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CLINICAL INTERPRETATION. of LABORATORY TESTS Fifth Edition Preface to the Fifth Edition There have been numerous changes and advances in clinical pathology smce the last edition of this book appeared 1 have attempted to evaluate these changes and adyances and to incorporate those that appear to be both pracucal and valuable mto the text At the same ume I have removed tests that have been superseded by newer and better methods I hope that this has resudted in a book that will be a useful reference for physicians, medical students, and technologists ‘The advances in enzyme chemistry warranted a separate chapter for this subject. The use of radioactive isotopes, parucularly in the diagnosis of thyroid disease, resulted in the addition of much new material Discussion of abnormal hemoglobm and newer concepts of the blood-clotung mechanism were also imcluded Blood groups and tests pertaming to the musculoskeletal system were separated and placed in new chapters I wish to express my deep appreciation to Margaret K Johnson for her painstaking work in the preparation of the manuscript Lam also deeply grateful to the publishers for therr cooperation in preparation of the reviston RaymMonp H Goopare, MD vu Preface to the First Edition The numerous advances m xi branches of clinical pathology have created a demand for ready information on interpretation of labora tory tests and on the most helpful laboratory procedures applicable to the diagnosis of a gren disease It 1s hoped that ch1s book will fill such a need by bringrng together the laboratory and the clinic Medical students :mterns and medical technologists as well as chnicrans should find it a valuable reference work for their particu Jar problems in tlus field Part 1 deals with the physiology normal values and the signifi cance of abnormal values of the various body fluids and excreta Thus section also includes chapters on bas1l metabolic rates I:ver function tests bacteriologic virus and mycotic examinations shin tests and poisons Chemotherapy and anttbiotic therapy in relation to bicterrology are discussed in Chapter 13 Bacteriologic Rami nations In Part 2 diseases with associated laboratory findings are dis cussed according to the various systems eg the Blood and Bone Marrow Lach disease which lends tself to laboritory diagnosis 1s bmefiy outhned ‘Tins 15 followed by a hst of changes to be expected in the pertinent laboratory examinations These examinations are given in the order of importance with the most important first 1” x Preface to First Edition Although it is hoped that other atomic bombs will not be used, the blood changes resulting from those dropped on Japan ate listed. These sequelae are similar to those which have been seen in radia- tion sickness following overexposure to x-rays. Part 2 ends with methods of preparing body fluids, excreta, and tissues for the laboiztory. The proper methods of preparing speci- mens is important in order that accurate results may be obtained. For example, the blood glucose level drops when tt 1s Kept at room temperature unless a preservative is added. Although: certain tests are pathognomonic, clinicians should not rely entirely upon the laboratory for a diagnosis, The laboratory and x-ray department are helpful adjuncts in the modern doctor's diagnostic armamentarium, but they should not replace a careful history and physical examination. The history and physical exam- ination are also important so that the laboratory tests requested will be pertinent to the diagnosis. Certain routine tests are desirable for the protection of the patient because certain diseases may be symptomless at the time. Thus, most hospitals perform routine urinalyses, blood-count estimations, and serological tests for syphi- ls However, it should be remembered that frequent venipunceures may work a hardship on the patient. Dr. Alexander §. Wiener was hind enough to help me in organiz ing the material on blood groups and the Rh factor. For this, I am most appreciative. Dr. William T, Carleton reviewed Chapter 12, Liver-Function Tests, and made several valuable suggestions. T wish to thank Mfrs, Isabel Baker Carleton for her painstaking work in preparing certain illustrations. The original photographs were taken by Mr. Matthew D. Carrigan, who has always been most cooperative, . Here J wish to express my gratitude to my secretary, Miss Agnes M. O'Hare, who spent many hours in preparing the manuscript and in checking references. Finally, 1 wish to express my appreciation to the publishers for their help in the many details about which I have asked their advice. Raymonp H. Goopace, M.D. Table of Contents Part 1 Body Fluids, Excreta, and Functional Tests HEMATOLOGY Bone Marrow Blood Erythrocytes Hemoglobin Leukocytes Platelets Coagulation of Blood Tests for Hemorrhagic Disorders New Terminology of Blood Cells Broop CHEMstry Grouping of Chemical Constituents Physiology of Acid-Base Equilibrium Chlorides Sodium . Potassium Magnesium Calcium Phosphorus Tron xi ii Table of Contents CHAPTER PACE 2 Broop Cuexusrey (Continued) Tron Binding 105 Copper 106 Todine 106 Protein Bound Iodine 107 Lipids 107 Bilirubin 110 Therapeutic Drugs 14 Miscellaneous Tests 1G 3 Bioop Cuemistry (Continued) 127 Enzymes 197 4 SPROLOcY . 140 Acquired Antibodies 140 Cold Autohemagglatinins 44 Nonspecific Antibodies 144 Bacterial Agglutnation Tests 145 Nonspecific Agglutination Tests 150 Complement Fixation 157 Flocculation Reactions 162 Precipitin Tests 166 Antistreptolysin 167 Antihyaluronidase 168 ’ 5 Bioop Grours 172 6 Urine 192 Physiology of Urine Formation 192 Normal Urine 192 Volume 192 Color 195 Odor 196 Specific Gravity 196 Reaction 200 17-Ketosteroids (Usinary Androgens) ae Proteins . Table of Contents veces xiii a4 oI CHAPTER PAGE 6 Urine (Continued) Nonprotein Nitrogenous Products tee 205 Sugars ae ee nee . 207 Products of Acidosis oe 220 Pigments eee ee 2u1 Inorganic Constituents : : 215 Sediment + : os 217 Casts . . 219 Crystals . 222 Bacteriological Examination : 225 Ova and Parasites soe 225 Special Tests 225 Kidney-Function Tests . os : 228 Drugs and Poisons . : 235 7 Gastric aNp DUODENAL CONTENTS 238 Physiology of the Stomach oe 238 Normal Gastric Contents 239 Tubeless Detection of Gastric Acidity (Diagnex Blue Test) 242 Abnormal Findings in Gastric Contents 243 Duodenal Contents : 247 Abnormal Duodenal Fluid : 248 Biliary System 249 Bacteriological Examination of Gastric and Duodenal Contents oa 250 8 GEREBROSPINAL FLUID : 251 Physiology - . 251 Spinal Puncture 252 Physical Characteristics . . 254 Chemical Composition 257 Precipitation Tests... - : : 260 Miscellaneous Tests . : 263 Bacteriological Examination . 264 9 Sruruat AND SALIVA . : 266 Source of Sputum . 266 xiv CHATTER 9 10 MW 12 13 14 Table of Contents Spurun anp Sativa (Continued) Collection : Physical Characteristics Microscopic Examination Bacteriological Examination Fungus Examination Saliva Frers Macroscopic Examination Chemical Examination Microscopic Examination Bacteriological Examination Ova and Parasites Helminths ‘TRANSUDATES AND EXUDATES Transudates Exndates Bacteriological Examination SPERMATOZOA Ongin Examination ‘Tests ror Liver AND Bruiary Funcrigy Physiology of Liver Function Classification of Tests Changes in Urine and Feces Secondary to Liver Damage and Biliary Obstruction Liver Biopsy BAcTERIOLOGICAL EXAMINATIONS Chemotherapeutic Drugs in Relation to Bacteriology Antibiotic Drugs in Relation to Bacteriology Resistance of Organisms to Chemotherapeutic Agents Blood PAGE 266 267 268 272 272 272 275 275 277 280 281 281 284 292 292 295 296 297 297 297 302 302 309 323 326 329 329 529 332 335 Table of Contents x catAPTER PAGE 14 BACTERIOLOGICAL Exaninations (Continued) Urine coke eee 337 Gastric and Duodenal Contents 339 Cerebrospinal Fluid 340 Sputum 342 Feces 344 Transudates and Exudates 347 Orifices and Tissues 349 15 RIcKETTSIAS AND VIRUSES 357 Rickettsias 357 Viruses 361 16 MycoLocicaL ExaMInaTIONS 378 Dermatomycoses : : 378 Fungous Infections of Skin or Mucous Membrane with Frequent Systemic Involvement 383 Primary Systemic Infections 386 17 ‘Tesrs FoR ALLERGY AND IMMUNITY 389 Types 389 For Primary Allergenic Diseases 391 For Bacterial Infections 392 For Fungous Infections 396 For Parasitic Infections 397 For Viral Infections 399 18 Toxico.ocic EXAMINATIONS 402 Gaseous Poisons 403 Inorganic Poisons 405 Organic Poisons 410 Part 2 Diseases with Associated Laboratory Findings 19 Tue Broop ann HEMATOPOIETIC ORGANS 417 The Anemias Al7 434 Hemoglobinopathies Table of Contents xvi CHAPTER PACE 20 9 THE Bioop axp Hesatoroirtic Orcans (Continued) Hemorrhagic Diseases 457 Polycythemia 472 Leukemias 476 Leukemoid Reactions 485 Agranulocytosis 485 Effects of Atomic Bomb Explosion 487 Infectious Mononucleosis 490 Acute Infecuous Lymphocytosis 492 Lymphoblastoma 492 Parasitic Diseases 493 21 HEART anv Boop VEssELs 500 Infections of the Heart 500 Diseases Causing Cardiac Insufficiency 504 Diseases of Blood Vessels 506 22 Tilt Respiratory SysTes 510 Infections of Throat and Larynx 510 Lungs 513 23 GASTROINTESTINAL TRACT AND PANCREAS 530 Mouth and Pharynx 530 Esophagus 530 Stomach and Duodenum 531 Intestines 534 Pancreas 550 24° Liver Ann Brurary TRAcT 557 Selection of Tests 557 Diseases of the Liver 562 Parasites 569 Extrahepatic Biliary Discases 569 25 THe GentrouriNary SYSTEM 572 Kidneys 572 Bladder 592 Genitalia 593 599 Pregnancy Table of Contents xvii CHAPTER PAGE 26 MuscuLoskeLeral SYSTEM aan » . , 603 ‘ Myasthenia Gravis : . oe 603 Muscular Dystrophy . 