces extreme hemoconcentration. Here the blood mu~t actually he
diluted since hemoglobin will cease combining with oxygen when its concentra-
tion rises above 130 per cent. Before extensive operative procedures where the
possibility of shock should be anticipated, its occurrence may be prevented by
increasing the patient's reserve of fluid and protein. In all of these conditions
plasma rather than )"hole blood transfusion is the procedure of choice.
Improvement of hemostasis is a third function of transfusion. This is a
b_road concept involving several types of deficiencies all of which may eventoate
in tbe same clinical manifestation, namely hemorrhage. Therapy effective in one
bleeding disease may be relatively useless in another, therefore rational treat-
ment of these states necessitates an understanding of the underlying hematologic
or vascular defect. In thrombocytopenic purpura, the deficiency is one of
576 M,SCELLANEOUS

platelets with hemorrhage due to delayed retraction and poor quality of the
clot. Transfusion is beneficial in producing remission and only fresh whole
blood should be used sinc~ stored blood soon loses its platelets and plasma of
course contains none. In secondary or allergic purpura without platelet deficiency
transfusion appears beneficial possibly as a source of easily available vitamin
C. Either fresh or stored whole blood, or plasma is equally effective. In
hemophilia the abnormality is one of prolonged coagulation. The effective sub-
stance in blood has been shown to be either a globulin or related to globulin
and is contained in the plasma. Therefore whole blood or plasma may be used;
preferably the latter if tbere is no co-existent anemia. The hemorrhage so
commonly associated with obstructive jaundice has been largely attributed to a
deficiency of prothrombin as evidenced by prolonged prothrombin time. Since
the prothrombin content diminishes rapidly in hoth stored whole blood and
plasma, ouly fresh blood or plasma should be used. Many investigators feel
that decreased fibrinogen is also a factor in icteric hemorrhage, and that this is
secondary and proportional to the degree of liver damage present. Transfusion
therapy directed toward such a deficiency may utilize either whole blood or
plasma.
In improvement of the immunologic defenses of the body those factors in the
blood likely to be effective are the antibodies, such as antitoxins, agglutinins,
opsonins and bacteriolysins, and non-specific agents as complement, leukins and
plakins. In addition, it is likely that transfused leukocytes are equally effective
as pbagocytes in the recipient. Specific antibodies would be effective only if
blood were taken from an immune donor, wbile the opsonin, complement,
phagocyte and possibly agglutinin content of random blood samples should be
beneficial. The therapeutic effectiveness of these agents depends largely upon the
age of the blood given. Complement is most stable, being well preserved for
periods up to 14 to 2 I days. Bactericidal activity is almost as stable, but the
leukocytes show definite degeneration within 72 hours. Kolmer believes this
latter factor to be most important and therefore recommends that for trans-
fusion in septic states without anemia, preferably fresh whole blood should
be used and in any event stored whole blood that is no more than three days old.
The stimulation of hematopoiesis by transfused blood appears illogical. It
is more likely that the administration of red cells actually inhibits the pro-
ductivity of the bone marrow since it tends to lessen any degree of anoxemia
which may be present and therefore eliminates a potent· activator of tbe eryth-
ropoietic tissues. Specific stimulating agents such as iron and the erythropoietic
factors are present in plasma but these substances can be given far more effectively
as liver extract or iron salts than by transfusion. ,
Finally, attention to indications is perhaps most important with respect to
plasma transfusion. It must be emphasized that plasma is a substitute for whole
blood only where certain specific effects are desired, namely (1) to replenish
deficiencies of blood protein, (2) to build up or sustain blood volume, and (3)
to reduce edema by hypertonic action.
 BLOOD GROUPS AND BLOOD TRANSFUSION 577
The types of blood preparations most effective in certain outstanding patho-
logic conditions in which transfusion is indicated are listed in Table XXX.

TABLE XXX
Condition Deficiency Blood Preparation
Anemia, chronic Red cells Fresh whole blood
Bums Fluid, protein Plasma
Hemophilia Unknown Whole blood or plasma
Hemorrhage, acute Fluid, protein Plasma or whole blood
Hemorrhage, with jaundice Prothrombin-Vito K Fresh~whole blood
Hemorrhage, of newborn Prothrombin-Vito K Fresh whole blood
Poisons (CO, etc.) Red cells Fresh whole blood
Purpura, secondary Vitamin C (?) Whole blood or plasma
Purpura, thrombocytopenic Platelets Fresh whole blood
Septic states Phagocytes and antibodies Fresh whole blood
Shock fluid, protein Plasma or whole blood
In addition to the above conditions, transfusion has been used in the treat-
ment of almost every known disease. This indiscriminate use of a valuable pro-
cedure may throw it into disrepute; so when resorting to it, there should be a
definite and valid indication. Blood transfusion should not be used as a procedure
of last resort merely to satisfy the desires of the family that nothing be left un-
done. Contraindications to transfusion are few. Two, which are absolute, are
the presence of pulmonary edema and cardiac fail nre. In known myocardial
weakness without failure, the procedure may be used if blood is given slowly.
To these may be added embolism, septic thrombophlebitis, .advanced nephritis
with anuria and possibly extreme allergy.

DOSAGE
The dosage of transfused blood for adults is generally placed at SOD ce. al-
though there is no rationale for the absolute use of this amount. Actually the
amount should vary from 200 CC. to as much as 800 cc. depending upon the need
and type of disease. Where an individual is exsanguinated from hemorrhage, it
may be necessary to give as much as 1000 CC. over a short period of time. In such
instances the patient's response must be the ultimate guide to dosage. Jones and
Rathmell feel that the amount of blood given should vary according to the square
meter body surface of the individual.
In children, dosage has long been calculated on the basis of body weight.
Wiener, quoting Halberstma, recommends IS cc. per kilogram of body weight as
an amount sufficient to increase the red cell count about one million per cubic
millimeter.
With respect to plasma, the dosage roughly parallels that of whole blood.
In effectiveness, 250 cC. of normal ,strength plasma or 500 cc. of dil uted plasma
is equivalent to 500 cc. of whole blood. Hill recommends, however, that no more
than 100 CC. of four times concentrated plasma be given at a single transfusion,
but that this amount may be given repeatedly during a period of a few hours.
 M,SCELLANEOUS

He emphasizes that plasma in any concentration must be administered as many

times as is necessary to produce the desired effect and that overloading of the
circulation may be prevented by withdrawal' of sufficient blood to compensate
for the volume of fluid added.

REACTIONS
Untoward reactions from all causes during or following transfusions may
be classified according to several types. Jones and Rathmell give a full classi-
fication based on the clinical features observed. Polayes and Lederer differ
in their approach, being more concerned with classification according to the
causes involved. Reactions, for simplification, may be divided into two gen-
eral groups; immediate and delayed. The immediate reactions are usually the
most severe and frequently eventuate in death. In the majority of such occur-
rences gross incompatibility through mismatched blood can be demonstrated.
Signs of this type usually become apparent after the injection of as little as 10
cc. of the donor's blood. Therefore, it is well to give the first few cubic centi-
meters slowly and cautiously. The first evidence that all is not well may be
flushing of the face followed by pain in the abdominal and lumbar regions. This
is succeeded by pallor, cold perspiration, t espiratory difficulty and may eventuate
in a condition not unlike shock. The patient may show rapid fall of blood
pressure, lapse into unconsciousness, and die in spite of repeated injections of
epinephrin. Fortunately, this type of reaction is rare but in the course of a
transfusion, whenever a patient becomes restless or complains of any discomfort,
the procedure should stop until the cause of the difficulty can be ascertained.
Transfusion of grossly incompatible blood, bowever, does no~ always result in
serious reaction. Wiener and his associates report two such cases in which the
degree of reaction was only slight and we have seen two persons each receive
500 cc. 01 blood a! a dillerent group (other than group 0) with no resulting
reactions.
Delayed reactions may be of several types. These sometimes follow non-
fatal reactions of the type described above, but more often come on one to twelve
hours after the completion of what appeared to be an uneventful and perfect trans-
fusion. The most commonly occurring type is associated with some degree of
hemolysis and is usually nonfatal. It is cbaracterized by chilliness and a slight
elevation of temperature, or in the more severe form by frank chills and a tem-
perature rise to 1030 to 104 This is followed by hemoglobinuria lasting twenty-
0
•

four to forty-eight hours. In some instances such a reaction may be accompanied

by anuria and terminate fatally. This added complication is thought by some to
be due to pr~viously existing kidney damage but its fatal occurrence has been
observed in persons whose kidneys have been shown to be normal clinically and
histologically. Baker and Dodd feel that it is due to the blockage of the kidney
tubules by precipitated hemoglobin in the form of acid hematin. Histologic
examination of kidneys from such fatal cases shows the tubules to be completely
filled with this material. DeGowin and Hardin, and Bushby and his coworkers
recommend alkalinization of the urine to pH 7.0 to prevent th~ formation of acid
 DLUUlJ \.:JRUUPS AND nLOOD lRANSFUbION 579
hematin and some investigators routinely alkalinize all patients as a pretrans-
fusion precaution, particularly where stored blood is to be used. The hemolytic
types of reactions are frequently due to technical error in grouping or cross
matching, indiscriminate use of the "universal donor," or to the presence 01
undetected subgroups. Wiener and Peters have recently reported three instances
of such reaction, one of which was fatal, due to intra-group incompatibility
from formation of anti-Rh agglutinins after repeated transfusion_
The milder types of reactions without remarkable hemolysis, not infre-
quently follow the administration of dtrated blood, and at times are caused by the
use of unclean apparatus but more often are due to improperly prepared salt
solution, Such reactions due to the presence of pyrogens (usually dead bac-
teria, non-pathogenic in nature) in distilled water generally are not hemolytic
in type. These consist of chilly sensations or frank chills followed by a rise
of temperature of short duration to 104°-r06° F., and are the types of reactions
which, in the past, have been attributed to citrate where indirect transfusion
was used. Such reaction does not interfere with the beneficial effects of the
transfusion.
Anaphylactic or allergic reactions are not uncommon, many instances hav-
ing been reported by Carrington and Lee, Duke and Stofer, Coca, and others,
These most frequently come on within two to twelve hours after transfusion and
are characterized by one or more manifestations of an allergic nature, such as
urticaria, angioneurotic edema of the face and hands, laryngeal or pulmonary
edema, and asthma or vasomotor rhinitis. Such an occurrence may be the result
of an extreme sensiti~ity on the part of the recipient to some food recently
eaten by the donor. Some writers feel that the reaction may be due to the effect
of altered protein in an already hypersensitive person and there are others who
question the belief that this type of reaction is necessarily allergic.
rolayes and Lederer include the transmission of disease under the head of
reaction to transfusion, and cite instances in which asthma, measles, small pox,
malaria and syphilis have been transferred in this way.
The rapid introduction of blood into the circulatory system is sometimes
attended by an abnormal response which has been called a "speed reaction,"
This may be mild in type characterized by distension of the peripheral veins,
cough, a sense of fullness in the head with vertigo, or acute cardiac dilatation
may result with collapse of the patient. This type of reaction is more likely
to occur with direct transfusion than with the citrate methods, since the latter
may be given as slowly as it is necessary.
Finally, persons with certain hemolytic blood dyscrasias, notably hemolytic
jaundice, are reputed to show frequent post-transfusion reactions which can not
be attributed to any of the usual factors, Sharpe and Davis feel tbat the inci-
dence of such reactions is perhaps not high when one considers the frequency
of transfusion in this type o! patient. They report two occurrences and we
have seen one patient die with anuria after transfusion in an hemoclastic crisis.
There are several views concerning possible causative factors. Sharpe and
Davis suggest that the delicate nature of the constructive-destructive ezythro-
58 0 1\.{rSCELLANEOUS

cyte equilibrium in hemolytic jaundice is so sensitive to varied influences that

transfusion may intensify the destructive function of the spleen. Dameshek
and Schwartz have observed an increased incidence of reaction when the uni-
versal donor was used and feel that this is due to the action of strong agglutinin~
on already fragile cells. As a second possibility, they suggest that the patient
with hemolytic jaundice may have sufficient hemolysin to destroy the donor's
cells. They recommend for all such patients the use of blood from homologous
groups only, a test for hemolysins in the patient's serum, and the avoidance, if
possible, of repeated transfusions to a group A patient. Other workers feel that
the answer lies not in any peculiarity of the disease but in the fact that these
patients are more likely to receive repeated transfusions and therefore may' be
included in that group of persons showing reactions due to the formation of
anti-Rh agglutinins. This seems likely when one considers the infrequency of
such reactions in the acute hemolytic anemia of Lederer where transfusion effects
such prompt cure of the condition that repeated administration of blood is rarely
necessary.
No difference in the incidence of reaction has heen noted regardless of the
use of fresh citrated whole blood or of stored preserved blood. Diggs, however,
feels that this is true only if careful attention is directed to the length of time the
blood remains in storage and to the presence or absence of hemolysis.
DeGowin and Hardin, on the basis 01 2423 transfusions, find that no types
of reactions occurred from preserved blood which are not recognized as com-
plications from transfusion of fresh blood. The liberation of potassium from
the cells of stored blood has been cited as a possible source of reaction. This is
insufficient in amount to influence the normal serum potassiurii~le;el of the
recipient and therefore would be unlikely as a cause of toxic effects.
The above considerations in connection with transfusion make it evident that
certain qualifications are necessary in the selection of donors, particularly where
professional donor bureaus are maintained. Such persons should be grouped not
only for convenience but also should be cross matched with the recipient imme-
diately before each transfusion. In addition to their general health being good,
their freedom from syphilis and malaria must be ascertained. Some investigators
feel that allergic disease in an individual is sufficient basis for exclusion as a donor.
Young adult males are most desirable. If they are giving blood at fairly fre-
quent intervals, periodic examinations should be made to determine that they
have the normal red cell and hemoglobin content. Such donors, when selected to
give blood, should not eat within two hours immediately preceding the transfusion.

MEDICO-LEGAL APPLICATIONS OF BLOOD GROUPS

Determination of the four blood groups AB, A, Band 0, and the three types
1\1, N, and MN has had extensive application to the medico-legal problem of
exclusion of parentage as well as to the identification of blood and other types
of stains. The more recent work with agglutinogens Rh and P suggests the pos-
sibility of similar application with these blood types. Tests involving exclusion
 BLOOD GROUPS AND BLOOD TRANSFUSION

of parentage are based upon the mode of inheritance of the agglutinogens. These
are transmitted in such fashion that knowledge of the parents' blood group or
type permits prediction of the groups or types possible in an offspring.

INHERITANCE
Two theories have been advanced to account for the inheritance of the isoag-
glutinogens. That of von Dungern and Hirschfeld, postulating the Occurrence of
two independent pairs of genes, enjoyed general favor for many years but since
1925 the Bernstein theory has been generally accepted. This theory bases the
transmission of these characteristics upon the existence of three allelomorphic
genes, A, B, and R (called 0 in Levine's discussion). Agglutinogens A and Bare
transmitted as dominants over the recessive R (absence of both A and B) and
therefore must be present in at least one parent to appear in a child. The com-
bination of any two of these genes (one from each parent) gives rise to a child
of a certain genotype from which the phenotype or blood group is determined.

TABLE XXXI

. . . . . . . ..
Genotype Phenotype or Blood Group
AB ............... . AB
~! A

BR ............... . B
RR ............... . o
Considering all combinations of blood groups in parents, there arises the
possibility of the occurrence of certain blood groups and the impossibility of
others in the resulting children. These are listed in Table XXXII.

TABLE XXXII
Parents Cbildren Possible Children Not Possible
OxO o A,B,AB
OxA O,A B,AB
OxB O,B A,AB
AxA O,A B,AB
BxB O,B A,AB
AxB O,A,B,AB
OxAB A,B O,AB
AxAB A,B,AB o
BxAB A,B,AB o
ABxAB A,B,AB o
The genetic relationships involved in the above summary are considered in
excellent discussions by Wiener and by Levine. From the above table two laws
of heredity may be postulated: first, that agglutinogens A or B cannot appear
in the blood of a child unless they are present in one or both parents; and sec·
ond, that a group AB parent cannot have a group 0 child or a group 0 parent a
 M,SCELLANEOUS
group AB child. Exceptions to these laws are extremely rare, there being orlly
a single valid one reported in wliich a mother belonged to group AB and ller
child to group O. Others appearing to be exceptions can be explained OIl" the
basis of a subgroup in one parent being mistaken for another group, or actllal
illegitimacy of the child in question.
Agglutinogens M and N are inherited as two allelomorphic genes, both domi-
nants, and give rise to the following genotypes and phenotypes (blood type!').

TABLE XXXIII
Genotype Phenotype (blood type)
MM M
NN N
MN MN

According to the theory of Landsteiner and Levine, determination of M and

N bodies in the red cells of parents makes possible the prediction of the blood
type of the offspring (see Table XXXIV).

TABLE XXXIV
Parents Children Possible Children Not Possible
MxM M N,MN
NxN N M,MN
MxN MN M,N
MxMN M,MN N
NxMN N,MN M
MNxMN M,N,MN

rrom 't'nese possitiii)'iles, 'two iaws 01 nereol'ty, appllca'oie'to a'fu'ila'60n ca~"tfl>

may be stated; first, agglutinogens M or N must be present in one or both parepts
to appear in an offspring; and second, the combinMions (type M parent and
type N child) or (type N parent and type M child) cannot occur.
No valid exceptions to these two .laws have been reported so they may be
accepted without qualification.

LEGAL APPLICATION
The practical application of these laws in the solution of the medico-legal
problems of affiliation cases offers considerable possibility, but obviously, with
respect to the accused man, such evidence can be valid only in a negative wl).y·
It may prove that he could not have been the father hut unless all other men of
his blood group can be definitely eliminated, such information cannot be used
as positive evidence. If all .men were falsely accused, the average chance of
proving non-paternity by determination of the A-B groups is about 16 per cent
and by the M-N types ahout IS per cent. Thus if both are determined the
chances of exclusion are increased to between 35 and 40 per cent.
Several instances have been reported in whicb newborn infants were acci-
dentally assigned to the wrong mothers or in which this occurrence was suspected.
 BLOOD GROUPS AND BLOOD TRANSFUSION

In a certain number of these (about 40 per cent) the determination of the A-B
groUp makes it possible to clarify such situations. The additional determination
of the MeN types raises the possibility of solution to almost 70 per cent.
Similarly, determinations of blood groups and types may be applied to the
identification of blood stains in criminal cases. Considerable stability of the ag-
glutinogens and agglutinins, in respect to ordinary conditions of drying and aging,
has been demonstrated by Landsteiner and Richter. Levine cites tbe instance
of the demonstration of agglutinins a and b in a group 0 blood stain four years
old. In spite of such stability it is desirable to perform these tests on the freshest
possible material so Wiener suggests that in all homicide cases, blood grouping
and typing be performed immediately on all blood stains as well as on the blood
of the deceased, and this information filed for future possible need.
Agglutinins identical with those characterizing the blood of an individual,
are present in saliva and seminal fluid as well as in all tissue fluids. The de-
termination of only the A-B groups is possible but this may be of value particu-
larly in the identification of seminal stains in rape cases.
The tecbnic for the examination of stains is based upan those principles al-
ready described for blood grouping but differs with respect to details of pro-
cedure since such small quantities of material are available for examination.
The procedure for these tests is given in detail in Wiener's work on Blood Groups
and Blood Transfusion (see bibliography).

ATTITUDE OF COURTS
The attitude of the courts toward such evidence varies considerably in this
country and abroad. The application of these principles medico-legally is widely
accepted in all European countries, Brazil and Japan, but in the United States
it is rarely admissible as evidence. The judiciary generally feels that these pro-
cedures are still in the stage of experiment and development which precedes their
acceptance as scientific fact. This attitude may be summed up by the following
excerpts from Woodward's discussion of Muehlberger's paper. The Court of Ap-
peals of the District of Columbia stated, "Just when a Scientific principle or
discovery crosses the line between the experimental and demonstrable stages is dif-
ficult to define. Somewhere in this twilight. zone the evidential force of the prin-
ciple must be recognized, and while courts will go a long way in admitting~expert
testimony deduced from a well recognized scientific principle or discovery, the
thing from which the deduction is made must be sufficiently established to have
gained general acceptance in the particular field in which it belongs." Also,
directly concerning the proving of paternity or nonpaternity, the Supreme Court
of South Dakota, in affirming a judgment of a trial court excluding such evi-
dence, states, ((Without endeavoring to arrive at any decision on other ques-
tions involved in connection with this particular claim of error, we hold that
the learned trial judge did not abuse his discretion in refusing to order the blood
test requested by appellant. We base such holding specifically upon the proposi-
tion that it does not sufficiently appear from the record in this case that modern
medical science is agreed upon the transmissibility of blood characteristi~s to
 MISCELLANEOUS

such an extent that it can be accepted as an unquestioned scientific fact, that if

the blood groupings of tbe parents are known, the blooj group of the offsp(ing
can be necessarily determined, or that, if the blood groupings of the mother and
child are known, it can be accepted as a positively established scientific fact that
the blood group of the father could not have been a certain specific characteristic
group. In other words, we think it insufficiently appears that the validity of the
proposed test meets with such generally accepted recognition as n scientific iact
among medical men as to say that it constituted an abuse of discretion far a
court of justice to refuse to take cognizance thereof, as would undoubtedly be the
case if a court today should refuse to take cognizance of the accepted scientific
fact that the finger prints of no two individuals are in all respects identical."
Some progress has been made in establishing the reliability of the procedure
in the mind of the courts since two states, New York and Wisconsin, have re-
cently enacted laws authorizing the courts to order blood grouping tests anel to
receive the results in evidence. However, remedy for the general situation ptill
lies first in the fostering of confidence of the courts in the procedure and in its
underlying principles; and secondly, in the enactment of legislation compelling
individuals to submit to blood tests. To effect these changes, the performance
of these tests must be limited to qualified persons who are expert in their (leld
and to this end, the American Medical Association Committee on Medicolegal
Blood Grouping Tests has set up a standard for technic to be employed in the
tests, and has recommended that some medical body such as the American Mtdi·
cal Association be authorized to examine and judge the qualifications of persons
purporting to be experts in the field.

BIBLIOGRAPHY

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BELK, W P .• HENRY', N. W., and ROSENSTEIN, F.: "Observations of human blood stored at 4 to
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 BLOOD GROUPS AND BLOOD TRANSFUSION

DEGOWJN't E. L., and HARDIN, R. C.• and HARIHS. J. E: "Studies on preservea human blood.
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DEGOWlN', E. L., and HARDIN, R. C., and SWANSON, L. W.: "Stuwes on preserved human blood.
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Dl:GoWlN, E. L, HARRIS, J. R, and Puss, E. D : "Studies on preserved human blood. I. Various
factors influencing hemolysis.1.l' Ibid, J 14, 8so, 1940. "II. DIffusion of potassium from the
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Jour. Amer. }.{ed. A.ssn., 115, 389. 1940.
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serums and other biological substances" Jour. lmmunol, 29, 389, 1935.
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JANSKY, J.: uHaematologkk6 studie u psycbotiku (Etudes hematologiques, dans les maladies
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KOLMER, J. A, and HOWA:RD, M; "Studies on the preservation of human blood.1I Amer. Jour.
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fektionskrankheiten~ 27, 35i, 1900.
58 6 M,SCELLANEOUS

LA..."iDSTEINt:R) K.~ and LEVINE, P.: "On the cold agglutinins in human serum." Jour. Immun"
12,-441, 1926.
Jour. Exper. Med J
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Report of two cases." Jour. Amer. Med. Assn., 110, 2°53, 1938.
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the significance of the buffy coat in the shed blood." Jour. Path. Bact., 6, 303, 1900.
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WIENEl\, A. S.· "Blood groups and bloo-d transiu5ion." :md Ed. Charles C. Thomas, Baltimore,
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 BLOOD GROUPS AND BLOOD TRANSFUSION

\VIENtR, A. S., and S~RJ G: ULimitations in the use of preserved blood for transfusion"
}.fed. Clin. North Amer., 24, 705, l:940.
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'VUOIN, S. S: uTransfusions of cadaver blood." Jour. Amer. Med A.s$oc., 10,6, 997, 1936.
 PLATE LIII

BLOOD PICTURES IN VARIOUS LABORATORY ANIMALS

Upper left' Norroal human blood sbowing rcd cells as round tl1cQncave dis.cs, non-nuclea.ted, aver-
age dIameter .,.'1. micra. Cell I a lymphocyte. cell 'Z a. monocyte, cell 3 a mulblobed neutrophil.e,
cell 4 a norma.l red cell, cell 5 a. group of thrombo(:ytes or blood platelets. Monkey blood is
identical.
Upp(:( right: Blood of a large white rat, one of Wistar strains for laboratory purposes. Red cell!
round, biconcave discs as in human Average dIameter 6.3 mirra. Neutrophiles :27%, lympbo-
. cytes 65%. White ceUs sirnilar to- human. Similar blood picture seen in mice, other rats,
guinea pigs, cats, dogs, horse, mule, ass, pig, cow and ox, except for percentage variatIOns in
white cells
CeU 7, large irreguJar lymphocyte, teU :2 a small lymphocyte, cell 3- a normal red cell. No
platelets shown since they are scarce.
Center left: Blood pkture of a n-ormal rabbit (New Zealand) used for laboratory purposes. The
red l:eUs are round, biconcave discs, average diameter 6"J micra, and identical with human
Rabbit does not have typical neutrophi.1s, but has 400/0 cells with multilobed nuclei and upseudo-
eosinophllid' cytoplasmic granules These are called amph-ophlls, as seen iit cell z. Cell:;J is
a basophile, cell 3 a nonnal red cell, and cell 4 a group of platelets. • ~
Center right; The blood of a camel, drawn fr-om actual smear. Shown because the cameJ,and
members of the cam(>] family are the .only animals with oval, non-nucleat-ed red
celIs._ Average
ceI1 site 5 x 8 micra. The sheep and goat have mainly round biconcave discs with a few oval
non-nudeated ceIls. CeIl I a lymphocyte, cen 2 an eosinophu, cell 3 the normal;red cell,
cell 4 a group of plateleb sUnilar t{l human,
Lower left: Rlood -of a <,-hlcken; and $imilar to other fowls Most red 1:ells at!? (lvl\l ~nd nudE'ated
Average size 7 x 12 micra. A few round cens may be seen. No typi-cal neutrophlles About
30% of white cells are multilobed with spindle shaped cytoplasmic eosin stained bodies as lD
cell 2. Cell 1 a group of platelets, cell 3 a lymphocyte, cell 4 a normal red cell, cell S an
eosmopbile. , ' ~ "';.-
LoN:~t:~i~iesrr:~:l~~. °hJJ f:O:'Iy!~go~~, ~~li12a~~ob~~;a~~ym~h:C~1: r::;% ~;:: ;i~
type), cells the normal red cell.
 CHAPTER 45
THE BLOOD PICTURE OF NORMAL LABORATORY
ANIMALS
Many times we have had occasion to seek information concerning the blood
picture of normal laboratory animals. This had always been difficult to find,
since apparently few studies have been made on various animals in a sole effort
to establish the normal. SUcil studies that are available are usually incorporated
as control findings in various experiments.
Such was the situation until Robert A. Scarborough published his monograph,
"The Blood Picture of Normal Laboratory Animals,"* which was submitted as a
thesis at the Yale University School of Medicine 'in 1927. In this excellent and
comprehensive review, obtained from nearly 600 sources, this information is now
available. We have obtained most of the figures from that source, and added
some comments of our own, based on ten years' experience in studying the
hematologic picture in rabbits, guinea pigs, dogs, chickens, rats, sheep and mon-
keys, under various experimental conditions.
We feel that'the inclusion of this chapter would make available this data
which is so difficult to obtain by one who would investigate some hematologic
problem involving one or more of the laboratory animals. 'Furthermore, such
information is not_.entirely without practical value. On one occasion we received
a blood film from a colleague who requested a "diagnosis" without providing any
informati07'-as to the source of the blood. Much to our surprise all of the red cells
were oval and non-nucleated. We made a correct diagnosis of "blood from a
camel," much to the amazement of our colleague, who did not know that he had
sent blood (obtained at a circus) from the only animal whose red cells are oval
and non-nucleated. Thus, one never knows when such academic information may
bave practical application.
The red cells in man and in all other mammals are bi-concave circular discs
without nuclei, with the exception of the camel and other members of the group
camelidae. This would include, of course, the cells of the dog, horse, monkey,
sheep, rabbit, guinea pig, rat, and mouse, which are often used in experimental
studies.
In the chicken, an example of the avian group, most of the red cells are oval
and nucleated, and tIrose of the frog, representative of the amphibian group, are
also oval, nucleated and bi-convex.

THE RABBIT

The rabbit is an excellent animal for many types of experimental work,

mainly because of the adequate sources of supply and also because of the
docility of the animal. However, there are certain disadvantages in using rab-
• SCARBOROUGH: uThe Blood Picture of Normal Laboratory Animals.~' Yale Jour. Bio. and
!ftd., 1930-31-32. Can be obtained from Yale University Press ($1.50).
59 0 M,SCELLANEOUS

bits, the chief one being that it is entirely herbivorous and it follows that its
metabolic activity and hematopoietic factors affecting the blood picture may
be quite different from those of the carnivorous animal. Therefore, the results
obtained in experiments on rabbits are always open to question because of
that reason.
Blood in small quantities can best be obtained froIll rabbits by puncture
of the marginal ear vein. It is wen to first shave the area after washing it with
soap and water. The blood for leukocyte counts is available by puncture of
the vein with an ordinary needle. When larger quantities of blood are required
this can be obtained by cardiac puncture which seems to cause the animal little
discomfort. This can be done without anesthesia although a light anesthesia
is not harmful. The following table shows the normal blood findings for this
animal.

Erythrocyte count-Normal range 4,500,000 to 7,ooo,ooo--Average 5,620,000

The erythrocytes are similar to human red blood ceUs with considerable anisocytosis,
poikilocytosis and polychromatophilia, but no nucleated cells.
Hemoglobin .................. Normal range 60% to 90% (Sahli)
-Average 75% (Sahli)
Leukocyte count. ... Normal range 4,000 to 13,ooo-Average 7,<)00
Differentia! count:
Range Average
N eu trophils ............... . 30-So % 43·4%
Lymphocytes ............. . 30-50 % 41.8%
Monocytes ................ . 2-16% 9. 0 %
Eosinophils _.............. . o·S- s% 2.0%
Basophils ................. . 2- 8% 4·3%
In general, the blood is very similar to that of the human, the chief differences
including marked anisocytosis, poikilocytosis' and'polychromatophilia of red cells
and the amphophilic quality of the nentrophllic leukocytes. The blood of
rabbits normally contains no nucleated red cells. The reticulocyte count is
from one to two per cent and the average cell size is 6.7 micra. Compared to
the human the lymphocytes are slightly increased. The total number of leuko-
cytes varies more widely and there seems to be a rather marked immaturity of
granulocytes in the normal animal. The normal platelet count ranges from
200,000 to 1,000,000; the average is about 400,000. The coagulation time ranges
from three to six minutes; the average is three to four minutes. The resistance
of the red cells to salt solution is about the same as that of the human.

THE GUINEA PIG

Blood is ordinarily obtained from the guinea pig by cardiac puncture. This
is best done under ether although it can be done without anesthesia. On numerous
occasions we have withdrawn blood from the heart of a guinea pig without
anesthesia and it appears that the procedure is attended by very little discomfort
to the animal. Apparently cardiac puncture affords little trauma of a serious
 THE BLOOD P,CTURE OF NORMAL LABORATORY ANIMALS 591
nature because of the fact that this procedure can be dohe repeatedly over a long
period of time and apparently without the animal suffering ill effects. We have
bled guinea pigs to obtain complement for the Wassermann test as often as once
weekly for periods of over a year. The following table gives the normal figures
for the guinea pig.

Erythrocyte count-Normal range-4,Soo,ooo to 6,Soo,ooo-Average 5,750,000

In general the erythrocytes are similar to human red blood cells, with considerable
anisocytosis and polychroma tophilia, and no nucleation.
Hemoglobin. . . . . .. . ....................... 80%-100% (Sahli)
Leukocyte count. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6,000 to 20,000
Differential Count:
Range Average
Pseudoeosinophils (Neutrophils) 3-5 0% 41.8%
Lymphocytes ............... . 3S-SS% 45.3%
Monocytes ................. . 1-20% 8.4%
Eosinophils ................. . 2-1 5% 4. 8%
Basophils ................... . 0-2% 0.7%
There are certain differences in the leukocytic picture of the guinea pig as
compared to that of man, including a definite increase in the total leukocyte COUllt
as the animal grows older. It has been said that the young guinea pig is rel<t-
tively leukopenic..Pregnancy causes a moderate leukopenia with relative lympho-
cytosis. There is also a definite digestive leukocytosis. The platelets avera!!e
about 500,000 per cu. mm. Coagulation time is about ,five minutes and the
resistance of red blood cells ranges from 0.3.' to 0.42.

TREMOUSE

The usual method of obtaining blood from a mouse is from the tail tip. How-
ever, this is open to the same criticism as stated concerning the rat, that is, the
process causes an acute inflammatory reaction at the tail tip. Therefore, f()!
cellular studies it is better to introduce a needle either in the femoral artery or In
the heart. The following table shows the normal figures .for the mouse:

Erythrocyte count-Normal range-8,ooo,ooo to II,ooo,ooo-Average 9,7OO,0C)0

The erythrocytes are round biconcave discs, generally non-nucleated, with sligbt
anisocytosis and marked polychromatophilia.
Hemoglobin ................. , ... , .. , ..... 90% to 100% (Sahli)
Leukocyte count. .. ... . .... , .. ',. ,... . .... ,Average 8,450
Differential count:
Range Average
Neutrophils ..... , , .... . 20-40 % 262%
Lymphocytes. . ... , ..... , 55-75% 67. 8%
Monocytes ..... , ....-: ... . 1-15% 7.5%
Eosinophils, . , , ............. . 1- 5% 2.0%
Basophils ................... . 0-,% 05%
59' MISCELLANEOUS

There appears to be very little information available concerning blood plate.

lets, coagulation time, resistance of red cells, cell volume, etc., of the mouse.

THE DOG

Blood in small quantities can be easily obtained from the marginal ear vein
of the dog after first washing the area with soap and water and then shaving
it. It is usually necessary that the animals be restrained during this procedure,
but oftentimes they are trained readily to submit to this type of puncture with
little or no resistance. Large quantities of blood can be drawn from the femoral
vein of the dog or from the heart. The following table shows the normal figures
for the dog:

Erythrocyte count-Normal range-S,5°O,ooo to 8,ooo,ooo-Average 7,220,000

The red cells are round biconcave discs, rather pale, often appearing as ring forms.
There is considerable anisocytosis and nucleation.
Hemoglobin ............................. , 90% to 100% (Sahli)
Leukocyte count. ... Normal range 6,000 to 20,000-Average II,840
Differential count:
Range Average
Neutrophils ...... , .......... , 60-75% 69. 0 %
Lymphocytes. . . . . . . . .. . ... . 10-30 % 20.0%
Monocytes .................. . 2-12% 6.1%
Eosinophils .............. ,' .. . 2-10% 5. 0 %
Basophils .................. , 0-2% 0·7%
The reticulocyte count averages 0.6 per cent and ranges'from 0 to 1 -4 per cent.
The average red cell diameter is 7.0 micra. The leukocy,tes in general are very
similar to those of human blood. Normal dogs oftentimes have what appears
to be an immaturity of granulocytes. In young puppies the leukocyte count is
approximately double that of the normal adult. There is a definite digestive
leukocytosis. The effect of anesthesia on the dog has been carefully studied and
the leukocyte count is elevated by the excitement and struggling incident to
this procedure. The platelets average about 350,000 per cu. mm. Coagulation
time varies from eight to ten minutes. The resistance of red cells ranges from
0.33 to 0.36.

THE MONKEY

Blood is best obtained from the monkey by puncture of the ear with a
spring lancet. The red and white blood cells of the monkey are very similar to
those of man, with the exception that the lymphocytes are somewhat larger. The
following table gives the normal figures for the monkey:
 THE BLOOD P,CTURE OF NORMAL LAllORATORY ANIMALS 593

Erythrocyte count-Normal range-s,ooo,ooo to 7,ooo,ooo-A~erage 5,590,000

The red cells. are biconcave discs showing some variation in the size, with mo-derate
polychromatophilia, and no nucleation.
Hemoglobin ..................................... 90% (Sahli)
Leukocyte count. .. . Normal range 8,000 to :25,ooo-Average 16,210
Differential count:
Range Average
Nentrophils .............. , . 30-5°% 4 2 • 2%
Lymphocytes ............. . 4o-<iO% 5 2 • 8%
Monocytes ................ . I-12% 1.5%
Eosinophils ....... , , , .. ,., , 1- 5% 3·7%
Basophils .. ,." ... , , .. ,," O.I-O·S% 0.3%

The reticulocytes average 0.3 per cent. The average red cell diameter in
one species is 7.r micra, in another 6,7 micra, and in another 64 micra. The
blood platelets average 250,000 per cu. mm. The resistance of red cells is about
the same as that of the human.

THE RAT

The usual method of obtaining blood from the rat is by snipping off a tip
of the tail with either a sharp razor blade or pair of scissors. This can be
done repeatedly; however, it should be pointed out that the consistent repetition
of this soon results in an inflammatory reaction in the tail tip, Therefore, leuko-
cyte studies from rat tails are of little value for this reason. Blood can also be
ohtained by cardiac puncture: The following tahle shows the normal ligures
for the rat.

Erythrocyte count-Normal range 7,000,000 to Io,ooo,ooo-Average 8,500,000

The red cells are hiconcave discs showing moderate anisocytosis, marked 'poly-
chromatophilia and an occasional nucleated cell. •
Differential count:
Range Average
NeutrophiIs"., .... , , .. , .. " . 15-40 % 27. 0 %
Lymphocytes, , , ............ . 50-80% 67·9%
Monocytes ... , ... , ..... ,.,. 2- 7% 5·3%
Eosinophils ................ . 0- 4% 2.r%
BasophiIs,. , . . . . . . . . .. , .... . 0-1 s% 0·7%
I
The blood platelets average from 600,000 to 700,000 per Cll. mm. The coagu-
lation time is three minutes plus. The outstanding difference in the leukocyte
picture of a rat and a human is the marked relative lymphocytosis which also
is an ahsolute lymphocytosis.
594 l\IISCELLANEOUS

THE CHICKEN

Blood may be readily obtained from a chicken by puncture of the veins in

the wing. It seems to coagulate quickly. The following table gives the normal
counts in a chicken:
Erythrocyte count-Normal range 2,800,000 to""'4,5°o,ooo-Average 3,440,000
The red cells are oval and nucleated. However, there are a few round nucleated
and non-nucleated forms.
Hemoglobin .... ........................•.... •. 60% (Sahli)
Leukocyte count ... Normal range '0,000 to 40,ooo-Average 25,900
Differential count:
Range Average
N eu trophils ................. . .0-4°% 31.1%
Lymphocytes ............... . 40-60% 51 . 6 %
Monocytes._. . . . . . . . . . .. . .. . 5- 5%
1 10.0%
Eosinophils. . . . . . . .. . ...... . 2-10% 5-9%
Basophils ................... . 1- 4% 2.6%

THE FROG

There seems to be only one method of obtaining blood from a frog and that
is to kill the animal and blood can then be taken from the heart or any other
source. The following table shows the normal count of a frog:
Erythrocyte count ........ Normal range] 400,000 to ·600,ooo-Average 460,000
The red cell of the frog is oval and biconvex, with an oval nucleus. Some of the
cells are small with a small nucleus. Non-nucleated forms may also be found.
Hemoglobin ................................ (' ... 80% (Sahli)
Leukocyte count (Average).... . . . .. ....... . . . . . . . . . .. 18,310
Differential count:
Average
Neutrophils . . . . . . . . . . . . . . 7%
Lymphocytes. . . . . . .. ...... ... . .. .... 59%
Eosinophils ............................... 27%
Basophils.......................... ...... 7%
There are very few or no reports on the number and type of platelets, coagu-
lation time, resistance of red cells, etc.
NOTE.-AU of the ngures given in this summary of the blood picture of laboratory animals
have been taken from Scarboroug~'s Monograph (reference previously cited).
 SECTION EIGHT
HEMATOLOGIC TECHNIC
 SECTION EIGHT
HEMATOLOGIC TECHNIC

CHAPTER 46
HEMATOLOGIC TECHNIC
The number and variety of technical methods increase as efforts are mllde
to perfect hematologic procedures. Almost every worker devises meth~ds of his
own and various modifications of standard procedures, so that mote confusion
exists in this phase of hematology than in the field of terminology.
For example, in a survey of the various methods for the determination of
hemoglobin and the conflicting results that are obtained, it becomes evid,mt
that when one worker reports a hemoglobin percentage of 80 by a certain methpd,
there is no assurance that this corresponds to the same figure obtained hy ;,n-
other worker using a different method, or even the same method. Many physi-
cians doubt the accuracy of their hemoglobin determinations and are at a !IlSS
to know what method to use. Many of them then revert to the use of the
TaIlquist §Cale, believing that they can make as accurate determinations with
this as with the more complex methods. The failure to adopt a standard metl:lo d
throughout the country is largely responsible for this confusion. From time to
time, new methods have appeared which, with few exceptions, have been modi-
fications of the same old principle of, conversion of hemoglobin into acid hematin,
the estimation of color change with the eye, and comparison with a color thaI· is
considered tbe average standard. The diversity o! metnods is evidence, bow-
ever, that laboratory workers have endeavored to provide better and more
accurate procedures.
Similar situations exist through the entire range of technical procedures in
hematology until a collection of these would fill many volumes_ In the following
pages, devoted to hematologic technic, we shall present only one or two methOill
for each procedure, giving those that have proved most successful in our hands.
Perhaps others could be included with more justification, as being more accurate
though more complex, but we have chosen these not only for accuracy, but be-
cause of simplicity, ease of execution, and reliability. For example, in our
laboratories we use Wright's stain exclusively for routine work with the full
knowledge that many excellent workers prefer Giemsa's stain, and knowing' also
that under certain circumstances and for the identification of certain cells
Giemsa's stain is probably superior. Wright's stain is easily prepared, simple
to use, even for the inexperienced, and when proper technic is employed, the prepa-
rations are adequate for detailed study. The only time we use Giemsa's staiIl is
when we have a large number of slides (roo or more) to stain simultaneously,
In presenting a collection of technical methods we would emphasize only
090
 FIG. 39.-A suitable tray for hematologic work constructed of light wood, size appro:x:matdy
12 x 14 inches. !\-Qte bottles for alcohol, diluting fluids and tourniquet in first compartment,
sterile wrapped syringes in second compartment, dirty syringes and s.oiled cotton in thir.d com-
partment. Also note rack for holding pipettes., steri1e needles kept in cotton plugged tubes. The
holes are perforated in three different sizes. Slide ~lot5 are at ODe end in which two slides are
placed b.'1ck to back; clean cotton beneath small pad on (:orner.