604 + Periodic Muscular Paralysis 605 Dermatomyositis . 605 Osteomyelitis 605 Paget's Disease (Osteitis Deform man ) : 606 Osteogenic Sarcoma . 606 Osteitis Fibrosa Cystica . 606 Multiple Myeloma. 608 Arthritis 609 7 EnpocrixE GLANDS 618 Pituitary Gland 618 Placenta 626 Ovaries : 627 Adrenal Gland : 629 . Thyroid 642 Parathyroid 658 Islands of Langerhans 660 28 CENTRAL NeRvous SYSTEM 669 Bacterial Infections 669 Mycotic Infections 675 Parasitic Infections . 676 Viral Infections 677 Tumors G8t Trauma 682 Subarachnoid Hemorrhage . 683 Ruptured Disk 683 Multiple Sclerosis 684 Cerebral Hemorrhage and Thrombosis : 634 Intrathecal Injections soe 685 29 BacrexioLocican Discasrs avp Diseases oF DousrruL Cause . . .. . §87 Systemic Infections 687 Infections of Intestines 703 Collagen Diseases . 708 xviii CHAPTER 30 31 32 INDEX, Table of Conte Viraman Dericiency Vitamin-A Deficiency Vitamin-B Deficiency Vitamin-G Deficiency Vitamin-P Deficiency Viramin-D Deficiency Vitamin-K Deficiency Merasouic Disorpres Fluid and Electrolyte Balance Fluid and Electrolyte Imbalance Edema Shock Burns Glycogen Storage Disease Hemochromatosis Miscellaneous Meliturias Alkaptonuria Poiphyrra Primary Essential Xanthomatoses Kwashiorkor COLLECTION AND Care or Lanoratory SrrciMEeNs Hematology Blood Chemustry Serology Blood Cultures Urine Gastric and Duodenal Contents Cerebrospinal Fluid Sputum Feces Transudates and Exudates Qulrarass nd. Sears Cytological Studies for Tumor Cells Surgical Specimens Part 1 Body Fluids, Excreta, _and Functional Tests 1 Hematology BONE MARROW Anatomy: The bone marrow lies in all of the bones and is surrounded by the endosteum, The functioning bone marrow is made up of young and adult blood cells and their precursors, blood vessels, fat cells, and reticulum. The reticulum is connected with the endos- teurn and is closely bound with the blood vessels. Most of the reticulum cells are fixed, but they are capable of rounding off to become large free macrophages. Lymph vessels have not been found in the bone marrow, and nerve structures have been demon- strated only in connection with the larger blood vessels. Lymph follicles have been seen and are generally considered to be normal bone marrow structures. The blood supply of the marrow is made up of a series of branching thin-walled vessels which connect with the nutrient artery of the bone and its vein. ‘The blood supply is further derived from, the axteries af the shaft af the hone and af the eniphysce. The finer divisions of these vessels are capillaries, some of which are collapsed, while others are dilated. The intravascular or extra- vascular origin of blood cells has not been definitely settled. Many investigators believe that all blood cells have an extravascular? origin in the bone marrow. Almost all of the bone marrow is red and is functioning during the first few years of life. From five years of age, fat cells appear 3 4 Body Fluids, Exereta, and Functional Tests in the marrow of the long boncs so that ultimately hematopoietic marrow is found only in the proximal ends of the femur and humerus. The red marrow in adult life is limited mostly to the bones of the trunk and head; namely, the skull, vertebrae, ribs, sternum and innominate bone. In adults, the total bone marrow has been estimated to weigh from 1600 to 3700 gm. (314 to 8 pounds). However only about half of it is in an active state in the normal adult. The nonfunctioning marrow of the long bones retains the essen- tial hematopoietic structures; and with proper stimulation it will start to function. Exiension of hematopoiesis to the long bones is stimulated by pernicious anemia, myelogenous leukemia, and to a certain extent during residence at high altitudes. Extramedullary Hematopoiesis: Extramedullary hematopoiesis may on occasion supplement the formation of cells by the bone marrow, Foci of hematopoiesis have been seen in the spleen, lymph nodes, adrenals, cartilage, broad ligament, organizing thrombi, and fat tissue. These blood forming foci may be made up of erythroblasts or their precursors, myelo- cytes, megakaryocytes or all three types. Extramedullary hema- topoiesis occurs particularly in anemia of infants and young chil- dren,? pernicious anemia,3.4 macrocyte anemia of Jiver disease, metastatic carcinoma,?, ° malignant granuloma (Hodgkin's disease) of the bone marrow,® bone marrow injury by a toxin, anemia resulting from severe sepsis,27 leukemia,¢ & and osteosclerosis.? Function of Bone Marrow: The bone marrow forms erythrocytes, granulocytes, and plate- Jets These cells are formed from special precursor cells, and nor- mally enter the blood stream only after they have matured. The method whereby the cells enter the circulation is stil] debated. One view2° contends that the red cells arise within the endothelial lin- ing, while the leukocytes are formed outside the vascular spaces. One of the reasons advanced to support this thcory is the fact that the erythrocytes possess no means of locomotion and could not Hematology 5 reach the blood stream if they were produced extravascularly. It is held by Klima and Rosegger1? that all blood formation is extra- vascular, the new cells being released into the circulation by diapedesis or by temporary dissolution of the capillary wall. Sabin!2 found that granulocytes enter the blood stream at regular intervals with small hourly increases and larger daily in- creases in the afternoon and at midnight. Chemotactic factors probably play an important role in drawing the leukocytes out of the bone marrow. As for the erythrocytes many contend that anoxemia is the stimulus which brings them into the blood stream. Such anoxemia may be brought about by deficient pulmonary ventilation, deficiency of oxygen in the air as in high altitudes, fixation of hemoglobin (¢.g., carbon monoxide poisoning), or by anemia. This, however, is apparently not the sole factor, as is sug- gested by observationst? which show that there is imperfect corre- lation between anoxemia and marrow hyperplasia. Control of Hematopoiesis: There is also evidence that certain hormones influence hema- topoiesis. According to Gordon and Charipper!4 castration of female rats raises the red cell count and, of male rats lowers the red cell count. Evidence that other endocrine glands influence erythro- poiesis is seen in the anemia which is associated with hypethyroid- ism, hypoadrenalism (Addison's disease) and hypopituitarism (Sim- monds’ disease) and by the increased red cell count which may be found in hyperadrenalism (Cushing’s syndrome). Influence of endocrine glands on other blood cells is noted in the lymphocyto- penia, the decrease or disappearance or eosinophils and rhe mncrease in the number of polymorphonuclear leukocytes which follow the administration of adrenocorticotropic hormone or adrenal cortical hormones. It ts possible that these hematopoietic effects are mani- festations of their action on general metabolism which influences the growth of cells. The specific influence of iton and liver extract on erythro- poiesis in various types of anemia must also be recognized. They are, however, not governing factors in erythrocyte formation; rather 6 Body Fluids, Excrefa, and Functional Tests they are materials of which the red cell is composed or are concerned with its construction and development. Examination of the Bone Marrow: Frevious investigators had studied the bone marrow from trephine specimens of che long bones and sternum, but Arinkin?® in 1929 was the first to propose the technic of needle operation. The aspiration technic has been improved and is generally pre- ferred over trephining. In fact trephine specimens are taken only when repeated aspirations have failed. Aspiration specimens are usually taken from the sternum because of its accessibility and because active bone marrow may be obtained at all ages. Other sites for bone marrow aspiration are the iliac crests, the spinous processes of the vertebrae, ribs, and in children the upper tibiae. Technic: The skin over the midsternum is shaved if necessary, and carefully sterilized with an appropriate antiseptic. Aseptic technic is observed throughout by the operator. The skin, subcu- taneous tissue, and periosteum are anesthetized. A short, beveled 18 gauge needle with a guard and a stylet is used. The guard 1s set at Tem from the tip for an adult and 0.6 to 0.2 cm. for a child. When the anesthesia is effective, insert the needle vertically into the middle of the sterntum between the second and third ribs, using a boring motion. When the cortex is pierced, a “give” is felt as the point encounters the soft marrow. The stylet is removed and a 5 cc. syringe 1s attached. Suction is then applied and marrow pulp is pulled into the syringe. If no specimen 1s visible in the syringe, the stylet is replaced, and the needle is forced deeper into the marrow. After remova! of the needle, a sterile bandage is placed over the puncture wound. Smears, contact smears or imprints, and supravital stain prepara- tions may be made from the material. Satisfactory tissue for sec: tioning is nat usually obtained by this method, but small bits of tis- sue may be fixed and blocked together. The smears may be stained by Wright or Giemsa stains. Sternal puncture is contraindicated in serious hemorrhagic con ditions including hemophilia. Hematology 7 Normal Bone Marrow: : Erythrocyte counts and leukocyte counts may be made on mar- row material, but there are marked fluctuations. Erythrocyte counts and hemoglobin determinations on marrow material are about the same as the patient's peripheral blood or slightly lower. The white cell count, however, ranges from 10,000 to 190,000 per cu. mm. according to Segerdahl.1¢ The leukocyte count tends to be higher the Jess the amount of marrow withdrawn. It would appear, there- fore, that marrow cel! counts are of little or no value. The differential count yields the most information. For a sat isfactory differential count, at least 300 to 500 cells must be counted. Table I shows the ranges to be expected in the normal adult, It must be realized that the ranges of the normal bone marrow cells as given by different investigators vary widely. For example, Osgood and Seaman have lower values for myeloblasts (0-2.4), promyelocytes (0-8) and erythroblasts (3-21), and higher values for lymphocytes Tapis 1; Percenraces or Cenis 1x Bone Manzow or Norman Aputts* Range Average Myeloblasts ... . woe +. [003 to 50] 2.0 Promyelocytes... eae . - $10 to 80) 50 Myelocytes: Neutrophilie.. . . ws . . . | 50 to19.0] 12.0 Eosinophilic.... -.. - : : : . 7005 to 80] 15 Basophilic.... 0. wee eee . 000 to 00.5] 00.3 Metamyelocytes (juyenile” forms). - -}13.0 to320} 22.0 Polymorphonuclear neutrophils. . . «f 78 to800) 200 Polymorphonuelear posinop his soe oe --- 00.5 to 4Q} 20 Polymorphonuclear basophils. . «+» {00.0 to 00.7; 00.2 Lsinpboeytes. . . 3.0 to17.0] 100 Plasma cells . hoe +» +-/00.0 to 2.0; 00.4 Monocytes .. . oe + . 005 to 50] 20 Reticulum cells . nae soe 00.1 to 2.0} 00.2 Megakaryoeytes wee . . 00 03 to 30 00.4 Pronormoblasts (maeroblasts) . -| 1.0 to 4.0 Normoblasts (basophilic, polychromatophilic, andacidophi lic)! 7.0 to Py ° isd * Wintrobe, M. M,: Clinical Hematology. Fifth Ed. Lea & Febiger, Philadel- phia, 1961, p, 65, 8 Body Fluids, Excreta, and Functional Tests (7-30). Custer on the other hand uses as normal! a higher range of myetocytes {up to 34), and a lower range of polymorphonuciear neutrophils (up to 20) and lymphocytes (up to 7). Bone Marrow Changes in Disease: Careful microscopic examination and intelligent interpretation of bone marrow smears yield information in certain blood disorders in which the peripheral blood shows little or no change. In evaluat- ing a bone marrow smear, attention should be given to the follow- ing criteria: (I) The myeloid: erythroid ratio (MLE. ratio). The normal M:E ratio excluding adult leukocytes is 0.56:1, A normal M:E ratio may be found in myelosclerosis, aplastic anemia and multiple myeloma as well as in the normal smear. If the proportion of myeloid cells to erythroblasts is increased, leukopoiesis from infection, leukemoid reaction or leukemia may be suspected. The altered ratio may also be due to decrease of erythroblasts. If rhe proportion of erythroblasts is increased, it may be due to depression of myeloid activity as in agranulocytosis, or to hyperplasia of the erythropoietic tissue. Such erythroblastic hyperplasia may be nor- moblastic and due to blood loss, increased blood destruction, iron deficiency, polycythemia vera, plumbism or cirrhosis of the liver. It may be megaloblastic such as is seen in pernicious anemia, sprue and other macrocytic anemias. (2) The number and shape of megakaryocytes. It should also be noted whether or not they are breaking up to form platelets. (3) The presence of cells which are normally not found in marrow smears, ¢.g., tumor cells from meta- static neoplasm such as carcinoma or Hodgkin's disease, plasma or “myeloma” cells, Gaucher's cells, lupus erythematosus, (L-E.) cells and foam cells of Niemann-Pick disease. (4) Note the presence of parasites such as malaria, Leishmania, and Histoplasma capsulatum. (5) Note the presence of pathological processes such as sarcoidosis, tuberculosis and brucellosis. (6) 1f inadequate material is obtained alter repeated aspirations, a bone marrow biopsy with a trephine is indicated. Kolmer!? has summarized the important changes in many of these diseases as follows: Hematology Disease Pernicious and related anemias .... Aplsstic anemia Aeute hemolytie anemias Chronie hemolytic anemias Sickle-cell anemia .. Hypochromic microeytic anemia Congenital hemolytic jaundice . ‘Thrombopenic purpura. .. .. Erythremia........ - Important Changes Untreated or during relapse: (1) increase of nucleated erythrocytes; (2) preponder- ance of megaloblasts; (3) increase of reticulum (Ferrata) cells; (4) abnormal lenkopoiesis, especiatly lymphocytes; (5) reduction in megakaryocytes Chiefly red blood corpuscles: relative lym- phocytosis constituting from 60 to 100 per cent of the nucleated cells; striking immaturity of the red and white cor- puscles; they may be normally cellular or hyperplastic Markedly hyperplastic: 60 per cent or more of the nucleated cells belong to the eryth- rocytic series; leukocytes relatively re- duced. . Normoblastie hyperplasia characteristic (normoblasts or macroblasts): no megalo- blasts . Largely nucleated red cells (chiefly normo- blasts): there may be moderate shift to the left of the myeloid leukocytes; eosinophils relatively increased; megakar- yocytes may be increased Hyperplastic: increase of normoblasts; no megaloblasts; granulopoiesis usually normal Erythropoietic hyperplasia. of the normo- blastic type: no megaloblasts or giant, abnormal leukocytes . Many megakaryocytes: usually an increase of erythroid elements due tosevere hemor- rhage and anemia Dark red and very cellular: hyperplasia of all elements, moderate increase of nucle- ated erythrocytes; sometimes an increase of megakaryocytes, myelocytes, and royeloblasts Myeloblastic and myelocytic leukemia 9Marked hyperplasia» crowded with myelo- blasts and more primitive cells; in eosino- philic leukemia, preponderance of cosino- phils; in monocytic leukemia, myelocytes and myeloblasts; also “monoblasts” and “monocytes” in some cases; in chronic myelocytic leukemia, the differential count is similar to that of the blood 10 Body Fluids, Excreta, and Functional Tests Disease Lymphocytic leukemia . Aleukemic Icukemia Tafectious mononucleosis Agranulocytosis. Mahgnant granufoms (Hodgkin’s disease) Multiple myeloma, Composition of Blood: Important Changes . .-May be only shghtly changed: ‘usually, however, a sell-marked lymphorytosis (80 to 90 per cent of the cells} Of great value in diagnosis’ frequently the changes are identical with those of typical blood findings; sometimes musleading when only a few cells are obtained, but pronounced unmaturity of the leukocytes may be observed when cellularity is reduced; in leukopenic cases of lymphocy- tic leukemia, marrow lymphocytosis may ‘be shght or absent. Increase of lymphocytes or moderate shift to left of myeloid leukocytes chiefly of value from » negative standpoint in the sense that findings characteristic of leu- kernia are absent Normal erythropoietic tissue and normal numbers of megakaryocytes. striking lack of granulocytes; plasma cells, lym- phocytes, and reticulum cells may be increased Findings variable and nonspecific. there may be slight shift to left in the myeloid celis; also sught monocytosis or moderate eosinophilia; 50 lymphocytosis; relative reiiuction in nucleated ted cells Various types described such as myelo- blastic, lymphoblastic, and erythroblastie, with “plasms cells” the usual designation: myeloma cells most characteristic, can- stituting from 5 to 65 per cent of all cells present BLOOD The blood is a fluid in which cells are free and suspended. The fluid part is “plasma™ before clotting occurs and “serum” after clotting accurs, Plasma is made up of water from 91 to 92 per cent and solids from 8 to 9 per cent. The solids consist of serum albumin, serum globulin, fibrinogen, sodium, calcium, potassium, magnesium, phosphorus, the nonprotein nitrogen group, neutral fats, phospho- Plate 1 Smear of bone morrow as obtained by sternal puncture. (From Tice: Practice of Medicine. W. F. Prior Co., Hagerstown, Md.) Key: Mt, myeloblast; Pro, promyelocyte, Me, myelocyte (differentiated), J, juvenile or metamyelocyte; E, ecsinophit, 1, lymphocyte; P, plarme cell; N, normoblan. Hematology nN lipids, glucose, cholesterol, oxygen, and carbon dioxide. Plasma also contains such substances as antibodies, complement, hormones, and enzymes. In adults the plasma constitutes from 53 to 58 per cent of the blood volume. The cellular part of the blood consists of leukocytes, erythrocytes, and platelets. As determined by centrifugation these cells constitute 45 to 47 per cent of the total blood volume in mates and 42 per cent in females, This percentage of packed blood cells is Known as the hematocrit. Blood Volume: Keith, Rountree and Geraghty!