FIG. 4~.-Same tray as shown above. It can be constructed by any good carpenter with
variations to meet the requirements of any particular laboratory. Designed by Clara M. Becto::1,
St. John's Hospital, Tulsa. Okla. If the above tray is cop:;;tructed with a handle it can be car-
ried more conveniently. Various types of hematological trays are now available and can be
purchased on the open market.
 HEMATOLOGIC TECHNIC 597
one point, which would apply to any technical procedure, that is, careful atten-
tion to detail in the execution of a test. If a blood film is too thick, no staining
method wiII be satisfactory, and if a worker stains a blood film and fails to mix
the stain properly with the diluent and obtains an unsatisfactory preparation, he
should remember that the fault is not with the method.
In presenting hematologic methods, we shaIl attempt to point out the under-
lying principles of the various tests, since it is obvious that the Jaboratory worker
should understand these; also, we shaIl endeavor to specify the more exacting
details to be foIIowed, and the usual sources of error. A simplified procedure for
making a thorough examination of blood is presented in the chapter on normal
blood, page 115.

OBTAINING BLOOD FROM ADULTS

For ordinary laboratory procedures blood may be obtained in sufficient

quantities from the finger or the lobe of the ear in an adult, and from the plantar
surface of the heel in an infant. If the ear is used the edge of the lobe, and not
the side, should be punctured. The finger is more convenient with bed-ridden
patients, though the ear is less sensitive.
Edematous, cyanotic, or traumatized areas should be avoided. For ordinary
purposes the middle or ring finger is generaIly used, and is held between the
thumb and forefinger of the operator. It should be held tightly enough that
the area becomes slightly engorged with blood or else stroked briskly to promote
circulation. If the finger is cold, a warm hand bath is indicated.

FINGER PUNCTURE

~faterials and Equipment

I. Instruments for puncture should have a sharp cutting edge. Some of the
instruments for this purpose are:
Hagedorn needle (a cutting needle).
Automatic blood lancet.
Bard-Parker knife blade (size eleven) pushed through a cork stopper.
The cork may be used for the alcohol bottle.
A needle or pin with a round point should never be used because the
wound is deep, seals quickly, and little blood is obtained. If the
point of a puncture instrument becomes rusty, it should be discarded
immediately.
•. Cotton.
3· Alcohol (70ro).
4. Instruments to collect blood for examinations desired.

Procedure
1. Rub the finger or immerse in warm water to promote circulation of blood.
2. Cleanse the end of the finger with ethyl alcohol and aIlow to dry. The finger
should be dry .. if it is wet, the blood will not form a round drop.
3. Puncture with a quick stroke deep enough to produce an immediate 'flow of
 HEMATOLOGIC TECHNIC

blood. A deep puncture is no more painful than a superficial one. The blood
should flow freely with gentle pressure since hard pressure liberates tissue
juices and dilutes the drop of blood.
4. Discard the first drop.
5. Allow the blood to flow out one drop at a time for (I) hemoglobin estimation,
(2) erythrocyte pipette, (3) leukocyte pipette, and (4) smears for the differ-
ential leukocyte count. Wipe the finger dry before each procedure. A large
drop of blood is required to fill the leukocyte pipette.
6. If the blood is collected away from the laboratory, stretch wide rubber bands
over the ends of the pipettes. This prevents the contents from escaping, but
the sharp tip of the pipette may perforate the stretched rubber band. If
blood is collected in the laboratory the pipettes are placed on the table with
the tips slightly elevated. We also use the Fisher Multiuse Pipette Holder,
which holds two pipettes and prevents their contents from escaping. This is
an important detail, well appreciated by one who has had to go a long dis-
tance from the laboratory to refill pipettes for a blood count.

VENEPUNCTURE*

Materials and Equipment

I. Sterile, dry syringe and needles from 18 to 21 gauge.
Instead of a syringe, one may employ other devices for securing blood
from a ,vein which possess the advantage that the blood can be drawn
directly into any desired reagent or culture .medium. If a syringe is
not available, a sterile needle and tube can be used.
2. Cotton.
3. Iodine.
4. Alcohol.
5. Tourniquet (rubber band, rubber tube, towel, etc.).

Procedure
1. Wipe the area to be punctured with tincture of iodine, and allow to dry.
Remove the iodine with 9S per cent alcohol and allow· ,to dry.
2. Adjust a tourniquet- about two inches above the bend of the elbow so that
a slight pull will promptly release it. Do not tie so tightly that the arterial·
circulation is impaired and do not leave on the arm for more than two minutes.
3: Instruct patient to open and close his fist a few times so that tbe veins will
become engorged. '
4. Pull back tbe plunger of the syringe and force air through tbe needle to
make sure that the:lumen is open. Be sure that the needle fits tightly.
S. Insert the needle with the bevel pointed upward, first through the skin and
then in to the vein. '
6. Withdraw the required amount of blood, release the tourniquet, and then
withdraw the needle. If the tourniquet is not released before the needle is
* In aU venepunctures it is desirable that the patient be in a recumbent position, since this
obvia.tes the danger of falling from a. cbair, with the po5Sibllity of a scalp wound o-t e'Ven skull
fracture.
 HEMATOLOGIC TECHNIC 599
withdrawn, a hematoma will be produced. When an accident occurs and
a hematoma is formed, release the tourniquet immediately, massage the area
for a few minutes, and give the patient instructions to apply hot applica-
tions. The area becomes discolored after some hours and the hematoma
usually disappears in a few days.
7. As the needle is withdrawn, press the point of entry immediately with sterile
gauze, or cotton immersed in alcohol, to prevent the escape of blood from the
vein. If bemolysis or coagulation is to be prevented, the syringe and recep-
tacles for the blood are rinsed with sterile saline before the vein is punctured,
or a dry syringe is used.
8. Transfer the blood immediately to the proper receptacle. Do not use pressure
to force blood from the syringe. Do not allow blood to clot in the syringe.
9. Rinse the syringe and needle immediately with cold water and dry thoroughly.
10. If the patient should faint !luring the process, avoid his falling and possible
injury. Lower the head between the knees in a sitting position, or elevate the
feet in a reclining position. Allow patients to inhale aromatic spirits of
ammonia, and fresh air.
I I. If the syringe barrel and pi unger become stuck or "frozen," (I) soak in
50 per cent alcohol and 50 per cent glycerin over night; or (2) heat the
barrel car~fulJy; or (3) use the instrument, "Plung-ejector"* which we find
very satisfactory.
Blood is taken from a vein when more than a few drops is necessary for the
laboratory examination. The veins in the bend of the elbow, especially the
median cephalic, and the median basilic, are used for this purpose·. In most in-
dividuals these veins are close to the surface, may be easily engOl ged, and are
generally bound down firmly with fascia which helps to prevent their rolling.

OBTAINING BLOOD FROM INFANTS

The following points are emphasized by Nicholson. In infants under ,8

months of age, blood is usually taken from the longitudinal sinus, particularly
when the neck is fat and the external jugular is difficult to find. In older in-
fants, under four years of age, the external jugular is the site of choice when
other veins are not large enough to enter with ease. After the fourth year, the
veins of the elbow are usually developed sufficiently to be easily located. It is
frequently possible to find veins in the scalp large enough to enter with a hypo-
dermic needle. As a last resort, a few cc, of blood may be obtained by incising
the side of the great toe.

FROM THE EXTERNAL JUGULAR VEIN (Nicho!sont)

Materials and Equipment
The same equipment as for venepuncture .
• A. S. Aloe Co., St. Louis.
t NICHOLSON: "Laboratory Medicine," 1934.- Lea and Febiger, PhlladeJpbia, Pa.
600 HEMATOLOGIC TECHNIC

Procedure
I. Wrap the child in a small sheet and pin and hold securely.
2. Turn the head to one side and hang well over the end of the table. (Tile
external jugular stands out when the baby cries.) Sterilize the area.
3. Hold the skin with the thumb and forefinger and introduce the needle di-
rectly over the vein surface. Deep puncturing is unnecessary as the vein is
directly under the skin. There is no danger of puncturing vital structures in
this region.
4. Fill the syringe and then remove the needle, placing cotton with alcohol on the
punctured area immediately.

FROM LONGITUDINAL SINUS (Nicholson*5

The anterior fontanelle of a child under' 18 months of age is triangular in
shape. The superior longitudinal sinus runs from the anterior to the posterior
angle just under the scalp. It is largest at the posterior angle; therefore, punc-
ture should be made here.

Materials and Equipment

1. Same needle and syringe as for jugular puncture.

Procedure
1. Have assistant hold the head.
2. Shave and outline the bony margins of the- fontanelle with a thin line of
mercurochrome or other dye.
3. Sterilize the skin.
4. Insert needle not more than one eighth of an inch at an angle of 20 degrees.
Puncturing the brain wm not prove latal although, 01 course, it should al-
ways be avoided.

ANTICOAGULANTS

An anticoagulant is a substance that will prevent the coagulation of blood.

A satisfactory anticoagulant should prevent blood from clotting without producing
cellular changes or aiterations in cell volume. The use of oxalates, that are most
widely employed, is based on their capacity to form a chemical union with the
blood calcium, forming calcium oxalate and preventing the calcium, therefore,
from exercising its function in blood ~agulation.
Heparin and hirudin are not widely used because of their variability of action
and because inorganic salts will serve as well for practical purposes.
When I cc. of a 2 per cent solution of potassium or sodium oxalate is placed
in a tube and dried, it will prevent clotting in 5 to 20 cc. of blood. This amount
will not interfere with the precipitation of protein and is satisfactory for most
determinations for which whole blood is employed (Magath and Hurn),
• NICHOLSON: "L~boratory Medicine," :1934. Lea and Febiger, Philadelphia, Pa.
 HEMATOLOGIC TECHNIC 601

Anticoagulants Can be used in blood' for fragility tests.

For sedimentation and cell volume tests an anticoagulant that does not
affect the volume of the erythrocytes should be used. There is no unified
opinion as to what anticoagulant or what amount should be used, but it is known
that there is considerable shrinkage caused by the use of dry oxalate.
Relative to anticoagulants Magath and Hmn conclude that:
I. Heparin produces no swelling, crenation, or laking.
2. Dry oxalate produces a shrinkage of erythrocytes of about 11.3 per cent
when 22 mg. per 10 cc. of human blood is used; the average hematocrit
value is 5.16 per cent less than that obtained with wet heparin.
3. Sodium oxalate in 1.1 per cent solution gives a hematocrit value equal to
that. obtained with heparin and does not alter human trythrocytes. This
solution does not cause human blood to become laked provided the cen-
trifuging is done before two hours have elapsed. For practical purposes, this
anticoagulant is considered suitable for human blood, using I cc. of the solu-
tion to 5 cc. of blood.
Heller and Paul recommend the use of a ~ixture of four parts of potassium
oxalate and six parts of ammonium oxalate, based on their studies that this
mixture does not affect cell volume. The mixture can be prepared according to
the following formula.

Potassium oxalate........ . .... 0.8 Gm.

Ammonium aula te. .. .....•.•.. I 2 Gm.
Water to .................... 100. ce.

Use one cc. of the above to 10 cc. of blood, after evaporation of the water
in an oven.
We use approximately the same concentration of potassium or sodium oxalate
as suggested by Magath and Hum, but prefer to use I drop of a 20' per cent solu-
tion to each 5 cc. of blood. Regardless of the type of anticoagulant employed,
the proper amount can be placed in a large number of wide-mouth bottles with
suitable corks, these then placed in the hot air oven until the fluid has evaporated,
leaving the dried anticoagulant in the bottles.

USE OF OXALATED BLOOD

When a series of blood studies are to be performed, it is sometimes necessary

to collect a quantity of blood from the vein and examine it later at a more con-
venient time. Many tests require both whole blood and blood serum. This
necessitates the use of a satisfactory anticoagulant.

Materials and Equipment

I. Equipment for venepuncture.
2. A wide mouth bottle.
3· Solution of anticoagulant.
602 HEMATOLOGIC TECHNIC

Procedure
I. Place the necessary amount of anticoagulant into the wide mouth bottle.
For blood chemical determinations, where cell changes are not importallt,
a few crystals of oxalate can be used.
2. Draw blood from the vein. Remove the needle from the syringe, and a~d
the blood to the oxalate. If pressure is exerted to force the blood throuph
the syringe, partial hemolysis may occur.
3. Shake the bottle immediately to insure proper mixing with the oxalate aJld
then stopper.

Unless a preservative is used the various examinations should be carrird

out the same day, preferably within an hour. If the test is not perf~rmed within
a short time, the blood should be kept on ice.
Tests in which oxalate is used in regard to the time element for accural'Y
(Osgood) :

I. Hemoglobin esthnation ... '.' ..... . 24 hours

2. Erythrocyte enumeration ........ . 24 hours
3. Platelet count. ................. . I hour
4. Red cell volume .............. , ., 3 hours
5. Color index ..................... . 24 hours
6. Volume index .. ' ................ . 3 hours
7. Saturation index ............... . 3 hours
8. Icterus index. . . . . . . . . . . .. . .... . 4 hours
9. Van den Bergh test ............. . 4 hours
IO. Leukocyte count. . . .. . ........ . 24 hourS'
II. Differential smears ........... . I hour
12. Peroxidase stains .............. . 3 hours
13. Fragility test. . .. ........ . .... . 3 hours
'4. Sedimentation test. . . . . . . . . . .. .. 3 hours
IS. Reticulocyte count .............. . 24 hours

Thus, from the above table, it can be seen that all procedures should be c~r­
ded out early, but that hemoglobin estimation and cell counts can be done even
24 hours later. 'Ve do not recommend the use of oxalated blood as a routine
procedure in a fragility test. Furthermore, erythrocyte counts on the blood of a
patient with hemolytic jaundice are inaccurate after about 3 hours.

OBTAINING BLOOD SERUM

Materials and Equipment

I. Equipment for venepuncture.
2. Dry, clean test tube.
3. Wood applicator or glass rod.
4. Centrifuge.
5. Capillary pipette with rubber bulb.
 HEMATOLOGIC TECHNIC 603

!lLOOD EllAMlllATIOHS
NAME AGE-CLINICAL DIAGNOSIS HI.5TORV No

DATE BLOOD TAKEN EXAMtNEQ..___DOCTOR

HEMOGLOBIN .1 METHOD
I~
014 METHOD I 0/0 OM COLOR INDE;)(

1~t- 5-0 ,,, Sa. 4th ~Q. ToTAL RED CH.L. c.oUNT

RED COUNT
<"
'" ..5th SGo AVERAGE

CELL CHECtl
COUNT
RE-CI-IECKI I I I
1st SO eoo Sa. sa 4th SQ. AVE AGE aT AI- AV RAGE WI1I 10 I'Ll- (:OU,..T

WHITE COUNT "

CEll CHECK
COUNTRE._CHECKI
I
DI FF ERENTIAL WHITE COUNT ,:::~'c,,% --:-
I~ "GM'NT<O@ @i@
POLYS _
,I p..6<
~
~ me u",@ @ (SJ
(PODNVCL'....... ~ ,
,-s
~
:::: '1::,·,~; ... ®@~
'" 0-1
t-'lYI!LG('.VTU) --.

"
~ M'1'fLOC'fTE-S'i) ®~ 0

EOS1NOPH1L$
~e~ 1-'

~~!W Ps-'
B-A$OPH1LS

LYNPHOC'fTES(Qj) ~ @1 20i.2

.. ,.
MONOCYTE:.S
~@)
OEGENE:.R.ATED 0
Cr.LLS

ATYPICAL
FOq/"1S

tl
10.eo 'OM 50 "

POL.VCHROMOQa
7080 901 1020 3<>40 '" ,0 7000

f\£.MARKS
.. ""
AND CONCLUSIC)N,5
>::
~!:i
ANI$O-
C'YTOSIS
oO>:? ATO~"-HLJA~

s~ POI KILO- E'Ryn-l~O-

@@
CYTQSIS
(?Pd BLASTS

~~"
~
PAFl.ASfTES
-. 00
M<eALO·
CVTE~
Rt.6~~u~c;.CYTE

BLEEDING TlME
@

PL.ATELET3 SE01ME:N"TA,ION
RATE

CJ..OTTINQ TIM.E. VOL.UME \NDE.X ElI:.AMINFO

"'
FIG 4I.-A satisfactory cbart for reporting blood examinations in the bospitallaboratory. Thi!
can be used either in color or in black and white. Note that It shows aU figures involved in compu-
tation of blood counts. (Reproduced by courtesy of the Sharp & Dohme Seminar, 2. No. I.)
 HEMATOLOGIC TECHNIC

Procedure
I. Withdraw blood and place into the test tube.
2. Allow this to clot at room temperature (5 to IS min.).
3. Loosen the clot around the edges with the wood applicator or glass rod.
4. Centrifuge from 5 to 10 minutes at high speed.
s. Withdraw the clear supernatant serum with the capillary pipette and dis-
card the remaining clot.
In tests such as the icterus index and van den Eergh reaction where the
serum must not be discolored with hemolyzed red cells, hemolysis may be pre.
vented by rinsing both the syringe and receptacle for blood with sterile norn1a1
saline before the vein is punctured, or by using a ,syringe and tube that is ahso·
lutely dry.

DETERMINATION OF HEMOGLOBIN

(DIRECT METHODS)

T ALLQUIST HEMOGLOBIN SCALE

Materials and Equipment

I. Equipment for finger puncture.
2. A series of 10 color bands in various shades representing the color of ble od
with 10 to 100 per cent hemoglobin.
3. Absorbent paper supplied with the scale.

Procedure
1. Blot a drop of blood from the finger with the absorbent paper; allow to Jry.
2. Compare at once with the color bands against a white background. There
may be an error of as much as 20 per cent if the finger is used as a'backgrOlmd
in the insertion of the blood drop. One hundred per cent is equivalent to
15.8 Gm. of hemoglobin per 100 cc. of blood. •

The Tallquist hemoglobin scale is the simplest, most inaccurate, and prob-
ably the most universally used method for hemoglobin estimation. It can be
used to determine gross changes in herpoglobin, but not absolute or accurate
values. Physicians find it convenient to determine whether a patient is ane[)lic,
but not to calculate color index as it is susceptible to error of as high as 40
per cent.

DARE HEMOGLOBINOMETER

:Muterials and Equipment

I. Equipment for finger puncture.
2. A circular disc of various shades of glass tinted red against which.is matc)led
a fresh specimen of blood draWl!- between two detachable glass plates, one of
white ~porcelain and the other transparent.
 HEMATOLOGIC TECHNIC 60S

Procedure
I. Clamp glass plates tightly together and place in contact with a large drop
of blood which becomes evenly distrihuted between them through force of
capillary attraction.
2. Place this in the instrument and match against the color scale. The number
appearing in the rectangular opening represents the per cent of hemoglobin.
One hundred per cent is equivalent to 13.77 Gm. of hemoglobin per 100 cc.
of blood (16 Gm. in newer instruments). When rotating the round disc, always
rotate in one direction until a match is obtained, then in the other direction,
and compute the average of the two readings.
This method is more accurate than the Tallquist scale, but the instrument
has the disadvantage of being cumbersome and expens;ve. The principle is the
same as that of Tallquist, that is, the comparison of fresh red blood with a red
standard.
(INDIRECT METlioDS)
Blood is mixed with tenth normal hydrochloric acid, thereby converting the
hemoglobin to acid hematin, a brown colored solution which is more easily
matched than the red color of blood.

SAHLI HEMOGLODINOMETER

Materials and Equipment

1. Equipment for finger puncture.
2. A small removable graduated tube inserted between two rods of brown colored
glass which serve as standards. (The standard originally consisted of a tube
containing a suspension of acid hematin.)
3. A Sahli pipette for collecting the blood, 20 cu. mm. capacity.
4. 0.1 normal hydrochloric acid. (A solution approximating o. I normal is
sufficiently accurate and may be prepared by adding I cc. of concentrated
HCI to 99 cc. of distilled water.)
5. Distilled water.

Procedure
1. Fill the graduated tube to the mark 10 with the hydrochloric acid.
2. Draw hlood to the mark on the pipette, insert into the bottom of the tube
and thoroughly mix.
3· Add distilled water drop by drop until the color of the solution nearly matches
the standard.
4. Then again draw the mixture into the pipette and wash out the remaining
acid hematin.
5· Carefully add distilled water until the unknown tube exactly matches the
standards.
6. The amount of solution is read on the graduated tube and corresponds to the
percentage of hemoglobin. One hundred per cent is equivalent to 13.8 Om.
606 HEMATOLOGIC TECHNIC

of hemoglobin per lOa cc. of blood. Until recently the higher figure of 17.3
Gm. was used as a rstandard for 100 per cent hemoglobin.

SAHLI·HELLIGE HEMOMETER
Tbis hemometer is a modification of the Sahli hemoglobinometer. Diluting
tubes may be obtained with graduations on one side representing per cent of
hemoglobin and, on the other side, representing values in grams. With this in·
strument, 100 per cent corresponds to 14.5 Gm. of hemoglobin per 100 cc. of blood.

NEWCOMER HEMOGLOBINOMETER

Materials and Equipment

I. Equipment for finger puncture.
2. The Newcomer instrument.
The color standard is a brown glass disc about I mm. thick, which may
be purchased and attached to a Duboscq colorimeter. There is also a
Newcomer hemoglobinometer, accurately and carefully calibrated, which
reads hemoglobin directly in Gm. per 190 cc. of blood.
3. Special Newcomer pipette.
4. One per cent hydrochloric acid.
Procedure
I. Draw blood to the first mark on the Newcomer pipette and dilute to the
mark above the bulb with the hydrochloric acid.
2. Allow to stand for 30 minutes.
3. Transfer to colorimeter cup. The other cup under the side containing the
color disc is filled with water. The specimen is matched against the color
disc. One hundred per cent is equivalent to 16.96 Gm. of hemoglobin per
100 cc. of blood.
This method is very accurate but quite expensive.
Other methods utilizing the principle of conversion of hemoglobin into acid
hematin are those of ''''introbe, Haden, Osgood, and Berheim.
Sources of error in acid hematin methods (Musser and Wintrobe):
I. Instruments are not uniformly standardized. Each instrument should be
standardized by making readings on a number of healthy adults with a red
blood count of five million cells per cu. mm. In this way instruments may
be corrected for error. All of the subsequent readings should be corrected by
this correction factor. Every instrument should he standardized.
2\ The color of acid hematin gradually increases in intensity during the first
30 minutes. By allowing the dilution to stand for that length of time be·
fore matching, this difficulty may be overcome.
3. Acid hematin standards are not stable and must he replaced frequently. In
additiou to this, colored glass does not exactly match the color of acid hematin
and may fade, hut these errors are only slight.
Despite these disadvantages the acid hematin methods are preferable to
the direct methods of hemoglobin determination.
 HEMATOLOGIC TECHNIC

Comparison of the four methods given above:

I. Newcomer's method is quite accurate, but is the most expensive.
2. The Sahli hemoglobinometer is both accurate and inexpensive.
3. The Dare hemoglobinometer is accurate but expensive and cumbersome.
4. The Tallquist scale is inexpensive, but entirely inaccurate unless only gross
values are required. In some instances its simplicity is a decided advantage.
To translate grams of hemoglobin for each lOa cc. of blood into terms of
percentage or normal, multiply by 100 and divide by the number of grams of
hemoglobin which has been adopted as representing the normal in the method
used.
Other miscellaneous methods include tbe carboxyhemoglobin method, quite
accurate and too complex for practical work; Van Slyke's oxygen capacity
method, also very accurate and used frequently for standardization of instru-
ments; Osterberg's iron content method; and the photoelectric methods of which
the Sheard-Sanford is given below in detail.
. In recent years the photoelectriC methods have almost supplanted entirely
the older methods for hemoglobin determination, particularly in larger labora-
tories where the volume of work justifies the expenditure for the instrument, or
in any laboratory where the most accurate method is required. The recording of
color changes on the photoelectric cell obviates the wide variation in reading
obtained by a group of individuals using their eyes for the same purpose. The
human error, therefore, is eliminated in these methods. Several excellent instru-
ments are now available, each manufacturer producing a satisfactory type. Re-
gardless of the type of photoelectric instrument employed it is necessary that it
be carefully calibrated before it is put into clinical use. In some instances it is
necessary that the purchaser do this himself, whereas in others the instrument
is properly calibrated for a large number of chemical procedures before it leaves
the factory. Photoelectric instruments are now available that will not only
determine hemoglobin content but also all other chemical procedures carried out
in the average clinical laboratory. Among the best of these instruments is that
manufactured by the Leitz Corporation, this instrume:-lt being carefully stand-
ardized hefore .ale for a large number of chemical procedures.

SHEARD-SANFORD PHOTELOMETER (From Todd and Sanford)

Materials and Equipment
I. Equipment for venepuncture.
2. 0.1 per cent solution of sodium carbonate.
3· Pipettes to measure 0.1 cc. and 20 cc. accurately.
4. 50 cc. flask or centrifuge tube.
5. Photelometer.

Procedure
1. Dilute o. I cc. of blood in 20 cc. of 0 I per cent solution of sodium carbonate,
thus making a I to 200 solution of oxyhemoglobin.
2. "The photelometer has a green glass filter in front of the photronic type of
608 HEMATOLOGIC TECHNIC

" photo"electric ceIl. This filter transmits light in its maximal intensity at that
- portion of the spectrum where the maximal absorption occurs in one of the
oxyhemoglobin bands. The light intensity through a standard spectroscopic
cell, which is filled with 0.1 per cent sodium carbonate, is first adjusted with
an iris diaphragm so that the reading on the meter is roo. The specimen
of diluted blood is placed in a similar spectroscopic cell, is then moved into
the path of light in the carrier and the reading is made on the meter. This
lower reading really represents the decrease in current from the photronic
cell which is the result of the light absorption of oxyhemoglobin in the green
portion of the spectrum. This reading is translated directly into a value for
grams of hemoglobin per 100 cc. of blood, by referring to a chart which is
prepared individually for each instrument by the manufacturer, based on
the oxygen capacity determinations" (Todd and Sanford).
This method has proved very satisfactory and accurate, its only disadvantage
being the cost, which is more than a colorimeter. However, it may also be used
as a colorimeter.

ADVANTAGES
I. Accurate and rapid.
2. Calibrated by Van Slyke oxygen capacity method.
3· Readings are interpreted directly in Gm. per roo cc. of blood.
4· No occasion to match colors; therefore, SUbjective errors are avoided.

ENUMERATION OF ERYTHROCYTES
l\Iaterials and Equipment
~. <AttQR ';lads Q< ste<,le gaU7.e.
2.Instrument for making puncture.
3· Water, alcohol, and ether for cleaning pipettes.
4. Microscope.
s· Diluting pipette for erythrocytes.
A Thoma red cell diluting pipette consists of a graduated capillary tupe
leading into a bulb containing a glass bead. The fifth graduated line is
marked 0.5 and the tenth is marked 1.0. Above the bulb there is a JiIle
marked lOr.
These pipettes are so constructed that if blood is drawn to the 0.5 mark
and diluent drawn to the 101 mark, the dilution is I to 200 in all red cell
pipettes, regardless of size of bulb or stem.
Trenner Automatic Pipettes are available for inexperienced workers
who have difficulty in stopping the flow of blood exactly at the 0.5 mark on
the capillary stem.
6. Counting Chamber.
The 'Bilrker type of counting chamber with double Neubauer ruling is in
general use at the present time. The chamber is made of heavy glass with
two central platforms on the surface of which fine rulings are delicately eJl-
 HEMATOLOGIC TECHNIC

graved. These two platforms are separated centrally by a transverse moat.

A longitudinal moat on either side divides the platforms from elevated bars
which are built so that they are 0.1 mm. higher than the central platforms.
When a special coverslip is placed over these bars, there is a space of 0.1 mm.
deep between the surface of the coverslip and the ruled areas. The ruled
portion on each platform has a total surface area of 9 sq. mm.
The 4 corner large squares are subdivided into 16 smaller squares. The
remaining 4 outside large squares are not so divided.
The central square is divided by double or split boundary lines into 25
squares, each of which contains 16 small squares, making a total of 40"
squares in the central square millimeter. The last division is indicated
on one side of the counting chamber, showing the smallest division to b{·
1/400 sq. mm.
The Hausser counting chamber consists of a bakelite holder in which a
small removable counting chamber can be placed. It has the advantage
of being non-breakable and in it can be placed other ruled areas for count-
ing platelets, bacteria, etc.
There is also a counting chamber available called the Bright Line Cham-
ber* having a dark metallic background, thus making the lines of the
ruled area stand out in sharp relief. We use this routinely and believe it
represents a decided improvement over other instruments. The Bass· Johns
ruling is preferred by some workers because of its simplicity.
7. DilutiJng fluids:
(a) Hayem's Solution.
Sodium chloride. . . . . . . . . . . . . . . I .0 Gro.
Sodium sUlphate. . . . . . . . . . . . . .. . 5,0 Gm.
~er~~l1;c chloride .. .......... .. 0,5 Gm.
DIstlllea water. . . . . . .. ......... 2'00 ce.
(b) o.8s1'>er cent sodium chloride.
The leukocytes are also included in all of these diluents uut may
be excluded in the count by their size. (Erythrocytes are about 7 micra
in diameter. Leukocytes are about 12 micra in diameter.) The number
present is ordinarily so small that the inaccuracy is of little importance.
(c) Toisson's Fluid.
Sodium chloride ...... .. . . . . . . I 0 Gm.
Sodium sulphate ........... 8.0 Gm.
Glycerine .......... ........ 30 0 cc.
DIstilled water .......... ..... 160.0 ce.
Methyl-violet, sufficient to give a purple tinge.
(d) Forkner has pointed out that Gower's solution is more satisfactory than
any other diluting fluid. The formula is as follows:
Sodium sulfate. . . . . . . . . . . .. .. 12.5 Gm.
Glacial Acetic Add. . . . . . .. . .... 33.3 ce.
Distilled water to ...... ......... 200 ce.
A hemacytometer consists of a counting chamber and cover glass with white
cell pipette, red cell pipette with rubber tubing and mouthpieces, all in a suitable
• Spencer Lens Co.
610 HEMATOLOGIC TECHNIC

case. When purchasing a !J.emacytometer it is well, despite the additional cost,

to secure one which has been tested by the U. S. Bureau of Standards.
New counting chambers and pipettes should be cleaned before using and
thereafter both instruments should be cleantd immediately or soon after each
count.
I. \Vith a pledget 01 gauze, wash the counting chamber with soap and water,
until all traces of blood and serum are removed .
•. Draw water through pipettes by suction until all traces of blood and serum
are removed.
3. Remove water in pipettes with alcohol by suction.
4. Dry pipettes with ether by suction until the bead of the bulb moves freely.
If blood dries in the stem of a pipette, remove the clot with a horse hair, fill
the pipette with nitric acid or cleaning fluid (potassium bichromate and sul-
phuric acid) and allow to stand over-night. Clean thoroughly 'as described
above. A suction pump for cleaning pipettes is a time saving convenience and
should be included in the equipment of every laboratory.

Procedure
A measured volume of blood is diluted with a measured quantity of suitable
diluting fluid (Gower's). The cells are then counted over a portion of an accu-
rately marked counting chamber 'and the total number per cubic millimeter is
estimated.
I. Draw blood exactly to the 0.5 mark of the Thoma pipette marked 101. This
pipette contains a red bead in the bulb. Remove excess blood on pipette tip
with a blotter or cotton.
2. Without allowing the blood to flow out, draw the diluting fluid to mark 101,
thus making a dilution of I to 200. Rotate the pipette while it is being filled.
3. Before placing the fluid in the counting chamber, shake the pipette at least
2 minutes, by holding it between tbe thumb and the middle finger, first
placing the thumb over the calibrated end of the pipette. Shake sideways.
Mechanical shakers are available.
4. Place the cover slip in position over the ruled area of the counting chamber.
5. Blowout and discard 3 or 4 drops from the pipette and, as quickly as possible,
touch a drop to the edge of the platform of the counting chamber and allow it
to flow under the cover glass. The fluid should not flow into the moats
on any side, and ·there should be no bubbles under the cover glass.
6. Allow the cells to settle for a few minutes.
7. Examine under the microscope. Lower the substage and partially close the
diaphragm. Focus with the 16 mm. objective, locating the ruled lines. The
cells should be evenly distributed over the, chamber. The microscope should
be continually adjusted with the fine adjuster to bring all of the cells into
focus.
8. For the actual count the 4 mm. objective should be used. 'Vhen a second
chamber is filled from the same pipette, it must be shaken again immediately
before filling and a few drops discarded.
 HEMATOLOGIC TECHNIC 611

CALCULATION
The large central square used for the red cell count contains a volume of
0.1 cu. mm. since the surface area is I sq. mm. and the depth is 0.1 mm. It is
necessary to calculate the number of red cells in 0.1 cu. mm. of diluted blood,
then the number in I cu. mm. of diluted blood, by multiplying by 10, and finally
the number of cells in I cu. mm. of undiluted blood, by multiplying by the dilu·
tion in the pipette.
This large central square millimeter is divided by double lines into '5 blocks
of 16 squares each. The red cells are counted in only 5 of the • 5 squares, prefer·
ably the 4 corner ones and a central one picked at random. The total number
of cells is added together and this number multiplied by 5 to determine lbe
number in the entire '5 squares (400 small squares) which constitute 0.1 cu. mIll.
The resulting figure is multiplied by 10 to obtain the number in I CU. mm. of
diluted blood, and then by '00, the dilution. This gives the number of cells
per cu. mm. of undiluted blood.
In counting the cells under the microscope, each of the 5 squares is focused
under the high power lens. The markings are such that one of the double-ruled
squares flts exactly into the high power field. There are 16 small squareS,
bordered on all sides by a double line. On the top row, cells are counted from
left to right, on the second row from right to left, etc., until the cells are counted
in all four rows. Cells lying on the left and upper outside lines are included in
the count, while those on the right and lower outside lines are ignored.
For routine work and only when the dilution is I to 200, four ciphers may 'be
added to the total number of cells counted in the 5 squares designated above to
obtain the number of red coq~uscles per cu. mm. of blood (5 x 10 x .00 ~ ~o,ooo).
EXAMPLES OF CALCULATION
,. By long method:
Upper left hand block. (16 small squares). . . . . . . . . . . . . . .. " . . . •.•. 101 cells
Upper right hand block. (r6 small squares).. .. .. .... . ................ . 97 cells
Lower right hand block (16 small SQuares)..... ..•.•... . ...........•.•. lor cells
Lower left hand block. (16 small squares). . . . . .. . ....•......... .. . •.•.•. 98 cells
Central block (16 small squares) ................ ,'.. .. ................ . T03 cells
Total (So small squares or lis of 0 J cu. mm) .. .....•............ . .... 500 cells
Total for 0.1 cu. mm. diluted blood (25 blocks or 400 small square-s) - 5 X 500 = 2500.
Total for I cu. mm dIluted blood (multiply by 10) = 10 X 2500,- 25,000.
Dilution is 1:200.
Total for I eu moo. undiluted blood = 200 X 25.000 = 5,000,000.
2. By short method:
Add four ciphers to the total number of cells (500), making 5,000,000
cells per cu ..mm. of blood.
The difference between the number of cells found in any 2 blocks of 16
small squares should not exceed 20. A greater difference is evidence that the
pipette bas not been shaken well or that the chamber is dirty. Different counts on
the same individual should check as closely as 200,000 to 300,000 cells per cu. mm.
NORMAL EH.YTIIROCYTE VALUES PEB CU. ll1l. o:r BLOOD
Men.. ... ........ ...... ... 4,$00,000-6,000,000
Women... ... .. ........... 4,000,000-5,500,000
Infants soon after birth. •••••...• .$,500,000-1,000,000
612 HEMATOLOGIC TECHNIC

SOURCES OF ERROR
I. Inaccurate diluti.m, because of faulty technic. This error usually comes about
by permitting an air bubble to enter while drawing up the blood, or inability
to stop the blood on the mark.
2. Failure to place cover glass properly 0;1 counting chamber. This is espe-
cially true of the closed type.
3. Over filling of the counting chamber.
4. Failure to shake the pipette sufficiently for an even mixture.
5. Uneven distribution of the corpuscles in counting chambers that are dirty,
and improperly cleaned.
6. The presence of yeast in the diluting fluid.
7. Auto-agglutination or clumping of red cells for some other reason.

COLOR INDEX

This is an expression which indicates the amount of hemoglobin in the aver-

age red corpuscle compared with the normal amount.

Color Index = Hemoglobin per cent.

Number of red cells per cent.
When hemoglobin is read in grams per 100 cc., the percentage is calculated
from the number of grams of hemoglobin given as 100' per cent by the type of
hemoglobinometer used. For example, 8.5 Gm. of hemoglobin by Newcomer's
method is 50 per cent (8.5/16.96 X 100 - 50).
Five million per cu. mm. is considered 100 per cent red cells. Per cent of
normal may be calculated by mUltiplying the first two figures of the red cell
count by 2.
Theoretically, the normal color index is 1.0. Actually, the average normal
is slightly lower than 1.0, since the hemoglobin content is generally lower than
100 per cent.
I. Normal color indel{ example:
Red cell count. . . . . . . . . . . .. ..... 5,000,000
Hemoglobin. . . . . . . . . . . . . . . . . . . .. ]00 per cent
Color index = ~ = I
SoX 2
2. Low color index (found in hypochromic anemias):
Red ceU count. . .. ............... 3,000,000
Hemoglobin. . . . . .. ............. ,30 per cent
Color index ""' 3
0
3~ 2 ... 0.5
3. High color index (found in hyperchromic anemias):
Red cell count. . . . . . . . . . . . . . . . . . . .. 2,000,000
HerooglQb-in. . . . . . . . . . . ......... , 60 per cent
Co!orindex = 20~ Ii "'" I·S
Nicholson gives the normal color index as 0.9 to 1.1$. We find that it aver-
ages 0.8S to 0.90 among healthy Southern women (nurses) and 1.0 in medical
students.
 HEMATOLPGIC TECHNIC 613

ENUMERATION OF LEUKOCYTES

Materials and Equipment

I. Same as for red count.
2. Thoma white cell pipette.
It consists of a graduated tube which runs into a bulb, -containing: a
movable white bead. The fifth line on the graduated tube is marked <)·5,
the tenth line 1.0, and a line directly above the bulb is labeled II.
3· Diluting fluid. II r1, :~1
I cc. of glacial acetic acid and I cc. of I per cent aqueous solution of
gentian violet in laO cc. of distilled water. This solution has been fotlnd
very satisfactory. The solution should be filtered frequently, or made fresh.

Procedure
I. Draw blood to the 0.5 mark of white cell pipette.
2. Draw diluting fluid to mark II, making a dilution of 1:20.
3. Shake sideways for at least 2 minutes immediately before making the count.
4. Discard 3 or 4 drops and fill the counting chamber.
S. Allow the cells to settle for 3 minutes.
6. Examine under the 16 mm. objective of the microscope. A single ruled square
millimeter exactly fits into the low-power field (I6 mm.). Reduce the in-
tensity of light by adjusting the diaphragm.
7. In performing a complete blood count, shake both the erythrocyte and leul<a-
cyte pipettes simultaneously; insert the red cells into one side of the counting
chamber and the white cells into the other.

CALCULATION
The white corpuscles are counted in the four large corner squares, each of
which has a volume of 0.1 cu. mm., making a total volume of 0-4 cu. mm. 'The
number counted is divided by 4 to find the average per 0.1 cu. mm., multiplied
by 10 to find the number of cells in I cu. mm., and multiplied by the dilu-
tion, 20.
For routine purposes and only when the dilution is I :20, add the cells in the
4 outside large squares and multiply by 50 (20 : 10 •

EXAMPLE OF. CALCULATION OF WHITE CORPUSCLES

I. Long method:
Left upper square mm .... ........... " .... 36 cells
Right upper square mm.. .. . . . . . . . . . . . . . . . . . 40 cells
Lower left square mm.. . . . . . . . . . . . . . . . . . . . . 31 cells
Lower right square mm.. . . . . .. ......... .. 37 cells
Total for 0.4 cu. mm. diluted ......... .. . ISO cells
Total for 0.1 cu. mm. diluted ...... , ... ISO + 4 - 37 5
Total for I cu. DlID. diluted., ', ..... 10 X 3'.5 - 315
Dilution-I: 20
Total for I cu. mDl, undiluted., ..... 20 X 315 - ',500
614 HEMATOLOGIC TECHNIC

2. Short meth~d:
ISO X 50 - 7.500 cells p:er cu. mm.
The difference between the largest and smallest number in
any two squares should not exceed ten.
The normal leukocyte count is between 5,000 and 10,000 cells per cu. mm.
of blood. It may vary between much wider limits than the erythrocyte count.
Normal fluctuations may appear according to age and time of day. The number
is generally lower in the morning after a period of- bed rest and before breakfast
(basal count). In doing daily, consecutive counts on a patient,the count should
be made each time at the same hour by the same worker and with the same
pipette. In itself the total leukocyte count is of little diagnostic value. It is,
however, one of the best laboratory aids when used in conjunction with the
differential count to determine the type of leukocytes involved in the disease.
In some cases of leukemia with very high counts it may be necessary to use
the red cell pipette with a dilution of I :100. If a white count is made on a
patient without knowledge that the case is leukemic, fill the chamber as usual,
count or estimate the cells in the central square millimeter, and mUltiply by
200. (10 X 20 = 200.) ,
In a case of leukopenia, the blood may be drawn up to the mark 1.0 on the
diluting pipette instead of 0.5, making a dilution of I:IO instead of 1:20.