® introduced a simple dye dilution method for the estimation of plasma volume. They first used brilliant vital red, Evans’, but this had the disadvantages of incomplete mixing of the dye and blood, elimination of some of the dye during the mixing period, and difficult matching in the pres- ence of hemolysis. These difficulties were largely overcome by the substitution of a nontoxic slowly diffusing blue dye (Evans’ blue dye or T-1824), The technic of using Evans’ blue has been modi- fied and improved by Gregersen, ef al.,1° Price and Longmire,?¢ and Morris.2 Whereas this method is useful for comparing values obtained in the same patient at different times, it cannot be con- sidered a precise method for measuring plasma volume, according to Courtice and Gunton.?? The use of radioactive elements has added other means for determining blood volume. Radioactive phosphorus (P?2) with a “half-life” of 14.8 days can be introduced into red cells in vitro and then they can be put back into the circulation where the extent of their dilution can be measured.2* Serum albumin has been tagged with radioactive iodine?4 °5 (1251) with a “half-life” of eight days, and by this method the plasma volume rather than the erythrocyte volume has been determined. Another tracer substance, radioactive chromium (Cr®1) with a “half-life” of 26.5 days will tag both plasma protein and erythrocytes according to Gray and Sterling.?6 Blood volume determination has been further refined by the use of radioac- tive iodine attached to normal serum albumin. A known amount of 12 - Body Fluids, Exereta, and Functional Tests radioactive iodine with albumin is given intravenously. After sufficient mixing time, a sample of blood is withdrawn and placed in a scintiHation detector such as the Volemetron, The blood volume in liters can be computed in 15 to 20 minutes by this method. Normal Values: In normal human adults the total blood volume ranges from 60 to 88 cc. per kilogram of body weight, depending on the method used for its determination. It represents from 6 to 8 per cent of the total body weight, Table 2 shows the normal values of plasma volume and total blood volume for man by various methods. Taste 2. Normar Vatues or Torar Broop Votume 4Np Pruasma VOLUME In Man Total Blood Plasma Autharity Afethod Volume vince. | Volume ince per kg body wt. per kg . body wet. Rountree, Brown, and Roth” | Vitel red 88 51 Sunderman and Austin®......f Vital red = 43 (scrum) Gibson and Evans®. . . . | Evans’ blue 7S (males) 43 (males) 66 (females) { 42 (females) Hurtado, Merino, and Delgado") Evans’ blue 85 (males) 46 (mates) Hopper, Taber, and Winkler! | Evans’ blue 80 45 Courtice and Gunton’, .... Evans’ blue _ aL Hopper, Taber, and Winkler™ | Carbon monovade ; 80 45 Salvesen™.. ca ee Carbon monoxide | 60 = Chang and Harrop® .. Carbon monoxide ) 67 (males) ~ Berlin et alts... . 2. ++ | Radioactive phosphorus 69 (males) 38.7 (males) 44 (females) } 27 (females) The Application of Blood Volume Determination: Blood volume determinations are of most value in surgery, ¢.g., surgical or traumatic hemorrhage, surgical or traumatic shock. They are also of value in gastrointestinal hemorrhage, chronic infection or chronic anemia. From the preoperative and postoperative blood volumes and the amount of blood and plasma transfused, the amount of blood, red cells, and plasma lost can be calculated. Hematology . oe Berlin et al.34 have computed the total blood loss, red cell loss, at plasma loss in a series of surgical cases representing both thoracic and abdominal surgery as shown in Table 3. As a result of blood volume determinations, transfusions, fluids and plasma expanders can be used quantitatively with some degree of precision as to the needs of the individual patient. ~ Taste 3, Bioop, Rep Cert, AND PLasmMA Loss IN THoracia AND ABDOMINAL SURGERY No. of Blood Red Celi | Plasma Operation Cases Loss Loss Loss ce. ee. ec. Thoracoplasty.. . 12 910 467 468 Pneumonectomy. . 4 2840 1267 1603 Lobectomy .. .. 8 2680 1156 1628 Gall bladder procedure... 6 690 270 420 Large bowel procedure..... 4 1630 460 1190 Gastric resection..... : ir 1870 440 1490 Specific Gravity: The specific gravity of blood is the ratio of the weight of blood to the weight of the same volume of water at a temperature of 4°C, The normal specific gravity of blood is reported as 1.048 to 1.066 with averages given as 1.052 to 1.063. Men have a slightly higher specific gravity (1.057) than women (1.053). The specific gravity of the blood depends upon the number of red cells and their hemo- globin content, also upon the amount of protein in the plasma. The specific gravity of serum is 1.026 to 1.031. Phillips ef al.88 developed a simple method of determining the specific gravity of whole blood, plasma, or serum by the use of copper sulfate of graded specific gravities. A drop of blood or plasma entering the copper su/fate solution becomes surrounded by a layer of copper proteinate and remains as a discrete drop for 15 t0 20 seconds. During this time it rises or falls and thus reveals its specific gravity relative to that of the solution. If the drop is 14 Body Fluids, Excrelo, and Functional Tests lighter than the test solution, it will rise a few millimeters and will subsequently sink to the bottom. If the drop is of the same specific gravity as the test solution it will become stationary and then fall, TE the drop is heavier, it will continue to fall. In addition to the specific gravity of the blood, the plasma protein level, volume of picked red cells, and hemoglobin concentration can be determined. Functions af Blood: The blood has numerous functions, which under normal condi- tions maintain proper body metabolism. These functions are listed by Best and Taylor36 as follows: Respiratory: The transportation of oxygen from the air in the lungs to the tissues, and of carbon dioxide from the tissues to the lungs. Nturitive: The conveyance of food materials, glucose, amino acids, and fats from the alimentary canal to the tissues. Excretory: The removal of waste products of metabolism, for example, urea, uric acid, creatinine, and so on. Maintenance of Water Content of Tissues: Though the blood itself is contained within vascular channels, a constant interchange of fluid through the vessel walls takes place. This fluid, which has left the blood vessels and has come into direct contact with the tissue cells, is known as the “lymph,” or tissue juice. It closely re- sembles the blood fiuid in chemical composition, The final stage in the transportation of oxygen and food materials to the tissnes, and the first stage in the journey of carbon dioxide and waste products from the tissues, arc made through the medium of the transuded fluid. It is the very high solvent and ionizing powers of water, of which the blood fluid and lymph are chiefly constituted, which make these such admirable media for the carrying our of the complex chemical processes of the body. Body-Temperature Regulation: The body owes its ability to regulate its temperature largely to the water of the blood and the tissue fluids, Water possesses three qualities that fit it preeminently to Fulfill this purpose: Fe oe we ARROW. _— CRS Merge ERAN Ree ORL Fae maT Da we et be 'E-CELIS OF THE BONEM oe AORMOBLAST C” A nosmaitacr wf t Z v, of Yor Se 4 th RETICULOCYTE nonce Plate 2 Celis of the Bone Marrow Red Cells The hushocyte or hemohisticblast is probably the forerunner of all the blood cells erythreblastic, myeloblaste, lymphoblastic, and monocytic. The Pp 2 red cell—erythrogone—is seen with red cell hyperplasia. Normoblast “A“ i: usually called mocroblast, “B," erythreblast, “C,” nermoblast. The approximate proporhons of these celts in the normal morrow are these erythrogones, 0, normoblast “A,” © to 3 per cent; “B," 10 10 20 per cent, “C" 70 t9 90 per cent. (Dameshek.) THE CELLS OF THE BONE-MARROW MYELO woke 4 ; MATURE FOLYMORFHONUCLEAR IE” MNETAMIYELOCYTE femop Plote 3 Cells of the Bone Morrow: White Cells The opprozimate proportions of theve cells tn the normal marrow are there myeloblasts, 1 te 5 per cent, promyelocytes, 2 fo 10 per cent, myototytes, 40 to 80 per cont, metamyelocytes, 20 to 30 per cent, meture palymorphonuclears, Sto 10 percent. Ar the cells moture, the cytoplasm becomes more pink ond the nucleus more ond more shrunken Knowledge of these cells becomes Important in the atudy of acute Enfections with “ght to the left" (Dameshek ) . ERYTHROGENESIS IRON DEFICIENCY HYPOCHROMIC, ATEN TIORMAL HYPERPLASIA am \ ERYTHROGONE tegen normostasT °A” Wi “LIVER periciency PERNICIOUS ANEMIA SPRUE VITAMINE’B’ DEFiclEncy NORMOBLAST “B* (@) \ NORMOBLAST C a Ss None ’ MEGALOBLAST ‘C” , g wt OK NORMOCYTE menocyte aout MACROCYTE Plate 4 Erythrogenesis. Two types of red blood cell formation may be seen in the marrow: (Q) Normoblastice—seen normally and in normal hyperplasia, (2) megaloblastic, os in peratcious anemia and ossociated states (Cf. Dameshek and Valentine. Arch. Path. 23:359, 1937). The most primitive cell in either cose Is the erythrogone (promegaloblest). Nermoblast “A,” Macreblast, Nor moblast "8," Erythroblast, Normoblast “C,"" Nosmoblast, : 0 CS 7 Plate 5 Abnormal erythrocytes. (From Davidsohn, |., ond Wells, B, B.: Tadd- Sanford’s Clinical Diagnosis by Laboratory Methods. Ed. 13. W. B. Saunders Co., 1962.) All drawn from actvol specimens end off stained with Wright's ston except where noted 1,000) (1 mm = micron), 1 Varlations in atze, shape and hemaglobia eantent, fram cases of pernicioun anemia and chlorosis. 