CORRECTION OF THE LEUKOCYTE COUNT FOR NUCLEATED

RED CELLS

When a large number of nucleated red blood corpuscles is present, it is

necessary to correct the leukocyte count for these cells as they are easily mis-
taken for the white cells in the counting chamber, thus causing an apparent
increase in the leukocyte count. This can be done as follows:
1. Examine the stained smear, counting 1000 red blood cells and noting the
number of nucleated cells.
2. 'Divide the total erythrocyte count by 1000 and then multiply by the num-
ber of nucleated red cells found per 1000 red blood cells. This is the number
of nucleated red cells per cu. mm.
3. Subtract the total number of nucleated red cells from the total leukocyte
count to obtain the actual leukocyte count.
This correction is made only when such a large number of nucleated red cells
is found in the stained smear that the leukocyte count is consideraqly increased.

EXAMPLE
Erythrocyte OO\Ult ••••.••••••••••••••••••• 2,000,000
Leukocyte count ...... ' ., ..... ........ 47,000
Nucleated cells per 1,000 erythrocytes.. . . . . . IS
IS X 2,000 - 30,000 nucleated red cells per cu. mm.
47,000 - 30,000 - 17,000, actual leukocyte count.
Another sborter and simpler method for correction of the leukocyte count for
nucleated red cells can be carried out by counting the number of nucleated red
 HEMATOLOGIC TECHNIC

cells while doing a differential cell count on the leukocytes. The nucleated red
cells counted should be in addition to the one-hundred leukocytes in the differen·
tial. This is shown in the following example:
Number of leukocytes counted. . .. ....... . . 100
Number of nucleated red cells.. ........ ..... IS
Uncorrected leukocyte count ...... . ......... :2~~OOO

Corrected leukocyte count ... :~ X :22)000 "" 18)644

EXAMINATION OF FRESH BLOOD

lUaterials and Equipment

1. Clean slide.
2. Clean cover glass.
3. Instrument to puncture finger.
4. Microscope.
5. Vaseline.

Procedure
It frequently becomes necessary to examine blood in the living, unstained
state to ohtain a quick survey of the blood.
1. Place a small drop of blood no larger than a pin head on the central portion
of the cover glass and invert gently on a clean slide.
2. Rim the edges with vaseline.
3. Examine specimen with oil immersion lens. Reduce the intensity of light by
adjusting the diaphragm of the microscope.
The procedure is found to be of value for the observation of size and shape
of living corpuscles, of rouleau formation, and of motility of white cells. This
type of examination is especially valuable for the detection of sickle cells, since
the sickling trait is often seen in fresh blood, when it is not demonstrable in
a stained smear.

PREPARATION OF BLOOD SMEARS

The first requisite for a good smear is clean glassware entirely free f~om
oil and dust particles. For blood smears new slides are preferable.
Cleaning slides and cover glasses:
I. New slides
(a) Wash with soap and water and rinse thoroughly.
(b) Immerse in alcohol (95 per cent).
(e) Polish with a soft cloth, preferably linen.
(d) Flame over a bunsen burner.
2. Dirty slides
(a) Boil in 5 per cent sodium bicarbonate solution, scrub with soap and
gauze, allow to stand over night in a cleaning sol ution (potassium
bichromate-sulphuric acid), and then treat as above.
 HEMATOLOGIC TECHNIC

3. Cover glasses
(a) The same procedure is followed as for new slides, omitting the flaming
process.
In preparing a smear, utmost care must be taken to avoid pressure, which
may result in injury to the blood cells. Smears may be made on either covel
glasses or slides; the latter are used, as a rule, for routine work.

COVER GLASS METHOD

Materials and Equipment

1. Equipment for finger puncture.
2. Large cover glasses. (No. I, frB in. square, or rectangular, No. I.) The new
plastic cover glasses are not satisfactory for this type of work.

Procedure
I. Have cover glasses thoroughly clean. Place small drop on center of one
glass. Drop the other glass on, and the blood then spreads in a thin film be·
tween. Carefully pull them apart on a plane parallel to their surfaces, holding
them at diagonal corners with thumb and forefinger.
The chief advantages of the method are: (I) a thin film is obtained; (2) the
leukocytes are evenly distributed. Sources of error are: (I) the drop of blood
may be too large; (2) when pulled apart, a tremor or unsteadiness will cause
"ridges" or "waves" in the film; (3) they are more fragile, easily breakable, and
more difficult to handle in staining; (4) possible cell distortion.
'Ve do not use the cover glass method, since we believe that leukocytes may
be distorted by being crushed between the sliding surfaces, and also, for the
reason that a properly prepared slide film results in a preparation equally as
satisfactory.

SLIDE METHOD

Materials and Equipment

I. Equipment for finger puncture.
2. Clean slides free from grease, acid and alkali.

PrQcedure
1. Puncture the finger as described before.
2. Place a drop of blood on one end of a clean slide.
3. Hold the slide between the thumb and third finger with the drop on'the upper
surface next to the finger or place it on the table top.
4. Place the narrow edge of a second slide over and in front of the drop of blood
at an angle of about 30 degrees.
5. Pull the "spreader" slide back until it comes in contact with the drop of blood
wbich then spreads outward to both edges.
 HEMATOLOGIC TECHNIC

6. With a firm but steady movement, smear the blood toward the opposite end
of the bottom slide. The rapidity of the movement as the drop is smeared
determines the thickness of" the film; that is, the slower the movement, the
thinner the smear. Also,lhe angle of the spreader determines the thicKness
of the film. The smaller the angle, the thinner the film. Therefore. i~ order
to obtain a thin film, (I) have a small drop of blood; (2) bave a small
angle to the spreader slide; (3) smear slowly and, smoothly.
7. Allow to dry in the air. Do not blot.
In some cases of severecanemia it is difficult to make good films because of
the large proportion of plasma, which leads to slow drying with consequent dis-
tortion of the r~d cells a'ld the appearance of artefacts. To overcome this, the
films should be made very thin and dried quickly Qver a low flame.
A good smear should be smooth and without serrations. It should have
even edges and should be uniformly distributed over the middle two thirds of the
slide. At least two smears should be made with every routine blood count, one
of ordinary thickness for the leukocyte count and a thin one for the st1.1dy
of the red cells. eare should be taken to avoid thick smears, since the staining
of thick areas of blood is never satisfactory.
Regardless of care in making smears, the end where the smear is begun is
generally the thicker. This area should never be examined. In a study of either
tbe red or white cells, the examination under the microscope is begun on the
thinner end of the smear.
After a smear is allowed to dry, it may be. stained within l! few ,!,inutes.
If smears are placed in a box away from the dust they may not deteriorate for
weeks. The best preparations, however, are those which have been stained within
the first 24 hours. SpeCial stains such as the peroxidase stain g;ye best results if
applied within an hour alter the smear is prepared.
Occasionally for teaching purposes it is necessary to make a large number of
slides at one time. Oxalated blood is not satisfactory' for this. It is necessary
to withdraw a small amount of blood from the vein and rapidly make sOlllll
drops on a large number of previously prepared slides, having a second person
to spread them as rapidly as po~sible. In this way 100 slides can be prepared.

CONCENTRATION METHOD FOR LEUKOCYTES

Materials and Equipment
I. Equipment fa'r venepuncture.
2. From 10 to 12 slides.
3. Ten ce. pipette, dropper, and capillary pipette.
4· Centrifuge and tube.
S. Anticoagulant 1.1 per cent potassium oxalate or equal parts of ammonium
oxalate and potassium oxalate. See page 600.
Procedure
I. Place 10 ce. of blood in a centrifuge tube containing 4 drops of the mixture
of ammonium oxalate and potassium oxalate. Invert gently 3 times.
 HEMATOLOGIC TECHNIC

2. Cen trifuge for IS or 20 minutes at high speed.

3. Remove the supernatant plasma carefully with a capillary pipette.
4. Remove and transfer to glass slides drops of the leukocyte cream,
forms in a thin layer at the top of the packed red cells. Smear as if making
a blood smear.
5. Prepare 10 to 15 smears in this manner. Some of these smears will contain
a large number of leukocytes.
The preparations should be made immediately after the blood is withdrawn,
since the leukocytes of oxalated blood soon become distorted and difficult to
classify.
In the leukopenic states, blood cells are frequently so scarce that it is diffi-
cult to find sufficient number on the usual smear for an accurate differential
count. This is a simple and satisfactory method for obviating a long search over
a number of smears.

FIXATION OF BLOOD FILMS

METHODS OF CHEMICAL FIxATION

J. One to two minutes in pure methyl alcohol or absolute ethyl alcohol.
2. Fifteen minutes or longer in equal portions of absolute alcohol and ether.
Chemical fixation is adequate for practically all work and is not necessary'
at aU in those stains suspended in either methyl or absolute alcohol (Wright's
stain).
In the absence of the alcohols, blood films can be fixed with a mild degree
of beat, ::.imilar to the fixation of bacteria on a slide.*

STAINING OF BLOOD SMEARS

The cellular elements of the blood have selective affinities for acid, basic, or
neutral aniline dyes. The nuclei of these cells take the basic dye::., such as
methylene blue. Acid dyes, such as eosin, act upon the cytoplasmic elements.
Polychrome or neutral stains, which are mixtures of acid and basic dyes, stain
not only acidophilic and basophilic constitutents, but other components that are
neutrophilic in reaction.
This group of stains utilizes the essential principle of a combination of eosin
(acid stain) and methylene blue (basic stain) in varying combinations and
types of mixtures, usually contained in a solvent of methyl alcohol for fixa-
tion. Of this group, 'Wright's is the most widely used .

.. The granules of the basophil ace generally known to be water soluble. Therefore, in order
to demonstrate these cells in either peripheral blood or tissues, it is necessary that a.n alcoholic
type of fixation be used rather tban the aqueous form. Otherwise the basopbiis will not appear
at all in suc.b fixed stained tissue"_
 FIG_ 42.-Showin~ centrifuge tube containing oxalated blood after ccntrifuging_ Note ted
cells packed to lhe bollom with blood p\a>ma at the lop and the thin white layer of leukocyte,
bctween_
 HEMATOLOGIC TECHNIC

WRIGHT'S STAIN

Materials and Equipment

r. Staining racks.
2. Staining bottles (50 cc. fiat top dropping bottles).
3. Wright's stain.·.
Wright's powder (Grub!er preferred). . . . • • • • • • • . • . . 0.3 Gm.
Glycerin......... .................... ........ 3.0 cc.
Methyl alcohol (Mallinckrodt, absolute acetone free) 97.0 ceo
Mit in a mortar, put in bottle and allow to stand over night.
Filter and allow to stand a few days before using. It seems to
improve with age.
4. Buffer solution.

t~~~:~t~p~~~o.~~~:.::::::: .. :::::: ~.~3 g::

Distilled water. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. 1000 ce.
• Wright's stain in solution and b1:lffer solu.tion can be obtained from any dealer in laboratory
reagents. We have found these preparations quite satisfactory.
620 HEMATOLOGIC TECHNIC

S. Cover glass and Canada balsam, if preparation is to be permanent.

6. Microscope and immersion oil.

Procedure
1. Cover the dried smear completely with stain for I to 3 minutes. This fixes the
blood film.
2. Add the buffer solution to the stain on the smear, drop by drop, until a green-
ish, metallic scum appears on the surface. -The quantity of solution should
completely flood- the smear witIJout running over the edges of the slide.
Blow gently through a pipette to insure mixing of the water and the stain.
Allow to stain for 3 minutes. (This varies with different lots of stain.)
3. Wash off the stain with water. The washing is begun while the stain is still
on the slide to prevent the settling of the scum-::like precipitate which can-
not be removed. The specimen has a purple appeara,!ce and should be
washed until it is a lavender-pink.
4. Allow the slide to stand on end to dry which is preferable to drying be-
tween filter paper. -
5. For permanent preparations mount the smear in Canada balsam. For ordi-
nary purposes this is unnecessary.
6. Examine the smear with oil immersion 1ens.*
A blood smear stained with this dye shows a color, range from the bright
red of acidophilic material to the deep blue of the basophilic material. Between
these two is the lilac color of neutrophilic granules, and other varying color com-
binations depending on tIle pH of the different constituents of the blood.
We find Wright's stain satisf.actory for practically all types of hematological
work.

GmMSA's STAIN

Materials and Equipment

I.-Staining jars" (Coplin jar is good).
2. Giemsa's stain..
(a) Stock solution:

Giemsa powder (GrUbler) (Best to purchase already prepared). 0.3 Gm.

Glycerin ....................... : ......................... 25 ..0 ce.
Methyl alcohol (Mallinckrodt)J absolute, acetone free ......... ~5.0 ce.

(b) Dilute stain (ready for use) :

I cc. of stOck solution to 10 cc. of distilled water~
3. Methyl alcohol.
• A satisfactory type of immersion oil with the proper refractive index' can be prepared ac-
cording to the following formula:
Alpha.-bromonaptha1ene ......... .... .. 19 cc.
ll.1:ineral oil .....•.. _............ _.•... 8t CC.

Nujol b. DlOIe !a.tis.factory than ordinary mineral oil.

 HEMATOLOGIC TECHNI« 621

Procedure
I. Fix the smear with methyl alcohol for 3 to 5 minutes (in Coplin iar).
2. Dry in the air.
3. Immerse in diluted stain for 20 to 30 minutes (in Coplin jar).
4. Wash with distilled water.
s. Stand on end to dry.
6. Examine under oil immersion.
This method for staining blood films probably gives more uniform results
Ihan Wright's method. It has the disadvantage of a longer and more complex
technic, but the cnd results are good, quite uniform and dependable. It is not
recommended, however, for routine work in preference to Wright's stain, since the
simplicity of the latter renders it more suited to routine procedure. We find the
Giemsa method quite useful .in staining large numbers of slides at onc time.

PEROXIDASE STAINS (method of Sato and Sekiya)

I\laterials and Equipment
I. Staining equipment.
2. Solution A.
0.5 per cent copper sUlphate.
3. Solution B.
0.1 per cent benzidine solution.
Filter.
To filtrate add 2 drops of a commercial hydrogen peroxide.
4. I per cent solution of safranin.

Procedure
1. Apply solution A to a fresh, dry blood smear for IS to 30 seconds.
2. Pour off excess and do not wash.
3. Apply solution B for 2 minutes.
4. Pour off excess and do not wash.
5. Counterstain with safranin ·for 2 minutes.
f Wash and allmy.t6 dry.
With this staiu the red cells are pink, the lymphocytes are red, and the
granulocytes are stippled with blue-plack granules with the single exception of
the myeloblast which contains no granules and is oxidase negative.
The peroxidase reaction is dependent upon the presence in granular myeloid
cells of an oxidizing ferment which causes the granules to take a blue stain when
brought in contact with benzidine, copper sUlphate and hydrogen peroxide.
At times it becomes necessary to resort to this staining procedure in the
differentiation of many of the abnormal immature types of cells. The peroxidase
stain serves to facilitate the differentiation of the granular cells of the myeiocytic
series from the non-granular cells of the'lymphoid series. We believe that the
granular monocyte, that is, the monocyte with visible granulation, is peroxidase
622 HEMATOLOGIC TECHNIC

positive. Frequently in myeloblastic leukemia this stain will show the presence
of granular premyelocytes which are difficult to identify with Wright's stain due
to the delicacy of granulation. This finding may be the only means of establish-
ing the identity of the predominating myeloblasts, that is, by their association
with older myeloid cells.

INDOPHENOL BLUE SYNTHESIS (modification of Schultz's Method)

Materials and Equipment
I. Same equipment.
2. Absolute alcohol.
3. 40 per cent solution of formaldehyde.
4. Solution A.
I per cent alpha naphthol in physiologic solution
of sodIum chloride . ....... ~ . . . . . . . . . . . . . . .. 100 CC.
0.1 normal sodium hydroxide. '" .. , ....... .. I ct.
s. Solution B.
I per cent sol. of paradimethylparaphenylenediamine base (Merck) in
physiologic solution of sodium chloride.
6. Physiologic solution of sodium chloride.
7. I per cent aqueous solution of safranin.

Procedure
I. Fix smear for 3 to 5 minutes in equal parts of absolute alcohol and the
solution of formaldehyde.
2. Stain for 3 minutes with solution A.
3. Wash with physiologic salt solution.
4. Stain for 3 minutes with solution B.
S. Wasb and counterstain for 3 minutes with safranin.
Myeloid and monocytic granules stain 'a deep and intense blue. Lympho-
cytes, red cells and platelets are stained bright red.
Naegeli recommends this synthesis for the differentiation of immature cell
types. He states that with Schultz's method myeloid granules, myeloblasts, and
monocytic granules stain blue; lymphocytes are not stained blue but are counter-
stained with safranin.

DIFFERENTIAL LEUKOCYTE COUNT

Materials and Equipment

I . Those ne<:essary fm: a finger pun<:ture and prepa.ration and staining of blood

smears.
2. Wright's stain.

Procedure
1. Prepare several smears as previously described.
 HEMATOLOGIC TECHNIC

,. Stain with Wright's stain.

3. Examine with low power lens to determine if the cells are equally distributed.
4. Examine with oil immersion lens, recording eacb type of leukocyte seen as
the slide is moved from one field to another. Schilling recommends the "four
field meander" technic. Four fields of the slide in the thinner half of the
smear are examined, }4 of the total desired number of leukocytes being ex-
amined. in each of these areas.
c. At the same time a special differential count of neutrophils may be made,
" called the Scbilling's nuclear index (described later);
6. For routine work, classify 100 leukocytes. The larger the number of cells
counted, the more accurate are the resulting percentages. Kolmer and
Boerner suggest that the total leukocyte count should determine the number
of cells included in a differential count. They recommend the following:
For total counts under 5,000.. ........ . ... .. .. classify 50 cells
For total counts of 5,000-10,000................ classify 100 cells
For total counts of lo,ooo--r5,000 .... ... ......... classify 200 cells
For total counts of .5,000-20,000.. . . . . . . . . . . . .. classify 300 cells
For total counts of 20,000-25.000 . . . . . . . . . . . . ', . . . classify 400 cells
For total counts of over 25.000 .. ........... ... classify 500 cells

RELATIVE AND ABSOLUTE VALUES

By the usual differential count, the various blood cells are listed with the
percentage found for each type. This merely indicates a relative or proportional
number. More information is obtained by converting this ratio into actual
or absolute numbers for eacb type of ·cell. The absolute number is computed
from the total number of leukocytes per cu. mm. of blood. If, for example,
the total count is 10,000 with 90 per cent neutrophils, the actual .number of
neutrophils per cu. mm. of blood is 0.90 X 10,000, or 9,000.
The following are the figures for the normal adult expressed in percentage
and absolute numbers.
Per Cent Per cu. nun. of Blood
Neutroehils <aU types). • .. ... .. .. ... 60 to 70 or 3,000 to 7,000
Basophils. . . . . . . . . . . . . . . . . . . . . .. .. 0 to I o to 100
Eosinophils. . . . .. ...... .......... I to 3 or 50 to 300

~~~~~:.'.'.::::::::::::::::':: 2~ ~ 3~ ~~
1.000 "to 3,500
100 to 600

When a differential count shows an increase above normal in the percentage

of any type of cell, without an increase in the absolute number when calculated
from the total count, the increase is called a "relative leukocytosis," and may be
a "relative lymphocytosis" or "relative neutrocytosis," depending upon the type
of cell involved.

SCHILLING'S NUCLEAR INDEX

Material and Equipment

Equipment f9r a, differential count.
62 4 HEYATOLO(HC TECHNIC

Procedure
Classify the leukocytes in the following manner:
Myelocytes Lymphocytes
Juveniles Monocytes
Bands Eosinophils
Segmenters Basophils
With the ordinary method of estimating the total number of cells and the
differential counting there are many cases in which the findings do not fit the
clinical symptoms. When there is an entirely normal total count and differential
count, it is necessary to classify the cells according to their maturity in order
to obtain additional information. Schilling has found, in studying the nuclei of
neutrophils, that the severity of the infection is oftentimes proportional to the
number of immature cells found in the peripheral blood. If, then, the neutrophils
are classified according to their maturity, this immaturity indicates a rapid bone
marrow output. While the Schilling classification is o(special value in such cases
it easily becomes a matter of rou!ine to list the presence of any immature forms
found on the blood smear. For the interpretation of Schilling's nuclear index, see
page '"9.
Arneth divided the neutrophilic leukocytes into five classes according to the
number of lobes in the nuclei.
Nonnal
Pet Cent
Clas5 I-Dne round or indented nucleus .... ~ .......... , ., • 5
Class 2 -Two nudear di.visions ... .. _........... ~ . . . . . . . . . 35
Class 3-Three nuclear divisions... . . . . . . . . . . . .. ......... 41
Class 4-Four nuclear divisions ... -; .................. :;. 17
Class s-Five or more nuclear divisions. . . . .. . . . . . . •. .... 2

This system classifies the neutrophils ,according to their age, the youngest
cells being included in class I. Myelocytes, metamyelocytes, and bands are in
class I. The clinical value of an Arneth count is questionable since only the
older types are classified. We prefer the Schilling classification and use it in all
differential counts.

FILAMENT AND NON-FILAMENT COUNT

In recent years, the so-called filament and non-filament count has become
popular. This consists in counting the neutrophils that have a well divided
nucleus with two or more lobes separated by a tbin filament-like strand. These
are filamented cells. Those not having such are so-called non-filamen ted. The
non-filamented cell is the band, juvenile, or myelocyte of the Schilling classifica-
tion. Tbe method is a short cut and abbreviation of the Schilling classification.
The upper limit of the normal for young forms or non-filament forms is 16
per cent. The average for normal adults is 8 per cent. The filament and non-
filament count may be interpreted the same as regards the "shift to the left" in
the Schilling count. This method is simpler than that of Schilling and has
 HEMATOLOGIC TECHNIC

grown in favor in the United States, but as in the Arneth count, the immature
cells are not classified.

VOLUME INDEX

CENTRIFUGE TUBE METHOD (Haden)

Materials and Equipment
1. Equipment for venepuncture.
2. Graduated IS cc. centrifuge tube.
3. Centrifuge.
4. 2 cc. pipette.
S. 1.4 per cent sodium oxillate. (1.1 per cent solution is preferred.)

Procedure
I. Remove 10 cc. of blood from the vein and place in a graduated IS cc. centri-
fuge tube which contains 2 cc. of the oxalate solution.
2. Invert the tube several times to completely oxalate the blood.
3. Centrifuge until the cells are completely packed. The centrifuge must be
tested to find the length of time required to completely pack the cells of nor-
mal blood. This length of time is used for centrifuging subsequent specimens.
Each centrifuge, therefore, must be standardized, and its efficiency checked at
frequent intervals. Normal packing is 4.6 cc. to 4.8 cc.

CALCULATION
Volume of packed cells (per cent)
Volume Index = ~umber of red cells (per cent)
Five million red cells is the arbitrary figure for 100 per cent of red cells,
The red cell.percentage is calculated· by dividing 5,000,000 into the patient's red
cell count and mUltiplying by 100, or more simply by mUltiplying the first two
ligures of the count by 2.

EXAMPLE
With a red cell count of 4,500,000 per cu. mm. and the volume of packed
cells,4 cc. in 10 cc. of blood, the volume of packed cells in per cent is 40/48 X 100
or 83 per cent. This calculation is based on the assumption that normal cells pack
to 48 per cent of the whole blood volume using a certain centrifuge speed. The
red cell percentage is 45 X 2 or 90 per cent.
Volume Index _ 83 per cent ... 09
90 per cent
The normal volume index is 0.85 to 1.0.
The volume index in pernicious anemia and other macrocytic anemias is
above 1.0.
The volume index in microcytic anemias is below 1.0.
 HEMATOLOGIC TECHNIC

WINTROBE'S HEMATOCRIT METHOD

Materials and Equipment

I. Equipment for venepuncture.
2. Wintrobe hematocrit tube.
A small, glass, rubber-stoppered tube, 10.5 cm. in length, graduated in mm.
from the bottom.
3. Special capillary pipette for filling the tube.
4. 20 per cent potassium oxalate (, drop for every 5 cc. of blood).
5. Centrifuge.

Procedure
I. Witbdraw 1 to 2 cc. of blood from the vein in the usual manner and place in a
receptacle containing I drop of the anticoagulant.
2. FiJI the graduated tube to the mark 10 with the oxaJated blood using the
special capillary pipette. Insert. the rubber stopper to prevent evaporation.
3. Centrifuge· until the blood cells are completely packed, usually IS to 20 min-
utes depending upon the centrifuge used.
4. Note the volume of packed cells.

CALCULATION
Use 4.24 cc. per 10 CC. or 42.4 CC. per 100 cc. of whole blood as the normal
volume of packed cells.

EXAMPLE
A sample of blood contains 3,200,000 red cells per cu. mm. and the volume
of packed cells is 2.1 cm. (21 cc. per 100 cc. wbole blood) on tbe graduated tube.
The volume of packed cells in per ceut is 21/42.4 X 100, or 49 per cent. The per
cent of red blood cells is 32 X 2 or 64 per cent. The volume index is 49/64 or 0.76.
This method bas the advantage of requiring only a small quantity of blood.
Other tubes devised for determination of volume index that are equally
satisfactory, are those of VanAllen, and Sanford and Magath.

THE MEASUREMENT OF RED CELL DIAMETER

PRICE-JONES CELL DIAMETER CuRVE (.rjicho!son)

Materials and Equipment
I. Equipment for finger puncture.
2. Equipment for preparation and staining of blood smears.
3. Hemocytometer.
4. Microscope.
5. Immersion oil.
6. Micrometer eye piece.
 HEMATOLOGIC TECHNIC

Procedure
I. Insert the micrometer eye piece in the microscope and place the hemo·
cytometer in position on the stage.
2. Under oil immersion, hring into focus one of the smallest squares in the
central'Square millimeter of the hemocytometer.
3. Adjust the tube length of the microscope so that the distance encompassed
by 50 graduations of the micrometer coincides with the distance across the
smallest hemocytometer square which is 50 microns across. In this position,
one division of the micrometer is equal to one micron when using the oil
immersion lens.
4. Prepare and stain a thin blood smear in the usual manner.
5. Remove the hemocytometer and examine the smear under oil immersion.
Measure to the nearest 0.5 micron at least 200 erythrocytes selected from
different areas of the smear.
6. Record by tally each erythrocyte according to its diameter and total the
cells in each group.
1. Plot a curve using the diameter in microns as the abscissa and the number of
cells as the 9rdinate of the graph.
A majority of the cells in normal blood measure 7.0, 7.5 or 8.0 microns in
diameter. A curve of normal blood is an acute angle with slightly outcurving
bases. In microcytic anemias, the curve is less acute and reaches a peak to the
left of the normal peak. In macrocytic anemias, the curve is less acute and
reaches a peak to the right of the normal peak.

HADEN·HAUSSER ERYTHROCYTOMETER

Principle
The principle of the erythrocytometer depends on the fact that the size of
the coronas (or concentric spectrum), produced by the aggregate of light dif·
fraction at the edges of the individual cells of a blood film, varies with the size
of the red blood cell and its distance from the light source. If the size of the
coronas is kept constant, then the size of the particles varies directly as their
distance from the light source.
The light source in this instrument is a small standard 50 watt projection
lamp above which is an opaque disc with a small central aperture. This light
source is surrounded by three sets of smaller apertures, which are large enough
to be visible but do not create 'separate spectra. A slot is provided for holding
the slide or cover glass on which the blood film has been made. A rack and
pinion is located on the right side of the instrument for varying the distance
of the film from the light source. As the film is moved away from the light
source, the colored rings of the spectrum become larger. The position of the
film can be thus adjusted until the inner red ring, used for the measurement,
directly overlies the inner circle of small holes.
 HEMATOLOGIC TECHNIC
Preparation of Blood Films
The accuracy of the readings depends largely on the preparation of the blood
films. The films should be thin so that the cells touch but do not overlap.
Tbey should not be so thin as to flatten the cells. Haden recommends a thin,
uniform film made on a coverslip and stained with Wright's stain. A few
drops of 0.9 per cent sodium chloride solution are placed On the dry stained
preparation and the coverglass is inverted on a clean glass slide. A film may
be made on a slide and stained. A cover glass 'is mounted over tIie thin area
with the salt solution. The dry 'stained preparation mounted in salt solution
gives a more brilliant spectrum than the dry unstained preparation which is
often used.

Method of Reading
Place the slide containing the blood specimen in the slot above the stage
at the top of the instrument so that the selected area of the film or the wet
preparation is directly over the opening.
Press the button to connect the light source. Tum the hand wheel which
operates the pinion, until the spectral rings are sharply defined. If the -film is
too thick, the spectrum will be blurred and the smaller apertures in the per_
forating disc, will be scarcely visible. If the film is too thin, the colored rings
will be faint, with t4e. outside red ring sqtrcely visible.
When the film is satisfactory, the colored rings will appear sharp and clear,
with a white ring surrounding the central ap,erture. This white ring will be
peripherally edged with yeJlow, red and blue, successively in the order named.
The blue ring fades into yellow and the yellow into an ol!tside red ring. The
dividing Iioe between the inside red ring and the blue ring is the sharpest in
the spectrum. .
Move the spectrum upward and downward by turning the hand wheel until
the inside red ring coincides with the inside. set of four apertures. The outer
edges of tbese apertures will then touch this sharp dividing line between the
inside red and, blue ring. The mean ~ell diameter is then read directly in r/sth
micron from the scale on the hand wheel.
We have found the Haden erythrocytometer quite satisfactory for the meas-
urement of the average diameter of red cells. It appears to be accurate within
one-half of a micron of the average diameter. It is certainly sufficiently accurate
for clinical purposes and has a marked advantage over plotting a Price-Jones
curve in that the readings can he made quickly. The price of the instnunent
is about $so.oo.

SUBSTITUTE FOR HALOMETER (Pryce)

This is a. simplified method utilizing the same principle of halo formation
as described in the preceding method. This apparatus may be constructed in
the laboratory by placing 2 electric bulbs about 2 feet apart on a black wall,
lahoratory table, or any black hackground. The exact distance between the lights
is determined by looking through normal blood smears at a distance of 7 fcct
 HEMATOLOGIC TECHNIC

from the lights and moving the lights back and forth until the red margins of
the , halos around the red cells come in contact. The floor is marked off at
right angles to the lights at 1 foot intervals up to a distance of 10 feet. (One
foot corresponds to I micron.)

Procedure
I. Prepare a thin ,blood smear.
2. Hold the thin end of the smear ip front of, the eye until a halo appears aroujld
each light.
3. Walk toward and away from the lights until the red borders of the halos touch
as described in the preceding metbod.
4. At this point the distance,on ~e floor in feet represents the average cell diam-
eter in microns. This method provides a rough estimate of cell size but is
relatively inaccurate.

MEAN .cORPUSCULAR VOLUME (WINTROBE)

This is a simple calculation to estimate the volume of the average red

corpuscle.
Mean Corpuscular'Volume ~ Volume of'packed red cells.(i~ 7c. per 1000 cc. of blood)
Number of red cells (ill millions per cu. rnm.)
EXAMPLE
A sample of blood contains 4,300,000 erythrocytes per cu. mm. and 31 ce. of
packed red cells per 100 cc. of blood. .
:Mean Corpuscular Volume - 314~/o - 72 ~bic microns, the average volume per red cell

MEAN .CORPUSCULAR HEMOGLOBIN (WINTROBE)

This expresses the amount of hemoglobin in- the average red corpuscle with-
out comparison to an arbitrarily fixed normal.
M C I H I b' Amount of hemoglobin (in Gm. per lOCO ce. of blood)
ean orpuscu ar emog 0 m = Number of red cells (in millions per Cll. rom.)

EXAMPLE
A sample of blood contains 4,300,000 erythrocytes per cu. mm. and 10.6
grams of hemoglobin per 100 cc. of blood.
Mean Corpuscular Hemoglobin _ IO.~~ 10 =a 24.6 mic:romicrograms of hemoglobin per average red c:ell·

MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION

(WINTROBE)

This expresses in per cent the proportion of hemoglobin i~ the average ted
corpuscle.
• A lnicromic:rogram hi .. millionth, millionth Pitt of-a. Gm. or (Gm.. X 10-12 ),
63 0 HEMATOLOGIC TECHNIC

Amount _of hemoglobin (in

Mean COlpuscular Hemoglobin Concentration = Gms. per 100 cc. blood) X 100
Volume of packed red cells (in
cu. em. per 100 cc. of blood)

EXAMPLE
A sample of blood contains 10.6 Gm. of hemoglobin per 100 cc. of blood
and 3 I cc. of packed red cells per 100 ce. of blood.

Mean corpuscular hemoglobin concentration _ 10.6 X tOO ... 34.1 per cent.
31

SATURATION INDEX (OSGOOD)

Per cent Hemoglobin = Saturation index.

Per cent Cen volume
The figures below, by Wintrobe, express the normal values with upper and
lower limits of the mean corpuscular volume, mean corpuscular hemoglobin, mean
corpuscular hemoglobin concentration and mean corpuscular diameter. We have
not found these determinations necessary in hematological work, since we believe
that the same information is available in other determinations.
NORMAL VALuES yoa SIZE AND HEliOGLOBIN CONTENT 0)' EllYTllltOCYUS"
Average Minimum Maximum
M~an C~rp~ar Votume (C.V.)
lD cubiC Inlcrons. . . . . . . . . . . . . .. ................ 87 So 94
Mean Corpuscular Hemoglobin (C.H.)
in mlcromicrograms .. .................... .-. . . . . . 29. S '7
Mean Corpuscular Hemoglobin Concentration (C.C.)
in per cent. . . . . . . .. ........................... 3S
Mean Corpusculax Diameter
in microns ............................. ,. .....• 7.$ 8.0

RETICULOCYTE COUNTS

Materials and Equipment

1. Equipment for finger puncture.

2. Carefully cleaned coverslip and slide.

3. One per cent alcoholic solution of brilliant cresyl blue.

Procedure
1. Smear across a clean slide a few drops of the solution of brilliant cresyl blue.
Smear the stain as if making a blood smear.
2. Allow to dry in the air.
3. Place a small drop of blood from the finger in the center of a coverslip and
invert on the slide. The preparation lasts about one hour and may be pre-
served for a longer period of time by rimming the edges of the coverslip with
vaseline .
• Frcm Wintrobe: Jour. Lab. eli". Med., 17. 899, 1932.
 HEMATOLOGIC TECHNIC

4. Allow to stand for 10 minutes for complete staining.

5. Examine under oil immersion. The film of blood should not be too thick.
6. Select a thin area in which the cells are scarce enough to count easily.
7. Count 1000 red cells and note the number of reticulated cells.
Micrometer ruled discs have been devised to facilitate the counting of
reticulocytes. A serviceable one is the reticulocyte disc of Johns; A. S. Aloe Co.,
St. Louis.
The count is expressed in per cent. The normal count is 5 to 10 reticulocytes
per 1000 red cells or 0.5 to 1.0 per cent. The count may be as high as 10 per cent
in infants during the first few days of life. It is also increased in pregnancy.
MallY variations of the technic given are in use. The cresyl blue can be
smeared on the cover glass with a small drop of blood, placed on the slide and
the cover glass inverted on it. Also, a drop of cresyl blue (10/0 solution in 0.85
per cent sodium chloride) can be placed beside a drop of blood, the two mixed
with a rod, smeared, and then stained with \Vright's stain, giving a permanent
preparation. We have found this method satisfactory. There is also a more
complex centrifuge method.

ENUMERATION OF PLATELETS

FONIO'S SMEAR METHOD

Materials and Equipment

I. Equipment for finger puncture.
2. Equipment for erythrocyte count.
3. Slides for smears.
4. '4 per cent magnesium sulphate.
5. Wright's stain.

Procedure
1. Puncture finger and place a drop of 14 per cent magnesium sulphate over the
site of the puncture before the blood begins to flow.
2. Allow the blood to flow into the drop until the proportion is about one part
of blood to five parts of magnesium sulphate solution.
3. Transfer a drop to ~ clean glass slide and make a ,thin smear.
4. Wipe away the sulphate solution and do a red blood count in the usual manner.
5. Stain the smear with Wright's stain and examine under oil immersion.
6. Count 1000 red cells in fields over the various parts of the slide, noting the
number of platelets in the different fields.

CALCULATION

Number of platelets X erythrocyte coun~ = the number of platelets per cu. mm.
1000

The normal platelet count by this method is 250,000 to 350,000 per cu. mm.
of blood. It is elevated by hunger and exercise, and lowered after eating.
63' HEMATOLOGIC TECHNIC

OLEF'S METHOD

Materials and Equipment

1. Equipment for finger puncture.
2. Equipment for erythrocyte count.
3. Warm water.
4. Soap, alcohol, and ether.
s. Slides and coverslips.
6. Paraffin cup.
Prepared by melting the center of a small cube of paraffin, about 2 em. in
diameter, with the heated end of a glass rod.
7. Wood appiicator, the end of which is coated with paraffin.
8. Diluting fluid:
SodiuDl Dletaphosphate (Rowe & French) . ......... _ .. . 1.0 Gm.
Sodium chIonde .... ................................ , 0.4 Gm.
Dextrose ............ _.... _._ ..................... :. 0.1 Gm.
Sodium bicarbonate . . ~ .................. " ..•....... 0.1 Gm.
Brilliant cresyl blue ... '" ....... , ................... . o.lsGm.
Distilled water. .. . ..•......•.............•......... 100 0 ce.

In this solution all blood elements are well preserved and both platelets and
reticulocytes are stained so that a reticulocyte and thrombocyte count can be
done simultaneously. Solutions must be kept in a cool place and filtered every
few days.

Procedure
I. Immerse hand in warm water to accelerate local circulation.
2. Make a deep puncture on the palmar surface ot'the finger without applyin!
pressure, after cleaning the part with soap and water, alcohol and ether. Th!
lobe of the ear is not a satisfactory site for a puncture' due to the presence 01
fine hair.
Recently, there has been described an instrument for making an 'incisiona
puncture to insure a uniform cut. It is quite valuable in determining the bleed
lng time, and could also be used for punctures for platelet determinations.
3. Discard the first drop of blood,
4. Place a drop of diluting fluid over the puncture before the blood reaches th.
surface of the, skin. Turn the hand over quickly so that palmar surface i,
directed downward.
s. Apply the mixture to the surface of three or four drops' of diluting fluid con
tained in the small paraffin cup, thus diluting the blood approximately I:S
6. Stir the contents of the cup gently with the wood applicator.
7. Allow the mixture to stand for a minute or two. Stir again and transfer to ,
glass slide by means of the applicator. Three preparations can be made a,
the fluid in one cup yields three large drops.,
8. Place the coverslip over the drop and allow to stand ten or .fifteen miD1:ltes
9. Examine under oil immersion and count both platelets and eryt1!rocytes it
 HEMATOLOGIC TECHNIC

fields taken at random until one thousand red cells have been counted. Prep-
arations made too thin or too thick yield inaccurate results.
10. Do an erythrocyte count in the usual manner and calculate the absolute num-
ber of platelets per cu. mm.
(Number of platelets X erythr~= count)

The average number of platelets'in a large group of normal individuals by

this method was 514,000 per cu. mm. This method is probably more accurate
than that of Fonio.
These indirect methods of counting platelets are preferred to the direct
method, which makes use of a pipette, spe~ial diluting fluid, and the COUllting
chamber.

DIRECT METHOD OF COUNTING PLATELETS (Todd and Sanford)

Materials and Equipment
I. Equipment for finger puncture.
2. Erythrocyte pipettes.
3. Counting chamber.
4. Diluting fluids:
Wright and Kinnicutt:
Aqueous solution of brillia.nt cresyl blue (1 :300) ••••• 2 parts
Aqueous solution of potassium cyanide (1:1400) .•••• 3 p~
Keep solutions in separate bottles; mix and filter just before using. The
cresyl blue solution keeps well, but molds may,grow in it. The cyanide solution
must be made fresh every ten days.
Rees and Ecker:
Sodium citrate, 3.8 per cent aqueous solution •....•.•..•• 100.0ce.
Formalin, 40 per cent SOIUtiOD4 ••••••••••••• _ . . . . . . . . . . 0.2 c:c.
Brilliant cresyl blue ...•..•••..••• 0................... O.I Gm.
This solution preserves the erythrocytes which may be enumerated in tbe
same preparation.
Leake and Guy:

~=~:!e;~~'~~t' ~1~ti6~:::::: :':::: :::::::::::::: 9~:~ ~~

Sodium oxalate ......................... : ,............ 1.6 Gm.
Crystal violet ... : ................................ :._ .•. 0.01Gm.
The fluid is warmed, filtered, and preserved in stoppered bottles. This solu-
tion is highly recommended; it is permanent, and erYthrocytes are well pre-
served.

Procedure
I.Draw the diluting fluid to near the I mark in the erythrocyte pipette.
2.Draw blood from'a freely bleeding puncture to exactly the 0.5 mark.
3· Then quickly draw the diluting fluid to the lor mark. This gives a blood
dilution of 1 ::ioo.
 HEMATOLOGIC .TECHNIC

4. Mix by shaking for about 2 minutes.

5. Fill counting chamber at once and allow 10 minutes for the platelets to settle
before counting. The count is made with a high dry (4 mm.) objective and
a lOX ocular; the platelets are counted in 80 small squares and the total
'number is multiplied by 10,000.
6. Make a control count with the hlood from a normal person, at the same time,
using the same dHuUng fluid and same technic. Rapid work is necessary to
prevent clumping of platelets.
This direct method which employs the hemocytometer gives lower figures
than the indirect method.
Some workers make a practice of counting the platelets'on an ordinary stained
smear, comparing with the number of red cells, and computing the number by
comparison. This is inaccurate because of clumping of platelets, but one may
look at a stained smear and at once get an approximate, idea of the number of
platelets. It has been said, with some truth, that the most satisfactory method of
platelet determinations is to study an ordinary smear ana report the platelets as
increased, normal, or decreased, and it must be admitted that this is adequate for
clinical purposes.

COAGULATION TIME

SLIDE METHOD

Materials and Equipment

I. Equipment for finger puncture.
2. Clean slide.
3. Needle.
4. Watch.

Procedure
1. Cleanse the finger with alcohol and puncture as for a blood count (a deep
puncture is necessary).
2. Place a few drops of blood on the glass slide.
3. At half minute intervals draw a needle through the blood. When a fine tIrread
of fibrin can be picked up with the needle point, coagulation has begun.
4. The time between the flow of blood at tire site of puncture and the formation
of fibrin on the slide is considered tire coagulation time.

CAPILLARY TUBE METHOD

Finely drawn capillary glass tubes (1-2 mm. diameter) can be filled from
the drop of blood and small pieces broken off at half minute intervals. When
fibrin strands appear at the broken end, this represents the coagulation time. We
prefer this to tire slide method.
The normal coagulation time varies from 2 to 8 minutes. The slide :and capil-
lary tube methods, as well as all others using blood from a finger puncture, are
 HEMATOLOGIC TECHNIC

subject to error because of the mixture with tissue fluids when blood flows through
subcutaneous tissue.
There are many methods which require special instruments, of which the
best known are those of Biffi-Brooks and Boggs. Directions for the use of these
can be obtained from the makers. They offer no great advantages over the
methods given above.

VENEPUNCTURE METHOD (Lee and White)

Materials and Equipment
1. Equipment for venepuncture.
2. Normal saline (sterile).
3. Test tube (8 mm. in diameter).
4. Watch.

Procedure
1. Rinse the sytinge, needle, and test tube with sterile normal salt solution.
2. Puncture a vein and remove one cc. of blood, avoiding suction,
3. Remove the needle from the syringe and place the blood in the test tube.
4. When the tube can be inverted without the blood flowing out, coagulation is
complete.
5. The time interval between the flow of the blood from tbe vein and the forma-
tion of the clot is considered the coagulation time. The greater the diameter
of the test tube, the slower the clotting time.
The normal time is from 5 to 10 minutes.
This method is far superior to any method using a finger puncture since
there is no admixture of blood with tissue fluids.

HOWELL'S METHOD

Materials and Equipment

1. Equipment for venepuncture.
2. Mixture of ether and petrolatum.
3. Test tube about 2 I mm. in diameter.
4. Watch.

Procedure
1. Draw a mixture of petrolatum and ether into a dry syringe and needle; expel
and fill with air a few times until the etber evaporates and leaves a thin coat-
ing of petrolatum.
2. Puncture a vein and withdraw 2 to 4 cc. of blood and place it in the test tube.
3. When the tube can be inverted without the blood flowing out, coagulation
is ~onsidered complete.
By this method the blood of healthy persons coagulates in 10 to 30 minutes.
The average time is about 20 minutes. If results are to be relied upon, the blood
 HEMATOLOGIC TECHNIC

must be taken from the vein. The chief difference between this and Lee and
White's method is the use of a petrolatum coated syringe, and a larger tt:~l tube.

BLEEDING TIME

Bleeding time may be defined as the time required for cessation of hemorrhage
when blood flows from a cut in the skin of approximately measured depth and
length.

DU:KE'S METHOD

Materials and Equipment

I. Equipment for finger puncture.
2. Filter paper.
3. Watch.

Procedure
I. Incise the skin or malte a deep cutting puncture in the finger or lobe of the ear.
2. Note the time the first drop appears.
3. Blot with filter paper each drop as it flows out or blot every half minute.
4. The interval betweeu the first drop and the last is considered the bleeding
time.
Normally there are about 6 drops on the filter paper gradually decreasing
in size. When the time required for the Ctlt to cease bleeding is longer than IO
minutes, the bleeding time is said to be prolonged. The normal bleeding time is
r to 3 minutes. This is an inaccurate method.

THE IVY ME1'HOD

In this metbod an ordinary blood pressure cuff is placed around the arm and
inflated to 40 mm. of mercury. The surface of the forearm is cleaned with
alcohol and a small cut made approximately 2 mm. in depth. The drops of
blood are wiped away at intervals of 30 seconds and the duration of bleeding
recorded. The normal bleeding time by this method is 2 to 6 minutes. As
Diggs points out, the skin bleeding time should not be confused with the bleed-
ing time of deeper tissues or from larger vessels; for e;l{ample, a patient with
thrombocytopenia may have a skin bleeding time that is remarkably prolonged,
yet the spleen can be removed without undue bleeding, and conversely, a hemo-
philiac may bleed to death from a tooth extraction, but may have a perfectly
normal skin bleeding time.

CAPILLARY RESISTANCE TEST OF RUMPEL-LEEDE

Materials and Equipment
r. Skin pencil.
2. Blood pressure apparatus.
 FIG . .+ ..... - Showing pt'lt·chi:ll ~puts- 011 ion,..:arlll in a pu:--iLivc G1.pilbry t-c~blJ.lh.~c U.:~l art·t.:r the
di!J,~tolk prt:':~surl' ha::;. ul'cn ma inta ined lur fiy-t minute=-. Some wurkers prefer to maintain the
pre~surc mid\\"ay hd ....-ecn the diastolic and :-;y~to] ic for thrr(' minutes_ There is no rca:-on to do
this. test on a paticnt who :::ho,,,s: ~pOlltan('ous. purpuric .:-pots.
 HEMAl"OLOGIC TECHNIC 637
Procedure
I. Examine the patient's arm for petechiae; if any are present, mark the Spots
with a skin pencil.
2. Place the cuff of the blood pressure apparatus on the arm of the patient and
hold at the diastolic pressure for 5 minutes.
3. Examine the arm for the appearance of fresh petechiae.

CLOT RETRACTION -TIME

Materials and Equipment

I. Equipment for venepuncture.
2. Test tube or a blood vacuum tube.
3. Incubator.
4. Watch.

Procedure
I. Place 2 cc. of blood in a dry test tube.
2. Incubate at 37 C. for 24 hours.
0

3. Observe at the end of each hour for 6 hour~ and at· intervals of 6 or I 2 h~urs
thereafter.
Under these conditions the clot normally- retracts completely within I~ to
'4 hours after it is formed. Beginning retraction is noticed in normal blool! in
I to 2 hours. There is no relation between the coagulation time and the clot
retraction time. Delay in retraction, 9r failure of the clot to retract, is USUally
associated with a decreased number of platelets.

YJWTHRDM.BJJ'IT TJJ\f.E

HOWELL'S METHOD (From Todd and Sanford)

Materials and Equipment
I. Equipment for venepuncture. Syringe rinsed with physiologic salt solution.
2. Centrifuge tube which contains 0.25 cc. of I per cent sodium oxalate in physi.
ologic salt solution.
3. Physiologic salt solution.
4- Dropper.
s. Four small test tubes.
6. 0.5 per cent solution of calcium chloride.
7. Watch.

Procedure
I. Obtain 2 cc. of blood from vein, avoiding suction.
2. Place the blood in the centrifuge tube at once.
3. Invert the tube several times and centrifuge.
4. Place 5 drops of the plasma in each of the 4 small test tubes.
638 HEMATOLOGIC TECHNIC

s. To these tubes add the calcium chloride as follows: 2 drops in tube 1; 3

drops in tube 2; 4 drops in tube 3; 5 drops in tube 4; mix gently.
6. Coagulation usually occurs in all tubes but not at the ,same time. The tubes
are inverted to determine the time of coagulation and the time of the tube
which clots earliest is considered the "prothrombin time."
7. With each unknown blood run a normal hlood specimen.
The average "prothrombin time" by this method is 10 minutes. The "pro-
thrombin quotient" can be determined by dividing the prothrombin time of the
unknown specimen by that of a normal control.

THE QUANTITATIVE PROTHROMBIN METHOD' OF QUICK

Principle
Blood coagulation takes place in two stages:
1. Prothrombin + Thromboplastin + Calcium - Thrombin
2. Fibrinogen + Thrombin - Fibrin
Fibrinogen is rarely decreased sufficiently in hemorrhagic diseases to alter the
clotting time. By controlling the thromboplastic and calcium factors and leav-
ing prothrombin as the only variable factor, the clotting time of the blood be-
comes a measure of the concentration of prothrombin.

Reagents
t. Sodium oxalate solution.
Sodium oxalate anhydrous .....•... 0............. 1.34 Gm.
Water ........................................ 200 CC.
2. Calcium chloride wluuon.
Calcium chloride anhydrous..... . . . . . . . . . . . . . . . . . I I I Om.
Water........... . ........................... 400 ce.
3. Sodium chloride 0.85%
4. Thromboplastin (from rabbit brain). Each ampoule contains 0.15
Gm. of stabilized, desiccated rabbit brain which is sufficient for the
prepa.ration of approximately 2 cc. of throm.bopla.stin -extract. It
can be obtained from Difco laboratories, Detroit, Mich.
Extraction of Thromboplastin
Empty the contents of an ampoule into a test tube containing 0.05 cc. of
sodium oxalate solution and 2.5 cc. of sodium chloride solution. Mix well
and incubate in a water bath at 45° C. for 10 minutes, stirring the suspension
with a glass rod several times during the extraction period.

Filtration
Place a thin layer of absorbent cotton over the mouth of the test tube and
carefully push it to the bottom of the tube with a pipette. Keep the tip of the
pipette against the cotton, so as to insure filtration of the solution through the
cotton, and withdraw as much extract as possible.
It is recommended that the thromboplastin extract be used promptly, but if
not used immediately after preparation, it should be kept in the ice box and be
used within 24 hours.
 HEMATOLOGIC TECHNIC 639

Procedure
Obtain 4.5 cc. of blood by venepuncture and immediately mix with 0.5 CC.
of sodium oxalate solution. The oxalated hlood is centrifuged. Mix 0.1 cc. of
the oxalated plasma with o.r CC. of the thromboplastin and quickly add 0.1
ce. of calcium chloride solution. The time required for the clot to form after
the addition of the calcium chloride is noted.

Normal Values
Normal plasma on recalcification in the presence of an excess of thrombo-
, plastic substance will clot in 12 to 20 seconds. With a decrease in prothrombin
the clotting time is delayed.

PROTHROMBIN METHOD OF SMITH, ZIFFREN, OWEN AND HOFFMAN

Principle
Thromboplastin is added to whole venous blood. A normal fibrinogen and
calcium concentration is assumed. Prothrombin is the only variable. The clotting
time is considered as a measUre of the prothrombin content.

Reagents
Tbromboplastin solution. 'Prepare as for Quick's test and dilute 25 times
(2 cc. diluted to 50 cc. with distilled water) so that the clotting time of normal
blood will be around 30 seconds.

Procedure
Place 0.1 cc. of thromboplastin in a small serologic tube (10 X 75 mm.).
Venous blood, freshly drawn, is run into the tube to a 1.0 cc. mark. The tube
is inverted once over the finger to mix, then is gently tilted every few seconds
until the clot forms. The time taken for the clot to form is noted. The test
is repeated on a normal individual, and the unknown expressed in percentage
of normal.
Cl' '" f al Clotting Time of Normal
ottlllg activIty In per cent 0 norm = Clotting Time of Patient X 100

A MICRO TEST OF PLASMA PROTHROMBIN

Materials and Equipment

1. Kato's Microhemopipet.
2. Thromboplastin (Use same reagent as in Smith's tests).
3. Watch glass.

Procedure
Puncture e"", finger or heel and with microhemopipet collect 30 to 40 c. mm.
of blood. Transfer to watch glass and immediately mix with IO c. mm.<'oJ
 HEMA'IOLOGIC TECHNIC

thromboplastin. Tilt glass back and forth until clot forms. Run a control on a
normal individual.·
. ... Clotting Time of Nonnal
Clottmg acllvlty m per cent of normal = Clotting Time 01 Patient X 100

CAJ,.CIUM TIME (TODD AND SANFORD)

Materials ~nd Equipment

J. Equipment for venepuncture.
2. Two test tubes, 8 to 10 mm. in diameter.
J. One cc. volumetric pipette.
4. Dropper.
5. One per cent aqueous solution of calcium chloride.
6. Watch.
Procedure
I. Place I cc. of blood in each of the test tubes.
2. Add 3 drops of I per cent solution 'of calcium chloride to only I of these tuhes.
3. If coagulation occurs in both tubes at about the same time, the calcium con-
'tent may be assumed to be normal. If it is delayed or does not occur in the
tube with no calcium added, then the blood is assumed to be deficient in
calcium.

FRAGILITY TEST

SANFORD'S METHOD

Materials and Equipment

I. Equipment for venepuncture. Syringe and needle must be dry.
2. Twelve small test tubes of approximately the same diameter are placed in a
rack and labeled 25, 24, 23, 22, 21, 20, '9,'18, '7, 16, 15, and 14.
3. Capillary pipette. (The syringe and needle used lor the venepuncture may be
used instead in order to have all drops the same size.)
4. o.S per cent solution of sodium chloride made volumetrically.
5. Fresh distilled water.

Procedure
J. Using the capillary pipette, drop as many drops of 0.5 per cent sodium chlo-
ride into each of the sm\lll test tubes as indicated by the number on the tube.
Hqld the pipette at the same angle for every tube to insure equality in the
size of drops.
2. With the same pipette at the same angle, add drops of distilled water to every
tube, except the first, o[ sufficient number to make a total of 25 d~ops in
each tube. Invert to mix. The percentage strength of tbe solution of sodium
chloride in each tube is equal to the number on the tube multiplied by 0.02.
 HEMATOLOGIC TECHNIC

3. Withdraw I to 1.5 cc. of blood from a vein and place one drop of blood into
each tube. (When oxalated or citrated blood is used, wash the cells in 0.7 per
cent salt solution; make a SO per cent suspension in the salt solution j and
place one drop in each tube of the fragility set-up. Prepare a control in a
similar manner using the blood of a normal individual.)
4. Mix by inverting and allow the tubes to stand at room temperature for 2
hours.
s. Determine the point where hemolysis begins and the point where hemolysis
is complete.
6. Prepare a similar set of tubes, using the blood of a normal'individual for test-
ing the accuracy of the solutions. This control must be made witn every test
and should be done before the patient's fragility is determined.
Normal blood shows beginning hemolysis in 0.44 to 0.42 per cent salt solu-
tion and complete hemolysis from 0.34 to 0.32 per cent.
A positive test should be repeated at least 3 times and a more detailed tech-
nic used at least once.
The following~ technic, modified from Sanford's method, gives a wider range
of hypotonic salt solution.
Materials and Equipment
1. Equipment for venepuncture.
2. Thirty-two small test tubes placed in a rack and labeled 60, 59, 58, etc.
3. 0.6 per cent solution of sodium chloride.
4. Burette or pipette.
5. Distilled water.
Procedure
1. Using the same technic as before, drop as many drops of 0.6 per cent sodium
chloride as indicated by the number on the tube.
2. Add drops of distilled water to every tube, except the first, of sufficient number
to make a total of 60 drops in each tube.
3. The number on each tube multiplied by 0.01 indicates the percentage strength
of sodium chloride in each tube.
4. Place 2 drops of the patient's blood into each tube as described above.
5. Prepare a control in a similar manner.

PIPETTE METHOD

Materials and Equipment

I. Equipment for finger puncture.
2. Thirteen red cell pipettes.
3. Hemocytometer.
4. Twelve bottles of sodium chloride solution of the following percentages: 0.5,
048, 0.46, 0,44, 0.42, 0.40,0.38, 0.36, 0.34, 0.32, 0.30 and 0.28. These solutions
should be carefully prepared by volumetric methods.
5. Microscope.
64' HEMATOLOGIC TECHNIC

Procedure
r. Make a red cell count, using the customary diluent, to establish the normal
count.
2. Fill the r2 pipettes, using the patient's blood and the various solutions of
sodium chloride as diluents.
J. Allow all pipettes to stand for r hour with occasional shaking.
4. Make a red blood count from each pipette.
By this method it is possible to find out exactly how many cells have been
hemolyzed. In an occasional border line case of hemolytic icterus, this method
demonstrates increased fragility where the results of the tube metJlOd are question.
able. ,
The extent of fragility can be shown in the form of a curve, using the number
of red cells as the ordinate and the saline dilutions as the abscissa of a graph.

SEDIMENTATION RATE

When red blood cells are 'permitted to stand in their, own plasma to which
a suitable anticoaguiant has been added, they tend to sink to the bottom of the
tube. This occurs first in the form of individual cells and within a few minutes
after the cells have become clumped together (rouleaux) they sink in groups or
aggregates. The greater the degree of clumping the larger the masses and tbe
faster will be the settling to the bottom of the tubes. The factors influencing
the sedimentation rate include (I) the number of cells available for sinking,
(2) the tendency toward aggregation and (3) the physical or chemical composi.
tion of the plasma. Thus, if there is a tendency toward autoagglutination or
rouleaux formation the cells will sink at a much faster rate.
The rate of settling has been divided by s;;~e)nto three arbitrary stages;
the first being tbe settling of the individual cells"before clumping, secondly,
the marked accentuation of settlhlg from five to twenty.five minutes during
which time cell aggregates are formed, and thirdly, the period of constant set·
tling with slowing of the rate as the cells accumulate at the bottom of the tube.
When the aggregates sink they displace the plasma which consequently forms a
current in an upward direction and this in turn hinders the sinking of the clumps
of cells. Therefore, the settling rate is greater in plasma that has a: greater vis-
cosity. This introduces the factor of fibrinogen and globulin content of the
plasma since these substances have a greater viscosity tha_!l serum albumin.
The more diluted the blood corpuscles the greater is the sedimentation
rate. Dilution of the cells with plasma increases the sinking velocity. There
is a complete parallelism between rouleaux formation and rate of settling. Some
red cells, like those of the horse, settle rapidly even when placed in the serum
of another animal. The serum globulin and fibrinogen is known to vary, par·
ticularly in pregnancy and infection, where they are increased in amount. The
fibrinogen content seems to parallel the sedimentation rate. The sinking velocity
is increased if oxygen is bubbled through dtrated blood and conversely, carbon
dioxide decreases the velocity. This suggests a possible cause for the low
values in cyanosis.
 HEMATOLOGIC TECHNIC

The sedimentation rate is not specific for any disease. It depends upon
physiological conditions 'and not pathological conditions. It is rapid in acute
general infections and in certain chronic infections like syphilis and tuberculosis.
It is also rapid in pregnancy, probably because of plasma disturbance. In
evaluation of sedimentation rates it is important that all of these factors be
taken into account and to remember tbat increased sedimentation is not peculiar
to any particular disease. Furthermore, the technical execution of the test must
be extremely meticulous, The precautions that should be included in the execu-
tion of the test include those that are given below.
It is not known why the sedimentation rate of red cells is variable in different
diseases, particularly the increased rate in the acute infectious states. It is known,
however, that many factors influence the rate when the test is carried out in the
laboratory. These include the type of anticoagulant, the bore and length of the
tube (the longer the tube, the faster the rate), the inclination of the tube (the
more inclined, the faster the rate), the temperature (the higher the temperature,
the faster the rate), delay in carrying out the test (the more delay, the slower
the rate), and the cellular concentration (the more dilnte, the faster the rate).
Therefore, cell volume determinations must be made at the same time and the
sedimentation time corrected for the number and size of erythrocytes.
From the above, it is generally agreed that the test should be done with cer-
tain precautions. These are:
I. Use of proper anticoagulant.
2. The proper sized tube.
3. Immediate performance of test after withdrawal of blood.
4. Sedimentation at room temperature (22°_27° C.).
5. Correction for cell volume.
6. Tube always in absolute vertical position.

WINTROBE* AND LANDSBERG METHOD

Materials and Equipment

I. Equipment for venepuncture (dry syringe),
2. Capillary pipette.
3. Special hematocrit tube (Wintrobe).
4. Anticoagulant.
Mixture of 6 mg. of dry ammonium oxalate and 4 mg. of potassium oxalate for
each 5 cc. of blood.
5. Tube with anticoagulant prepared by placing in it the proper amount of
oxalate in solution, and then evaporating the water in a hot air oven.

Procedure
I. Withdraw 5 cc. of venous blood and place in the tube containing the anticoag-
ulant; shake for a few minutes.
2. With a capillary pipette fill a Wintrobe hematocrit tube to the 10 cm. mark .
• Wintrobe states that a tube illdlnation of :1,3 per cent causes a 30 per cent acceleration of
the rate.
 HEMATOLOGIC TECHNIC

3. Place the tube of blood in a vertical positign at room temperature, and observe,
at r 5 minute intervals, the point on the scale to which the corpuscles fall for
one hour.
4. At the end of I hour centrifuge the tube at high speed and obtain the volume
of packed red ceUs.
5. Then correct the sedimentation rate, according to the volume of cells by using
the correction chart shown below.

Volume of Packed Red Cells, ce. per 100 cc. blood

FIG. 4s.-Conection chart for sedimentation time. (From Wintrobe and Landsberg: Ame,.. Jour.
Med. Sci., Jan., 1935. Lea and FebigerJ Publishers.)

USE OF THE CHART

First, find the horizontal line that corresponds to the mm. of sedimentation
for I hour. Then, follow this line until it intersects the vertical line representing
the volume of packed red cells. At this point, follow the nearest curved line llown-
ward until it intersects the dark line at 42, if the patient is a woman; if a man,
follow the curved line until it intersects the dark line at 47. This point of inter-
section represents the corrected sedimentation rate, when read on the horizontal
line.
We use the Wintrobe tube and correction chart, exclusively, because a volume
index may be estimated at the same time. Many differ{nt~kinds of tubes and
apparatus have been recommended for this determination: including those of
Cutler, Walton, Westergren (in wide use in Europe), and recently tbat of Brooks,
recommended because it is constructed to fit into the 'butt of a, needle.
The normal average sedimentation in one, hour by the Wintrobe method is
3.7 mm. for men, and 9.6 mm. for women, with a maximal range from 0 to 9 mm.
for men, anll from 0 to 20 mm. for women.
 HEMATOLOGIC TECHNIC

We agree with Todd and Sanford that it is preferable to report sedimenta-

tion rates as fast, very fast, or normal.
Cutler and his associates have concluded that anemia has little to do with
sedimentation rates. 'They point out that the rate of sedimentation depends on
the size of the cell aggregates which may be produced by rouleaux formation.
They suggest that it is not necessary to make corrections for anemias.

WESTERGREN'S METHOD

Materials Needed
1. Westergren's sedimentation stand.
2. Westergren's tube--2.5 mm. in diameter graduated in mm. (0-200).
3. 3.8 per cent sodium citrate (sterile).
4. Sterile syringe and needle.
5. Specime'l ~ottle.

Procedure
I. Place 0.5 cc. of 3.8 per cent sodium citrate into syringe.
2. By venepuncture draw out blood to the 5 cc. mark.

3. Empty mixture into specimen. bottle.

4. Mix thoroughly and run test immediately if possible. Not more than one
hour should elapse before tbe test is run.
5. Draw blood up to the 200 mm. mark in the Westergren tube. Avoid
bubbles.
6. Set upright in stand and record the time.
7. Read red cell level at end of one hour.
8. Report results as the distance sedimented in mms. by the top of the red cell
column.

Normal Values
Men: 3 to 5 mm. in one hour.
Women and children: 4 to 7 mm. in one hour.

A MICRO-METHOD FOR DETERMINING SEDIMENTATION RATE ON

CHILDREN

Materials Needed
I. A I per cent solution of 'heparin (store 'in refrigerator).
2. Hanging drop slide (depression IS mm. in diameter and 3 mm. in depth).
3. Petri dish containing moist filter paper (used only when blood has to be
transported to laboratory or there is any delay in running test. -
4. 20 cu. mm. micropipet.
s. Combination microhemopipet with metal holder and rack.*
• ){icrohemopipet can be purchased from Fisher Scientific Co., 711 Forbes St., Pitt.sburgh.
 HEMATOLOGIC TJ:CHNIC

Procedure
I. Place exactly 20 cu. mm. of a I per cent heparin solution in the depression
of the hanging drop slide. Allow to dry at room temperature.
2. Puncture patient's finger and allow 4 or 5 drops (about 0.2 ce.) of freely
flowing blood to drop into prepared depression.
3. Mix blood and anticoagulant thoroughly with fine glass rod.
4. Place slide in moist petri dish with cover, if test is not to be run im-
mediately.
5. Fill clean dry microhemopipet to top mark (50 cu. nun.) with blood.
6. Close two ends of the pipette with the rubber-cushioned cups of the metal
holder and place in vertical position on the rack.
7. Readings may be taken at any desired intervals during sixty minutes obser-
vation and recorded in mm. or percentages.

Normal values: for children (I mO.-I4 yrs.): 0-20 per cent.

This method is recommended when bloed cannot be obtained by venepunc-
ture. Kato's microhemopipet can also be used to determine the volume of packed
cells and the icterus index on the same sample of blood used in the sedimenta-
tion rate.

TEST FOR BILE PIGMENT IN BLOOD SERUM

ICTERUS INDEX

Materials and Equipment

I. Equipment for venepuncture (syringe and needle either dry or rinsed with
sterile normal saline).
2. Centrifuge and centrifuge tubes.
3. Wooden applicator.
4. Pipette.
5. Dichromate standards (Kolmer and Boerner).
Prepare 100 cc. of a I per cent aqueous sol ution of potassium dichromate.
Add 2 drops of concentrated sulphuric acid. Into II chemically clean test
tubes place 10, 5,3, 2, I.S, 1.2, I.O, 0.1, 0.5, 0.3 and 0.1 cc. of this solution.
Dilute the last 10 tubes to a total volume of 10 ce. with distilled water.
Place 5 cc. of the solution in each of these tubes into I I small tubes (10 X
lOa mm.) and label laO, 50,30, 20, IS, 12, IO, 7,5, 3 and I. Stopper each
tube thoroughly. The labels represent icterus index units_
Sets of icterus index standard tubes can be purchased from laboratory supply
houses. \Ve prefer to prepare our own standards_

Procedure
I. Withdraw 5 cc. of blood from the vein using a dry syringe.
2. Transfer to the centrifuge tube and allow to clot.
3. Loosen the clot around the edges gently with a wooden applicator.
 HEM.ATOLOGIC TECHNIC

4. Centrifuge for IS to 20 minutes and withdraw serum with a pipette. There

must be no hemolysis.
5. ,Compare with the standard tubes. The deeper the color the higher the index.
If there is hemolysis the serum 3Vill not match the standards.
The normal range is from 3 to 5 icterus units. "Latent icterus" is from 6
to 16 units. When the icterus is above 'S, the patient invariably shows the clin-
ical signs of jaundice. Values as high as 100 or more may occur in severe jaun-
dice.
A diet of carrots may give a peculiar color to the blood serum and skin.
This condition, known as carotinemia, is often confused with bilirubinemia.
The blood for this test should be taken preferably before breakfast to pre-
vent the cloudy appearance of the serum due to a high lipoid content.

VAN DEN BERGH REACTION

The addition of sulphanilic acid and sodium nitrite to a solution of bilirubin
results in the formation of a colored pigment, azobilirubin. When large quanti-
ties of bile are absorbed in the blood, the addition of the above reagents causes a
characteristic color change within 10 to 30 seconds. This is called a "direct reac-
tion" and occurs mainly in obstructive jaundice. When bilirubin appears in the
blood as a result of- hemoglobin destruction there is no color change upon the
addition of the sulphanilic reagent unless bilirubin is extracted with alcohol.
When an alcoholic extraction is necessary to bring about the color change, the
reaction is called "indirect" and occurs in hemolytic jaundice and to a lesser de-
gree in normal serum. Any serum giving a positive "direct" reaction will give a
positive "indirect reaction."

MODIFICATION OF THANNHAUSER AND ANDERSON (Todd and Sanford)'

Reagents required
(a) Standard artificial bilirubin solutton:
(I) Dissolve 0.I508 Gm. of ammonium iron alum in 50 cc. of concentrated
hydrochloric acid, and add water to make 100 cc. This solution repre-
sents 0.5 mg. of bilirubin in 100 cc. and will keep indefinitely.
(2) To 10 cc. of (I) add 25 cc. concentrated hydrochloric acid, and water
up to 250 cc. This solution keeps about 6 months.
(3) To 5 cc. of (2) add 5 cc. of 20 per cent potassium sulphocyanide in a
glass-stoppered cylinder (50 ce. capacity) ; add 20 cc. of ether. Shake
well and transfer ether extract to a colorimeter cup .. This standard solu-
tion must be prepared each day a test is made.
(b) Sulphanilic reagent.
This is freshly prepared Ehrlich's Diazo reagent. It is made of 2 solu-
tions each of which keeps well, but the mixture must be made immediately
prior to the test .
.. Todd and Sanfordts modification is so satisfactory that we have found it unnecessary to alter
the text or the order of procedure.
 HEMATOLOGIC TECHNIC

Solution A: Sulphanilic acid, 5 Gm.; concentrated hydrochloric acid, SO

cc.; distilled water to make 1000 cc.
Solution B: Sodium nitrite, 0.5 Gm.; distilled water, 100 ce. Take 0.8
cc. of solution B and make up to 25 cc. with solution A.
(c) Saturated s01ution of ammonium sulphate.
(d) Ethyl alcohol, 95 per cent.,

Procedure
I. To 2 cc. of clear serum add I cc. of freshly prepared sulphanilic reagent (b).
If there is a marked color change this may be compared at once with the
standard; the "direct reaction."
2. Add 2 cc. saturated ammonium sulphate (c).
3. Add 10 cc. 95 per cent alcohol (d).
4. Centrifuge at high speed.
5. Compare with standard (a) in a Duboscq colorimeter, setting standard cup
at 20 mm ..

CALCULATION
Reading of Standard (20) * f bilirub' . f
Reading of Unknown X 3·75 = mg. 0 In m 100 cc. 0 serum.

Normal serum contains less than 1.5 mg. of bilirubin per 100 cc. and never
gives a direct reaction. The immediate direet reaction occurs in obstructive jaun·
dice. According to some authorities a delayed direct reaction occurs in severe
jaundice of the hemolytic type, while the milder cases give an indirect reaction.

ELTON'S RING TEST

This test is simple and practical and a positive direct reaction can be c1e~rlY
shown.

Materials and Equipment

I. Equipment for venepuncture.
2. I cc. pipette.
3. IS cc. graduated centrifuge tube.
4. 0.5 ce. pipette.
5. 'Ehrlich's Diazo reagent. (For preparation, see van den Bergh reaction.)

Procedure
I. Place I cc. of clear serum in the centrifuge tube.
2. Slant the tube to an almost horizontal position and overlay the serum with 0.5
ce. of Diazo reagent.
3. Restore the t.rl:.e to a vertical position and examine the contact zone of the
two fluids for the development of a Teddish ring .
• The serum, 2 cc. is diluted 75 times by the addition of 13 cc. of reagents (b) (c) (d). It Is
matched against 3 standard of 0.5 mg. to 100 cc. Therdore for 1 mg. to 100 ce. the factor is
one·half of ).5 or 3.75.
 HEMATOLOGIC TECHNIC

4. If no color appears within 60 seconds, gently shake the tube, and observe for
anotller 10 minutes.
This test demonstrates the presellce or absence in the serum of bilirubinate
which is a water soluble salt formed by tile liver in excreting bilirubin.

QUANTITATIVE DETERMINA'l'ION OF UROBILINOGEN IN FECES

SPARKMAN'S METHOD

Principle
The urobilin is converted to urobilinogen by tire addition of ferrous sulphate
and sodium hydroxide. This is followed by filtration, addition of Ehrlicb's re-
agent and colorimeter comparison with the artificial standard after a suitable
period has been allowed for tire development of color.
Reagents
J. Ehrlich's reagent:

Cone. Hel.. ...••••.••••••...•••..•.•••..••• 75 cc.

Distilled water ............................. 75 ce.
PaJadimethylaminobellZllldehyde... . . •. •• . . . •. 10 Gm.
2. Ferrous sulfate: (tlris reagent must be freshly prepared)
Fenous sulfate.... . ... ...•. . . . . .... . . . . . . . . . 8 GIll.
Distilled water. . . . . • • . . . . . . . . . . . . . . . . . . . . . .. 40 ce.

3. NaOH 10 per cent.

4.5 N HCI.
S. Artificial standards:
A. Stock solutions:
( I) 4 per cent aqueous Solution of gold chloride.
(2) 10 per cent aqueous solution of sodium bromide.
B. Working standards: (These standards keep one year).
(r) Strong Standard: One volume of sodium bromide is added to I
volume of gold chloride, alld the resultant solution is made up to IS
volumes with distilled water. This standard is equivalent to 8.175 mg.
of urobilinogen per roo cc.
(2) Intermediate Standard: Dilute I volume of the strong standard
witlr an equal volume of distilled water. This standard is equivalent
to 2.365 mg. per 100 cc.
(3) Weak Standard: Dilute I volume of tire intermediate standard
with an equal volume of distilled water. This represents 0.935 mg. of
urobilinogen per roo ce.

Procedure
I. Emulsify exactly 5 cc. of tire stool in 100 cc. of distilled water by gradual
addition of the water and repeated transfer to a 250 ce. Erlenmeyer flask.
2. With mixing, add the ferrous sulphate dissolved in the 40 ce. of water.
050 HEMATOLOGIC TECHNIC

3. Add slowly 40 cc. of 10 per cent sodium hydroxide, rotating the flask during
the addition.
4. Incubate in a water bath at 50· C. for IS minutes.
5• .cool to room temperature and filter (Wbatman no. 2 filter paper).
6. To 5 ce. of the filtrate in a test tube add 5 cc. of distilled water, 0.3 cc. of
five normal HCI and Ice..of Ehdich's reagent.
7. Allow 5 minutes for color development and compare the solution in a col-
orimeter with the standard of the nearest depth of color.

Calculations
Reading of Standard .. .
Reading of Unknown X F X 3. 85 X 20 = mg. of Urobilmogen 10 100 Gm. of feces.

F = strength of standard used.

Normal values
ISO to 300 mg. of urobilinogen per 100 Gm. of stool.
Extreme limits of normal values are 70 to 600 mg.

QUANTITATIVE DETERMINATION OF UROBILINOGEN IN URINE

Principle
The method is applicahle either to a single specimen of urine or to specimens
excreted during twenty-four hours. Single specimens should be freshly voided.
Twenty-four hour specimens are collected in a brown glass hottle to which has
been added about IOa cc. of purified petroleum benzine and 5 Gm. of anhydrous
sodium carbonate. Bile pigments are removed by the addition of anhydrous
calcium chloride. The ~ldehyde reaction is produced with the, addition of
Ehrlich's reagent.

Reagents
(I) Ehrlich's .reagent.
(2) Anhydrous calcium chloride.
(3) Working Standards (see quantitative 'determination 'in feces).

Procedure
(I) To 50 ce. of urine in a flask add 2 Gm. of calcium chloride.
(2) Mix and filter. The residue on the filter contains the bile pigment
which may be detected by Gmelin's test.
(3) To 10 cc. of the filtrate add I cc. of Ehrlich's reagent.
(4) Mix and allow 5 minutes for color development.
(5) Match in the colorimeter against the standard which has, the nearest in-
tensity of color.
 HEMATOLOGIC TECIINIC

Calculations
Reading of Standard X F. = mg. of urobilinogen in 100 cc. of urine.
Reading of Unknown
F = strength of standard used for comparison.

Normal values
Any specimen of urine yielding a color in the range of the weak or of the
intermediate standard may be regarded as containing no increase in urobilinogen.
It should be reported as "not increased." Values exceeding 8 mg. per 100 CC.
represent pathologic urobilinogenuria.

STERNAL PUNCTURE

REICH'S METHOD

Materials and Equipment

1. Tincture of iodine and alcohol.
2. I per cent novocaine.
'3. A needle with a 10 gauge hore, and an adjustable guard.
4. 20 cc. sterile record transfusion syringe.
5. IS cc. centrifuge tube.
6. 2 cc. pipette and a capillary pipette.
7. 1.4 per cent sol ution of sodium oxalate.
8. Tubes made by sealing off one end of ,a short piece of glass tubing of slightly
larger diameter than the pipette.
9. A small hammer.
10. Slides.
II. Equipment for staining.

Procedure
1. Prepare the area with iodine and alcohol. Infiltrate the skin with I per cent
novocaine in the middle of the sternum iust below the iuncture of the body
with the second rib.
2. Continue to inject straight down to the sternum and infiltrate the periosteum.
3. Push the sterile needle vertically or at an angle of about 45 degrees, until it
reaches the periosteum. Continue downward pressure by hand, but if the
outer plate is thick and the resistance is too great a few taps with a small
mallet will serve to drive tbe needle into the spongy bone.
4. Remove the stylet, attach the sterile syringe, apply suction and if the needle
is deep enough, bloody fluid will appear in the syringe. If not, replace the
stylet, raise the guard a few turns and drive the needle down until ahout 10
cc. of bloody fluid are aspirated. .
5· Place the bloody fluid in a IS cc. centrifuge tube which contains 2 CC. of 1.4
 HEMATOLOGIC TECHNIC

per cent solution of sodium oxalate and centrifuge at moderate speed far
minutes.
6. Using a capillary pipette, remove the supernatant plasma and transfer the
buffy coat of cells to another tube.
7. Centrifuge again, remove the plasma, and pipette off the buffy coat for the
second time.
S. Prepare smears on slide in the usual manner.·
9. Make a differential count of roOD cells.
NOlWAL DIFFERENTIAL COUNT ON BONE MAU.OW SMEARS (REICH)
Neutrophils (mature) ................. ................. .. 25 o~
Neutrophil! (young forms) ............ o............ ....... 5.0 0
Neuuophils (band fOrID..!l) ••.•...••..••.•..•••.•••••••••••• 10 0 0
~=~;:".'.::::::::::::::::::::::::::::::::::::::::: I~.~~
Monocytes......................... ..................... 1.0%
Mye1oblasts. ... . . . . . . . . . . . ..... .. . ... . .. .... . . . . . . . .... . 1.0%
Premyelocytes and Myelocytes .........................•.. 20.0~

!=~~:.::::::::::::::::::::::::::::::::::: ::::: :;:~~

YOUNG AND OSGOOD'S METHOD
This method is similar to that of Reich except for the following variatinns :

Materials and Equipment

I. IS gauge spinal puncture needle 3 to 4 em. in length.
2. No guard on the needle.
3. 2 to 4 mg. of powdered oxalate in a 4 by Y:l inch test tube.
4. Wright's stain.

Procedure
I. Only I to 2 cc. of marro;vis aspirated since it is thought that the removal of
large amounts of fluid causes the patient considerable discomfort. Proc3,ine
hydrochloride is used for local anesthesia.
2. The needle is inserted at the sterno manubrial junction at an angle of 60 de-
grees; it is. braced by the finger against the breast to prevent going enthely
through the sternum, and depressed to an angle of about 30 degrees as it is
forced into the marrow cavity. Care is taken not to exceed a depth of 1.5 tIn
3: The marrow fluid is added to the oxalate and smears are made directly fI.o~
this mixture.
4. Wright's method for staining is used with a buffer phosphate at a pH of 64.
5. 200 to 500 cells are counted.

The ratio of _total myeloid cells to the total nucleated cells varies fl'om
8.29:1 to 2.00:1 and averages 3.61 :1. The predominant cell is the neutrollhil
staff cell (band). Reticulocytes occur in the same per cent as in normal blGod.
• Reich recommends a combina.tion of Jenner's and Giemsa's stain. Other methods sucl:J. u
Wright's and Giemsa's are satisfactory.
 HEMATOLOGIC TECHNIC

Average differentia!\l:elI counts on sternal marrow from 28 normal persons

(Young and Osgood).
Segmented neutrophils ...•........ .................. 7·4t025. 2
Segmented eosinophils ................. ............. . 0.0 to r.o
Segmented basophils .....•...••••......•........•.•. 0..0 to o.~
Neutrophilic staff cells ............................. . 15 8 to 33.0
Eosinophilic staff cells ............... ............•... 0..0. to 1.6
Basophilic sta.ff cells. .. .. .. ........ . ........... . ccto 0.6
~~=&l:11f~ :::::::::l~:::::::::::::::::::::::::::
1.8 to 9.8
o oto 2.0
~~~°fr~:hllk:'~fo~rs~::::::::: :::::::::::::::::: c.eto 2.6
EOSlDOphilic myelocytes .... ........................ . 0..0 to 0..4

~::Jy~l~~T~ i::::::::.:::::::::::::::::::: 0..0 to 4.0.

Promyelocytes-Type II .... ....................... . 0..0 to 3.8
Myeloblasts (stem cell) .....•..........••.......••.•. 0.0 to 1.:1
Lymphocytes. . . . . . . . . . . . . .. . •.........•.....••.• 4.8 to 16.0.
Monocytes (reticulo-endothelial) ..................... . 0..0 to 4.2
Normoblasts ..................................... . 5.4 to .20.0
Megaloblasts. . .• ...•...•....•.....•.... . .....•... 0..0to 4.2
Megakaryocytes.. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. " 0.0 to 0 2
o.cto 1.0
~lW::;~~T~:~~~ ~~.u~~~): .. :::: ::::::::::::::: 12.8 to 31.8
Reticulocytes . . .................................. . I.~ to 5.:11

COMPLETE EXAMINATION OF BONE MARROW

A thorough examination of the bone marrow should include the following

procedures:
I. The removal of a button of bone and marrow from the sternum. (Other bones
may be examined at autopsy.)
2. Gross examination of marrow at the time of removal.
3. An accurate count of the total number of cells per cu. mm. of marrow and an
estimation of the number per cu. mm. of leukocytes, nucleated erythrocytes,
mature erythrocytes, and reticulocytes in some instances.
4. A differential count of leukocytes and nucleated erythrocytes from a serum
spread, stained with a polychrome dye.
5. Examination by histologic section.
6. Supravital stain of marrow cells in selected cases.
The removal of a button of bone is superior to the sternal puncture method,
since the marrow is frequently diluted with sinusoidal blood when aspirated with
a needle. A puncture does not provide material for either a gross examination
or a histologic section. These procedures are considered essential features of a
careful bone marrow study.
Isaacs has devised a method in which bone marrow is studied quantitatively
and architecturally by cell counts and histologic section, and qualitatively by the
use of a serum spread stained with Wright's or Giemsa's stain. The method given
below is essentially that developed by Isaacs with some additions and a few minor
modifications. Custer's technic for histologic section, a slightly different method
for preparing serum spreads from curetted marrow, and a list of materials needed
654 HEMATOLOGIC TECHNIC

f(jr sternal biopsy are added to Isaacs' specifications. Reticulocyte counts (p.
630) and supravital stains (p. 658) are used when additional cellular identification
becomes necessary.