2. Polychromatephilia and besophske cranvter degeneration in cares of lead paxtoning and pernicious anemia 3. Normablosts, reticulated erythrocytes and one qicrablast, the top row represents stoges in the development of the normablast The two Feticulated erythtecytes org stained wih brlliont cresyt blus, 4 Maegolsblosts in pernicious anemia. Two show polychromatophila and fairly typical nuclei, two have condensed nuclei ond one of these hox boscphulic oyioplasmic granules 5, Nuclear porticles er Howell Jolly bodies, One cell olso shows batophite granular degeneration. 4. Mitohe Figures, bvo from myelogenous leukemia and one with polychtometophalie cyloptoem, from von Jelath’s onemia Tha tout wer sioined with Leishman's stole. 7. Cabor's ring bodies in von Jaksch’s enema Two cells olso eon suctene particles and one shows bosophilie granuior degenerstion {Leuhhmar’s slain). Hematology 15 HIGH srEciric HEAT: The specific heat of water is considerably higher than that of any other liquid or solid. Because of this great heat-storage power of water, sudden changes of bady temperature are avoided. Even a cold-blooded animal, such as the frog, has— because of this purely physical quality—some ability to maintain a relatively constant body temperature against transient fluctua: tions in environmental temperature. A man of average weight develops 3000 Calories in 24 hours. This amount of heat is capable of raising the temperature of his tissues (which are mostly water) only about 89.60°F. (32°C). Heat elimination (radiation and so on) is able to keep pace with heat production, and the body temperature varies but slightly within normal limits. It has been pointed out by L. J. Henderson, however, that if the tissues had the low heat-storage capacity (specific heat) of most substances, an amount of heat equal to 3000 Calories wold raise the temperature of the tissues and fluids of the body by from 212° to 302° F. (100° to 180° G). HIGH ConDucTIVITY: The thermal! conductivity of water is greater than that of any other ordinary liquid. The advantage of this in the dissipation of heat from deeply situated regions of the body is obvious. HIGH LATENT HEAT OF EVAPORATION: More heat is required for the vaporization of water than for that of an equivalent amount of any other liquid. About 0.6 Calorie (large) is required for the vaporization ot 1 cc. of water. This figure is 50 per cent higher than that of water's closest competitor. Fluid is constantly lost from the body through evaporation from the lungs and skin. A large amount of heat is lost in the process. These physical properties of water, which make it ideal as a heat-regulating medium, are enhanced by other purely physiologi- cal factors. The mobility of the blood and the readiness with which it may be quickly redistributed in the body, combined with the unique physical properties of the fluid itself, make it highly efficient as a regulator of body temperature. The blood may, in a moment, be brought from deeper to superficial regions and spread out in 16 Body Fluids, Exereta, ond Functional Tests fine vessels over a broad axea just beneath the skin. In this way, it greatly increases the radiation of heat. At another instant, in order that heat may be conserved, the Muid is drained from the surface areas and collected in the deeper parts of the body, in the internal organs, muscles, and so on. Protective and Regulatory: The blood and lymph contain cer- tain chemical substances of a complex nature, antitoxins, lysins, and other antibodies, which are the basis of the body's defense against injurious agents of various hinds. The circulating fluids also bring the endocrine principles {rom the ductiess glands into direct contact with the cells of the tissues. Taste 4: Sumstary or Normat Boop VaLurs ADULTS Erythrocytes : Temales 4,600,000 to 4,800,000 per cu. mm Males. . 5,400,000 to 5,800,000 per cu, mm, Retiewlorytes 05 to 15 per cent of erythrocytes Hemoglabin ... . Females. 14 (£2) Gm per 100 ec. of blood Mates 16 (£2) Gm. per 100 ce. of blood Hematocrit (volume of packed erythrocytes). 1, Females 42 (4:5) ce per 100 ce. of blood Males .... . 47 (27) ce per 100 ce. of blood Mean corpuscular volume Females, .. . : 86 1u. merons Males 82 cu. microns Color index. vee 1 (in absolute terms) Mean corpuscular hemoglobin 29 (2) micromicrograms Mean corpuscular hemoglobin concentration . S4 (2) Gin, per 100 ce, Saturation index . 09 to 12 (in absolute terms) Sedimentation rate. . Males Females (mm fall in i bour) . .. Otv8 mm. 0 to 10 mm, (Cutler) 0 fo 15 mm. Oto 2mm (Westergren} Oto 651mm. Oto 15mm (\Wentrobe) Fragilty of erythrocytes... . Minirstns resistance (slight hemolysis) 0,48 to 0.4 per cent sodtum chlonde ‘Maximum resistance (coraplete. hemolysis) 0 36 to 0 3 per eent sodtum chloride Leukocytes. ..-. -. - —- 6000 to 16,000 per eu, mm. Hematology 7 Tasix 4; Suamrary oF Normat Boop Vatues (Continued) {per cent) Percentages of leukocytes. Nonsegmented neutrophils (metamyelocytes)..........6.0068 2tod Segmented neutrophils (mature neutrophils) 54 to 60 Eosinophils 1to3 Basophils 0.25 to 0.5 Lymphocytes... -. 25t033 Monocytes (arge mononuclear ‘cells) 3to7 Platelets .. . . . 250,000 per cu. mm. Figures vary with different technies; for example, Dameshek’s method’? gives 500,000 to 900,000 per cu. mm. Bleeding time ~ . 1 to 3 minutes (Duke) Coagulation time. Capillary tube .. . . 1to7 minutes Howell method. 10 to 30 minutes Lee and White method 5 ta 8 minutes Clot retraction time .. Begins in | hour; marked in 18 hours Prothrombin time... - -10 to 20 seconds (Quick**); 12.5 per cent plasma, 37 to 42 seconds Prothrombin level. . 300 units per ce. of plasma Prothrombin index . . 70 to 100 per cent Capillary fragility... 2. . Suction cup .. ~-20 to —35 em. of mercury “LE.” Capus erythematosts cells... 0 0.0... . «Normally negative Tagce 5: Summary oF NorMaL BLoop VaLues INFANTS AND CHILDREN Erythroblasts . ... . Atbrth. . 1 to § per 100 WBC After seventh day , 0 per 100 WBC Erythrocytes .... 0... .. . Under 2weeks of age.. .. 5,100,000 to 5,660,000 per eu. mm One to 15 years.... 4,600,000 to 4,700,000 per cu. mm’ Reticulocytes | At birth. 2 to G per cent Second week and Tater. 0.5 to 1.5 per cent Hemoglobin ........ . . At birth: capillary . 19.85 Gm. per 100 cc, venous.... 17.0 Gm. per 100 ce. Children: males. . 16 (+2) Gm. per 100 ec. females.... 14 (42) Gm, per 100 cc. Hematocrit (volume of packed erythrocytes).......-.-2.05 Infants. .49 to 54 (4:10) ce. per 100 ec. of blood. Children, .35 to 39 ce. per 100 ec. of blood 18 Body Fluids, Excreta, and Functional Tests Tate 5: Suntary or Noamar Broo Vauues (Continued) Mean corpuscular volume.... Infants. . Sf eu. microns Children. 2. 2... . . 72 eu microns ‘Mean corpuscular hemoglobin .. Chitdren.,., 26 to 28 micromucrograms Mean corpuscular hemoglobin concentration . wee Children... 32 to 34 Gm _ per 100 ec Sedimentation rate Landau’ . method}. oe Under 2 years. . + 1to6mm. (rom. fal} in 4. hour) Over 2 years .. . te 9mm. Fragility of erythyvooytes . .. Minimum resistance. . O48 fo 0,42 per cent of sodium chloride Maximum resistance 0.39 to 03 per cent of sodium chloride Leukocytes. «. » Infants .. 10,008 to 22,500 per cu, mm Children. , 8300 to 10,800 per cu. mm Percentages of leukocytes Nonsepmented neutrophils per cent (metamyelocytes) . stos Segmented neutrophils (mature neutrophils}. . 16 to 60 Eosinopluls 1to3 Basophils. : 025 00.5 Lymphgeytes. . ns . 42 tods Monocytes (Jarge mononuclear cells), 3to 5 Platelets. At birth 140,000 to 290,000 per cy mm. Atl month. 200,000 to 370,000 per cl. mm Ata months 200, 000 to 480,000 per cu. min Bleeding time Atbirth. . 2minates Second week 2 to 3 minutes Coagulation time : _At birth 2 ta 3 minutes (occasionally up fo 12 rainutes} Second week 2 to § minutes Prothrombin time At birth . 43 seconds (Kato & Poncher') Second week —-25 seconds Children, 13-20 seconds ERYTHROCYTES Life Cycle of Erythrocytes: The normal erythrocyte is a non-nucleated round cell which originates in the bone marrow from a nucleated cell called an “erythroblast.” Although the nuclei are normally not present in the circulating blood, a few cells retain a blue reticulum when Hematology 7 stained with a vital stain such as brilliant cresyl blue. These are called “reticulocytes” and are found in normal blood in values varying from 0.5 to 1.5 per cent. Opinions differ on the length of the life span of erythrocytes. Dekkers*? has estimated it to be from 50 to 75 days, while Ashby#? believes they function for from 80 to 100 days. Vischer#3 has Fig. 1: Red corpuscles of normal blood. Wright's stain (photograph, x 750). (From Davidsohn, |, and Wells, B. B.: Todd-Sanford‘s Clinical Diagnosis by Laboratory Methods, Ed. 13. W. B. Saunders Co., 1962.) calculated that transfused erythrocytes function only 12 to 13 days. Shemin and Rittenberg!