Materials and Equipment

r. For sternal biopsy.
The usual surgical sterile drapes, towels, sponges, clamps, and materials
for skin incision (iodine, alcohol, I per cent novocaine, syringe, needle.
and scalpel).
Eight to 10 small, curved hemostats.
Two small, self·retaining mastoid retractors, or thyroid retractors.
Periosteal elevator.
Trephine.
Bone wax.
Curette.
Plain catgut (00) for ties.
Black silk for skin closure.
2. For counting bone marrow cells and preparing serum spreads.
Sahli hemoglobin pipette or leukocyte pipette.
Erythrocyte pipette.
A few cc. of blood serum from the patient or from an individual of the same
blood group.
Two watch glasses.
Two blunt glass rods.
Hayem's solution as a diluent for total cell counts.
One per cent acetic acid as a diluent for nUdeated cells.
Slides or cover glasses.
Hemocytometer counting chamber.
Microscope.
Immersion oil.
Wright's or Giemsa's stain.
One per cent alcoholic solution of brilliant eresyl blue when reticulocyte
counts are made.
Supravital stains when desired (p. 658).
3. For histologic section of the button of bone.
ReIly's fluid; or ro per cent formalin.
Helly's fluid is the same as Zenker's fixative except that formalin is sub·
stituted for glacial acetic acid.
FoRllUtA FOR HELLY'S FLUID
Bichromate of potassium... . . .. . .................... . :2.sGm.
Bichloride of mercury (corr.osive sublima.te)..... .. . .... . S oGm.
Sodium sulphate. . . . . . . . .. .. . ..................... . I.oGm.
Water ......•...................................... 100 ceo
Formalin ........................................ . 5 cc.
Decalcifying agent. (Sodium citrate-formic acid preferred.)
 HEMATOLOGIC TECHNIC 655
Formula:
Equal parts of 95 per cent alcohol, 85 per cent aqueous formic acid,
and 2 a per cent aqueous sodium citrate.
Routine materials for dehydrating, embedding, and cutting paraffin sec-
tions:
Maximow's Azure II-Eosin stain; or the stains used in the routine hema·
toxylin-eosin methods.
Formula for Maximow's Azure II-Eosin stain:
Stock solutions:
A. I per cent aq. solution eosin (W. G.).
B. I per cent aq. solution azure II.
Working solution:
Add 10 cc. of solution A and 10 cc. of solution B to lOa cc. of distilled
water.

SURGICAL BONE MARROW BIOPSY

I. Under local anesthesia"remove aseptically a button of bone and the attached
marrow from the sternum. The periosteum is incised and retracted, and the
bone exposed. The button is delivered by use of·a trephine. (Cells are counted
from the marrow removed in this manner and the remainder of bone and
marrow is used for, histologic section.)
2. With a curette, scoop portions of marrow from the hole in the sternum and
mix in a watch glass with equal parts of blood serum. (Smears are' made
from this preparation.)
3. The same procedure is followed for studying'bone marrow at autopsy. Mate-
rial may be obtained, also, from other bones of the body. The earlier the
marrow is removed after death, the more satisfactory the preparations. Isaacs
states that red blood cells are not altered within a reasonable time after
death, but the myeloid leukocytes tend to disappear in a short time.
4. When 'it is not feasible to remove an entire bone button for histologic section,
a satisfactory procedure consists of the use of a small perforator and burr
with a bandle for penetra~ion into the marrow cavity. The marrow can then
be removed by a small curette or by aspiration with a syringe and needle.
Gradwohl has devised an instrument for this purpose.

GROSS EXAMINATION
A gross examination consists of observing the color, amount, and consistency
of the bone marrow. ,

COUNTING BONE MARROW CELLS (Isaacs Method)

I. Wet the stem of a leukocyte pipette with serum., (A Sahli pipette or an
erythrocyte pipette may be used if desired.)
•. Work the tip of the pipette back and forth among the bony trabeculae, draw-
ing the bone marrow into the tube up to the I mark; draw serum up to the
4 mark; expel contents into the watch glass. If the Sahli pipette is used,
 HEMATOLOGIC TECHNIC

draw up 1 volume of bone marrow, place into the watch glass, and add quickly
3 volumes of serum.
3. Add 8 more volumes of serum to the mixture in the watch glass and mix thor-
oughly with the glass rod. This makes a dilution of t :12.
4. Draw this mixture to the 0.5 mark of the red cell pipette and dilute with
Hayem's solution.
s. Estimate the average number of cells in 1 sq. mm. in the counting cbamber
10 x U X 200 )
( and multiply by 24,000. No. of sq. rom. used in the count' This procedure
gives the total number of cells in the bone marrow which include leukocytes,
nucleated erythrocytes, and non-nucleated erythrocytes.
6. To find the number of nucleated cells (leukocytes and erythroblasts), follow
the same procedure but use I per cent acetic acid solution as the diluent, thus
dissolving the mature erythrocytes. The total number of leukocytes and of
erythroblasts may be determined by estimating the percentage of each type
from the stained smear.
7. Make several counts in this manner and average the results. Isaacs states
that the variation in counting cells from approximately the same region rarely
exceeds 5 per cent in normal marrow but considerable variation may be found
in pathologic marrows. There is a particularly uneven distribution in aplastic
marrows; some areas are non-cellular and others byper-cellular.
8. Reticulocyte counts may be made on bone marrow in the usual manner
(p. 63 0 ).

PREPARATION OF SERUM SPREADS

1. Mix the bone marrow removed by curette with equal parts of,serum; smear
on a slide in the same manner that blood' smears are made. (Isaacs recom·
mends cover glass preparations, but smears prepared on slides are more
easily stained.)
2. Stain the smears witb Giemsa's or Wright's stain, When the latter stain
is used, allow the diluent to stand on the slide for 10'minutes before washing,
3. Examine under oil immersion and make a differential count of 500 cells, in-
cluding nucleated erythrocytes. In some instances supravital studies are
necessary for the identillcation of cells. The technic is identical with that
described for peripheral blood (p. 658).

PREPARATION OF HISTOLOGIC SECTION (Custer's Method)

.1.Fix the deep surface of the button of bone for 24 bours in Helly's fluid.
2. Wash for 10 to 24 hours in running water. Place in 35 per cent alcohol colored
to a wine red with tincture of iodine until the iodine color no longer disap-
pears.
3. Decalcify in sodium citrate-formic acid solution for a few hours to several
days. Wash in running water for 16 to 24 hours.
4. Follow the routine proceoure for dehydrating and embedding the tissue in
paraffin.
s. Section at 4 microns.
 HEMATOLOGIC TECHNIC

6. Stain in Maximow's Azure II-Eosin stain for 6 to 24 hours; p'!Ss quickly

through the lower grades of alcohol, and complete the differentiation in 9S
per cent alcohol; dehydrate, clear in xylol, and mount in balsam.
Custer's procedure is recommended as the most satisfactory method. If these
reagents are not available, sections may be prepared by formalin fixation and
routine decalcification and hematoxylin-eosin staining methods. The latter
method, although inferior regarding cytologic differentiation, is adequate if sup-
plemented with serum spreads.
Isaacs states the total number of nucleated cells (erythroblasts and leuko-
cytes) varies from 900,000 to 1,000,000 per cu. mm. The differential count of these
cells is shown in Table XXXV.
TABLE XXXV

Per Cent of All

Nucleated Cells
Undifferentiated "lymphoid" cells. "Primitive blasts" . .... . 23· I ±8.0
Megaloblasts .......................................... . 3.0± 1.0
Basophilic normoblasts ................ '" .............. . 7· 2 ±2·5
Eosinophilic normoblasts. . . .. . ........................ . I2.0± 7 0

Polymorphonuclear neutrophils-aduit. .................. . 7·5± 5·5

Polymorphonuclear neutrophils-young .................. . I3.0± 6.0
Metamyelocyte•........•............................... 20.8± 3.0
Myelocytes ... .' ..........•............................. 2.8± 1.8
Myeloblasts .............•............................. 1.8± 0.8
Eosinophils (immature and mature) ...................... . 5·5±3· 0
Lymphocytes ......................................... . I.O± 0.5
"Endothelial cells." Phagocytes, ........................ . 0·9± 0·5
Hemohistioblasts and hemohistiocytes .................... . 2·3± 1.3
Megakaryocytes ....................................... . 0.5± 0.1
Basophils ............................................. . Present
Monocytes ........................................... . Present
Bone marrow findings in illustrative cases of various anemias are shown
by Isaacs in Table XXXVI. For details of bone marrow findings in normal
and pathologic states see Chap. 4r, by Custer.

CULTURE OF BONE MARROW CELLS

Osgood bas devised a method by which he claims bone marrow cells can be
successfully cultured. The aspirated marrow is placed inside of a semipermeable
membrane past which nutrient medium flows. The medium diffuses inward and
the waste products outward. His original apparatus permitted control of tem-
perature, composition and rate of circulation of the medium, oxygen and carbon
dioxide tension, and elimination of waste products. In his second simpler method
the aspirated material is placed in a 50 cc. vaccine vial containing citrated salt
solution. The buffy coat is removed, mixed with balanced salt solution, a total
cell count done, and sterile human cord serum added to a final concentration of
3S per cent. This can then be divided into separate vials and incubated at 37° C.
 HEMATOLOGIC TECHNIC

TABLE XXXVI

HEMATOLOGJC DATA IN ILLUSTRATIVE CASES OF VARIOUS ANEMIAS (ISAACS)

Blood Bone Marrow

ConditiOD RDC. W]3.C. Rb. Total Prim. ~rlm. Poly Me. Megalo- Daso. Eosino.
MilL ThoU!! gm. Nucleated Blasts· Blasts. Series Series blast9 Norma.- Norm-o·
per P<' % Cells per per % % % % blasts blasts
CU mm. cu. UlDl CU.tnnJ.. cu. tnm. % %
- ---I - - -
u6,ooo ,6,600
-
Apla!ltic: anemia 35 0 5'5 07 04·

Hodgkin's roentgen
ray 2.80 192,000 22,800 Il 0 61.0 IS 0' 0 5 13·5
-----I---I-'--I-r-- - - - - - - - - - , - - -
_C_h"'_n;_c_",_p_hn_·'_;'_f_ _.I___ I_'_.5_2 660,000 349,800 53 -0 28.0 18.0 ,. 0

Pernicious anemia 6.72 1.440,000 336.900 230.4 46.2 11.1 1.0 3.0 t.r
----1--1---1--1---1---1·--1-'-- - - - - - - -
Cirrhosis of liver 5.04 1.448,000 434,000 30.0 :22.5 24·9 II.5 67 61
-----I---I--I--I-------------
Cirrbosis of liver 504 882.000 506,000 57 5 130 4.0 6.0 3.0
-------I---I----I--f-----r----r--I--.~--r---r--
Acute hemorrhage 6 16 222,000 32,190 14 5 62.5 14.5 :2 5 6·5 soS
--------I----l----I--~--------------
HetJ:l.o1ytic anemia 4..06 840,000 494,800 470 170 33.0 40 7..0 22.0
--------I----I---I--- - - c - - - - - - - - - - - -
Mytlob!a.stic leu-
ketnia 3-.06 191·4 6·44 588,000 40:r,~ 68.2 a.ot 14·5 .,0
~
.5
~ -
Cruorlic rnyeloge-
cousleukeniia ,80 175 0 9 66 800,000 .348,000 435 47·0 ,,5 0.' ,., 0.5

Chronic lymphatic
leukemia I·97 5,0 4.76 1,380-,000 1,276 ,ooot 9'5 '0 4,0 015 '5 0.75

Bronchopneumonia 4·50 .32·4 g.lo 720,000 100,800 140 50.0 17.0 '.0
-n.o- I---
.0

Gas ba.cillus infec·

--- - -- e--
tiOD 285 28.2 6.44 1,240,()QO 290,000 2,3 5 .31.0 II.S 30 1·5

s -. -s.s-, -'-"-'000---99-'300-~-4'-0 -'0-,5 -'.5--'-'.5---7.5-

-Pn-eum-o-ru,-·.--I-.-so- - -
------1·
InfIuenzal pneumo.-
nia; beginning
empyema ".50 80 8·40 go6,ooo 312,000 34.5 36.0 18,0 1·5 IS 5

.. Both erythroblasts and leukoblasts. t More mature than the prhnitive blast stage.
t Including Iynlphoid cells.
This medium is changed every 48 hours. He states that there are cells in mitosis
after 34 days' incubation and that sometimes there are from 40 to 60 times the
number of cells in the original marrow. Tbis entire work requires further con-
firmation before it can. be accepted without reservation.

SUPRAVITAL STAINING OF BLOOD CeLLS

(SAllIN, CUNNINGHAM AND TOMPKINS)

The various methods for supravital staining are modifications of Pappen-

heim's original technic in which he used neutral red in the study of blood cells.
 HEMATOLOGIC TECHNIC

Both Sabin and Simpson devised a modified procedure for staining white blood
cells by adding Janus green B to stain mitochondria. The technic outlined by
the various workers is essentially the same.

Preparation of Solutions
1. Stock Solutions
Neutral red: (GrUbler's)
Sabin-IOo mg. of dye to 10 cc. absolute alcohol.
Cunningham-Saturated solution of dye in absolute alcohol prepared several
days before use. Solutions are kept in glass stoppered bottles, in the
dark, and at room temperature.
Janus green-Saturated solution in absolute alcohol.
2. Dilute Solutions
Neutral red:
Sabin-Io cc. of absolute alcohol to 0-4 cc. of stock solution. The slightest
staining of the nucleus of leukocytes indicates tbat the stain is too strong.
Cunningham-2o to 30 drops of stock solution to 10 cc. absolute alcohol.
This is satisfactory for normal blood, but for leukemic blood, exudates, or
tissue scrapings, the strength should be doubled.
3. Neutral red and Janus green combined
Sabin-2 cc. of dilute neutral red to 3 drops of saturated solution of Janus
green. This preparation lasts only for a few days and is more toxic
than neutral red used alone. Neutrophilic leukocytes are not motile with
this combined stain.
Cunningham-3 drops of saturated solution of Janus green to I cc. of dilute
neutral red.

Technic (Cunningham and Tompkins)

I. Cleaning of glassware.
Wasb slides with soap and water; rinse in running water for several
hours; wash in distilled water; transfer to cleaning mixture made up of
equal parts of sulphuric acid and a saturated aqueous solution of potassium
dichromate; leave slides for several days; remove slides and leave in run-
ning water·for at least 24 hours; wash in distilled water; store in 70 per cent
alcohol; dry with a clean cloth, preferably linen, immediately before using;
flame and allow to cool. Coverslips are cleaned in the same manner but
are not flamed.
2. Preparation of smears.
Flood the slide with dilute stain and let the excess run back into the bot.
tle, holding the slide with forceps so that the fingers will not touch the
surface of the slide; place the drained slide on end in a holder and allow
to dry; do not hasten drying. Smears prepared in this manner do not
keep long. Each box of slides should be tested with normal blood before
using with abnormal blood. Any staining of the nuclei indicates poorly pre-
pared stains or cell damage.
660 HEMATOLOGIC TECHNIC

3. Collection of blood.
Prick the finger so that the blood flows freely; touch a coverslip to a
small drop of blood and place on a slide smeared with the dried stains;
rim with vaseline of a higber melting point than that of the hot box in
which the preparation is to be placed. Sabin uses vaseline mixed with
paraffin. Cunningham uses Salveline of the James Bailey Co., applied with
a camel's hair brush.
4. Observation of smears.
In order to obtain the best results, the preparations are observed in a hot
box at a temperature of 380 C. When it is necessary to go out of the labora·
tory for the collection of blood, the smears may' be kept warm by placing
them on a hot water bottle. The smears should be observed about 20 min·
utes after they are'made. A comparison between granulocytes and mono-
cytes is more striking at this time, since normal granulocytes do not begin to
show vacuolar degeneration until 30 to 40 minutes while the vacuolar
apparatus of monocytes is apparent after 10 to IS minutes. Slides should he
kept in the dark while not being studied.
For appearance and description of cells supravitally stained see plate XIV.

EXAMINATION FOR MALARIA PARASITES

FRESH BLOOD

Materials and Equipment

1. Equipment for finger puncture.
2. Cover glasses and slides.

Procedure
I. Place a drop of blood from the finger on center of cover glass.
2. Invert and place on slide so that the hlood will spread out in a thin film.
3. If examination is prolonged, seal the edges of the coverslip with melted vase-
line to prevent drying.
4. Examine at once with oil immersion objective, cutting out the light with
diaphragm of the microscope.
The best time for examination is about 8 hours after a chill, but the parasites
can be found at other times.
Wet smears are not recommended for routine work, though useful in study-
ing the motility of parasites.

THIN SMEAR METHOD

Materials and Equipm.mt

1. Equipment for finger puncture and preparation of smears.
2. Wright's stain.
 HEMATOLOGIC TECHNIC 661
Procedure
I. Prepare smears as for differential leukocyte count but so thin that the red
cells are well separated.
2. Fix and stain with Wright's stain as for a differential count:
3. Dry and examine with oil immersion objective.
4. The smears must be well stained for satisfactory results. When the nuclei
of leukocytes are deeply stained, the plasmodia will be distinctly outlined.
Smears should be taken a few hours before a chill and, if possible, before
therapy is administered. In latent and chronic cases, the patient may be given a
hot bath followed by a brisk rub immediately before the specimen of blood is
collected, in an attempt to draw the parasites into the peripheral circulation.
THICK SMEAR METHOD (Barber and Komp)
In many latent and chronic cases of malaria the parasites are not present in
sufficient quantities to be found in one thin smear of blood and it becomes neces-
sary to examine a thicker smear. When the blood of a great many individuals
is examined, as in an extensive malaria survey, the thick smear is used to deter·
mine -the presence of the parasite, and the type is then determined by means of a
thin smear.
Procedure
I. Puncture the finger and deposit 3 or 4 drops of blood on one end of a clean
glass slide. Allow to dry in the air.
2. With a toothpick, smear the blood to the size of a 10 cent piece with cen-
trifugal motion. Do this for at least 2 minutes. This insures the blood
sticking to the slide." Some work~rs use the concentration method of Bass
and Johns, but the thick smear methods are now in wide use, have been very
satisfactory, and according to James, the thick smear is cOlleentrated So times,
as compared to an ordinary smear. For appearance and description of
parasites see Plate 52.
3. Make a thin smear in the usual manner on the other end of the slide,
4. Insert the thick end of the smear into a jar containing freshly diluted
Giemsa's stain (1.3 cc. of stock stain to 75 cc. of neutral distilled water). Al-
low to stand for one hour. The solution should be deep enough to cover only
the thick smear without injury to the thin smear. (Other workers recom-
mend placing the smear for a few moments in distilled water before staining
in order to completely dehemoglobinize the red cells.)
5. ElCamine under oil immersion. The red cells are dehemoglobinized and en-
tirely obliterated without injury to the parasites and white cells which stain
in the usual way. Platelets, also, are not destroyed.
6. If the parasites are found, stain the thin smear with Wright's stain to deter-
mine the type of parasite present.
NOTE.-A number of thick smears may be stained simultaneously by separating them with
small squares of card board and bindmg them together with rubber bands. At least 75 CC. of dilute
stain should be used for 25 smears.
• MAcAm, T. B : Amer. Jour. CUn. Path., 6, 91, 1936.
662 HEMATOLOGIC TECHNIC

TESTS FOR INFECTIOUS MONONU~LEOSIS

(Technic of I. Davidsohn)
Tl1E Pro:SUMPTIVE TEST

Indication
The presumptive test is indicated in cases when the clinical or the hemato-
logical findings or both suggest infectious mononucleosis.

Principle
The test is based on the agglutination of sheep erythrocytes by the heteroph-
ilic antibodies in the serum of patients with infectious mononucleosis.

Materials Required
I. Test tubes 75 mm. long and 9 to 10 nun. in diameter.
2. 0.1 cc. blood serum inactivated for 30 minutes at 56° C.
3. A 2 per cent suspension of sheep red corpuscles. The sh~ep blood should be
not less than twenty-four hours old and not older than one week. For the
preparation of the 2 per cent suspension the sheep cells are washed three
times in physiological saline on the same day the test is run.

Procedure
The titration of the agglutinins is performed with 0.25 cc. of the serum
dilutions, ranging from 1:7 to I :7168. The best way to prepare those dilutions
is to place 0.4 cc. of physiological sodium chloride solution in the first test tube
and 0.25 cc. in the Iemaining 10 test tubes.
I. To tbe first tube add 0.1 cc. of serum.
2. Mix and transfer 0.25 cc. to the second tube. Mix the second tube and
transfer 0.25 cc. to the third, etc., until the last tube is reached from which
0.25 ce. are discarded after mixing.
3. Add 0.1 cc. of a two per cent suspension of sheep red, blood cells. The final
dilution of serum is considered the titer.
4. Shake the test tubes and leave at room temperature for two hours. Some
workers prefer to centrifuge the tubes at 2,000 r. p. m. for exactly 5 minutes
and read immediately. (The Strauss Technic.)
5. The results are read after gently shaking the test tubes. The shaking is con-
tinued until the entire sediment is suspended. The tubes in which the cells
remain in the form of a single clump are read as three plus. Those in which
the cells break up into distinctly visible clumps and the fluid is clear and
transparent, are called two plus. The reading of the one plus agglutination
is best carried out hy means of a low power objective of the microscope.
The test tube is placed horizontally on the stage after the tube is shaken
and in this way one can determine the end point of the agglutination with
the greatest accuracy. When time permits, it is advisable to repeat the read-
 HEMATOLOGIC TECHNIC 663

ing after an overnight incubation in the ice box. The titer is tben usually
one to two dilutions Wgher.
6. One control is used consisting of 0.25 CC. of physiologic sodium chloride solu-
tion and 0.1 CC. of the sheep cell suspension. The technic of the test is sum-
marized in the table below.
TABLE XXXVII
TECHNIC OF TEST FOR INFECTIOUS MONONUCLEOSIS

2 Per Cent Titer (Final

Saline Serum Serum
Tubes cc. cc. dilution Dilutions Sheep Cells Dilutions
CC. of Serum)
04 o. I "5 0.1 "7
2 0.25 0.25 oft :5 1:10 0.1 1:14
3 0.25 0.25 of 1:10 1:20 0.1 1:28
4 0.25 0.25 of 1:20 1=40 0.1 1:56
5 0.25 0.25 of 1:40 1:80 0.1 1:112
6 0.25 0.25 of 1:80 1:160 0.1 1:224
7 0.25 0.25 of 1:160 1:3 20 0.1 1'448
8 0.25 0.25 of 1:320 ,:640 0.1 1:8 6 9
9 0.25 0.25 of ,:640 1:1280 0.1 1:'79 2
10 o 25 0.25 of 1:1280 "25 60 0.1 1:35 8 4
II 0.25 - 0.25 of 1:2560' 1·5120 0.1 1:7 168
Control
12 0.25 0.1

• Discard 0.25 cc. from last tub_e.

Interpretation of Results
A titer of at least I :224 in a person who did not receive an injection of
horse serum or of horse immune serum in the recent past and who presents a
clinical picture and hematologic findings suggestive of infectious mononucleosis,
indicates the presence of infectious mononucleosis.
A titer of over I :224 should be c~nsidered positive even if there is a history
of horse serum administration, unless the patient is suffering at the time of ex-
amination from serum disease or unless he had recently gone through an attack
of serum disease. Such titers are mainly encountered in the course of serum
sickness and for relatively short periods afterwards. If a history of serum in-
jections is present, it is advisable to check the result witii the differential test.

THE DIFFERENTIAL TEST

Indications
I. A history of a recent injection of a horse immune serum· Dr of serum disease
in the recent past in a patient with a titer of heterophilic '\ntibodies of
I :224 or over as determined with ·the presumptive test for infectious
mononucleosis.
 HEMATOLOGIC TECHNIC

2. A borderline titer of heterophilic antibodies (1 :56 or 1 :112) as determined

with the presumptive test for infectious mononucleosis.
3. A low titer of heterophilic antibodies (1 :56 or less) as determined with the
presumptive test in cases suspected of having had infectious mononucleosis
in the past.

Principle
The heterophilic antibodies (antisheep agglutinin) in infectious mononucle-
osis are not of the Forssman type. They are not absorbed by a suspension of
guinea pig kidney. The heterophilic antibodies in serum disease are of the
a.
Forssman type and are readily absorbed by suspension of guinea pig kidney.
The antisheep agglutinins are promptly absorbed by boiled beef red cor-
puscles from the sera of patients with infectious mononucleosis and almost as
well from the sera of patients with serum disease, but not from normal sera.

lI'laterials Required
I. Test tubes: (a) for absorption-8s X 13 mm. with rounded bottom and no
lips; (b) for agglutination test, see presumptive test.
2. 0.6 cc. of blood serum inactivated for thirty minutes at 56· C.
3. A 2 per cent suspension of sheep red corpuscles. (See presumptive test.)

Preparation of Reagents
1. Guinea Pig Kidney. The kidneys of the guinea pig are kept frozen in the
refrigerator until needed. They are then thawed and washed repeatedly in
a physiological solution of sodium chloride until the washings are free of
blood. Tbey are now mashed into a fine pulp and used for absorption as a
~<> ?Ct \:cnt <;",<;?Cn<;iCln i.n Ilhy<;iClIClgical salt "'Cl\",tiCln. The ",u\;?en\;iCln 1\; ooiled
for one hour on the water bath and the loss by evaporation is made up with
distilled water.
2. Beef Cells. The beef red cells are washed three times, packed well in the
centrifuge, suspended in four volumes of physiological salt solution and
boiled for one hour on the water bath. The loss by evaporation is made up
with distilled water.
Enough phenol is added to the antigenic suspensions to make a 0.5 per cent
solution. The antigens may be kept in the ice box for many months withont a
noticeable change.

Procedure
Absorption with boiled guinea pig kidney antigen.
I.Place in a test tube (85 X 13 mm.) 1.0 cc. of the thoroughly shaken 20 per
cent suspension of boiled guinea pig kidney.
•. Add 0.2 cc. of patient's serum that has been heated for thirty minutes at
56 •• C .
3. Shake and let stand at room temperature for one hour, shaking at fifteen
minute intervals.
 HEMATOLOGIC TECHNIC 665
4. Centrifuge at 1500 revolutions for ten minutes.
5. Remove the supernatant fluid willi a capillary pipette.
6. To a row of six tubes (75 X 12 mm.) add 0.25 cc. of physiological salt
solution.
7. To the first tnbe add 0.25 cc. of the absorbed and diluted serum.
S. Mix and transfer 0.25 cc. to the second tube, etc. Discard 0.25 cc. from
the last tube. The serum dilutions are: 1 :10, 1 :20, I :40, etc.
9. Add 0.1 cc. of a 2 per cent suspension of sheep cells. Shake well. Final
dilutions of serum are I :14, I :28, etc. Let stand at room temperature for
two hours. Read.
Abs()rpti()n with Boiled Beef Corpuscle Antigen. Exactly the same pro·
cedure as above, using 1.0 ce. of the thoroughly shaken twenty per cent suspen-
sion of beef cells.
Absorption of Serums with Titers Below I :II2. If it is necessary to begin
with a final dilution of I :7 as may be the case in serums with titers below 1:112,
then add'0.2 cc. of serum to I ce. of the antigen suspension. For titration, omit
the physiological salt solution from the first tube, but in the other tubes, place
'the usual amount o( 0.25 cc. from the absorbed serum, add 0.25 cc. to the first
and to the second tube. Proceed as above. Final dilutions are: "7, I :'4, etc.
Control with Unabsorbed Serum. At the same time carry out a diagnostic
test on unabsorbed serum according to the previously outlined technic to have a
basis for comparison.

Interpretation of Results
In the case of infectious mononucleosis, the absorption of the serum with
the suspension of the guinea pig kidney will effect a partial removal of the
agglutinins for sheep red cells, but as a rwe, not less than one-fourth of the
titer will remain (the titer before absorption I :II2, after the absorption with
the guinea pig kidney 1:28). If all or almost all (more than 90 per centyof
the agglutinins were removed, then this speaks against infectious mononucleosis.
The absorption with beef cells is a confirmatory procedure. The agglutinins for
sheep red cells are completely or almost completely (more than 90 per cent)
removed by beef cells.
Recent research by Bernstein indicates that in leukemias of all forms these
heterophile antibodies are either absent or are found in low titer (generally but
not necessarily below the normal level). This facilitates the differential diagnosis
between certain forms of leukemia and infectious mononucleosis which in the
early stages are often clinically and hematologically identical. Leukemia may be
excluded with a high titer but a low titer does not establish its existence. A titer
of I to 128 in the absence of recent horse seruin therapy and in the presence of
suspicious symptoins almost invariably establishes the diagnosis of infectious
mononucleosis. A titer of above 1:112 is considered positive for this disease
even if there is a history of serum therapy, unless the patient has serum sick-
ness (urticaria, etc.) at the time of examination, or bas only recently recovered
from this condition (see chart below). In severe cases the titer may reach 1:4096.
666 H£MA1:'OLOOIC TECnNIC

HETEROPIDLE ANTIBODY TITERS UNPE:Q. VARIOUS CmCUMSTANCES. (BERN"STtIN)

Normal Infectious Mononucleosis

Zone I Zone 2 Zone 3

I; I I: ~ I: 4 :l; 8 I: 16 r : 3Z I : 64 I : 128 I : 256 I : 512

Leukemia Serum Therapy

TEST FOR HODGKIN'S DISEASE (GORDON)

Materials and Equipment

I. Equipment for obtaining lymph nodes by biopsy.
2. DisseCting knife.
3. Scales and sterile watch glass.
4. Scissors.
s. Mortar with a cover and pestle (sterile).
6. Nutrient broth containing 0.5 per cent phenol with a pH of 7.1.
7. Cotton stoppered tubes.
8. Icebox at 6· C.
9. Ether for anestbesia.
10. Rabbits.

Procedure
I. Remove lymph nodes aseptically from suspected cases and place in a sterile
boltle or test tube.
2. Using sterile precautions, remove a portion for histologic section.
3. Place about a Gm. of the remainder in a sterile watch glass and weigb. Trans-
fer to a sterile mortar with cover, mince with scissors, and grind in the
mortar. (Place the remainder in the refrigerator and freeze. This will re-
main active for several weeks.)
4. Add enough broth to the material in the mortar to make a 10 per cent suspen-
sion, and decant into a tube; store in the refrigerator for one week.
5. Test for sterility by culture on the day preceding and on the day of injection
into animals.
6. Using a light ether anesthesia, inject into a rabbit 0.4 cc. of the gland suspen-
sion intracerebrally and 0.6 cc. intravenously.
7. Examine the animal daily, at first recording temperature, and later the weight.
8. Examine when the animal shows abnormal signs. Continue for a month or
more.
The signs of a positive test in a rabbit include muscular rigidity, incoordina-
tion, ataxia, spastic paralysis, loss of weight and, in some instances, the more
severe symptoms of meningo-encephalitis (head retraction, paresis, and cqnvul-
sions). The disease produced in rabbits is often fatal, usually within ten days,
\Jut death may occur at any time from 3 days to a month or more. A large
number of rabbits recover gradually and, wh~n recovery is complete, are immune
 HEMATOLOGIC TECHNIC

to a second injection of the gland suspension. For this reason, an animal' should
never be used a second time for the test. Gordon found that tbis encephalitic
syndrome occurred in rabbits injected with a suspension of lymph gland removed
from patients with Hodgkin's disease. A negative test was found with .similar
suspensions from normal individuals and from patients with chronic adenitis,
leukemia, sarcoma, tuberculosis, mycosis fungoides infection, and allergic condi-
tions.

DETERMINATION OF THE SPECIFIC GRAVITY AND TOTAL PROTEIN

OF BLOOD OR PLASMA BY THE FALLING DROP METHOD

Changes in the density of whole blood or plasma are important in recognizing

the onset of shock and in determining the total protein content and the degree
of hydremia. With the falling drop method of Barbour and Hamilton the specific
gravity of blood or plasma may be rapidly determined on a 0.01 cc. sample with
a maximum error of ~ 0.0001 specific gravity. The determination is made by
comparing the falling time of a 0.01 cc. drop of the unknown with the falling time
of a similar drop of a standard K 2 SO. solution of known density. The drops
are timed by stop-watch as they fall over a distance of 30 cm. through a mixture
of xylene and bromobenzene (XBB mixture) contained in a tube of exactly 7.5
mm. bore. By use of the accompanying alignment chart (see Fig~ 46) the ap-
parent density difference between each falling drop and the XBB mixture is
determined and from these data the density (specific gravity) of the unknown
is calculated.
Moore and Van Slyke have demonstrated a very close relationship between
the specific gravity and the protein content of plasma so that if the former is
known, the total protein may be' determined by application of the formula
(according to Weech) P = 340.1 (G. - r.0069). P represents grams of total pro-
tein per 100 cc. of plasma and G is the specific gravity calculated at specific
gravity 20°C.j20°C. Translation of various plasma specific gravities into total
protein content is listed in Table XXXVIII.

Materials and Equipment

1. Falling Drop Densiometer.
This may be purchased from the LaMotte Chemical Products Company,
Baltimore, Maryland.
2. Dropping pipette (with compression clamp) calibrated to deliver exactly
0.01 ce.
3. Stop watc1r.
4. XBB Mixtures.
Varying proportions of xylene and bromobcnzcne result in mixtures of
different specific gravities. The mixture selected for use in any given instance
should be that in which the drop falls slowly. The following mixtures are
suitable for the uses indicated. (See Table xx.'CIX.)
 TAllLE XXXVIII
TRANSLATION OF PLASMA SPECIFIC GRAVITY INTO PLASMA PROTEIN
ACCORDING TO WEECH'S FOR.l\o:lULA

Plasma Protein = (Plasma Sr. Gr. - [.006<)) X 340.1

(four. Bioi. Chem., Vol. "3,167-[74, 1936)

1.0187 .••••. '" 4.01% 1.02.:3&......... 5.75% 1.oz89... · .••.• 7.48% 1·°340 .. ··•··· . 9 ~2-%
1.0188......... .f..oS 1.0239· ...... ·· 5.78 1. 0290....... 7·5::' 1.034-1 ........ . 9·'1.5
J_oI89~ ....•... 4.08 1.0240........ , 5. 82 1 0291......... 7.55 1.034-.:········· 9 .8
1.0190........ 4- u 1.0:141.· ..... ·· 5.B5 1.02~....... ,sit 1 °343 '" ... 9·3'
t 0191....... 4.IS 1.02..2 ......... 5.88 1.0293 ........ 7· 6 'l, [.0344- .. •·· .. 935
J 0192. .... 4.18 1. 0243 ........ 5.92 1. 02 94......... 7 65 1.034-5 ....... . 939
(.0193...... 4-.2'1 1. 02 44.. 5·% 1. 0295 ......... 7·t':Y) 1. 0 34-6 ....... . 9··P
r .0194_..... 4.25 1. 02 45. 5·99 10291')' ........ 7.72 1. 0 34-7 .. 94$
1.0195...... ....29 1.0246. 6 02 1. 0297..... 7·7~ I 0318 949
['01g6 .. _.... 4-31. I . 02 47. . 6.05 1.0298 ......... 1.7';) 1·°319 9·52
I.O[!I7. .••...• 4.3S 1.0248. 6 og 1.0299.. '. ... ·.· 1.82 I 0350 9.5 6
1.0198......... 4.39 1.0249....... 6 12 1.0300......... 1·86 1 0351.. 9·59
1.0199. •••••..• 4 42 1.0250......... 6.16 1.0301 ......... ·7·8<) 1.0352 ... 9. 6 3
1.0200.. •••••• 4-46 1.0251......... 6.19 1.0302 ......... 7·9'1. 1.0353 .... · .. · . 9. 66
1.0201......... 4.49 1.0252......... 6.22 1.0303 ........ · 7 96 1. 0354. .. .. 9·69
1.0202..... ... 4.52 1.0253..... ... 6.26 1.0304···· .. ~., 7·99 I.0355· ....... • 9.73
1.0203.. ...... 4-56 I 025'" .. ..... 6.29 10305 ... :-.... 8.03 1. 0356.. ...... 9 76
LOZ04-.... .... +-59 1.0255· ....... 6.33 1.0306... 8.06 1 0357....... 9.80
1. 0205......... 4 63 1.0256 ...... 6.36 1.0]07 . • . . . . 8.~ l.OHB....... 9.81
[.0206......... ....66 1. 0257. ..... 6·39 1.0308... ... 8 13 1.0359 9 86
1. 02 07 •..• ". ....69- I 0258... ..... 6 ..4.] 1.0309.. .... 8 lu 1.0]60......... 9.90
I 0209 •... ... 4.73 1.0259......... 6.46 1.0310 .... 8 20 1.036 1... •• 9·93
1.0209. .•...... 4-76 1.0260......... 6.50 I.03lI ....... 8.23 1.03 62 . . . . . . . . 9·97
I 0210......... ....80 I 0261.... ..... 653 I.03IZ..... S 26 1.0363 10 00
1.0211......... .. 83 1.0262......... 6.56 1.0313.. .... 8 30 1.03£4 ..... · ... 10.03
1.0212......... 4.86 1 0263 ........ 6.60 1.03l.4·.· ..... ·• 8 33 1 0365 ....... 10.07
1 O~13 .•.•• " .. 90 1 0:>64 ••••••••• 6.63 1.0315. .... .. 8·37 ] .0366... . .... 10 10
I.02I.f.. .•.•••.• f 93 [.0265......... 6.61 1.0316..... .•• 8.4-0 1.0367... . .. 10.13
1. 021 5......... ..,. 97 1.0266......... 6.10 1.°317......... 8.4) 1.0368 ........ 10 ]7
l.c:n6 ....... _ ~ 1. 02 61..... 6.73 1.0318. ..... 8·47 1.0369.. ..... 10.20
1. 021 7.. 5,03 [.0268 6.77 1.0319.. .. ... 8.5 0 1.037°......... 10.24
1.0218 5.07 1.01.69 . . . . . . . . . 6.80 1. 0 320......... 8·S4- 1.0371 ......... 10.27
J .0219 5.10 I 02']0. ... ,6.84' 1.03 21 . ... 8·57 1.0372. ........ 10.31
1.02Z0 S.1.f. 1.027[, .... ·· 6.87 I 0322..... 8.60 1.0373... 10.34-
I.022I... S 17 [,0272.. 6.\)0 1.0323.... 8.64- 1.0374-..... 10·37
1.02.22... 5 20 1.0273... .... 694- 1 0324-.. 8.67 1.0375.. . .. 10 ..4-1
1.0223·· ••••.• 5.24- 1.0274·.. ..•.. 6 97 10325. 8.71 1.0376... 10.44-
1.0224 ........ 5·27 1.0275 ... · .. · .. ~ 10326... 8.74- 1.0377 ........ 10.4-8
1.02-15.......... 5.31 1.01.']6........ ,.04- 1.0327......... 8.77 1.0378 ........ 10·51
I 0226....... 5.34- [,0277 ........ · 7.07 1.0328......... 8.81 I 0379 . .. .. 10.54-
I.02Z7......... 5.37 1.0278 ........ 7.11 1.0329......... 8.84- I.0380... . . 10 58
1.02z.8 ... "... 5.4( (.0279 ....... , 7.1~ 1.0330 ........ 8.S8- 1.0381.. 1061
1.0229.···.···· 5.44 1.0280 ......... 7.18 1.0331 ........ 8 91 1.0382 ........ 10.65
I.0230.... •• • .. 5.48 1.0281......... 7.21 1.0332....... 8·94- 1.0383........ 10 68
1.0231 ....... ,- 5·51 t .oa8z......... 7.1.4- 1.0333......... 8.93 1.038~ .... . .. 10 71
1.02]2 .... ,.... 5 ·54 Loz83 ......... 7.28_ 1.0334 ......... ~ I 0385 10.75
1.0233......... 5.5 8 I.oz84 ...... ·.. 7.31 1.0335 ......... 9·05 I 0386 ......... 10.78
1.0::.34.... . .• .• 5.6t 1.01: 85......... 7·15 1.0336........ 9·0S 1.0387 Jo.8:J.
1.02]5......... 5,65 1.0286... ...... 7.j8 1.0337...... · •• 9·II 1.0388 ......... 10.85
1.0::.36 .•••••.• :. 5·68 1.0.:87......... 7.+1 1. 0 338......... 9 IS 1.0389 ........ 10.86
1.0237 .. • ..... • 5.71 1.0288......... 7.45 1.0539......... 9. 18 1.0390 ........ IO.9:J.

TUE FORMULA FOR SERUM PROTEINS:

Serum Protein. = (Serum Sp. Gr. - LOOn) x 347.9

 HEMATOLOGIC TECHNIC

TABL1~ XXXIX
XYLENE-BROMOBENZENE "'fIXTURES

Sp.Gr. Xylene %* Bromobenzene %* Use

XBB#I 0·993 So.o 20.0 Cerebrospinal fluid, urine, exu-
dates, transudates.
XBB#2 1.003 78 .5 21.5 Plasma, serum, very anemic
XBB#3 LOI3 76 9 23. I blood.

XBB#4 I 023 75·3 24·7 Anemic blood and heavy secre-

XBB#S I. 033 73·7 .6·3 tions.

XBB#6 1.043 72. I 27·9 Normal and anhydremic blood·

XBB1h LOS3 70·S 29·5
• Eastman Kodak Company products recommended. No. T 27s-technical m-xylene or No. P 460-
histological xylene and No. 43-bromobenzene.
The choice of XBB mixture depends upon t}l:e character of the material to be examined.

s. Standard potassium sulfate solutions.

Distilled water and three different strengths of K 2 SO. solution are used as
standards depending upon the relative density of the material examined.
The concentration and uses of each are given in Table XL.
6. Heparin.
TAl'LE XL
STANDARD :g~SO. SOLUTIONS
Grn. per Specific Gravity T : T
Liter at Uses
20°C. IS' 20' 25° JO' 3S'
00.00 10000 1.0000 I. 0000 I. 0000 I. 0000 Cerebrospinal fluid, dilute urine, exudates
and transudates.
#1. :r8.84 I.OISt I.orso I 0149 1.0I48 1.0147 Plasma, serum and very anemic blood.
'2. 44 56 1.0352 1. 0 350 1.0348 1.0346 1.0345 Anemic blood and heavy secretions.
113. 70 .81 I.0553 1.0550 I 0547 I 0545 1.0542 Nonna! and anhydremic blood.
Since K 2 SO, dissolves so slowly, it is more convenient to prepare the solutions by boiling. If the
&olutions are stoppered to prevent evaporation, they are stable indefinitely.