# concluded that the average life span of human erythrocytes was about 127 days by following the isotope concentration of heme in red cells after feeding glycine labelled with N15. Bale e¢ al.45 calculated an average life of 115 days for the red cell protein of the dog by using C1 labelled di-tysine. Finch and his colleagues*6 followed transfused red cells tagged with radioactive iron in subjects in whom the reunhzation of the iron was blocked. Thus the life span of a red cell population of a single age could be followed. It was found that the red cell turnover in both man and 20 Body Fluids, Excreta, and Functional Tests in the dog was approximately 1 per cent a day. It would seem therefore that the length of life of human erythrocytes in the normal individua) is about 120 days. Apparently the nonfunctioning cells are removed by the phago- cytic cells of the reticuloendothelial system after they have under- gone fragmentation. The phagocytic cells of the liver, spleen, bone marrow, and subcutaneous tissues carry on this process. The hemoglobin of the erythrocytes is probably broken up into globin and an iron-containing pigment, hemochromogen in its reduced form, and hematin when oxidized. This pigment is freed of iron and as changed to bilirubin, which is a normal constituent of the plasma. Bilirubin gives plasma its yellowish tinge and is normally present at a level of 0.7 mg. per 100 cc. (quantitative van den Bergh test). The fate of globin is not known at present, but it is assumed chat it is converted into amino acids and used again in manufacturing hemoglobin. Normal Erythrocytes; The normal erythrocyte is 2 round non-nucleated cell with a thinner central portion. The normal mean diameter of red cells is reported as from 7.2 to 7.9 microns.47 The normal mean thickness varies, according to diflerent reports, from 1,84 to 2.14 micrens.48 Certain physiological changes take place in the erythrocyte count and hemoglobin level, There is apparently a diurnal “varia- tion equivalent to 11 per cent of the mean hemoglobin for the day. Such variation must be correlated with the activity of the per- son, since there is little or no variation in xed cell count and hemo- globin during complete rest.49 Muscular activity is usually asso- ciated with an increase in red-blood cell count and hemoglobin value. The ingestion of a large amount of water may temporarily lower the red-blood cell count, while dehydration will increase this value because of hemo-concentration, Excitement and fear cause an increase in the number of red blood cells, which is higher than would be expected from technical variations, Adult males have Hematology 21 higher counts than adult females. Low barometric pressure, which is experienced at high altitudes, causes anoxemia and an associated increase in both red blood cells and hemoglobin. Abnormal Erythrocytes (Plate 2): A differential count should not be considered complete unless a report is made concerning the presence of any abnormal erythro- cytes. Certain findings will help the clinician in making a diagnosis. Certain erythrocytes stain a light blue, polychromatophilia, which is seen in unripe macrocytes or in degenerated nucleated red cells, Variation in size, anisocytosis, is seen in both primary and secondary anemia. Variation in shape, poikilocytosis, is scen in more advanced anemias. A deficiency of hemoglobin is called “hypochromia.” It may be so marked that only a rim is left. These forms occur in hypochromic anemia, sickle-cell anemia, and erythroblastic anemia. Red blood cells may also contain an excess of hemoglobin, hyperchromia. These cells are seen characteristically in macrocytic anemia, such as pernicious anemia. Basophilic stippling is seen in certain cells. The action of toxins on regenerating red blood cells results in the formation of residual chromatin dots, which take the basic stain. These forms are present in lead poisoning, malaria, and in marked primary and secondary anemias. Nuclear remnants are sometimes seen. They may appear as blue dots; chromatin particles; spherical, eccentrically located, datk- staining granules; Howell’s bodies; and blue-staining, threadlike, twisted rings, Cabot's ring bodics. Confusing bodies are seen in degenerating red cells: (1) A round body, Maragliano body, has the appearance of a vacuole, and is in che center of the cells; (2) bacillary degeneration, rodlike hyaline areas with a vibratory motion; and (3) Ehrlich’s hemoglobinemic degeneration, dark bodies in the centers of red blood cells. A peculiar cell, called a “target” corpuscle, may be found in hypochromic anemia, sickle-cell anemia, Cooley’s anemia, and in 22 Body Fluids, Excreta, and Functional Tests jaundice without anemia, This cell is characterized by a central round region of pigmented material, surrounded by a clear zone without pigment, and this in turn is surrounded by the outer border of the cell. Erythrocytes that take the shape of a sickle when deprived of oxygen are found in sickle-cell anemia, which is peculiar to the Negro race. Decrease in Number of Erythrocytes: A reduction below normal of the number of erythrocytes per cu, mm., the amount of hemoglobin and the volume ot packed red cells is called anemia. The oxygen carrying capacity of the blood is reduced with the reduction in the number of erythrocytes. In anemia from sudden blood loss or from chronic slow blood loss there is a tendency ta make up the blood volume by increasing the fluid portion of the bload. Anemia has the time-honored division inte primary and second ary types, but such division is not considered satisfactory today. Primary anemia referred to anemia resulting from fundamental dis- ease of the hematopoietic organ itself and also to anemia of un known etiology. Secondary anemia included those anemias which are the indirect result of disease of other organs and also those anemias of known etiology. Anemia can occur as a result of (1) acute or chronic blood loss; (2) excessive blood destruction; (3) decreased blood production from deficiency of substances concerned in erythropoiesis or some fault in the construction of red cells; and (4) congenital “dystro phies” of the erythron. {The “erythron” includes the circufating red cells arid the bone marrow from which they originate.) Increase in Number of Erythrocytes (Polycythemia): Polycythemia is 2 condition in which the red cell mass is mcreased above norma}. There is also a concomitant increase in hemoglobin (more than 17.5 Gm. per 100 cc.), in hematocrit (more than 52 per cent), and red cell count (more than 6,000,000 per ct. mm.) , Hematology 23 The polycythemias can be conveniently divided into absolute and relative types as follows: Absolute 1. Primary Polycythemia vera 2. Secondary a. Compensatory polycythemia High altitude Alveolar hypoventilation Chrome pulmonary insufficiency Congenital heart disease Methemoglobinemia Carboxy hemoglobinemia b, Chemical agents and drugs Coal tar derivatives Cobalt Manganese Mercury Iron Arsenic Bismuth sulbnitrate Digitalis Caffeine Nicaune Testosterone . c, Neoplasms Neoplasms of kidneys, liser and uterus Subtentorial neoplasms d. Miscellaneous Relative (pseudopolycythemia) Hemoconcentration secondary to dehydration. See page 472 for discussion Reticulocytes: Reticulocytes are seen in the blood smear only when the blood is stained with a vital stain, such as briffiant cresyl blue. They are characterized by a faint blue reticulum in erythrocytes which are larger than normal. The reticulum may appear as a pale blue band, it may be distributed evenly throughout the cell as fragments or it may be so densely packed that it can be mistaken for a nucleus. As a rule there is a large amount of reticulum in reticulocytes which have just emerged from the nucleated stage and very little in those which are approaching maturity. Reticulocytes will last 24 hours in oxalated blood at room temperature, However, if the blood is Lept in a refrigerator, Heath 24 Body Fluids, Excrefa, ond Functional Tests and Daland5? report that the reticulocytes retain demonstrable reticulum for six months, The normal range for reticulocytes is 0.6 to 1.5 per cent in healthy persons, Friedlander and Wiedemer®! reported that they Fig. 2: Reticulated erythrocytes (x 1000). (From Davidsehn, 1, and Wells, B. Ba Todd-Sanford’s Clinical Diagnosis by Laboratory Methods, Ed. 13. W. 8. Saunders Co, 1962, are slightly increased in the spring. Newborn infants have from 2 to 6 per cent, but this drops to from 0.5 to 1.5 per cent in two to five days. an | An increased reticulocyte count is indicative of increased erythropoiesis, and is a helpful guide an proving the adequacy of liver therapy in primary macrocytic anemia. The reticulocyte response is in inverse ratio to the original erythrocyte count; that 1s, the lower the original erythrocyte count, the higher the reticulo- cyte response. Hematology 25 Reticulocytes are also increased in congenital! hemolytic jaun- dice, sickle-cell anemia, in cases of chronic blood loss of short dura- tion (from two to four months), and in anemic premature infants. There is a slight increase in leukemia, myelophthisic anemia, lead and mercury poisoning, malaria, and in the full-term infant at birth. There is a decrease below low normal (0.5 per cent) in both idiopathic and symptomatic aplastic anemia. Stippled Cells: Basophilic stippling is not seen in a smear of normal blood. It does occur in severe anemia, lead and mercury poisoning, leukemia, and chronic malaria. The granules of stippled cells are stained by Wright’s stain and are characteristically deep blue. They are not to be confused with the reddish granules seen in erythrocytes con- taining Plasmodium vivax. Heinz Bodies: Heinz bodies are refractile inclusions varying in size from 1 to 4 microns and are found in red cells under certain conditions. They may be demonstrated by dark field or phase microscopy and may be stained by 0.5 per cent methyl violet in 0.85 per cent sodium chloride. , Heinz bodies are found in patients with hemolytic anemia resulting from exposure to certain drugs or chemicals. Among these are Pamaquine and primaquine (antimalarial drags) which affect the red cells of susceptible patients, most of whom are Negroes. Adults habituated to acetanilid or phenacetin and children suffi- ciently exposed to fumes of mothballs may develop hemolytic anemia and positive Hemz bodies. Hemolytic anemia with many Heniz bodies with no exposure to drugs has been reported in 14 premature or underweight infants by Gasser.52 In postsplenectomy cases, small numbers have been found, according to Dacie.®% Siderocytes: Siderocytes are red blood cells which show blue granules after the Prussian blue stain. These granules are thought to contain 26 Body Fluids, Excreta, and Functional Tests jonized ferric iron and can be demonstrated in orthochromic normoblasts and occasionally in reticulocytes. Siderocytes up to 20 per cent or more are found in che blood of hemolytic anemias and in hematologically normal blood after splenectomy. Indices Derived from Erythrocyte Values: Hematocrit: The hematocrit or volume of packed cells in terms of cubic centimeters per 100 cc. is of value in determining alteration in size of erythrocytes. The normal values for cell volume are as follows: infants, 49 to 54 (+10); children from one to 15 years, 35 to $9; adult males, 4? (+: 7); and adult females, 42 (+ 5). It gives clinical infonnation similar to that given by the red-blood- cell count and hemoglobin, Mean Corpuscular Volume: The mean corpuscular volume may be determined from the volume of packed cells and the erythrocyte count as follows: volume of packed blood cells, ce, per 100 ce, red blood cell count, millions per eu, mm. The value for infants is approximately 84; children, 72; adult males, 82; and adult females, 86 cu. microns. The values are increased to more than 100 in macrocytic anemia and are decreased in microcytic anemia, Volume Index: The volume index may also be determined by a slightly different formula: volume of packed blood cells, ce. per 100 ec. x 23 red blood cell count, millions per cu. mm, x 20. The volume index is the relative mass of circulating red blood cells as compared to the normal. The values for children are from 0.68 to 0.82 and for adults from 6.8 to 1. In macrocytic anemias, values from 1.20 to 1.60 or higher are found. = number of cubic microns Sedimentation Rate: The sedimentation rate or index is the fall of the red cell column in one hour measured in millimeters. It is a nonspecific test which is accelerated in a variety of diseases and in pregnancy. ‘The underly- ing reason for this change in rate of fall is not clearly understood. Hematology 27 Various protein fractions such as alpha and beta globulin®+ have been added to plasma with some acceleration of the sedimentation rate. However, the effect is less marked than that produced by the addition of fibrinogen.35 This fact, plus the observation that plasma fibrinogen is usually increased when the sedimentation rate is accelerated, led to the conclusion that increased sedimentation rate SEDIMENTATION RATE, MM. IN ONE HOUR 30 70 a 60 50 40 a 5 "10 «15° «20 25 30 35 40 45 50 55 VOLUME OF PACKED RED CELLS, CC. PER 100 Cc, BLOOD Fig. 3—Chert for correction of sedimentation rate according to volume of packed red cells. The fogarithmie curve on which this chart is based fs heavily outlined. The mean pormal volume of packed red cells for men (47 cc} and for women (42 cc.) are ako heavily outlined and the range of normat sedimentation is represented by solid and open cofumns for each sex, respectively, To “correct” sedimentation rate, find on the chart the horizontal line corresponding to the sedimentation rate for the potient; find also the vertical line corresponding to the yolume of the packed red cells in the patient's bleed. Select the curve lying nearest to the point of junction of the horizontal ond the vertical fines and follow this to the sormal line for the sex of the patient, The horizontal fing corresponding to this lost potnt of juncture gives the corrected sedimentation rate, Since the rence hetween the normal sedimentation rates in men end women is largely due to the difference in volume of packed red cells, all sedimentation rates moy ho corrected to @ volume of 47 cc. and a single standard of normal (0 to 6 mm.) used. Thir chart should not be ysed to “correct” polycythemic blood fo a normal level. (Wintrcbe and landsberg, Am. Jour. Med. Sef) 90 80 70 50 20 10 238 Body Fluids, Excreta, and Functional Tests is the result of an increased level of fibrinogen.# 57 Since the addi- tion of pure albumin inhibits the fall of erythrocytes and the globulins and fibrinogen cause varying increases in rate of fall, it seems likely that the sedimentation rate depends upon the ratio of the various protein fractions to each other. It is customary to correct the sedimentation rate for anemia because there is an increased rate of fail when there are fewer erythrocytes. Sickle-cell anemia fs an exception because the peculiar shape of the red cells prevents the formation of rouleaux. In order that the clinician may evaluate the findings properly, it is advisable to Teport the uncorrected and corrected sedimentation rates together with the hematocrit. The method of correction of sedimentation rate according to volume of packed red cells (hematocrit) is shown in Figure 3, The normal results vary with the method used as indicated in Table 6, Certain precautions should be taken in carrying out a sedimentation rate, viz: (1) The test should be done within two Taste 6; Variations or Normat SrpiMENtTATION Ratrs Cutler Westergren Wrntrohe (Mm in One Hour) | (Aten.in One Hour) | (Mim. in One Hour) Children .. : — Oto 10 3 to 130 Men .. Oto8 Oto 15 O65 (average 37) Women. Oto 10 Oto 20 0 to 15.0 {average 9.6) hours of the time of collection of the specimen. (2) The test should be carried out within a temperature range of 22° C. to 27° C,, since the rate increases with increase in temperature. Blood which has been kept in a refrigerator should be allowed to reach this tempera. ture range befare starting the test. (3) The tube should be kept in an exact vertical position during the test because any deviation in- creases the rate of fall. The sedimentation rate remains within normal range in func. tional nervous disease, benign tumors such as uncomplicated Hematology 29 fibroids and cysts, asthma, hay fever, essential hypertension, diabetes mellitus, cirrhosis of the liver, simple peptic ulcer, hydronephrosis, uncomplicated cholelithiasis, and nephrolithiasis, early acute appen- dicitis, head colds, and hypertrophic arthritis. Changes in the sedimentation rate in various conditions are shown in Table 7. TABLE 7: CHANGES IN SEDIMENTATION RATE Increased Decreased Physiological. Malaria during paroxysms Pregnancy after second mouth Erythremis Menstruation Sickle-cell anemia, . Certain cases of severe liver disease Infectious processes. Acute general and localized infection Chronic active infections Tuberculosis Syphilis Rheumatic carditis Blood dyserasias Leukemias Anemias Poisoning: Lead Arsenic Alcohol Miscellaneous: Coronary thrombosis Hemorrhage Advanced malignancy Hyperthyroidism Nephritis and nephrosis Certain cases of severe liver disease Hemolysis of Erythrocytes: Hemolysis is a phenomenon which is sometimes preceded by agglutination. Red cells have a tendency to agglutinate under cer- tain conditions, and under other conditions they tend to hemolyze. 