Technic
r. Blood is drawn by venepuncture and added to an amount of heparin equivalent
to 1 mg. per cubic centimeter (with the LaMotte apparatus, the small device
in the cork of the heparin bottle holds approximately 1 mg. when the amount
of heparin is I/16 inch in height) .
•. _Centrifuge and remove the plasma for protein determiDations or mix the
specimen well and use whole blooc;! for specific gravity.
3. Fit pipette into the rubber stopper of the compression clamp with thumb screw
adjusted so that there will be sufficient suction on release to draw the material
above the upper graduation.
4. Fill the pipette with potassium s1)ifate standard and adjust to upper mark.
Care must he taken that no air enters the pipette and no solution adheres to
the outer surface.
 HEMATOLOGIC TECHNIC

5. Place the filled pipette in the clamp above the tube of XBB mixture and
lower until the tip is beneath the surface of the liquid. The tube and pipette
must be in perfect vertical alignment.
6. Expel exactly 0.01 cc. of the material (the amount between the two gradua-
tions) by slowly screwing in the thumb screw and lift the pipette ant of the
XBB mixture to release the drop.
7. By stop-watch, determine the exact time required for the drop to fall be-
tween the two graduations.
8. Repeat the procedure using either whole blood or plasma to determine the
falling time of the unknown and observe the temperature of tlie mixture.
9. Determine the apparent density difference of each falling drop by applying
a thread to the alignment chart (Fig. 46) according to the directions on the
chart.
10. Calculate the true density difference between standard and unknown by sub-
tracting the apparent density difference of the standard from the apparent
density difference of the unknown. The density (specific gravity) of the
blood or plasma is determined by either adding the true density difference
or subtracting it from the specific gravity of the K 2 SO. standard used, de-
pending upon whether it is a plus or a minus value.
II. If the total protein is to be determined, the specific gravity of the plasma is
substituted for G in the formula; P =
340.' (G - 1.0069), or reference is
made to Table XXXVIII. '
EXAMPLE-Using K 2SO. Standard No. , at 20°C.

1. If the plasma is heavier than the standard:

Blood plasma. falling time ... 48 sec,
Apparent density difference....................... ......... . 0 0040
Standard RISQ, falling time """ 62 sec.
Apparent density difference ......................... -..... 0 0028

True density difference ................................... +0.0012

Specific gravity of standard .......... , . . . . . . .. ... . ..... +.1 OISO

Specific gravity of plasma ............ ,. .................... 1.0162

Applying Weech's formula:
p - 340 r (G - 1.00(9)
p - 340.1 (I 0162 - I 0069)
p -= 340.1: X 0.0093 - 3.16293 Om. protein per 100 ce. plasma.

2. If the plasma is lighter than the standard:

Blood plasma falling t me = 62 sec.
Apparent dt:nsity difference ... _............................. 0.0028
Staudard K1SO, falling time ... ,p: sec
Apparent density difference. . . . . . . . . . .. .................... 0 0048
True density difference. .. ............. . . . . . . . . . . .. ..... - 0 0020
Specific gravity of standard ................................. +1.OISO
Specific gravity of plasma. ................................... +1.0130 r

p ~ 340.1 (G - 1 0069)
p - 340.1 (1.0130) - 1.0069)
p - 340 I X 0.0061 '"'"' 2'07461 Gm. protein per 100 ce. plasma..
 ...,

,,..
61212'

."

'f
 HEMATOLOGIC TECHNIC

VISCOSIMETRY (BIRCHER)

1. Viscosimeter of Hess. This apparatus is based on the principle that fluids

at equal pressure and temperature will pass through capillary tubes of equal
caliber at a rate proportional to their viSCOSity.
2. Materials for finger puncture.
3. Distilled water.

Procedure
1. Place a drop of fresh capillary blood in the blood receptor and distilled water
in the water receptor.
2. By the aid of the rubber bulb attached to the viscosimeter, draw the blood
and the distilled water up the two capillary tubes of the instrument until the
blood reaches the graduation I.
3. At this til1)e, measure the height of the column of water on the graduated
scale. This reading corresponds to the viscosity of the blood as compared
to water, since the volume flow of two liquids under equal temperature and
pressure through tubes of equal caliber is inversely proportional to their
viscosity.
4. The viscosity of blood, therefore, is expressed in viscosimetric units of dis-
tilled water at 20' C. The temperature should not vary more than 3 degrees.
Any greater deviation may be corrected by adding 0.8 per cent of the reading
for every degree above 20 degrees and subtracting 0.8 per cent for every degree
below 20 degrees.
The viscosity of normal blood, as compared to water, averages 4.5 units.

BASOPHILIC AGGREGATION TEST

The hasophilic aggregation test primarily demonstrates regeneration in the

peripheral circulation under conditions in which toxic agents exert an action on
the bone marrow. This is manifested by the appearance in the erythrocytes
of a basophilic substance, either in the form of polychromatophilia, punctate
basophilia (stippling) or reticular designs. In the process of laking and staining
the red cells in the aggregation test, the basophilic substance is artificially ag-
,gregated into readily visible masses. With respect to lead absorption and early
lead poisoning this test appears to be more accurate than stippled cell counts on
ordinary smears stained with Wright's. Pearlman and Limarzi have shown a
close correlation between the aggregation test and reticulocyte count using "vital"
staining.
In normal adults the aggregate count averages between 0.4 to 0.8 per cent,
not exceeding I per cent, McCord, Holden and Johnston, during the W34-1935
epidemic of lead poisoning in the automobile industry examined the blood of
6,900 workers using the basophilic aggregation test. From their observations,
they state that a basophilic aggregate count averaging 1.5 to 2.0 per cent in the
lead worker suggests lead absorption and the possibility of lead poisoning. In
 HEMATOLOGIC TECHNIC

chronic lead poisoning the reliability of the test diminishes as it is usually not
positive.
Reagents
I. Manson's methylene blue (modified)

Bol'8.l:.. •.. .•.•.•. .••••••••• ••• ••••.••••• 1,0 Gm.

~~mr~en;ab~~~.:::.':::::::::::.':.': :::::,' 1~'O ~~.
Dissolve the borax in boiling distilled water, add the methylene blue and
filter. The stain is stable for two weeks.
2. Methyl alcohol (acetone free)

Procedure
1. Make a thin, even blood smear on a clean slide and allow to dry for at least
I hour, but not more than 3 hours. If permitted to become excessively dry
the cells will not lend themselves to aggregation of their basophilic material.
'2. Fix a longitudinal half of the blood smear by placing on it a strip of filter
paper which is carefully wetted with methyl alcohol. Allow to dry until the
filter paper becomes loose. The remaining longitudinal half of the slide is
unfixed in order that laking may take place during the process of staining.
3. Immerse the slide in a jar of stain for 10 minutes. Wash the slide in di$tilled
water through 3 Or 4 rinses. Air dry.
4. In the unfixed portion of the slide, count 10 consecutive fields in two pa-rallel
rows, making a total of 20 fields. Moving to the fixed portion of the slide,
5 appropriately corresponding fields are counted. The basophilic ag~rega­
tions are then expressed as a percentage of the latter.
"EXAMPLE:
In twenty fields counted in the unfixed portion there were 76 basophilic
aggregations.
In the five fields counted in the fixed portion there were 1,000 erythro-
cytes or 4,000 in 20 fields.
Percentage: 76/4,000 - l.g per cent.

AUTOHEMAGGLUTINATION

Materials and Equipment

I. Equipment for finger puncture.
2. Slides.
3. Red cell pipette.

Procedure
r. Make blood smear and stain with Wright's stain.
2. Observe for rouleaux formation_
3. Draw blood into pipette and dilute with Hayem's solution.
4. Observe for clumping of red cells. .
 HEMATOLOGIC TECHNIC

Foard has observed that auto-agglutination of red cells occurs consistently in

multiple myeloma. He attributes the phenomenon to hyperproteinemia. In-
stances of auto-agglutination have been reported (Ordway and Gorham) in hemo-
lytic jaundice when the patient's red cells were mixed in a watch glass with his
own serum (I drop of blood to 10 drops of serum).

SPECTROSCOPIC EXAMINATION OF BLOOD

Either the direct-vision or the angular-vision spectroscope may be used in

the spectroscopic examination of the blood. CO,ntrols accompanying the exami-
nation of the blood for an unknown would facilitate its identification.
1. Oxyhemoglobin. Diluted defibrinated blood with distilled water sufficiently
to give a clear spectrum, exhibits two narrow bands between D and E. One
nearer the D line is more narrow. These are typical absorption bands of
oxyhemoglobin. Too much dilution will cause the bands to be very narrow
or disappear entirely.
2. Hemoglobin (Reduced Hemoglobin). Dilute defibrinated blood as for oxy-
hemogl!>bin (I). Examine spectroscopically. Reduced hemoglobin shows a
single broad band lying almost entirely between D and E.
Preparation of Control
1. To dilute blood, giving a typical absorption band of oxyhemoglobin add
a small amount of Stokes' reagent (2 per cent ferrous sulphate and 2 per
cent tartaric acid mixed in a test tube. Add sufficient ammonium hy-
droxide to dissolve the precipi~ate whi9h first fo~ms on the addition of
the hydroxide). The color changes from.a bright red to violet red.
2. Examine spectroscopically. Note singl~ broad band between D an!l E.
3. Carbon Monoxide Hemoglobin. Dilute defibrinated blood as in (,). Ex-
amine spectroscopically. If carbon monoxide is present there will be two
absorption bands between D and E, similar to oxyhemoglobin, but nearer
the violet end of the spectrum. Add Stokes' reagent (2) and again examine,
No change takes place in the presence of, carbon monoxide.
Preparation of Control
I. Pass ordinary illuminating artificial gas through defibrinated ox-blood:
2. Dilute with distilled water and examine spectroscopically.
4. Neutral Methemoglobin. Dilute defibrinated blood. Examine spectroscopi-
cally. A single, very dark absorption band lying to the left of D demon-
strates the presence of neutral methemoglobin. If the dilution is sufficiently
great, observe two rather faint bands between D and E.
Preparation of Control
I. Dilute defibrinated blood I !IO.
2. Add few drops of freshly prepared 10 per cent potassium ferricyanide.
Bright red color of the blood is displaced by a brownish red.
3. Dilute with distilled water.
4. Examine spectroscopicaily. Note spectrul!! bqnd as described for methemo-
globin.
5. If a few drops of Stokes' reagent is added the methemoglobin is converted
to oxyhemoglobin and then hemoglobin.
 HEMATOLOGIC TECHNIC

5. Alkaline Methemoglobin. Dilute defibrinated blood (I), and examine spec-

troscopically. The spectrum bands for alkaline methemoglobin will be seen
on either side of D, the one nearer the red end of the spectrum being much
fainter. A third, darker hand lies between D and E nearer E.
Prepacation of Control
1. Add a few drops of ammonia to a neutral solution of methemoglobin.
The solution becomes redder in color.
2. Examine spectroscopically. Note three bands listed ahove.
6. Alkaline Hematin. Examine diluted defibrinated blood spectroscopically.
Alkaline hematin will show a single absorption band lying across D and
mainly toward the red end of the spectrum.
Preparation of Control
I, Mix one volume of concentrated potassium hydroxide and two volumes of
dilute (I :5) defibrinated blood.
2. Heat gradually almost to boiling.
3. Cool and shake a few moments in air.
4. Examine spectroscopically and note the single absorption band.
7. Reduced Alkali Hematin or Hemochromogen. Examine diluted defibrinated
blood spectroscopically. A narrow, dark band lying midway between D and
E demonstrates presence of hemochromogen. If dilution is not too great a
faint hand will be seen in the green extending across E and b.
Preparation oj Control
I. Dilute alkaline hematin (6) to such an extent that it shows no absorption
band.
2. Add a few drops of Stokes' reagent. 'The greenish-brown color of alkali
hematin is displaced by bright red due to the formation of hemochromogen.
8. Acid H emalin. Dilute with distilled water a sample of defibr_inated blood.
On spectroscopic examination acid hematin will give a distinct absorption
band in the red between C and D, lying somewhat nearer C than the band
in the methemoglobin spectrum. Between D and F may be seen a rather
indistinct broad band which on further dilution will resolve itself into two
bands. Of these the more prominent is a broad, dark absorption band lying
in the green between band F. The second narrow band lies in the light
green to the red side of E. A fourth very faint band may be observed lying
on the violet side of D.
Preparation of Control
I. To 5 cc. of defibrinated blood add 2.5 cc. of glacial acetic acid and 5 cc.
of ether. Mix thoroughly.
2. Pour off the acidified ethereal solution of hematin.
3. Examine spectroscopically. Note spectrum bands described above.
4. If necessary dilute further with acid ether (1 part of glacial acetic acid
to 2 parts of ether).
9. Acid Hematoporphyrin. Examine spectroscopically a diluted solution of
blood (I). Acid hematoporphyrin gives a spectrum with an absorption band
on either side of D, the one nearer the red end of the spec~um being the
narrower.
 HEMATOLOGIC TECHNIC

Preparation of Control
1. To 5 cc. of concentrated sulphuric acid add 2 drops of blood. Mix thor-
oughly after addition of each drop.
2. Examine the wine red solution (acid hematoporphyrin) spectroscopically.
ro. Alkaline Hematoporphyrin. Alkaline hematoporphyrin in blood diluted as
in (,) and examined spectroscopically possesses fo!)r ahsorption bands.
(a) A very faint narrow band in the red, midway between C and D.
(b) Broader, darker band lying between C and D, principally to the violet
side.
(c) A band lying principally between D and E, extending for a short dis-
tance across E to the violet side.
(d) A broad, dark band lying between band F.
Preparatio'l oj Control
I. Dilute'acid hematoporphyrin (9) with an excess of distilled water. Cool.
•. Add potassium hydroxide until the solution is only slightly acid. A
precipitate which includes the principal portion of hematoporphyrin is
formed. Sodium acetate facilitates the formation of this precipitate.
Filter.
3. Dissolve precipitate in a small amount of dilute potassium hydroxide.
Alkaline hematoporphyrin prepared in this way forms a bright red solution
and spectroscopically has four absorption bands described above.
~~. .
Reference: Hawk, P. B. and Bergheim, 0.: Practical Physiological Chemistry, nth ed, 193'1i

METHODS OF DEMONSTRATING SICKLE CELLS

Detection of sickle cells (meniscocytes) cannot always be done on the

stained smear. Especially is this true if only the "sickle cell trait" is present.
It has been noted that oftentimes the sickling will develop only after cells have
been permitted to stand in the fresh state, with oxygen excluded.
The method of Beck and Hertz, given below, is satisfactory for this purpose,
and consists of fixing the erytbrocytes with formalin after they have assumed
their sickle shape in a test tube sealed with oil.

TUBE METHOD (Beck and Hertz)*

Apparatus and Reagents
I. Small test tube, 4 :x I ems .
•. Physiologic saline citrate. This may be prepared by mixing equal quantities
of normal saline and 3 per cent sodium citrate.
3. Paraffin oil.
4. Saline-formalin fixative. Add 0.85 grams of sodium chloride to 100 ce. of
10 per cent neutral formalin.
5. Glass slides, cover glasses, lancet to prick finger, tbree small bottles and three
small drawn glass pipettes equipped with rubber nipples, and a microscope.
*This technic ~ reproduced verbatim from the article by Beck and HertzJ Amer. Jour. eli",
Path., 5, 325, l"935.
 PLATE UV

The absorption spectra on the following two pages are reproduced

directly from observation with the use of the direct vision spectroscope
 Plate LIV

r. Spectrum
2. "H.emoglobin
3. Oxyhemoglobin
4. Carboxy bemoglobin
5. Neutral hethemoglobin
6. Alkaline methemoglobin
 Platt'" LIV (continued)

10

11

12

7. Acid hematoporphyrin
8. Alkaline hemaloporphyrin
9. Urobilin
10. Alkaline hematin
I I, Acid hematin
f2. Coproporphyrin I

NOTE: Thl! yellow band has been slightly enlarged to facilitate reproduction of absorption band~
occurring in this range. The scale in milli-micra has been adjusted t() compensate for this enlarge-
ment. (Plate reproduced from Kracke and Parkili:r: Textbo()k of Clinical Pathology, BaltimQre 1 Wm
Wood and Co., t940.)
 HEMATOLOGIC TECHNIC

"The saline-citrate, the paraffin oil, and the formalin are to be kept in
separate bottles, stoppered and labelled. One pipette is to be used for handling
the saline citrate solution, one for the paraffin oil, and one for the formalin.
Mark these pipettes appropriately because a trace of formalin in the blood prior
to sickling may prevent the deformity. Before starting the test, place from 0.2
to 0.5 c.c. of saline citrate in the tube to receive the fresh blood."

Procedure
"Clean and prick the finger as in collecting blood for a count. Wipe the
finger dry and collect one or two drops of blood in the test tube containing the
saline citrate. Invert to mix. Cover with sufficient oil to make a layer I cm.
thick. Make sure no bubbles of air are under the oil. Let the preparation stand
at room temperature for twenty-four hours, then introduce 0.2 to 0.5 c.c. of the
formalin solution beneath the oil layer by means of the formalin pipette. Thor-
oughly mix by forcing the liquidsoin and out of the pipette several times. Do
not break up the oil layer for fear of letting air in too soon. Two or three minutes
or more should be allowed for fixation. After this period the suspension is
mixed again with the formalin pipette to insure a uniform distribution of cells.
Remove a few drops from the tube, wipe away the excess oil from the tip, and
place a drop on a glass slide. Cover and examine. The percentage is calculated
in the manner of the differential leukocyte count.
"If it is desired to measure the blood and the solutions accurately, it may
be done with a hemoglobin pipette for the blood and I c.c. pipette, graduated
into tenths, for the solutions. It will be found that 20 cu. mm. of blood in the
solutions described above make a very satisfactory cell suspension for microscopic
observation.
"Permanent preparations are made from the suspension on cover glasses like
an ordinary fresh blood film. The smears are dried in air and fixed by flame.
Blood cells treated with formalin are not well tinted in Wright's stain. Excel-
lent stains may be made by using the hematoxylin eosin technic, but if it is de-
sired to stain only the erythrocytes, a simpler method consists of staining with
basic fuchsin or some similar dye. To use fuchsin, flood the smear with a I per
cent aqueous solution for 15 to 30 seconds. Pour off, wash in water, and dif-
ferentiate by flooding with 95 per cent alcohol for 30 to 60 seconds. Destaining
in alcohol is to be controlled microscopically before mounting. If the cells are
too pale, restain and differentiate less. When the staining is satisfactory, wash in
water, blot, dry, and mount in balsam."

SEALED MOIST PREPAllATlONS:

Materials and Equipment

• I. Equipment for finger puncture.
2 •.Cover glass and slide (cleaned).
3. Petrolatum.
4. Microscope.
s. Small rubber band.
 HEMATOLOGIC TECHNIC

Procedure
r. Place rubber band around proximal phalanx of finger for two minutes. Then
prick finger.
2. Place a drop of blood upon the cover ,slip.
3. Quickly invert upon the slide.
4. When spreading is complete, thickly seal the edges with petrolatum.
s. Examine for sickling at once, at 6, 12 and 24 hours.
BIBLIOGRAPHY
SECTroN ON TECHNIC
BARBER, M. A' I and KoMP, \V H. W.o "Method of prepa.ring and examining thick films for the
diagnosis of malaria." U. S. Pub. Health Rep" 44, 2330, 1929.
DAlUIOUR, H. G, and HAMILTON, W. F.: "The falhng drop method for determining specific gravity"
Jour. Amer. Hed. A.ssoc., 88,. 91.94, 1927.
BECK, J. S. P., and HERTZ, C. S .. Standardizing sickle cell method and evidence of .sickle cell traits.
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 HEMATOLOGIC TECHNIC

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680 HEMATOLOGIC TECHNIC

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A AgTanulocytosis--(Contin~ea")
Ab"ce!!!, Iterile. 126
~\bBcessel. leukocytosis of, 126
Absolute cell values, 15
Acetanllid, effect on blood, 497
methemoglobin from, 196
Acetylphenylhydrazine, hemolytic
anemia from, 241
in polycythemia, 494
Acblorhydna, absorption of iron,
176
in children, 341
In hypochromic anemia, 230
in pernicious anemia, 280
in pregnancy. 210, 303
In sickle cell anemia, 330
in tape worm infestation, 208
Acblorhydric hypochromic anemia,
22.
Acholurlc Jaundice, 2:59
Achrestic anemia. 307
Achylia In pernicious anemia, 282,
286
ACId, effect on iron, 218
effect on neutrophil!, 1Z$
hematin, :200
In renal tubules. 240, 242
hydrochloric. in idiopathic hy·
pochromic anemia, 234
in pernicious anemia, 295
Acidosi9. leukocytosis in, 126
Acquired hemolytic anemia, 244
Acute hemolytic anemia from
drugs, lSI, 241
infections. anemia of, 205
It ..kemia, 385
myeloblastic leukemia, 385
Addisin in polycythemia, 491
Addison's disease. leukopenia of,
1S2
lymphocytOSIS of, 136
Addisonian anemia, see Pernicious
anemia
Adrenal deficieneies, leukopenia
of, 153
Adrenahn, effect on leukocytes, 124
effect on platelets of, 99
effect on red cells, 94
Adult, blood volume in. 114
Adults, bone marzoo'W in, 5.()S
obtaining blood from, 597
Adventitial cells, 76
Affiliation cases, blood groups in,
582
Agglutinins, cold, in blood, 564
in blood groups, SS6
Agglutinogen Rh, 565
Agglutiuogens, in blood groups, 556
M and N. 56S
Agonal leukocytosis. 126
Agranulocytosis, IS3
blood findings in, ISS
in cats, 152
chnical findings of, 154
etiology of, 149, 153
history of. 153
incidenCe of, 154
n:.a.rrow findings in, 517
monocytes in, 137
mortahty rate in, 158
pa.tholoaic chanies in. IS6
INDEX
prognosIs in, 158
treatment of, 156
AleukemiC leukemia, 370, 405
leukopenia in, 159
Alignment chart for plasma pro-
tem, 671
Alimentary anemaa, 213, 340
Alkahne hematIn, 200
AllergiC paroxysmal hemoglobi~
rlUna, 255
readioDs in transfUSions, 579
Allergy, eoslDophilia of, 141
in fabismus, 241
leukopenia In, 123
to liver extract, 295
Altitude, effect on leukocytes, 125
effect on red cells, 94, 118, 496
role in hematopoiesis, 184
Amidopyrine, effect on blood, 147
Amoebiasis, anemia of, 215
eosinophilia Of, 141
macrocytic anemia in, 306
Anaphylactoid purpura, 458
Anemia, 165
achlorhyclr-ic, usc: of iC(Ja iu, 223
acbreltH:, 307
of acute hemorrhage, 204
of acute infections, 205
in agranulocytosis, 15S
aplastic. 311
in blood donors, 204
blood transfusions in, 217
bone marrow in, 512
causes of, 192
of childhood, 334
e1assification of, 337
symptoms of, 335
from vitamin deficiency, 343
in chIldren, treatment -of, 342
of chlorosis. 215
of cbronic hemorrhage, 205
of chrOhie infections, 206
classification of, 191
of coeliac disease, 341
congenita.l hc;mQlytic, see HelD
olytie jaundice
Cooley's. erythroblastlc, 353
copper in, role of, 170
of cretinism, 343
diet 1M', 224
experimenta1, copper in, 171
famlilal hemolytic, in clnldhood,
348
of first month. 334
auses of, 335
01 gastrectomy, 175
of g3stro-intestinal disease, 214
hemolytic, 236
acqUired type. 244
blood fiding'BI in, 237
caused by infections, 239
from chemicals, drugs, and
poisons. 240
of cblidbood, 345
dasslfiClition cf, 238
~erimeotal, 244
hemoglobinuria in, 251
from hem()lysios, 243
of Lederer, 245, 251, 347
m~hamsm of, 236
of newborn, 346
Anemia-(ContlnMt"d)
hemolytic-(Clmtuuc,-d)
from sulfonamide drup, 241
treatment of, 243
types of, 237
from hemorrhage, 166
in Hodgkto's dlSease~ 550
of hookworm disease, 208
hypochromIC, 202
• of infants, 213
of lead pOisoning, 272
liver therapy in, 223
symptoms of. 203
hypoplaStiC, I!plenectomy in, 167,
268
of hypotbyroidism, 212
idiopathic aplastic, 320
hypochromic, 229
blood findings in, 233
course and prognosis of, 234
incidence of. 229
marrow findings tn, 512
.ymptoms and physical :find-
ings, 230
treatment of, 233
()f IUfection5, 356
of iron deficiency, 212
achlorhydric tYJIe, 229
causes of, 170
macrocytic, diseases character-
ized by, 301
in liver disease, 178
tropical, 179
in malaria, 534
of malaria, 209
of mahgnancy, 206
of malnutritIOn, 213
marrow findings in, 658
of nepbritis, marrow findings in,
513
ostco:o-clerotic, 322
oxygen iD, role of, 184
of parasitic infestation, 207
pernicious, see PernicIOUS atlemia
porphyrins in, 199
of pregnancy, 182, 209
causes of, 210
.ymptoms of, 211
treatment of, 211
of prematurity, 338
ref'BeforT, 311
classification of. 313
of scurvy; 214
sickle cell, see Sickle Cell anemia
skID color in, 203
splenie, 356
from sulfonamide drugs, 151
of tbyroid dlsorden, 181
transfusions in, 573
treatment of hypochromic type,
215
types of, 191
use of fluids in, 218
of vitamin C deficiency, 213
von Jakscb's type, 349
Aniline, effect on blood, 147, 497
methemoglobin from, 196
Ammals. blood pietures of, 589
Hodgkin's disease in, 548
leukemia in, 363
Anopheline mosquito, 525
68.
682
Anoxemia, from carbon monoxide.
198
effect on reticulocytes, 96
Antianemic factor. 173
Anticoagulants, 600
Heller and: Paul mixture, 601
Anhtbrombm in blood clotting, 433
Anuria, from incompatible blood.
250, 578
in il.etl1alyti-c anemia, 240, 242
Aplasia of marrow. 14S
from arsenic, 317
from benzene. lIS
findings in, 517
Aplastic anemia. 311
from AT'enic, 316
etiology of, 313
idiopathic, 320
laboratory findings In, 321
symptoms of. 320
treatment of. 321
manow findings in. 313
neutropenia in, 159
from radiation, 147, 318
secondary, 314
Appendicitis, eosinopbtlia of, 141
Aregenerative anemia, 311
Arneth classincahon of leukocytes.
12.
Arsenic, aplastic anemia from, 316
in lymphahc leukemia. 3M
in treatment of leukc:rnia. 318,
420
Arsphenamine, aplastic anemia
from, 311
effect on blood. 147
Arthrttis. eosinopbilia of. 142
Artificial fever, leukocytosis from.
124
AspiratiOl1 of marrow, 509
Asthma. eosiOoph.itia of. 141
Atabrine. leukopenia from, 151
in malaria, 542
Atypical monocytes. 19
rnyelob13.sts. 63
types of leukemia, 405
Autoaggluhnins in blood, 564
AutohemagglutinatioD. 673
Autopsy. bone marrow at. 511
AVIan tuberculosis, relatIon to
IIodgkin's, 547
}i..viatora, red celts in. 94-
A~r2:a'l di~ea!le. 491
B composition of, 114
Band neutrophil, morphology 01,
56
Banks af blood, 568
BantL's disease, 356
leukopenia in, 160
macrocytic anemia in, 308
splenectomy for, 161, 268
Basal leukocyte caunt, 128
Basal metabolic rate in leukemia,
375, 383
Basket cells. 83
in leukemia. 381
Basophilia, 96
In lead poisonini', 273
Basoplnlic aggregation tcst, 215
technic for. 612
Basopbilic granules•. 143
lttanuIation in neutrophils, 132
leukemia, 310, 414
myelocytes in leukemia, 31'
morph010gy of, 57
significance of, 69
atip:pling, 96
INDEX
BasophUs, function of, 142
increased, 142
maturation of, 50
supravital stain of, 105
Bay sic1mess. 257
hemoglobinuria from, 251
Beck and Hertz method for aick.
ling, 676
Benign lymphadenosis, 473
Benzene. effect ,on blood, 146
for leukemia, 420
poisOtung. 313, 314
eo!inophilia of. 142
maIT<)W fin~ings in, 513
treatment of, 316
prepa:ration$, hemolytIc anemia
from, 241
relation to leukemia. 367
Biermer's anemia, .Ie, Pernicious
anemia
Rile in l'Jerum, technie for. 646
Biliary £stula. vitamin K iu, 463
BIlirubin, origin of. 93. 236
in serum, technic for, 646
Diopsy of bone marrow, 508, 655
Birth, blood findings at. 117
Black tongue, leukopenia in, 148
Black water fever. hemolytic ane-
mia in. 240
in malaria, S39
Dlack widow spider bite, 141
D1eedlng time, In bemophllla, 451
in non·thrombopenie purpura,
458
tecbnlc for, 636
in thrombocytopenic purpura, 442
Blood, bankll, 568
cells, destruction of, 501
devdopment of, 43
effect of beru:cue on, 146
enumeration of. 608
immature, terminology for. 12
in infectious mononUcleosis,
.80
morphology of. 55
origin of• .501
terminology of. 3
undifferentiated, 501
chemical 'Constituents of, 119
clot. formation of, 434
coagulation of, 433
in "hemophilia, 448
PTevtntioD of, 600
time, 634
counts, errors in. 6121
technic fcr. 608
destruction of. Ui6
diseases. terminology of, 3
'With fl.eutropel'lla, 159
donors. findings in. 204
health of, S80
preparation of l 580
dust. 113
effect of benzene on, 313, 314
effect .of chemicals on, 146
eJtamination of, 114
technic {ot'. 115
findings. after acute hemorrhage.
20'
In acUte leukemia, 392
in agranulacytasts. ISS
in animals. 589
in aplastic anemia, 321
at birth. 3.34
in catne1!o. SS9
in chickens, 594
in children. 111
Blood-(Contlful~d)
findmgs-{Coneinued)
in chronic lymphatic leukemia,
382
in Cooley'. anemia, 354
in dogs, 592
in frogs, 594
after gastrectomy, 175
after splenectomy, 268
in guinea pigs, 590
in hemolytiC anemiae, 231
in hemolytic jaundice, 263
in hemophilia, 451
in Hodgkin'a. diaea!oe, 55G
in idiopathic hypocbromic an....
mia, 233
in infancy, 117
in infections. 206
in infectious mononucleOSIS,
480
• in lead poisoning. 213
in ~deTer's anemia, 246
in mice, 591
in monkeys, 592
in monocytic leukemia, 400
in myelogenous leukemia, 375
in non.thrombopenic purpura,
458
in osteosclerotic anemia. 323
in pernicious anemia. 283
in polycythemia vera, 492
in raLbil:!l, 589
in rats, 593
in sickle celt anemia, 329
in thrombocytopenic P\lTpun,
442 .
in "Von Jaksch'l .nemis. 351
formation in the embryo, 46
!troup;ng, technic. of, 560
grOUps, 556
anomalies in. 558
attitude of courts to. 583
at birth. 55?
classification of. 556
inheritance of, 581
legal application of, 580. 582
paternity tests with, 58l
racial and geographic, 557
relation to sickling, 326
subtypes of, 558
universal donor in, 559
incompatible. hemog10binuril
from. 250
loss. anemia in, 166
matchIng of. 562
sources of error in, 563
normal. III
ox.alated. use of. 601
pigment, metabolism of, %37
plasma, 113-
in anemias, ~18
desiccation of. 511
hemoglobin in, 248
indications far, 572, 571
preparation of, 511
technic for protein in, 661
trandusion of, 510
platelets, 91
maturation of, 51
porphyrins in, 198
reports. chart for, ti03
&erum, 113
preparation. technic for, tiD2
.mears, cover glass metbod for,
616
£ntiou <)f, 618
preparation of, 61.5
.tide method {or, 616

Blood-(Com;nvt'd)
staining (If. 618
with indopbeno-l blue, 622
witb Gle.msa's stain. 620
with peroxidase stain, 621
with Wright's stain. 619
specific gravity of, 661
spectroscopIc examination of, 614
storage of, 568
studtes in "hemorrhagic diseases,
."
total pNltein In, 667
types, 556, 565
unstained, far sickling, 671
viscosity of. 6TZ
volume. 114
10 polycythemia vera. 493
transfusiCtn3 for, 575
Blood transfusion. 556, 566
allergic r~act1qa In. 519
dosage in, 511
of fresh whole blood, S66
hemoglobinuria from. 250
history of. 556
in acute leukemia, 394
In anemia, :217
In apiastic anemia, 322
indications for, 572, 517
in hemolytic jaundice, 519
in hemophilia, 452
in hemorrhage of newborn, 461
in infectious mononucle()Sis, 487
in Lederer's anemia. 246
in leukemia, 420
in 2ymphahc leukemia, 384
in Don-tbromoopenic purpura, 460
in pernicioUli anemia, 293
in lIil;kle cclI anemia, 331
in thrombocytopeniC purpura, 443
of placental blood, 570
reactJODS from, 578
of stored whole blood, 561
Bom::, changc.a, in cbloroma, 4o,
in Cooley's anemia, 354
in Jeukemia. J1f
in slclele celt anemia, 329
Pathology in leukemia, 429
tenderneu ill leukcDua, 314
Bone marrow, 500
in agranuleteytosis, 1 S6
aooxemia in polycythemia, 491
aplasia, 311
conditions producing, 320
in pernicious anemia. 319
autopsy study of. 511
in benzene POIsoning, 315
biapST Df. 508, 6SS
by aspiration method. 509
by drtll method, 510
by Tnphine method, 508
eompiete examination of, 65J
in Cool-ey's anemia. 354
taunting c:ells of, 655
erisis, 166
in hemolytic jaundice. 263
culture of, 657
delivery of leukOC)Us, 121
depression frem radi2,tiO'D. 318
development of, 503
difJeJ"tJJtW count of, 0506. 511,
.53
dlltribubon of, 46
eifeet 01 cltoemieaJ., 141
effect of temperature, 184
in erythroblastic anemia, 354
erythro-myeloid ratio in, 50S
examination of, 510
fu:ldlIlp. in ant'ttlia. 512, 658
in aplastic anemia. 313
INDEX
Bone marrow-(Ctmti"".d)
findlDgs-(Contifuud)
in aplastic marrow, 514
wjt}J displaced marrow, 514
in hemorrhagic diseases, 514
in hyperplashc marrow, 512
in hYPOplastic marrow. 514
in leukemia, 517
in leukocytosis, 517
in leukopenia, 517
in hpoid Itorage disease, 520
in polycythemia, 515
in 1umots, 520
in hemolytic jaundice, 262
in hemophllia, 451
hlstologic section of, 656
hyperplastic type, 14S
hypoplaSia, 1 S2
in leukopenia, 144
lere14. 28. SO
in malaria, 538
in osteosclerotic anemia, 322
oxygen tension in, 184
in pernicious anemia, 277
physiology of, 41
serum spreads of, 656
In sicld~ cell anemia, 330
stimulation cf, 128
structure of. 505
transfwnons of, 322
Trephine examination of. 653
votume of, 504
nrucellosis, relation to Hodgkin's
dil~, 547, 552
Buffer solution for blood Imears,
61.
Burns, hemoglobinuria from, 251
c hemoglobinuria from, 252
Cabot rin~ bodies,
Calcium, 220
9'
salts In blood. clotting, 433
time, technic for, 640
Camel, blood fillmngs .lu, 589
Capillary, fraglhty increase. 456
resistance tcat, technic for, 636
weaknCIIS, pUrJIura uf, 456
Carbon monoxide. effect 00 sick-
ling, 327
hemoglobin. 197
spectrosccpic bands of, 674
poi.soning, 198
Careinogenie agents, reIation to
l~ul&:emia. 368
Carcinoma, anemia of, 206
of .tom4ch, macrocytic anemia
in, 306
Cat, agranulocytosis in, 152
Cevitamic acid, '" Vitamin C
Chart, con-ection, for sedimenta·
tion,644
for blood reports, 603
Chemical C!Onstituents of blood, 119
COIltent c£ blood, 114
of er)'throcytes, 92
ChemicaIs~ effect on blood. 146
Chemilltr,r Qf hemoglobIn, 194
Chicken. blood :ftndingl in, 594
Childhood. anemias c£, 334
erythroblastic anemias of, 350
familial hemolytic jaundIce in,
348
hemolytic anemias ill, 345
Lederer's anemia in. 341
leukemia in, 364, 385
Slclde cui aDt"'mia in, 348
splenomega}r in. 358
Cluldren, aehlorbydria in. 341
blood v.aluea in, 111
iron deficiency in, 336. 341
lymphocytosU in, 13'
myelobla.sts in. 66
Chlorides In blood, 119
Chloroma, 406, 414
marrow findings in~ 520
Chlorosis. anemia (If, 215
Cholesterol, in blood, 119
in red eells. 93
Chronic, benzene poisoniog, 315
infectioDs, anemia of, 206
lympbattc leukemia, 381
myelogenous leuketaia, 311
neutropema. 158
Cirrhosis of Itver, anemia in, 178
neutropenia in, 160
Classification. of anemias, 191
of b~mo1ytic .l1~mia.s. 238
of hemorrhagic diseases, 433. 435
of leuketma, 369
of leukocytes, 129
of platelets, 99
cf red cells.. 95
Clot retraction time, technic for,
.37
Ccagwabon, of blood. 433
of blood in hemophilia. 448
Coagulation time, normal JilUtes
for, 117
techntc for, 634
Cobalt, in erythropoiesis, 220
polycythemia from, 491
Cobra vebom. hemoglobinuria from,
251
Coeliac disease, anemia of, 341
Cold, agglutinlDlI, 564
Colitts, aIlemia of, 214
ulcerative, JDaCTocJl'tJe anemm In,
30.
Color indt"'x, tecbnic for, 612
Composition of blood, 114
ConcentratloD of JeuJc-ocytu. 617
.,.
Congenital leukemia, 364
Congo red In pernicious anemia.
Cooke-Pon.der claliSification of
leukocyte.s, 71, 129
Cooley's erythroblastic anemia, 353
marrovr findings in. 513
Coppe-r. in blood. 11'0
in chIldhood anemia, 342
in idiopathic hypochromic aue-
rnia, 234
role of, 219
Coproporphyrin I, 199
Coproporphyrin III, 199
Corona-ry thrombosis, leukocyte. in,
12.
Counting, of platelet•• 631
of red cells, 608
of reticulocytes, 630
of white edb, 613
Counting chambers, Wle of. 610
Court., attitude to blood (1"OtlP',
583
Coverglass blood smt:an. 616
Cow's milk anemia, 340
Creatinine in red «lls. 9"3
CretinisID, anemia of, 343
Crisis, bone marrow, 166
Cross-matching of blood, 562
Culture of marrow eells, 657
Cyanosis, 196
in polycythemia Yera, 492, 497

D EpitheliOid cell, relation to mono-
Dare method for hemogloblD, -604
Darling, histoplasmosis of, 5SJ
DefimtIons of hematologIc terms,
15
Degenerated leukocytes, 83
Degenerative index of neutrophils,
III
Dehydration, effect on red eells, 94
Denmark, agranulocytosis in. 153
Denvation of blood terms. 5
Derivatives (If hemoglobin, 195
Desiccation of plasma. 571
Destruchon, of blood, 166
of blovd cells, 507
Deuteroporpbyrio. 200
Development of blood cells, 43
Dlab~e8J leukocytOSIS in, 126
Diarrhea, anemia. of. 214
Diet, In anemia. 224
pernicious, Z94
rich in vitamin K. 463
Differential count, normal figures
for. 116
of leukocytes. 129. 622
of marrow cells, 506
of platelets, 99
Digestive leukocytosis, 123
Diluting fluids, foOr platelets, 632
for red cells, 609
for white cells, 613
Dinitrophenol, effect on blood. 147
Diseases, With leukemOid r~ctions,
407
with porphyrinuria. 199
"l1tb .,.ita.min K deficien-CT. 465
Distribution of bone marrow, 46
Diurnal tide of leukocytes, 121
Dog. blood findlIlgs in, 592
Donath·Landstelner test, 252
Donon of blood. findings in, 204
Dorothy Reed Celli, 549
Double reticulocyte rise, 225
Dried plasma. 511
Drugs causing hemolytiC anemia,
239
causing moe.thernoglQbinemia, 491
c3-using thrombocytopenic pur-
pura, 443
containing amidopynne. 150
leukopenia from, 149
producing purpura. 457
Dualists' theory of cell origin, 44
Dule's method for bleeding time.
636
Duodenum, absorption of Iron in,
16.
in pernicious anemia, 279
Dysenteries, anemia of, 214
Dysphagia in hypochromic anemia.
231
E gastrie facton in, 172
Eltop's ring test for bilirubin. 648
Embryo, hematopoiesis in, 46
Eosinophilia, 139
ID Hodgkill's disease, 5!i0
in perniCIOUS anemia, 286
significance of, 142
Eoslnophlhc. grantll~., 139
leukemia, 370. 414
leukoe,.tosis, 119
myelocytes, Dlorpho]o" of, 56
SignIficance of, 68
Eosjnophds, matura.tlon of, 50
morphology of, 57
lupravital .tain of, 105
INDEX
cyte, 78
Errors, in blood counts, 612
in blood matching, 563
Erytbremia, 490
F... ythrobbst,87
In Lederer's anemiJ. 246
Erythroblashc anemia. Cooley type,
353
Erythroblastosis foetatis, 346
Erythrocytcs, 92
agglutinatIon of, 558
techniC for, 673
in aplastiC anemia, 321
after benzene poisoning, 316
cnemical content of, 92, 93, 169
.. description of, 92
destruchon of, 93, 166, 167. 236
development of, 4~
diameter of. measurement of, 626
effect of altitude, 496
m elderly -people, 165
elliptical, 332
• enumeration of, 608
~ factors affecting, 94
formation of, 506
fragliity of, 264
fragility test for, 640
.functlon of. 93
after hemorrhage, acute, 204
after hemorrhage, chrome, 205
hfe span of, 93, 111
in malaria, 534
maturation of, 47, 89
mean corpuscula.r V(l.\ume of, 629
in newborn, 117
norItlal figures ior, 116
nUcleated. morphology of. 61
number of, 93
origin of, 501
In pernicious anemia, 283
in polycythemia vera, 492
production of, 236
relation of age, 16$
Rouleaux [ormation of, 94
In SIckle cell anemia, 3Z9
sickhng trait in. 326
spherucytic types, 260
termlno]ogy of, 9
in urban people, 11&
"aTiatlOns in, 95
volume index of, 62S
volume of packed cells, 116
Erythrocytometer for cell diameter,
627
Erytbrocytosis, compensatory, 496
from hemoconcentration, 498
transient, 496
Erythroleukemia, 407, 413
Errthro-myeloid ratio, 505
Erythron, 111
Erythropoiesis, 87, 174-
factors influencing, 16!i
in liver disease, 178
oxygen tension in, 184
in ~rnicious anemia, 277
porphyrins in, 198
role of pituitary in, 181
role of yeast in, 180
temperature in, 184
vitamm B2 In, 180
vitrunm C in, lSI
Escphaaeal, hands in hypochl'omie
anemia, 231
stenosis, anemia of, 214
Essential thrombocyto'Penic purpura,
440
Estivo-autumn.al malaria, 528, 532
Estrogenic agents 1D hemophilia,
454
Etiology, of aneml3s, 192
of hemoglobinuria, 249
of Hodgkin's disease, 546
EXatnlnatlCln, of bleod, 114"
of bone marrow, 510, 653
ExerCise, effect on Jeukoc)' tes, 12i
dleet (}n red cella, 94
Experimental, infectious !bononU
cleosl., 476
leukemia. 367
leukemOid reactions, 407
leukocytosis, 127
spherocytosis. 261
Extramedullary hematopOiesis, 46
Extrmslc factor, in perniCIOUS ane'
mia, 277
nature of. 172
F
Fablsrn, hemolytic anemia from.
241
Fabismus, 2S5
Factor. hematopoietic, 173
Falling drop method for protem,
667
Familial, hemolytic Jaundic~, 259
IdIOpathiC methemoglobinen"ua, 19b
incidence of perniciOUS ;memla,
280
purpura simplex, 460
transmission of hemophilia, 447
of leuKemla. 364, 369
of ovalocytosis, 332
Fatigue producing leukopenia, ISJ
Favistn. 255
Feces, in perniciou!I anemia. 286
porphyrins m, 19"8
urobilinogen Ill, 649
Ferrous iron, advantages of. 219
Ferrous sulfate, advantages of, 222
Fetus, blood formation In, 46
Fever, leukocytosis from, 124
Flbrmogen 10 blood clotting. 433
Flbrmopenia, hereditary, 460
Fllament·Non·filament cour~t, 69,
129, 624
Filterable VirUS, leukemia frqm, 368
Finger puncture, techniC f<;lr, 597
Fish tape worm, anemia tlf, 207
Fixation of blood smears, .618
Fluids. effect on red cells, 94
Fontanelle, obtalJling blood from,
600
Foods, containing Vitamin 1\:, 463
effect on 1eu\ocyte$, 123
iron containlng, 224 ,
Forssman antibodies, 485, 664
Fowler's solution m leukerata, 37B,
395
FOWls. leukemia in, 367
Fraction G of Cohn, 289
FragIlity of red cells, in acquired
hemolytiC anemia, ;244
In hemolytic jaundice, 261
Fragtlity test, technic. for, 640
pipette method for, 641
Fresh blood, examination of, 615
Frog, blood findmgs in, 59"1-
Function, of bcl.sophils. 1<12
bone marrow, 47
erythrocytes, 93
hcmoglobm, 194
Iymphocyt~, 73
monocytet, 78
neutrophil., 125
 INDEX
Fundion-(Continued) Hemacytometer. 609
plasma eel!!. 19 HemagglUtination, 558
platelets, 434 Hematin, 200
acid, spec.troscopic bands of, 675
G alkaline, spectroscopic hands of,
675
Gal'iooek's disease, 492 HematOCrIt determtnatl-OD, 625
Gast"ectmny. anem~ from, 175 HematCllogic, claSSification of :me-
macrocytic anemta after, 305
Galltuc, atz-opllY, 175
mias, 191
standards, 116
cancer, anemia of. 207, 306 te<!hmc, 596
contents in p1'egnancy, 182
terminology, J
factors in hematopoiesIs, 172
terms, definitions of, 15
findlng$ In perniCIOUS anemia,
286
de1'ivation of, 5
tray, 598
jutce, hematop()ietic factor in, 176
Hematopoie$ts, 174, 500, 501
In polycythemia vera, 117
m bone marrow, 47
role in hematopOIesis, 173
lavage in polycythemia, 117, 494 10 d1seasc:d liver. 179
iD embryo, 46
pathorogy in pez-.oiCJOIlJJ :memia,
gastric factors in, 172
296 10 pernicious anemia, 27'/
Gutro-intestinal. disease, anemia
of, 214
porphYrins in, 198
principles of. 43
dysfunctions, macrocytic anemia
protem in, 172
In,305 transfusions for, 576
resection, anemia of, 214 Hematopoiettc factor, 173
tract 1n pernicious anemia, 282
1n liver, 171
Gaucher dtsease, marrow findings
nature of, 289
In, 520 in polyCYLhenua, 491
German measJeJ. ;!nd infectiou," storage of, 174
mononucleosIs, 485 10 urlOe, 286
leukopenia. of, 146 HematoporphynD, 198
plasma cells in, 82 spectrosC(lptc bands of, 675
Glemsa's stain for blood smears, HemochromatlD, 200
620 Hemoconce11tration, erythrocytosil
GingiVitis In monocytic leukemia, in. 498
397 Hemocorua. 113
Glandular fever, see Infectious Helllocytoblasts, 43
mononucleosis Hemoglobm, 194
Gloh1llin ~nh~ta.nce in 'hemophilia,
at birth. 117
44. constructIOn of, 195
Glomerular threshold fo-1' hemoglo- deficieocies, causes of, 170
bm,249 derivatlvoes, 194
Glossitis, in hypochromic anemia, destructl(ID of, 236
231 determination. by Dare mc:tLod,
in perDiClOUS anemia, 281
Glutatluone ID red eells, 93
60.
by Newcomer method, 606
Gold salts, effect on blood. 147 by photo-electric method, 607
Gordon test, for IIodgkm's disease, by SahlI method, 605
550 by- Tal1quist scale, 604
techni-e for, 666 technics fllr, 604
Gower's solution, 609 increase after iron, 225
Granules in- basophils, 143
1n malaria, 535
in eosinophlls. 139 mean corpuscular, 629
from leukocytes, 113 tllole...::ular weight, 201
10 neutrophJis, 125
Itt negroes, 118
Granuioc:ytes, formation of, 507
normal figures for, 116
maturation of, 49
phYSIologic vanattons ill, 218
morphology of, 55
in red cells, 93
origin of, 49, 502 rdatlO.ll of coppeI", 170
Granulocytopenia, acute, see Agran-
renal threshold for, 248
ulocytosis
role of tron In, 168
Granuloma, Hodgkin's, 546
Granulomata, monocytosis in, 138 role of salt in, 218
spectroscopic bands of. 674
plasma ce1ls tn. 83 Hemog-Jobltlen:tra, 237
GranulopOiesis, factors depre$Sing,
causes of, 248
145
Hemoglobinuria, 248
Guinea pigs, blood findings in, 590
allergIC parO-ltysmal type. 255
causes of, 249
H Heterophile antibodies, ddF~retttiaJ
fn hemoIytfc anemias, 237, 240
Haden-Hausser erythrocyto211eter,
In malaria, 539
627
Halometer for edt diameter, 628 With paralYSts, 256
Hand-Schuler-Christian disease, paralytic type, 256
marrDW findings in, 520 parwtysmal, cold type, 252
Hayem's solution, 609 March. type, 253
Hay fever, eosinol>htlia of, HI nocturnal type, 253
Heart disease, congenttal, erythro- Hemolysins, hemolytic anemia
cytes in, 497 from, 243
685
ITe! _olYSla, in pemicioul anemia,
Z86
mechaDism of, 238, 254
of mlcrospherocytes. 261
of red cells, 93, 236
Hemolytic anemia, 236
acqUired. 244
treatment of, 245
of childhood, 345
from drugs, 151
Without Icterus gravis, 346
of Lederer, 245
of newborn, 346
Hemolytic jaundice, 259
autoagglutmatlon tn. 674
famllt.al transmission of, 259
inCidence of, 260
laboratory findings in, 263
pOrpb}rlDS in, 199
.plcnectomy in, 167
.ymptoms and phY$ical findinis,
Z.,
transfUSions In. 579
treatment of, 264
Hemophilia, 447
blood tindtngs in, 151
diagnosis of, 452
incidence of, 450
inheritan~e of, 447
marrow findings in, 515
nature of defect, 448
pathology of, 451
prognOSIS of, 454
symptoms and physical findmrs,
450
transfUSion in, 573
treatmcot of, 452
Hemophiliacs, marriage of, 448,
452
Hemorrhage, in acute leukemia, S91
Hemorrhage. anemia from, 166,
204,205
tn hemophilia, 450
leukocytosis of, 126
In non.thrombopenlc purpura,
451
reticulocytosis after, 166
HemoTrbagic diseases, 433
anemias with, 358
classification of, 433, 435
hemophilia, 441
marrow findmgs in, 514
of newborn, 461, 466
non-thrombo-penic purpura, 456
I!rtudl~s {or, 437
thrombocytopenic purpura, 440
transfusion for, 573
Henoch's purpura, 456
Heparin as ant1coagulant~ 600
in basopluh. 142
in blood c1ottmg, 433
Hereditary, famihal purpura sim-
plex, 460
fibrinopetlla, 460
pseudohemophilia, 460
Herpes zoster in leukemia. 428
Heterophile antibody test, 485
interpretation of, 665
technic for, 662
test for, 663
presumptive test for, 662
HIatus ltukemlcus, 66, 365, 393
Histamine stimulation 10 pernicious
anemia, 28'
Htstiocytts, 76
Histologic section of bone mar-
row, 656
Histoplastna eapsulatum, 553, 554
686
Histoplasmosis, 553
leukopenia of, 146
treatment of, 554
Hodelcin'. diseue. 546
blood findings tn. 550
clInical findings 10. 551
diagnosis of, 552
etiology of, 546
as forDl of tubtrculosis. 547
Gordon test for, 550, 666
history of, 546
as infectlous eranuIoma, 547
lymphocytosis of. 13J
monocytosIS In, 138
pathology of. 549
relation to brucellosis, 541, 552
treatment of, .552
as virus dtsease. 548
Hookworm disease, anemia of, 208
eoslOophlha of, 141
nones, hemoglobilluria. iu, 256
Howell-Jolly bodies, 97
Howell method, for coagulation
bme, 635
for prothrombin, 637
Hydremia in anemia, 183
Hydrogen, arscmuretted. hemalytie;
anemia from, 240
Hyperchro:>ttu.a l.(J_ pernlc.lOUiI
mia, 28S
Hyper{llasta of marrow. 145
Hyperplastic bone marrow, find-
~ngs In, 5t2
Hyperprotewemla, autoa(:"glutina-
tion in. 674
HyperthyroidisM, anemra of, 181
ttythr~,tc:"" ~U. 497
lymphocytosis of, 136
Hypocilromic anenua, 202
of infants, 213
hver therapy in, 223
of pregnancy, 209
treatment of, 218
HypochromiC red cells, 95
Hypo:phyau, Tole in anemia, 181
Hypoplastic anemia, 311
splenectomy for, 26.8
HypothyrOldl!;.m, anemia of, 181,
212, 343
I 'I" Ii ,t
Icterus, gravis neonatorum, 346
hemolytic congenital, 259
mdex In hemolytIc anemtas, 237
test for, 646
IdiopathIc, aplastic anemia, 320
hyp.oehrOIJllC anemia, 229
IleItiS, macrocytic anemia in, 306
Index, degeneratJve, ]31
Schilling, for leukocyte., UO
Indications fDr splenectomy, 268
Indophenol blue synthesis, 622
Infants, anemia of, 334
blood findings in, 117
blood groUps in, 557
lead po-isoning in, 273
nutntlOnal anemia of, 339
obtainulg blood from. 599
Infections, ac.ute, anemia of, 205.
356
in agranulot:ytosis, iSS
blood plasma In, 573
hemoglobinuria In, '250
hemolytiC anemias from, 239
in leukemia, 318, 427
leukocytosis of, 125
marrow aplaSIa In, 320
INDEX
Infecbons-(ContiJuud)
relatIon to leukemIa, 366, 391,
384
sidle cell anemia, 321
trans£u"ion for, S7J '
Illfectiou, dlseases, myelocytes in,
68
Infectiou, mononucleosiS, 473
blood findlDi.s in, 4S0
diaposi~ of, 484
etiology of, 474
heterophile antibody test for, 485
history of. 473
monocytes in, 139
prognosIS of, 487
symptoms and physical findings,
476
test for, 662
treatment of, 487
In8ammatlon. plasma cells in, 83
IlifluCD'i:a, leukopenia of, 146
Inheritance of, blood groups, 581
famlual laundlce~ 260 -
hemophilia, 447
leukenu.a, 369
sickle cell anemIa, 325
Injection! of iron, 223
Intermediate monocytes, 52
International classtfication, blood
groups, 556
Intestinal parasitism,. eoslllophdia
of, 141
lntrmsi.c factor. I1J
In pernicious anemia, 277
p-toperties of, 174
Iroo, absorption of, 169. 218
Hi ad.llQrbydna., 17G
ID bookworm dIsease, 208
in a.t.blorhydnc patIents, 223
amount, for treatment, 219
in body, 169
for clJlldhood :lDeml3. 342
foods containing, 224-
In hemoglobin, 194
m hUllolytie Jaundice, 265
in idIopathic hypochromiC
mia, 234
injectable forms of, 223
in leuktrn13, 420
mJ.inteoance dose of, 223, 234
metahohsm, 169
in pernicious anemia, 294
preparallOn, -of, 221, 222
relation of copper to, 219
response to treatment with, 225
role ID hematopoiesis. 168
tissues containinK, 168
uS(! of, 218
polycythemia, 494
Iron deficiency, anemia of. 212
in children, 336, 341
m nails, 170
Isaacs refractIve granu1es, 96
Ivy method for bleedillg time, 636
Jansltv c1a.ssification, blood groups,
".
Jaundice, hemolytic, 259
of the newborn. 334
Jews, polycythem13 In, 490
J OIDtS, hemorrhage of in hemo-
philia, 451
in non-tbrombopenic purpura, 457
Jugular vein, obtaining blood from,
59.
Juvenile leukocyte, supravital stain
of, 103
Juvemle neutrophil, morphology (If.
56
K
Kato's method for prothrombm, 639
Kidney, excrttion of bcmoilob,o.
248
in leukemia, 383
KOilonychia, In hypochr-ornic
mIa, 231
iron defiCiency In, 170
L
Laboratory animals, blood findings
!n, 589
Landsteiner classification, blood
groUps, 556
Law, aUltude to blood grOups, 583
Lead PQisoning. aD.e[ll.La irom, 21a
basopbiltc aggrcp.tion test for.
275, 672'
hemolytic anemia in, 240
in mfants, 272
LeCithIn in red cells, 238
Lederer, hemolytiC anemia of, 24S,
347
Leg ulcers, i-o hemolytic jaund«:.e,
263
in Sickle cdl .meruia, 327
Legal applicatIOn of blood groups,
582
Leukemia. 363
acute, 385
dIagnosis of, 394
incldt:.nce c'f, 3B5
marrow findings in, 519
pathology of, 395
radioactive phosphorus in, 427
relaticn. to infection, 391
symptoms and phYSical find-
ings, 391
treatment and course of, 395,
.26
a1ettkellllc. 405
leukopenia tn, 159
in anImal., 36l
atyplcal types of, 4-0S
basal metabohsm in, J75
basopbilic, 310, 414
basClphllic myelocytes in, 69
bone findings in, 374
bon.e pathology in, 429
chloroma type, 406, 414
chronic lymphatiC, l81
blood findings in, 382
course and prognosis, 384
diagnosis of, 383
marrow findmgs in, 519
pathology of, 383
radiation treatment for, 421
symptoms and physical find-
ings, 381
treatment of, 384
chromc myelogenous, 371
blood findings in. 375
dJagoollis of, 3'17
marrow findings in, 519
prognosis of, 379
radiation treatment for. 424
symptom! and phySical find-
ings, 371
treatment of, 377
claSSIfication of. 369
chnical types of, 413
eosinophiilc" 414
experimental production of, 367
in fowl., J67
 INDEX
Leukemla-(Conti"Sled) Leukocytes-(Ctmtift14eti)
general measures for, 427 differential countlllg of, 129, 622
herJK,:s :l:ostt:I" jn, 428 effect of benzene un, 146
}lIatus leucaemicus in, 66 food on, 123
history of, 363 ellmmatlon of, 69
mfluence of mfectlOns, 378 emigratIOn of, 129
mherltance of, 369 enumeration of, 613
macrocytic anenna tn, 309 filament-nonfilament count of,
marrow aplasia 10, 319 62'
marrow findmgs in, 517 granules tn, 113
megakaryocytic, 414 unmature, Significance uf, 129
marrow findings m, 5+0 in tnfectlous mononucleosts, 4S0
mrnlce, 367 In malana, 535
mixed cen, 407 In myelogenous leukemia, 377
monocytic, 396, 413 In monocytic leukemia, 400
blood findings In, 400 m newborn, 117
dlagnostlc Criteria £Or, 401 in nOD-thrombopeDic purpura,
inCidence of, 397 • 458
marrow findmgs 10, 519 normal nutnber of, 116, 121
nature of, 396 in pernicious anemia, 285
patholDgy of, 401 in polycythemia v~ra, 493
symptoms and phYSical nnd- redlstnbuhon of, 124
mgs, 397 10 stored blood, 569
treatment and prognosIs, 401 supraVital stamlllg of, 103
nature of, 364 m thrombOl::ytopenic purpura, 442
neurologic findlllgs in. 374 Leukocythemia, 363
oral sepsIs In, <121 LeukocytOSIS, digestive, 123
plasma cell, 82. 406, 414 eosinophlbc, 139
marrow findings In, 520 of exerCise, 128
polynuclear, 414 experimental types of, 127
and pregnane,., 428 in Hodgkm's disease, 550
pnapism In, 429 in lead polsontng, 273
primitive cell, 394 in malaria, 535
relatlon, (If trauma, 364 marrow findings in, 517
to neoplasm, 365 neutrophthc, 121
smudge cell! In, 83 of newboTll, 123
subleukemic tYPe, 414 in polycythemia, 493
surgical operations in, 427 pathologic, 125
treatment of, i13 physiologlc, 121
with arseDlc, 420 of pregnancy, 123
With benzol, 420 from sulfonamIde drugs, 152
contact theral)y~ 415 from temperature, 12'\
for hemorrhages, 427 tranSient, 124
by Heublem method, 416 ty~s of, 121
general measures in. 421 Leukon. 111
Grenz rays lU, 415 LeukopeDla, 144
heliotherapy in, 427 m acute leukemia, 393
With high voltage, 416 in aplastic anemia. 321
mtermedlate voltage~ 416 ~hroDl(:. 158
interstitial radiation" 417 digestive, 123
intracaVitary radiation, 411 from drugs, 149
with min and hvcr, 420 of histoplasmosis, 553
local radium, 417 from madeQuate diet. 148
local x rays, 415 In mfectlOus mononucleosis, 480
low voltage therapy, 415 in malana. 209, 535
with radioactive uotopes. 418 marrow findlllgs m, 517
With rest. 420 marrow pathology 10, 145
segmental thet'apy, 416 in monocytic Icukenua, 400
super voltage, 416 in osteosclerotic anemia, 323
With thonum products, 418 in pernicious anemIa, 160, 285
total Irradtation. 416 of radiation, 147
with transfusions, 420 from radium, 318
With vltamms K and C, 420 tests for allergy, 123
Leukemoid reaction, 407. 415 Leukopenic diseases, 144
IQ hemolytic jaundice, 264 leUkopenic leukemIa, 405
In polycythemJa, 4-93 Leukopoiesis, 47
Le!J'knhla!!t.~, in cell det<~Jopm,..nt, 46 Lelfkos~reama_, lympb-2tl(~, 414
perOXidase reaction of, 66 LeukOSIS. acute, 385
Leukocytes, abs-olute numbers of,
Lipoids, monocytic reaction to, 78
11. Lipoid storage diseases, marrow
after acute hemorrhage, 204 findings in, 520
in acute: kukemia. 393 Liver, damage, rnacrocytl(: anemia
after benzene pOisoning, 316 10,308
bone marrow dehvery of, 121 Vitamin K tn, 467
in chronic lymphatiC leukemia, macrocytic anemIa in, 178
382 extract. advanta2"es of, 290
concentration of, 617 '" 10 agranulocytosis, 157
degenerated t,.pes of, 83 dosage of, 293
descnptive terma for, 6 intoleranCe to, 295
68 7
Liver-(Cont1nued)
extract-(Contlnucd)
intravenous use of, 295
refinement of, 289
10 tropical anemIa, 308
fraction for secondary anemia,
224
in leukemia, 420
preparations, concentration of,
28.
development of, 288
relation of mtnoslc factor, 173
role in hematopOieSIS, 173
storage of hematopOietic factor.
177
therapy, eosinophilia after, J4Z
JQ hypochromiC anemia, 223
Longltudmal SIllUS, obtammg blood
from, 60'1)
Lymph glands, In Hodgkin's diS-
ease. S4~, SSI
in acute leUkemia, 392
ID InfectIOUS mononucleosis, 476
ID leukemia, 381
m monocytic leukemia, 397
In myelogenous leukemia, 42.5
Lymphadenosis, benign, 473
marrow findmgs in, 519
Lymphahc, leukemia, chrome, 381
leukosarcoma, 414
Lymphoblasts. development of, 72
morphology of, 57
supravltal stam of, 105
Lymphocyte-monocyte ratio, 78, 137
Lymphocytes, SOJ
to aplastic anemia, 321
confus)(In with myeloblasts, 63
development of, 72
formatlOn of, 507
In infectiOUS mononucleoSIS, 480
maturation of, 51
morphology of, 57, 72
normal number of, 133
relation of p1allma cells, 83
supraVital staID of, 105
LymphocytOSIs, 133
of children, 117
diseases characterized by, 73, 133
JIl infectious mononucleosis, 180
in malaria, 209
m perniClous anemm, 285
Lymphoid diathesis, lymphocytosh
of,136
Lymphoidocytes, 43
Lymphogranuloma, Hodgkin's, 546
M
M and N agglutmogens, 565
Macroblasts, 89
development of, '"
Macrocytes, diseases characterized
by, 95
l\facrocytlc anemia, after gastrec-
tomy. 305
in achrestic anemia, 307
ID amoebiasis, 306
in coeliae disease, 344
diseases characterized by, 301
in gastrO-lntestinai dysfunctions,
.oS
in leukemta. 309
in liver disease, 179', 308
in malignancy, 207
marrow findmgs m, 513
1D newborn, 343
in pellagra, 302
in pregnancy. 182, 209, 303
031> •
Macrocytic anemia-(Continued)
in sprue, 301
18 thyroid disease, 212
MacrocytOSIS, dtseases with~ 285
Macrophage!, 76
j:.;n-watlon uf, 507
Mamtenance dose of hver. 297
Malaria, 522
anemia of, 209
'Black Watn {eve" oi, 539
blood picture in, 534
clinical symptoms of. 530
estivo-autumnal type, 532
bemogloblDurla in, 250
bemolytic anemia m, 240, 245
history of, 522
incubatJon penod of, 531
leukopema of, 146
DlonocytOf.:1S tn, 138
parasites, cycle in mosquJto, 5.25
dIfferentIal diagnosIs of, S,!9
exammatlon faT, 660
-exo-erythrocytlc stage, 526
morphology of, 527
Plasmodium !alciparwm. 528
Plasmods'um ma/ariae, 528
Plannodulm o'l1ale, 530
Plasmodium vivax, 527
5chlZogenous cycle, 525
sporogenous cycle. 525
pnthologic changes In, 536
plasmodia, 523
transmission of, 525
treatment of, 539
atabnue In, 542
plasmochin in, 542
qUinine In, 540
type of fever in, 533
Ma}Jguancy, anemia of, 206
Malignant neutropenia, ISJ
Mallgnant tumors, leukocytosis of,
12.
MalnutritIOn, anemia of, 213
lymphocytosis of, 137
Marblebotle disease, 322
MMCb. h~m~bt\:n_'\l'lh., 2S~
Marchlafava·MIcbeh syndrome, 253
Marmite in tropical anemIa, 180,
308
Marnage of hemophihacs, 448, 452
Marf"ow, bone, see Bone marrOW'
1.1art"ow, transfusions of, 322
Massage, effect on red cells, 94
Matching of blood, 562
Maturation, arrest, in agranulocy-
tosis, 156
in leukopenia, 145
of erythrocytes, 47, 174
of granulocytes, 49
of lymphocytes, 51
of monocytes, 52
of myeloblasts, 63
of thrombocyles, 51
Measles, leukopenia of, 146
M ecbamsm of leukopenia, 144
MedIcal group. agranulocytosis jtl,
."
lledico--Iega) aspects of blood
groups, 5S0
Mean corpuscular, hemoglobin, 629
vnlume~ 629
J..Iedlterranean anemia, 353
Megakaryoblasts, 98
Megakaryocytes, 502
In bone marrow. S1S
formation of, 507
in Hodgkin's dIsease, 548
maturation of, 51
INDEX
lIegakaryocytes-(Conflllued)
morphology of, 61
relatiOn to platelets, 97
Megakaryocytic leuk~I3, 414
Megakaryocytosis. marrow findin&,s
ln, 520
Megaloblasts. 500
in acute leukemia. 392
descnptIon of, 87
de.e1o-pment uf, 49
In hemolytu: jaundice, Z64
morphology of, 61
in pernicious anemia, 285
in Sickle cell anemia, 329
SIgnificance of, 44, 92
Melena neonatorum, 461
anenn.a With, 358
Men, eQthrocytes tn, 116
MeningItIS, Jeuk~ytosis of, 126
MeniscocytosIs, 325
M~struatlOn, an~mia of, 215
in hypochromic anemia, 230
relatiOn of blood findings, 165
Metabolism in leukemia, 315, 383
Metamyelocytcs, supraVital stain
of, 103
lrIethemoglobin, spectroscopic bands
of, 674
Methemoglobinemia, 497
causes of, 196
chronic, 498
Methylene ",lue for cyanosis, J97
Mice, leuke ua in, 367
MicrocytiC a~ 1tlorhydn-c anemia, 229
MIcromethod, for prothrombin, 639
for sedimentation rate, 645
Mic.romye.loh1.asts in. acute le-"llke-
mia, 393
MicrospherocytosIs, 260
Milk anemia, 213, 340
Milk injections, leukocytosis of, 12'
MI'C:ed cell leukemia, 407
Monkey, blood findings IU, 592
1.1onOOla8t9, in cell maturation, 52
morphology of, 60, 78
1>\.\'Jta~hal ~tai.n. of, IUS
Monocyte-lymphocyte ratio, 78, 137
lI.fonocytes, 503
in agranulocytosis, 137, ISS
descnptlOn of, 73
formation of, 507
function of. 78
in hematopoiesis, 52
in mala-rin, 535
maturation of, 52
in monocytic leukemia, 4-00
morphology of, 60
origlD of, 46, 73. 77, 503
relatIon to othe!' cells, 76
suptavital staIn of, 105
Monocytic leukemia, 46, 396
monocytes in, ]39
MonocytOid myelogenous lc:ukeJ:Dia,
".
Monocytosis, 137
In various diseases. 138
Mononucleosis, infectlous, see In-
fectious tnononuc1e6sis
Monophyletic theory, 43
Morphology of blood cells, 55
MOsqUlto as malaria cartier, 525
]l.10ss cl.asslfication, blood groups,
55.
Motlhty of neutrophils, 125
Mouse, blood findings in, 591
Mouth legions in m.onoclltic leu_
kemia, 397
Multiple myeloma, autoagglutina-
tion in, 674
Multiple myeloma-(Continued)
marrow findmgs in, 520
plasma cells' m, 82
Mumps, leukopema of, 146
Muscular achvlt)', leukocytosis of.
12'
Myeloblast:c leukemia, acute, 385
MyeloblastOSIs, Significance of, 66
Myeloblasts, 500
lU acute le-uken:lla. 393-
confusion with Iympbocytes, 63
descnption of, 63
identificatIOn of, 55
maturation of, 50
morphology of, S5
occurrence of, 66
with peroxidase stain, 66
recognition of, 55
supravita.l staIn of, 103
Myelocytes, 67
l.asoph111C, s1gnincance of, 69
-descnption of, 67
development of, 69
in leukemia, 377
maturation of, 50
sigmficanc::e of, 68:
supraVital stain of, 103
Myelogenous leukemia., 311
eoslnophlls in~ 141
Myeloid chloroma, 406
Myeloma, multiple, marrow find
in2's in, 520
plasma cells m, &3
MyelosclerOSIS, 322
Myelosis, chronic, 371
marrow findings in, 519
Myoglo.binUl:ia, -paralytic, 256
Myxedema, anemia of, 212
N
Nalls, brittleness of, 170
Nasal secretIOns, eosinophils in, 141
Negroes, agranulocytosis in, 154
leukemia in, 364
1:cl Cl:.lt~ -and noe.tl:I.Ggt(lb.tn \n, U&
perniCIOUS a.nemia 10, 279
Sickle cell anemia In, 325
sicklmg trait in, 326
Neoarsphenamine, leukopenia frorn,
'5'
Neoplasm, rela.tion to leukemia, 365
NeurologiC, changes in acute leu·
k~ia, 392
disorden in polycythemia vera.
492
findings in leukemia. 374
symptoms in infectIOUs mononu·
cleosis, 484
system in pernicious anemi.a, 281
Neutral hematin. 200
Neutrocytosis, J 21
mart".ow findIngs in, 517
pathologic, 125
Neutropellla, 144
in agranulocytosis, t SS
in 8-plastic anemia. 159
in blood dyscraslas, 159
chronic, 158
from drugs, 149
malignant, 153
marrow findings in, 511
SIgnificance of, 144
In various dlseases, 160
NeutrophIls, developmertt of, 69
hyp~~gm~ntat;on of; 285
maturation of, SO
morphology of. 56
motiGn of. 125

Neutrophlls-(ConrlKwed)
orlgm of. 50.1
supravItal staining of, 103
Netltrophlllc, graQules, 125
leukocytosis, 121
myelocytes. morpbology 001, S6
Newborn, blood findings in, 117
blood grollps in, S57
bone rnarfQW in, 50S
erythroblastOSls of, 346
hemolytic anemias of, 346
hemorrhagic du~easc (If, 461, 466
jaundice tn, 334
leukenua in. 364
leukocytosis (If, 123
macrocytic anemia of. 343
retlculocytes in, 96
vltanun K 10, 465
Newcomer IIlethad for hemoglobin,
606
Ni<:etinic add in pellagra, 302
Niemann-Pick disease. marrow find-
ings in, 520
NItrobenzene, methemoglo-bin from,
19_
Nocturnal hemoglobinuria. 253
Non-protem tutrogen Itt blood. 119
Non-thrombopellic purpura, 456
Normal blood, 111
hematologic standards, 116
people, eosinophils in. 141
Normoblasts, descI"lPtion of, 87
dev-e}opment of~ 49
morphology of, 61
Norm<IChronnc red cells. 95
Nuclear fenestrahons, 484
Nucleated red cells, correction for.
614
morphology of, 61
NucleiC acid. In agranulocytosis,
IS_
• leukocytosis of, 127
Nucleoli 10 smudge cells, 83
N utntlonaI anemia of infants, 339
o Phagocytosis Wlth neutrophils, 125
Ohstructlve jaundice, vitamin Kin,
467
Old's method for countiDi' pla.te-
lets, 632
Operatwns, Jeukocytosil after, 126
Origin of. blood eells, 43, SOl, S06
bemato]ogJC terms, 4
Oroya fever, hemoglobinuria in, 250
hemolyhc anemia ln, 240
Osguud's, method (If marrow cul-
ture, 657
terminology, 14
Os!er·Vaquez disease. 490
Osteopetrosis, 322
Osteosc1et'ottc anemia, 322
Ovalocytes, 95
Ovalocytosis. 331
Ovat'lan extracts in hemophilia. 4S3
Oxa.lated blood. for examination,
"5
lymphocytes in. 72
use of, 601
Oxalates as anticoagulants, 601
Oxahc acid in helhOphllia, 454
Oxygen tension, effect on erytht"D-
cytes, 496
effect on sickling, 327
in erythropoiesiS. 184
in polycythemia, 491
OJl;yhernoglobin. 195
spectroscopIC bands of, 674
INDEX
p Plasma cells-(CoOfttiJluecl)
Packed cell determination, 625
Pairltens, anent"" In, 272
Pallor In anemJ.a, 203
Pancreatlc -dlsease, anemia in... 306
Panmyelocytosis, 68
Paralybc mY"iJohlnurja~ 256
Parasites of malaria, 523
Parasitic infestation, anemia of,
2.7
eoslDophllia of, 141
~:~:~~e:~l ir~~~~~~~:~rf3 252
Marc:h. 253
Pathologtc leukocytosis. 12S
Paul-Bunnell test, 474, 4s5
Pel-Epstel.l1 fevet', 551
Pellagra, 302
Pentnuc1eohdes in agranulocyto5is,
156
Periartentis nodosa, eoslDophilia of,
142
Pernicious aneJl1u, 276
bJood findUlC'S ill, 283
dIagnOSIS of, 287
eosinopbdia itt, 142
gasttle atrophy in, 175
hntf)TJ' of, 2')6
mcidence of, 279
leukopenia in. 160
maintenance dose for, 297
marrow aplasia in, 319
marrow findings in, 512
pathogenesis of, 277
porphyrins ill, U9
of pregna.ncy. 211
relation to hypochromiC type, 230
symptoms and pbysieal findings,
2S0
treatment of, 288, 293
Peroxidase stain, 621
lymphocyte reactlon to, 72
monocyte reaction to, 60, 78
myeloblast 1'eactil)n to, 66. 394
Phenacetin. methemoglobin from,
19.
Phen),lhydraziue. hemolytic anemia
fro-m, 241
Phlebotomy In polycythemia, 494
Phosphorus, :radJated, for leulre-
nua, 418
Pbotelometer, Sheard-SanfoTd, 607
PhotoelectriC method for hemoglo-
bin, 607
Physiologic, ecy-throcytusis, 496
leukocytosis. 121
PhySiology, of bone marrow. 47
of Vitamin g. 464
PigeonS, reticulocyte response in,
m PT1api~m In leukemm, 374. 429
Pigment of blood, 237
Pipettes, blood counting, use of, 610
Pituitary. role in anemias, 181
Placental, blood, transfusion of, 570
extracts m hemophilia, 453
Plasma. of blood. III
.4.
globulm fractIOn in hemophilia,
indications for, 577
protein, relation to spedfi.c I[t'av,
Ity. 670
transfUSion of, 570
Plasma ('ell leukenlla, 82, 406, 4]4
Plasma eells, 79
function of, 79
iIt variOUS diseases, 406
morphology of, 61
occurrenCe of, 83
oriain of, 19, S02
PlasmatQl;;yte:s,76
Plasmocbin in malaria. 542
Plasmacytes, orJgm of, 502, 507
Plasmocytosls. marrow findings in,
52.
Plasmodia o£ malaria.. 523
PlastnQduml jalciparNm, 528
malanae. 528
ova.te,530
VWO";-, 527
Platelets. see Thronmocytes
maturation of, 51
Plumbism, chronic, 272
Plummer-Vinson syndrome, 231
PneuDloma, anemia..$ With, 357
e05inophllia in, HZ
leukocytosis of, 126
POlkllocytes, 95
Polyblasts. 76
PolycbTomatophtlia, 9S
Polycythemia, symptomatIc, .. 96
vera, 490
blood findings in, 492
etIOlogy of, 490
gastric juice In, 1'17
marrow filldwgs .in. 515
prognOSIs of. 495
.)'mptoma and phys.ical find-
Ings, 491
treatment of, 494
Polynuclear Ie:ukeml3, 414
Polypnyletlc theory, 44
Pons-Krumbhaar classification of
cells, 69
PorphYria, congenital, ZOO
Porpbynns, 198
rela.tlon to hemoglobin, 194
spectroscopic: bands_of, 675
Porpbyrinuria, 199
Post-InfectioUS lymphocytosis, 136
Post-operative. leukocytosis, 126
shock, blood plasma in, 5'12
PotaSSium tn red cells, 93
Pregnancy. anemias in, 182, 209,
42'
leukocytosis of, 123
macrocytic anemia in, 303
rehcuI()Cytes in, 96 •
Prematunty, anemia of, 338
Pretnortem leukocytosis, 126
Premyelocyte!., maturation of, 50
morphQlogy of, SS
Preparation. of blood serum, 602
of blood smearllo. GIS
Prepa rations, of iron, 222
of liver, 290
of vitamin K, 467
P1'eservahon of blood, 568
Prke.Jones curve, 626
Pnmary aplastic anemia, 320
Pnmitlve ceU leukemia. 394
Primitive cell, morphology of, 61
Primitive free cell, 46
Protein deficiency, blood transfu-
sion for, 575
Proteins In blood, 119
non·specific. leukocytosis of, 127
role in hematopoiesis, 172
totaJ, technic 10-(', 667
Prothrotnbin. in blood clottinlr, 433
In bemopbj]Ja, 449
properties of. 465
l"elatJon to vitamin X, 465
In stored blood, 569
hme, determination of. 631

Protoporphyrins, 198
psoriasis. eosinophilia of, 141
Pulmonary fibro.!l1s, erythrocytes ill,
497
Puncture of stunuro, SC9
Purpura, of capillary weakness, "56
drug. producing, 457
hemarfhagica, 440
nou-thrombopenic. 456
"blood fmdinJs Itt. 458
dmgnoais of, 458
symptoms and physical find~
ings. 457 I 8
treatment: of, 458-
simplex. familial, .460
thrombocytopenic, 440
Pyrogens, cause for reactions, 579 Sanford's J}1ethod for fragultt, 640
Q 241
Quartan mataria, 528
QUick method for protlir01llbtn, 638.
QUinme in malaria. S.. O
R interpretation of. 130
Rabbits, blood findings in, 589
effect of benzeoe on, 146
in acute leukemia, 39.5
In agranulocytosis, 156
RadlatlOD, in agranulocytosis, 156
effect ::to blood, 147
in polycythemia vera, 495
ptCdUCIDg a?lashc a.n~mia., 318.
ulaban ta leukemia, 367
in thrombocytopemc purpura. 444
treatment for leukemia. 415
RadJOactlve, Isotopes, 418
!iuhstanees, effect on blood, 141,
318
Radium, leukopenia from, 31S
Rat, blGCI-d findings. in, S93
Reactions from transfUSions, 57!
Red blood cells, see Erythrocytes
Reduced hemoglobin, 195
Refractive granules of Isaac" 96
Rdrac.to~,. auetnias, 311
porphYrins in, 199
Reich method for sternal puncture.
651
Relative lymphocytosi', 15, 133
Relative values of blood ceUs, 623
Renal threshold for hemoglobin. 24S
Retlculocytes, 96
in aplastIC anemia, 321
counting of, 630
development of, 49
effll'!Ct of lead on. 273
after hemorrhage, 204
increas.es of, 96-
In malaria, 534
Me of. 96
morphology of, 62
Telation of porphyrin~, 198
Retlculoc:ytosill, after iron, 225
in hcmolytu:= anemias, 237, 264
in lead poisoning, 273
lD Lederer's anemia, 246
after liver treatment, 294
in polycythemia, 493
In Biehle cell anemia, 329
tberapeutic test with, 287
Reticula-endothelial cytomyc::o~is, SSJ
Retu:uloendothehosill, 396
Retu:uloSlS, marrow fipdirlg'S In, 519
Reticulum cells, 46
Ret1":olction 01 clot, 6$1
Rh agglutinogens, 56S
INDEX
Rhythmic delivery of leukocytes,
IZ3
Rosenbach tC$t, 252
Rosc:ola In fantum, lytllphoeytosis of,
136
Rouleaux formation, in blood matcb-
lng, 564
~!la~~thr:yt::~!!ntatiOD' 6<43 ~
reticuloc1tU ln, 96
Rwnpel-Leedc test. 636
Sabin~s theory of cell odrin. 45
Sahlt method for hemoglobin. 605
Saponi.n, hemolytic anemla from,
Saturahon index, 630
Scarlet fe"Ve.r, eo5l1lOphiita af, 141
purpura m, 461
S.cbllhng classwcatioo, of celb, 38,
69, 130, 623
Sc.honle1n''S purpura, 4-%
Scurvy, anenua of. 214
Secondary atl!;lllia liver fracUon,
22.
Secondary aplastic •anemia, 314
SedunentatioD rate, 642
Sedorlmd producIDg purpura, 443
SC&1Dented neutrophd, morphology
of,56.
Serologic tests In infectious mono-
nucleosis. 487
Serum, of bIoed,' 113
Sickness,. hemophtle test in, 487
spreads of bone manow, 656
Sex, relaban of anesula to, 165
Sheep cell test, for infectious mono-
nudeosi9. -485
technic for. 662
ShIft to the lcf~ 38
Shock, blood plasma 10, 572
Sura-leal. hemoconcentration in,
«8
Sickle cell anemia, 325
in cll.lldbood, 343
laboratorl findIngs in, 329
pathology of, 330
prognosb of, 3J1
treatment of, 331
Sickle cells, techruc for, 676
SickleDna, 325
Stckhng, demonstration of, 676
trait, fa"tors producing, 326
Siderosis ill Sickle cell anenua. 330
Skin, color of, and anemia, 203
diseases. eos.inophiila of, 141
mfiltratton in monocytic lenke-
IllJa, 400
lesions in Hodgkin', disease,. S51
In rnfeGhoUS mononucleOSIS, 477
in leukemIa, 3c3a
Sme.ars of blood, preparatiol1 of,
61S
Smith rDethod for prothromhil1, 639
Smudge ceDs, 83
In leukemia, 382
~ Snake venom, actIOn (lin red cells,
238
ID hemophilia, 4$3
in non-thrombope:nlC purpura, 459
Sparkman', method {or urobilino-
gen, 649
Spectroscopic examination of blood,
67<
Spherocytosis, experimental produc-
tion of, 244, 261
in hemolytic jaundu:e~ 260
from s~h, 263
Spider bite. eosinophilia of, 141
Spleen, in blood destrnction. 161
effect on red cells, 94
extracts frGDl., 441
in histoplaamosis, 553
In Hodgkm'a disease, 5.f9
in infectious mononucleosis, 417
in leukemia, 366
in lym~h:atic leukemia, 3S1
ID malaria, 536
in monocytie leukemia, 397, 4-00
in myelogenous leukemia. 371
in polycythemia vera, 492
'in sloCkl.e cell atieDIia, 321, 33G
in thrO!l1boeytopenic purpura, 441
Splenectomy, In acq\lired hemolytIC;
anemia, 245
in Band's d1sease, 3$6
effects of, 261
{raSlhty of cells after. 265
for hemolytiC Jaundice, 265
indications for, 268
monocytosis after. 138, 139
in polycythemIa vera, 494
rauonate for, 161
results of, 265, 268
apherocytosl.! after, 261, 265
in thrombocyt.openic purpUJ"a, 444
Splente, anemia, 356
indes in malaria, 536
Splenomegaly In children, 3S8
Spray radiation. for leukenua, 416
in polycythetnia, 0495
Sprue, 301
Staining, of blood smears, 618
of reticulocytes, 96
by supravital method, 658
Stauda!'ds for nort1\at htQOd, 116
Status iymphatleus, Iymphoc:ytosi,
of. 136
Steatorrbea~ macrocytic anemia in.
306
Ster~e ah5c::eas, 126
In agranulocytosis. 157
productJoll of, 126
Stentat puncture, 509
technic for, 651
Sternberg cells. 549
Stippling, basophilic, 96
in lead pOl.soning, 273
Stomach, absorpouon of i.ron in, 169,
218
in perniCIOUS anemia. 279
in polycythemia, 491 ~
preparations, development of, 288
removal, anettlla from, 175
Stomatitis In monocytic leukemia,
400
Stc.rage of hlood, 568
Subleukemia, 414
Sulfanii1amide, hem01ytic anemia
from. 241
Sulfapyridine, hemolytic anemia
from, 243
Sulfathiazole, hemolytic' anemia
from, 243
produetng purpura, 443
Sulfhemoglobin, 197
Sulfonamide drugs, cyanosis from,
196
hemolytic anemia from, 241
leukocytOSISfrom, 151:
leukopenia from. 151
methemoglobin from, 196
neutropen~ from~ 149

SWlhght, effect on blood, 148, 496
,.3
Supravital stainlng. of leuk()C)'tcs,
technic f.or, 658
Sweet clover disease, 463
SymptolUatie erythrocytosis, 496
Sypluhs, In blood donors. 580
con}l:-emla.l, aDCIIUa. of~ 351
hemoglobinuna in, 252
hemolytic anemia In, 239, 245-
;and infectlous mononucleosis, (.87
lymphocytosis of, 133
plasma cdl! in, 83
Syrmges, stuck. 599
T in acute leuke:rnia, .192
Tallqrust seale for lIemogloblo, &04
Tapeworm, disease, eosinophilia of.
141
.fish, anemia of, 207
Techmc for, autaheman:1utination.
.r3
basopbilic aggregation test. 673
bJl~ m serum, 646
bleeding time, 636
blood examination, 115
for blood crouping, 560
calCium time. 640
capillary resistance test. 636
dot retraction tlma, 637
coairUlahon time, 634
color mdex, 612
conce!ltrattng leukocytes, 617
differential counts, 622
enumerahon of platelets, 631
:fiJarnent~nontilament count, 62f
finger puncture, 597
fragility test, 640
fresh blood eumination, 615
Gordon test, 666
halometer readings, 628
hemoglobin determmallon, 604
.heterophile antibody test, 662
malana -examinations, 660
mean corI)Uscular~ hemoglobin. 629
concentratIon, 629
volume, 629
.2.
measurement of wI dlamder,
obtaining blood. bom adults, S97
from infants, 599
serum, 602:
Price-Jones curve, 6Z6
prothrombin time b7 Howtll's
method, 637
by Kato'. method. (;39
by Quick's tnethod, 638
by Smith's method. 639
reticulocyte counts, 630
saturation index, tiJO
Schlllmgs count, 623
sedimentation rate, 642
by micromethod, 645
by Westergren's method, 645
by Wintrohe method, 643
.,.
sickle cdIs. 676
spectroscopic blood exammation,
st.ainill8' blood smt'ars. 618
s~rna1 punchlr~, 651
supravital staining, 658
trtphine of marrow, tiS3
total proteins, 667
urobilinogen in feeel, 649
in urine, 650
Van den Bergh reaction, 647
venepuncture. 598
INDEX
Technic for-(Cot"in.ued)
viiCOSlmetry, 672
volume inriex, 625
Technic. hetnatofogic. 596
Temperature, m hetpatopoiulS, 184,
50S
effect on leukaeytes, 124
Tcnninolog,. of, blood diseases, 3,
Osgood, 14
Tertian malana. 527
...
Thick smear fot' malaria, 661
Thorium dioxide. effect on blood,
Thrombobluts, 98
Thrombocyte., I} 7
after henzene poisotung, 316
in blood ClottilZ6, 434
counting of, 97, 631
forma.bon of. 507
In hemophilia. 449-
in infectious mononucleosis, 4S0
in leukemia. 99
marrow threshold for. 438
maturation of, 51
in monocytic leukemia, 400
in newborn, 117
In non-thrombopt:nic purpura, 458
Bonnal figure!! for. 117
occurrenee of, 99
ongm of, 502
ID pel'm(llOUS anemia, 286
in stored blood, 569
in thrombocyt()peDic purpura, 441
in various disease., 98
Tbrombocytopenia, m agranulocyto-
sis,ISS
from radlum. 318
Thrombocytopenic purpura, 440
blood findings in, '442
diagnosis of, 442
drugs caUsing, 443
mcidence and etiology. 440
marrow findings tn, 51S
splenectomy in, 167, 268
symptoms and physical findings,
«,
treatment of, 443
Thrombon, 1)3
Thromboplastin, In blood clotting,
."
in hemophilia, 449
for prothrombin time, 638
Thyroid, drsease of, anemia of,
343
disorders, leukopen.ia of. 152
secretion in anemias, 181
Thyroidectomy tn leukemia, 375
Tissue, deltruction of, effect on
neutrophlls, 126
e:;l(tract". JeuJwcyt051,s of, 121
Tohson's fluid, 609
Transfuiion. blood, 556
in agranulocytosis, 157
In anemia. 217
of bloOd plasma, 570
Transitional cells, 60
Trauma, retatlOD of leukemia, 364
Tray for hematologic work. 598
Treatment of, agranulocytosis, 156
anemia, 192
cOPJler In. 171
In Children, 342
of pregnancy, 211
rcsponlll"l With iron, 225
benzene poisoning, 316
erythroblastic anemia, 355
hemolytic anemla of kderer, 245
hemol)1:lc jaundice. 264
Treatment of-(COfftintml)
hemophilia, 452
hemorrhagiC di!iieale of newborn,
•••
blstoplastbosis, 554
HodgklD'. disease, 552
hypochromlc anemias, 215
idiopathic hYl;oOChromic anemia.
233
infedioua mononucleosis, 487
leukemia, 413
malana, 539
perniciollS a.JlemJ3, 288. 293
polycythemia vera, 494
sickle cell -an(!mia, 331
von. ]aqch'. anemia, 351
Trephine of marrow, 508
Trialists' tbeory of eell origin, 44
Trlchmiasis, eosinopbilta of, 141
Tropical, anemia, of pregnancy. 305
macrocytic anemias, 179, 308
'rubercn1osis, hemolybc anemia in~
245
leukopenia of, 146, 152
lymphocyte-monocyte ratio in, 78
lyttlphocytosi. of, 133, 137
relation to Hodgkin', disease. 547
Tumors, anemia of, 206
leukocytosis Qf, 126
marrow JindUlgs in, 520
Turk cell., morphology of, 73
in varlQUI diseases. 82
Types of blood, 565
Typing of blood, 560
TyphOid {ever, lymphocytosis cf,
133
mODocytosi!l in, 138
Typhus fever, leukopenia of, 146
lymphoc:ytosi, of, 133
u
UndIfferentiated cells, 501
Undulant fever, leukopenia of, 146
lymphocytosis of, 133
Universal donor for transfusions,
55'
Urine, h~matopoletie factor in, 175
hemogloblD in, 248
in malaria, 537
in pelT'licloul anemia, 28.6
porphYrins in, 198
urobihnogen in, 650
Urobilin, source of, 236
Urobilinogen, in feces, technie for.
•••
source of, 237
in urine, technic:: for, 650
Uroporphyrin, I. 200
V
Vaecination, lymph.ocytoais cf, 136
Van den Bergh reaction, 647
.,
Vasomotor control of bone marrow.
Venepundure, technic for. 598
Venesection In polycythemia, 494
Ventriculin, development of, 288
Venous blood for examinahon, IlS
Virus, relati(ln to Hodgkin's dis-
ease, 548
Viscosimetry, 672 ~
Viscosity of blood. 672
In polycythemia, 493
Vital stalDint of leukocytes, 103
Vitamin, B, in ~l1agn., 302
deiiciency, in pregnancy, 303
B1 in herpe., 429

Vltamin-(Conl","ed)
B, w hematopoiesis. 180
C in ant-mta.s, 22<4
deficiellcT, anemia of, 213, 343
effect tin leukocyte" 121
in hematopoiesl:11. 181
In leukt;mia, 420
In llon thromoopetlic purpura,
456.459
in thrombocytopepic purpnra,
.OS
G deficienc,", lc"kcpeni.a ham,
148
K, 463
deficiencies, 465
(hstnbution of, 463
in hemorrhage of Ilewborn, "61
in leukemia. 420
me.chattism of action.,. 464
preparations of, 467
INDEX
Volume iQdtx, technic {or, 625
Volume. af blood, 114
1D polycythemia vera, 493
bone lll&rrow, 503
of tlack~ .::ens. 116
von Jaksch', anemta. 349
w x
Weight, of blood, 114
mo]ecuhl.t, of hemoglobin, tOl
Westergret1's method for sedimen-
tation, 645
Whipple liver fraction, 224
Whlte blood cells, see Leuko-
cyte.
WbOOQing cou~b, Iymvhocytosis of,
.,6
Winlr~. sedimentattcn m~tboo.
643
hernatot:rit metllod, 626
Women, erythrocytes in, 116
hypochromic anemia In, 2JQ
\Vrlght'. stam for blood solears,
619
X_ray trea.tment. in agranulocytosis.
/let! Radiation
X-ray.s, effect on blood. 147. 318
y
Yeast. In :Illacrocytic .nerola. 179
in 'tlC.rtlI.c.lOU'l anemia,. 19Q~ 2.96.
role in hematopoiesis, 180
. . . . . III IIIIII I 1111' , I , , " , " , , " , , , " " , , , Lilli ,It" " " t

The Clinical Significance

of the Rh Factor with Comments
Cqncerning the Laboratory Problems
By
Roy R. KRACKE, M.D., and WIWAM R. PLAn, M.D.
,Epo'] Unwersity, Georgia

IN 1940, Landsteiner arid Wiener 1 discovered the existence of a nev"

blood group 'factor in man following the injection of rabbits with the blootl
of the ~Macacus rhesus !1Ionkey. The serum of the immtmized rabbitS
clumped not only the blood cells of' the Rhesus monkeys but also the blooil
of about 85 per cent' o'f hUttIGn beings irrespective 0: their blood groups; thuS
revealing the presence in man'of a newjblood factor designated as Rh, be-
cause it was first found in the Rhesus monkey.
Present knowledge of the Rh factor can be summarized as follows':
(a) it is an antigenic substance in human red b1~d cells similar in some
respects to other previously discovered antigenic factors, the mosf important
of which are those known as A, B, M and N; (b) it is inherited as a Men-
~'t.\:'<m ~=~'t\=\"""'" "",'t. 'In't \)\~...,"",~ \t) ~\ tt~-""'" \)'t\\'j ~'t\ 'In... ,'t'I> ~'h""'."tr
sembling in this respect M and N, but differing from A and B, which occur
in tissues and secretions of at least some persons; (d) the Rh agglutinogell
occurs ih about 85 per, cent of white people, in about 92 per cent of negro~,
in about 100 per cent of the Chinese' and in all Macacus rhesus monkeys;
(e) there are no normal agglutinins against the'Rh factor in man, agaill
resembling the factors M and N, whereas such agglutinins are present no(-
mally against A and B (e.g., natural isoagglutinins a and b); (f) on the
other hand, when blood containing the Rh factor (Rh~+) is introduced intO
a person without it (Rh-), agglutinins may develop against it'(called
anti-Rh agglutinins); (g) however, no anti-M or N agglutinins ever develoP
if blood containing the M or N factors is injected into persons without these
.. Read by the senior author before the Southeastern Sectional Meeting of the AmericV1
College of Physicians, Jacksonville. Florida, May 26, 1943. .
From the Department of Pathology, Emory University School of Medicine, Emory unl'
versity, G~rgia.

From ANNALS or INTERNAL MEDICINE, 20: 559-569, 1944, Copyright, 1944, by the
American ColJege of Physicians
1
 2 '1I0Y R: 'KRACKE AND WILLIAM 1I: PI!ATT

factors in their blood (i.e" M negative or ij negative recipients). "There-

fore, from the above it can be seen that the Rh factor has iso-immunizing
ability, whereas the M and N fa!=tors lack il, That is why the M a.!ld N
factors have no significance' wJth respect -to hemolytic reactions in blood
transfusions where;s the R~ f~c{or is very irpport,a,nt i~ this respect.

HEMOLYTIC REACTIONS AFTER TRANSFUSIONS,

~ ~ , ' - , ,- . "

Soon after the discovery o'f the Rh factor Wiener' and Peters' stlldied
the blood of patients who had hemolytic reactions after one or more preVious
uneventful transfusions of blood of 'the correct homologous group. The
serum of. these' patients ,contained, 'atypical isoagglutinins (anti·Rh ag-
glutinins) which clumped the -red blood cel!~ of donors containing the Rh
factor (Rh + ). These same sera, therefore, behaved in a similar W<l.y to
the sera of rabbits immunized with monkey blood (also Rh +). Therefore,
,it was ,concluded that the blood 'of these patients did not contain th~ Rh
f~~to;' (as they were Rh' ~egative), al)d, therefore, t~~ presence ,of tite atypi-
cal agglutinins (a,nti-Rh agglutinin~) found il) their serums was expl~ined
by assurping that Rh + blood w~s given'to lhem:during one or s.everal.of the
,transfusions whi~h. they had received, previ.qu~ly. The fact,~t ~hey ~itl not
pos~ess the,Rh factor in their'qwn blood ,made it,possible for the injected Rh
positive blood to act as an antigen and, therefore, to stimulate the production
of anti;Rh agglutil1ins. After the, ~ggl\ltinins~ h.a~ .developed in' thes~ pa-
tients" it. was then realized that Rh + blood from donors, for transf\lSion
purPoses was nor su'itable for theSe patie~ts even if. the donors did beloltg to
the identical ,blood group AB, A, B or O. Confirming.this concept was the
fact tru:.t whenever Wiener and Peters administered Rh + blood to these
patients, ih~y develop~d hc;molytic reactions. Sl1bs,qllent r~ports, by "ther
'authors, have confirmed theSe observations. That is, it has been found that
patients showing'hemolytic reacti~ns a.fter oi!e: or ~everal previous uneventful
transfusions of. Iilood of the correct. group AB,. A, ~ or 0 were as a rule
Rh - and that som~ _of them had anti-Rh agglutinins in, ,their blood.
Therefore, in order to prevent further hemolytic reactions following ttans-
fusion~, oply Rh- blood donors could'be usea.

PREGNANCY -AND ERYTHROBLASTOSIS

The same mechapism 'has been found'to be resP!>l)sibie for reactions alter
first transfusions given, to pregnant women, "In such instances,.h,qY'.ever, the
development of the anti-Rh agglutinin is explained by the presence of the Rh
factor'(-Rh +) in the blood of the fetus to whom it was transmitted by the
father as a,Mendelian dominant and by the Pilssage' ohhis Rh factor-through
the placenta to the mother-who was thus immunized during pregnancy
'(i:e" 'the mother developed anti-Rh' agglutinins). Bearing these' facts in
mind, Levine' suggested that erythroblastosis fetalis may be caused by a
similar mechanism, and postulated that the 'production of anti-Rh agglutinins
 CLINICAL SIGNIFICANCE OF RM FACTOR 3

in the mother is followed by placental passage of the agglutinins to the fetal

blood with subsequent hemolysis of fetal blood and the clinical state of ery-
throblastosis fetalis. Since the mother's blood contains the anti-Rh ag-
glutinins it reacts with. Rh +blood cells of the fetus and destroys them.
The correctness of this hypothesis is based upon at least four conditions:
I. The father must have the Rh factor as a Mendelian dominant factor
(Rh +) in his blood in order to transmit it to the fetus.
2. The fetus must have the inherited Rh factor in his blood (Rh +).
3. The mother must lack the Rh factor (Rh - ) in order to develop anti-
Rh agglutinins in response to the passage of the Rh factor from the
fetus through the placenta.
4. There must be a' free exchange of antigenic substances and of anti-
bodies through the placenta from the fetus to the mother and there
must be a mechanism whereby Rh +
fetal cetts can escape into the
Rh - maternal circulation. This mechanism probably is placental
injury. The fulfillment oHhe fourth of these conditions can be taken
for granted. The passage of protein substances from the fetus to
'the mother and of antibodies from the mother to the fetus has been
previously satisfactorily demonstrated. ."
The only s:lfe way to transfuse an Rh - person who h:lS received
previous transfusions, or an Rh - pregnant woman, or an infant with ery-
throblastosis, is to use known Rh - blood. An important problem is where
such blood can be obtained, arid it must be obtained from donors who are
known to be Rh negative. For this purpose an Rh - donor pool should be
established by determining the Rh negativity' or positivity of a large group of
people. The technic used for·that purpose is described later in this paper.
The following four patients were observed in a period of six ·weeks in
the Emory University Hospital, all presenting different' types of Rh problems.
The obseryal}ce. of this number in such a short time illustrates forcibly the
necessity for careful consideration of the Rh problem in clinical medicine.

CASE REPORTS

Case 1. Isoimmuni.:ration dwing Pregnancy.and It.: Role in ErylhroblasloSis

Fetalis: This patient was a two day old, severely jaundiced, eight-pound, white, male
infant who was delivered spontaneously at full t<fm on March 8, 1943. The mother
was a 28 year old mUltipara ,and had two children in perfect health, aged one and
three years respectively. In her two previous pregnancies she had an uneventful
prenatal, parturition, and puerperal course. Furthermore the prenatal course of this
third pregnancy was also normal. Examination of the placenta at the time of
delivery ~howed several small infarcts. Physical examination of the baby revealed a
well developed, two day old, white male with a spleen that was palpable 4 em. below
the costal margin and the liver enlarged 2 cm. below the right costal margin. No
enlarged lymph nodes were palpable. Blood examination showed: Red blood cells
2,350,000 per cu.mm.; hemoglobin 52 per cent; white blood cells 18,420 per cu.mm.;
(neutrophiIes 60, lymphocytes 39, and monocytes 1) The stained smear' showed
 ROY ll. KRACKE AND WILLIAM R. PLATT

marked variation in size, shape, and color of the red cells with many macrocytes and
microcytes. In addition a large number of immature red blood cells· were seen con~
sisting chiefly of normoblasts. Based upon these essentiaL criteria of severe jaundice,
m!arged liver and spleen, severe anemia with the presence of large numbers of nu-
cleated red cells, the diagnosis of erythroblastosis fetalis was considered to be estab-
lished. Because of the marked anemia which was caused by excessive hemolysis of
the fetal red cells, the infant was then given 50 c.e. of maternal blood, using 25 c c.
in each buttock intramuscularly. No improvement was noted in a 24-hour period and
then 100 C.c. of the father's blood Were given i~travenously. A subsequent blood count
revealed a red cell count of 2,100,000 per cu.mm., hemoglobin 42 per cent, and a COn-
siderable increase in the number of immature red celts in the blood. It was at this
time that Rh determinations were made and these revealed the father to be Rh +, the
mother Rh - , and the infant Rh +. Determination of the titer of anti~Rh agglutinins
in the mother was then made and found to be 1: 1024. All three members of this
family were type O. Two subsequent transfusions of 100 c.c. each from Rh - Type
o donors were entirely successful and the patient's red cells and hemoglobin rose to
normal limits with a disappearance of immature red cells and gradual reduction in
icterus, with decrease in size of the liver and spleen during a period of 10 days after
delivery. This case illustrates the fairly typical findings in an infant with erythro-
blastosis fetalis, born of a multiparous mother-the immunizing antigen or Rh +
fetal red cells entering the maternal circulation through a placental defect and pro-
ducing isoantibodies in the Rh - maternal blood. These in turn, since they are
sol uble products, were passed batk into the fetus in sufficient titer to cause severe
hemolysis of the fetal red blood cells leading to erythroblastosis, jaundice, hepato-
megaly, and splenomegaly. Also, this case illustrates why the mother's blood or the
father's blood is contraindicated for transfusion purposes into erythroblastotic infants
and why a donor list of Rh - donors should be available for such emergencies. It
should again be emphasized that neither the father's nor mother's blood is satisfactory
for transfusion into an erythroblastotic infant, although this is exactly the practice
that is followed in well over 90 per cent of such cases simply because of availability
of the blood. It is obvious that transfusion of the mother's blood simply confers upon
the infant more anti-Rh agglutinins, or the transfusion of the father's blood confers
more Rh + cdls to be hemolyzed. by tht! anti-Rh agglutinins that are already present.
Case 2. A Transfu.rion Accident Resulting from Anti-Rh Immunization during
Pregnancy: This patient, who was a 2S year old primigravida, was delivered on April
24, 1943, in the Emory University Hospital of a full term, normal white, male infant
During the course of delivery the patient sustained a lateral rupture of the placenta 5
em. from its margin which resulted in excessive hemorrhages during the remainder
of parturition. Since the patient was Type 0 a male donor of the same group was
obtained. When the crossmatc:h was set u'p at room temperature there was no ev idence
of agglutination. TIlerefore, a transfusion of 500 c.c. of blood was begun. After
nearly 200 c.c. had been given, the patient developed a severe shaking chill, headache,
elevated temperature, a rapid pulse rate, and severe pain in the flanks. Transfusion
was discontinued. Adrenalin was administered and heat applied, and these symptoms
subsided. in the course of one to two hours. The crossmatch was then again set up
and the blood was found to be wholly compatible. At that time, examination of the
mother's blood revealed that she was Rh - and in addition showed a titer of I :16 of
anti-Rh agglutinins whereas the blood of the donor was Rh + and also that of the
infant was Rh +. Following this the patient was given a transfusion of Rh - Type
o citrated whole blood without any reaction whatever.
It is characteristic that this type of hemolytic transfusion accident occurs
at the first transfusion of the pregnant woman or the woman who 'bas re-
 CUNICAL SIGNIFICANCE OF RH FACTOR

cently delivered. :rhese accidents, of course, are of great significance to the

obstetrician and gynecologist because ·of the relative -frequency with which
transfusion difficulties have always occurred in pregnant women in spite of
the fact that patient and donor may be of the same blood group and the blood
entirely compatible. It should be borne in mind that a pregnant woman !lIay
develop a rather substantial titer of anti-Rh agglutinins and at the same time
the titer may not be sufficient or they may not gain access to the fetal 'Cir-
culation in such amounts as to produce marked hemolysis of fetal red cells
and consequently the clinical picture of erythroblastosis fetalis. In this Case.
for example, there was no evidence of erythroblastosis in the newborn inf""t.
yet the mother showed a titer of 1: 16 of anti-Rh agglutinins, which, al-
though not sufficient to dest~oy fetal red cells, was quite sufficient to give a
marked trans fusion reaction.
Case 3. Immunization after Multiple Blood Transfu.ions: This patient was a
58 year old white female admitted to the Emory University HoS-pital on April 6.
1943, with an established diagnosis of aplastic anemia. Her past history reve"led
nothing remarkable until one year prior to admission when she experienced her
first evidence of gradual progressive weakness and fatigue. At that time a thorough
physical examination was negative except for slightly reduced hemoglobin and rfd
ceJls. Four months before admission, she noticed bruises on her lower e..'Xtrem1.ties
which were spontaneous purpuric spots. At that time she was hospitalized and her
blood showed the following: Red blood cells 1,200,000 per CU.mm ; hemoglobin 30 per
cent j white blood cells 2,600 per cu.mm., with a predominance of lymphocytes. Duting
this hospital stay which was in North Carolina, the patient recehed six transiusions,
one in February, four in March, and one during April. Careful questioning reye~led
that the first two transfusions were uneventful but that each succeeding one prodQced
a more severe reaction, including a shaking chilJ, elevated temperature, rapid pulse, and
pain in the flanks, and the last one produced, in addition, nausea and 'vomiting. On
admission to the Emory Hospital she presented a marked pallor of the skin and rnuc:ous
membranes with many diffuse and patchy ecchymotic areas over the entire bc)dy.
Her red cell count waS 2,000,000 per cu.mm. j hemoglobin 35 per cent, 57 gm.;
white cells 1,350 per cU.mm. with 85 per cent lymphocytes; reticulocytes 0.1 per ctnt;
and no blood platelets were seen on the stained smear. Because of the fact that she
had been given multiple transfusions with reactions of increasing severity. the patient,
who was a Type A, was tested for Rh factor. She waS found to be Rh - and ais() to
have a titer of anti-Rh agglutinins of 1: 64. She was then transfused from our
permanent Rh - donor list, the typing and crossmatching being carried out at room.
incubator, and refrigerator temperatures. Five hundred c.c. of blood were then
given without reaction. It is of interest that in this particular transfusion this blood
was administered by way of the sternal marrow since the patient·s \ eins were not
in suitable condition for the administration of blood. After this one other Rh-
transfusion was given but no further blood was administered after this time since we
felt that her prognosis was hopeless. This patient succumbed to her disease, aplastic
anemia, on the twenty~sixth hospital day, the red cell count having reached the low
level of 800,000 per CU.mm. at that time. This case demonstrates the productiol\ 01
anti~Rh agglutinins in an Rh - patient by means of multip1e transfusions of Rh +
donors of homologous blood groups. It serves to illustrate a principle which must be
emphasized, that is, Rh determinations should be done on aU medical a.nd surgical COlses
who are to receive or who have had more than one transfusion.
6 ROY R. lCRACKE AND WILLIAM R. PLATT

Case 4. The Developmen, of Anti-Ris Agglutinins as a S"'I1;Ca) Problem: ~is

patient was a 10 year old white female who was admitted to the Emory UniversIty
Hospital on March 14, 1943, with a history of progressive weakness, frontal headaches,
malaise, slow loss of weight, and pain in the left upper abdominal quadrant. Ex-
amination of this patient revealed a marked pallor of the skin and mucous membraoesf
a slight brownish skin discoloration, a visible superficial network of veins over the
chest and abdominal waU, numerous ecchymotic areas over the trunk and IQwer extre-
mities, and a marked enlargement of the spleen. The patient had known of the pres-
ence of this large hard mass in her upper left abdomen for a period of six years prior
to admission. A tentative diagnosis of Banti's syndrome was made and after this
tests' for liver function were carried out using the (bromsulfthalein) dye test, hippllric
acid synthesis test, and the cephalin' cholesterol test. Liver function was found te> be
normal, and the liver was not enlarged. Examination of the blood showed a hel"D-
globin of 40 per cent; red cells 2,500,000 per cu.mm.; and a leukopenia of 2,zOO ~1s
per ell.mm., with an essentially normal differential count. Based upon the presenc~ of
an enlarged spleen of presumably six years' duration, the rather marked anemia, the
presence of leukopenia, and a well-developed collateral circulation over the tronl', a
diagnosis of Banti's syndrome was made. It was then decided to remove the spl~en.
and because of her low hemoglobin and red cells it was considered necessary to "re-
pare her for splenectomy by two or more blood transfusions. .
The patient was the mother of three, living, healthy children, the last child Mmg
eight months old, and since she was only eight mon;hs postpartum it was decidec:l to
test her for Rh factor. She was lound to be Rh - and in addition to this, mud' to
our astonishment, she showed a titer 01 anti-Rh aggltltinins of 1: 1024. It was jelt,
+
of course, that it would be unwise to transfuse her with Rh blood. Whereupon she
was transfused with two Rh - transfusions without reaction and aftet this spler.ec-,
tomy was successfully performed, from ,which she made an uneventful recovery. At
this time the blood findings of this patient are entirely normal, the leukocyte count,
being 10,000 cells per cu.mm. The spleen wa•. characteristic 01 Banti's syndrome, that
is to say, it was an organ characterized by e.."<:cessive fibrosis. This case pres~;s,
an example of' the value of prophylactic measures to avoid transfusion reacti,m•.
An astonishing feature of this patient was t!;e fact tl:at she ca,ried such a high tite{ of
anti-Rh agglutinins which pres)l!Dably resulted from her last pregnancy which oc-
curred eight months before. We can aniy speculate as to what might have happened
+
to this patient if she had been given a transfusion of Rh blood.

TECHNIC OF PREPARATION OF REAGENTS AND THE DETAILS OF THE

TEST FOR THE RH FACTOR -

Since the advent of the Rh problem in clinical medicine and general

recognition of its importance, a justifiable demand has been made on
pathologists and other laboratory personnel to aid in the solution of these
problems as they arise in clinical practice. Thus, there must be available
materials and methods for determining Rh negativity and positivity; the
presence and titer of anti-Rh agglutinins in patients, and methods for pre-
transfusion crossmatching of blood that will detect rncompatibilities not only
of major blood grolips but of Rh faA:tor as well The following is an at-
tempt to summarize the procedures involved in these problems.
Before one can begin to determine the Rh positivity and negativity of a
given number of donors, there must be available a suitable. quantity ot a
 CLINICAL SIGNIFICANCE OF RH FACTOR 1
known serum containing the specific anti-Rh agglutinins. Such serum may
be obtained from three sources as follows:
1. By Purchase: Small amounts of Rh negative serum containing anti-Rh
agglutinins can be obtained from the Certified Blood Donor Service,
146 Hillside Avenue, Jamaica, New York at cost of approximately $5.00
per c.c. Then, by using the following technic with the above anti-Rh
serum, one can obtain a smallli.t of known Rh negative and positive donors:
"Prepare a fresh saline suspension (2 per cent) of the red blood cells to
be tested. Before proceeding with the test examine a drop of this suspension
microscopically to make sure that there is not peculiar agglutination or hemo-
lysis of red bloOd cells. Then using a culture tube of 3 X 3%", place on the
bottom of the tu»e one good sized drop of th~ fresh saline suspension pre-
pared and tested as above. This drop should be approximately .05 c.e.
Carefully place into the same tube 1 large drop of the known anti-Rh serum.
This drop should be as close as possible in size to that of the cell suspension
already in the bottom of the tube. If smaller amounts of serum and cell sus-
pension are used the result will be difficult to read. If larger amounts are
used, there will be an unnecessary waste of anti-Rh serum. Then shake very
lightly. Place in a water bath at 37° C., for I to 1% hours. Centrifuge at
SOO RPM for one minute and observe sedimented blood. Resuspend gently
-'Very gently. Read the result at the bottom of the tube macroscopically for
clumping. Where clumping is evident, theTesult is Rh positive (very rarely
a minor degree of clumping is seen due to anti-M isoagglutinins). Those
that seem to be negative macroscopically are then examined under the micro-
scope (low power). Those that show no clumping are now definitely Rh
negative. A small drop may be easily removed from the test tube by means
of a small glass rod or a plajinum loop. This drop may be placed on a micro-
scope slide and examined microscopically. Whenever doubt arises, check
again the original- suspension of unknown red blood cells, and start with a
definite unagglutinated suspension of red cells. This rules out pseudo and
cold or autoagglutinins."
. 2. Olltaini1lg Large Amoullts of SeTl,"' from Erythroblastotic Mothers:
The most reliable source of serum. is from women who have been immunized
during pregnancy or who have given birth to infants with clinical erythro-
blastosis fetalis. The Rh factor in the fetus is responsible for the iso-im-
munization ·in the great majority of cases, but other blood factors may also
immunize. In order to determine whether the mother's serum contains any
anti-Rh agglutinins (since in at least 50 per cent of the cases there are no
demonstrable antibodies of the Rh type'), the following is thought to be a
satisfactory working procedure:
Remove about 10 c.c. of blood from the patient and recover the serum.
First remove the isoagglutinins a and b present in the serum of group A, B
and 0 patients. This is done to prevent the interaction between the
8 ROY R. KRACKE AND WILLIAM R. PLATT

"natural" isoagglutinins of the serum, being tested and the agglutinogens in

the red cells containing agglutinogen A of Group A red cells and agglutinogen
B of Groups B red cells. According to Witebsky and his associates; the
addition of the recently isolated group-specific substances A and B leads to a
marked reduction or elimination of the isoagglutinins anti-A (a) and anti-B
(b) present in the specific blood groups Or present together in the serum of
so-called universal blood of Group 0.* For this purpose, it has been found
that the addition of approximately 1-2 c.c. of the AB containing solution
(prepared by Eli Lilly and available for experimental purposes only) to 5 c.c.
of the patient's serum is sufficient to neutralize the natural iso-agglutinins
"and b.
The serum must then be tested for neutralization of these isoagglutinins
and in order (0 do this the following titration,agglutination setup is thought
to be adequate. Serial dilutions of serum in decreasing amount (volume
0.2 c.c.) are mixed with 0.2 C.c. each of a 1 per cent suspension of human
red blood cells belonging to group A and group B respectively. After
standing for 30 minutes at room temperature, the tubes are centri fuged for
about one minute. The resulting agglutination is recorded as in the follow-
ing table:

P .... , Part 2
Before Addition of After Addition of
Group SpCC. Su b. Gro<Jp Spec=. Sl1b.
TuboeNo. Dilution

Group A Group B A B
Cells C,db Cells Ct'lI.s

1 Undiluted ++++ ++++ ++ +

2
3
1: 2
1 :4
++++
++++
++++
++++
+
-
--
4 1: 8 ++++ +++ - -
+++ +++
--- ---
5 1: 16
6 1: 32 ++ ++
7 I: 64 ++ +
8
9
1: 128
1: 256
+
+
+
-- -
--- --
10
11
12
I: 512
1: 1024
Saline control
-
-
- -- - ---
Note: If there is sull insufficient neutralization, I or 2 c.c. more of the Witebsky AB solu·
tiOD caDbe added. .

Having determined that complete neutralization of the natural agglutinins

has occurred, it is then necessary to determine the specificity of the anti-Rh
• Convenient sources for the isolation of group specific substance A are commerci2.1
pePSin. mucin and peptone. The addition of 25 mg. of specific substance A to SOO c.C. group
o blood reduces the titer of the isoagglutinin anti·A considerably or at times even com ..
pletely. The B substance was finany liwlated from the gastric juice of human ~in~ be ..
longing to group B. AI: 1,000 stock solution of group specific substances is prepared.
25 c:..e of stock solution of A substance a.nd 10 c e. of stock solution of B sub!!itance are kept
in vaccine bottles under stenle conditions and added to Soo c.e. of citrated blood five minutes
prerious to administtation of blood, i.e.• if one is to use universal donor blood safely (Group
0).
 CLINICAL SIGNIFICANCE OF RH FACTOR 9
serum obtained from the patient by determining not only the presence of the
anti-Rh agglutinins but their strength or titer as well. This should be done
according to the following table, using known Rh +
Cells, Group 0, since no
agglutinogens are present in that group.
TIle<
1:2 Normal saline (1aru.e drGP) +
large drop of anti-Rh serum - - very gently shake-Bi!!1'e:lr~:: sO~;~n~~~
1 :4 Large drop nonnal saline +
large drop of no. 1 mixture - - -
1:8 4rge drop normal saline +
large drop no. 2 mixture
I: 16 Large drop normal &aline +
large drop no. 3 mixture
1: 32 4ige drop normal &aline +
large drop no. 4 mixture
I: 64 Large drop normal &aline +
large drop no. 5 mixtuJ"e
1 : 128 Large drop normal &aline +
!arg:e drop no. 6 mixture
1 : 256 4rge drop normal saline +
large drop no.' 7 mixture
1 : 512 Large drop normal saline +
large drop no. 8 mixture
1 : 1024 Large drop normal saline +
brge drop no. 9 mixture
1 : 2048 Large drop normal saline +
large drop no. 10 mixture • remove
and discard one drop,
Salme control-Large drop of normal saline + large drop of 0.2% RBe saline SUspertSlon.
Shake all tubes very gently-place in water bath for 1 to 1% hours at
37° C. Centrifuge for one minute at 500 RPM and observe sedimented
blood. Resuspend gently, ,very gently (count the number of shakes required
to resuspend the tube number 11 and use the same number of shakes to
resuspend the others). Read the results under microscope (low power).
The highest dilution at which agglutination occurs is the titer of the anti-Rh
serum. It is preferable to use a serum with a titer of 1; 128 or above; there-
fore, for economical purposes it may be desirable to dilute strong titer solu-
tions to this strength, but on the other hand, this may not be wise since the
anti-Rh factor is lost rapidly and usually lasts no longer than three months.
The preservation of anti-Rh serum sometimes is rather difficult. It us-
ually does not keep well in fluid state, since there is a gradual loss of potency,
and should be kept either frozen or dried if the facilities are present. Since
desiccation facilities are usually not available, there has been the constant
problem of replenishing the laboratory supply of potent serum. This can
be done only by constant lookout for new cases of erythroblastosis and at-
tempts to obtain suitable serum from the mothers. When a large obstetrical
service is available, this can be done, but even so there is the constant problem
of being uncertain as to the titer of any serum in current use, and frequent
titrations against known Rh +
cells must be carried out. At the present
time we are testing serum that was air-dried, using one drop on a cover glass,
an_? after two months the potency seems unimpaired. If .such air·dried
10 BOY R. KRACKE AND WILLIAM R. PUTT

serum proves satisfactory after passage of sufficient time, this would promise
a solution of this problem.
After having determined that the serum contains a sufficiently high titer,
of anti-Rh agglutinins, the patient can be bled for a larger amount such as
100 to 200 c.co for sted< purposes. The agglutinins in the ftuid serum
should remain potent for about three months, although this is variable. The
serum, of course, should be used as early as possible to establish a large pool
of Rh - donors, using the technic as outlined previously. Anti-Rh serum
can also be obtained from Rh - patients who have developed the antibodies
from repeated transfusions of Rh + blood, but that from erythro-
blastotic mothers appears to be more satisfactory because of higher titers.
Anti-Rh serum may also be produced in laboratory animals by repeated
injections of washed cells from the Macacus rhesus monkey, but at this time
such sera are not reliable because of antigenic non-specificity.

THE MODIFIED COMPATIBIUTY TEST

In situations in which Rh - donors are'not available and an Rh problem

may be suspected, a modified type of cross matching, as ,recommended by
Levine, should be done. This is known as the modified compatibility test
and is carried ,out as follows:
An equal mixture of the patient's serum and prospective donor's cells is
incubated in a slriall tube in a water bath for 15 to 30 minutes at 37· C. This
mixture is then centrifuged at 500 RPM for one minute and the sediment
resuspended is examined microscopically for the presence or absence of ag-
glutination. In pregnancies of all types, for repeated transfusions, and in
erythroblastotic infants, this Levine compatibility test should be done
routinely.

SUMMARY

1. A summary of existing knowledge cOl1cerning the Rh factor is

presented.
2. Four cases illustrating variable Rh problems are presented.
3. Methods for obtaining and preparing anti-Rh serum are described in
detail.
4. The importance of having donors ready and classified for the Rh
factor is obvious; in fact, many workers in this field use Rh.negative. donors
routinely for postpartum patients and patients having transfusion reactions
of any kind unless there is time to study the case thoroughly and the Rh
factor'as a source of danger can be excluded.
5. Sera for testing for the Rh factor can be obtained (1) by purchase,
(2) from patients who have had transfusion reactions caused by the Rh
factor, (3) and from the mothers of erythroblastotic infants.
 CUNICAL SIGNIFICANCE OF RH FACTOR 11
6. The agglutination reactions with anti-Rh sera, even those 0 f high
titer, are much weaker than those of ordinary blood group sera and may hot
show up at all by the ordinary slide technic.
7. The modified compatibility test should be used in all instances in which
the Rh disturbances may be suspected.

BIBLIOGRAPHY
t. LANOSTEINEJt, te., and WIENER, A. S.: An agglutinable factor in llUtnan blood recognized
by immune sera for rhesus blood. Proc. Soc. Exper. BioI. and Med.. 1940, xlii~ 223.
2. DAVIDSOHN. I., and TOBARSKY, B.: The Rh blood factor, an antigenic analysis, Am. Jr.
Clin. Path., 1942, xi~ 434.
3. LEVI NE, p" and WONG. H.: The incidence of the Rh factor and erythroblastosis febUs
in Chinese, Am. Jr. Obst. and Gynee., 1943, xlv, 832.
-4. WIENER, A. S., and PETERS, H. R.: Hemolytic reactions following transfusions -of blood
or the homologous groups, with three cases in which the same agglutinogen was
responsible, ANN. INT. MED., 1940, xlii t 2306.
5.. LKVINE, P., BURNHAM, L, KATZIN', E. M_, and VOGEL,. P.: The role of isoimmtmization
in tIlt: pathogenesis of erythroblastosis fdalis, Am. Jr. Obst. and Gynec .. 1941, xlii, 925.
6. MOLCNEY, W. c.: Personal communication. March 31, 1943.
7. \VITEBSKY. E.: Prepa.ration and transfusion of safe universal blood. Jr. Am. Med. Assoc..
1941, cxvi, 2654.
8. WIENER, A. S., SILVERMAN, I. ]., and ARO~SON. \V.: Hemolytic transfusion reactiotJ.s-
prevention with ~~cial reference to a new biological test, Am. Jr. Oin. Path., 1942,
xii. 241.