30 Body Fluids, Excreta, and Functional Tests ‘The role of these peculiarities of red cells in the development of disease is not entirely understood, but helpful diagnostic procedures have been developed in which the agglutination and hemolysis potentials have been used. Agglutination of red cells will be dis. enssed in Chapter 5. Osmotic Fragility Test; The fragility test as originally described is a measure of resistance of red cells to hemolysis by hypotonic sodium chloride solutions, When normal venous whole blood is used, hemolysis just begins at 0.46 to 0.40 per cent sodium chloride; this is called “minimum resistance.” Hemolysis with such blood is usu- ally complete in solutions varying from 0,36 to 0 30 per cent sodium chloride; this is called “maximum resistance.” Increased Fragility in Hypotonic Sodium Chloride: Increased fragility is marked in congenital hemolytic jaundice (familial hemo- lytic jaundice). This test helps to differentiate this disease from various types of obstructive jaundice. There is some increase in fragility in aplastic anemia. This may help to differentiate it from pernicious anemia and hypochromic anemia. Decreased Fragility in Hypotonic Sodium Chloride: Decreased fragility is found in erythroblastic anemia, pernicious anemia, sickle- cell anemia, hypochromic anemia, hemolytic anemias, polycythemia vera (erythremia), and obstructive jaundice. In hemolytic jaundice, after splenectomy, the fragility of erythrocytes may be increased or decreased. Fragility in Other Reagents: It is sometimes helpful to employ a modification of the standard fragility test by determining the resistance of erythrocytes to dilute hydrochloric acid, lysolecithin, or saponin. Dilute hydrochloric acid reveals increased fragility in nocturnal hemoglobinuria. The reaction of erythrocytes in tyso- lecithin is the same as in hypotonic saline except in acquired hemolytic jaundice. In this disease saline fragility may be increased while lysolecithin fragility remains normal according to Singer.5% In pernicious anemia, saponin hemolysis is increased while saline Fragility is decreased. Mechanicai Fragility: Mechanical fragility may be determined by shaking blood in a flask containing glass beads."? Congenital Hematology 31 hemolytic jaundice and sickle-cell anemia show increased mechan- ical fragility. HEMOGLOBIN Hemoglobin Formation and Destruction: Hemoglobin gives the red color to the blood. The red blood cells are, for the most part, made up of water (64 per cent), and the remainder of hemoglobin. Hemoglobin is composed of globin, which is a sulfur-bearing portion (96 per cent), and hematin, a ferrous complex of protoporphyrin (4 per cent). Hemoglobin is formed in the bone marrow in the maturing red blood cells, but the detailed steps are not known. In the lungs, hemoglobin combines readily with oxygen, form- ing a compound known as “oxyhemoglobin,” which gives arterial blood its bright-red color, The oxygen is given up readily in the Ussues, since it is only in Joose combination with hemoglobin. Hemoglobin then becomes reduced hemoglobin and. carries carbon dioxide from the tissues to che lungs, which accounts for the dark color of venous blood. After erythrocytes have lived their normal span of life, they undergo fragmentation. Hemoglobin is released and destroyed by the cells of the reticuloendothelial system of the liver (Kupffer’s cells), spleen, and bone marrow. The destruction of hemoglobin consists first of separating globin from hematin. Globin is broken down into amino acids. Hematin is broken up by separating the iron, which is stored in the liver and spleen to be used again in the manufacture of hemoglobin, The remaining iron-free product, porphyrin, is converted to bilirubin and transported to the liver to be excreted in the bile. A small amount of bilirubin is excreted in the urine by the Kidneys. Normal Hemoglobin: In the past, the level of hemoglobin has been expressed in per- centage, but this is unsatisfactory because of the variation in dif- ferent sexes and at different ages, which would require the establish- ment of arbitrary normals to insure accuracy, In expressing hemo- a2 Body Fluids, Excreta, and Functional Tests globin in percentage, it is assumed that §,000,000 red blood cells per cubic millimeter of blood contain 100 per cent hemoglobin, Hemoglobin is preferably expressed in grams per 100 cc. of blood, using normal values that are corrected for age and sex. At birth the normal hemoglobin level in capillary blood is 19.85 Gm, and in venous blood is 17.9 Gm. Both cells and hemoglobin are about 15 per cent less concentrated in venous blood than in capillary blood at birth. However, this difference gradually dis- appears ducing the first week. In male children the normal hemoglobin level is 16 (2) Gm. and in female children 14 (2) Gm. These figures are the same in adults, viz: 16 (+2) Gm. for males and 14 (+ 2) Gm. for females, Abnormal Hemoglobin Values: Hemoglobin is increased (hyperchromernia) in those conditions in which there is an increase in erythrocytes. These are erythremia, compensatory polycythemia at high altitudes, and dehydration such as is associated with burns and diarrhea. Hemoglobin is increased in macrocytic anemia because each erythrocyte carries more hemo- globin than normat (hyperchromia), although the erythrocyte count is lower than normal. Hemoglobin values are reduced (oligochromemia) in all anemias. In hypochromic ancmias particularly, the hemoglobin is low, not only because there is a reduction in total erythrocytes, but also because the individual cells have a decreased amount of hemoglobin. Indices Derived from Hemoglobin Values: Golar Index: In computing the color index, it is assumed that 5,000,000 red blood cells per cubic millimeter of blood have a hemo- globin value of 100 per cent, The color index is determined by dividing the percentage of hemoglobin by the first two figures of the erythrocyte count multiplied by 2, A hemoglobin value of 70 per cent with a red-bland cell count of 4,000,000 would give a color index of eS ar 0.87, which would suggest hypochromic anemia. A color index greater than | is indicative of macracytic anemia. Hematology 33 Mean Corpuscular Hemoglobin: This index is computed by dividing the hemoglobin value in grams per 1000 ce., by the erythro- cyte level, in millions per cubic millimeter, and expressing the results in micromicrograms, The normal for adults is 29 (=: 2); for children, from 26 to 28. Mean Corpuscular Hemoglobin Concentration: This index is calculated by dividing hemoglobin, in grams per 100 cc., by the volume of packed cells, in cubic centimeters per 100 cc. of blood, and multiplying by 100. The results are given in grams per 100 cc. The normal for adults is 34 (+ 2); for children, from 32 to 34. The mean corpuscular hemoglobin and mean corpuscular hemo- globin concentration are decreased in hypochromic anemias and increased in macrocytic anemias. Saturation Index: The saturation index measures the concentra- tion of hemoglobin in the average erythrocyte, as if it carried hemo- globin in solution. The saturation index is computed as follows: hemoglobin, per cent (gm per 100 cc. % 6.9) Saturation index = Volume packed red blood cells (cc. per 100 ce. x 2.3) The saturation index is decreased in hypochromic and micracytic anemias, and increased in macrocytic anemias. Hemoglobinopathies: See page 434, LEUKOCYTES Types of Leukocytes: Leukocytes of the peripheral blood are divided into two gen- eral groups, the granulocytes and the nongranulocytes. The cells in these two categories are: Granulocytes Nongranulocytes Polymorphonuclear neutrophils Lymphocytes Eosinophils Monocytes (large mononuclear Basophils cells) 34 Body Fluids, Excreta, and Functional Tests Functions of Leukocytes: Polymorphonucleay Neubopkils: The chief function of these cells is chat of phagocytosis particularly of bacteria and tiny par- ticles. This function is well illustrated in the opsonocytophagic index for B. abortus. They also destroy antigen and distribute anti- body, “Their function in the production of antibody is problem- atical. Eesinophils: These cells are thought to have an important role in detoxification, and in the disintegration and removal of protein. Their function in relation to allergy and hypersensitivity is not yet clear. In persons with normal pituitary and adrenals, injection of 25 mg. of adrenacorticowropic hormone (ACTH) or 0.3 ce. of epinephrine will cause an eosinopenia.®® 6! The mechanism which brings about this fall in circulating eosinophils is not as yet com- pletely understood. It is known that ACTH stimulates the adrenal cortex which releases steroid hormones in lage quarttities, Of these steroids, those with an oxygen atom at I} and 17 carbon positions (“11-17 oxysteroids”) produce a decrease in circulating eosinophils by a mechanism as yet unknown.®? ~~ Basophils: Although these cells may be funerionless, there is some evidence that in inflammation they bring an anticoagulant (heparin) to prevent the clotting of blood in the inflamed tis- stig, 61, 62 Lymphocytes: The function of these cells is not completely understood. They may help to destroy the toxic products of protein metabolism.¢2- 03. ¢4 They are increased during convalescence from infections, and, therefore, must have some function in connection with the process of healing. They have been shown experimentally to participate in resistance to tuberculosis. Monocytes: These cells phagocytize some bacteria, pavticulste matter, and protozoa. In an inflammatory process the neutrophils predominate for about three days, then they break up and the monocytes remain to phagocytize fragments of cells, ete. Because monocytes contain lipases they are able to digest bacteria with a lipoid capsule such as Mycobacteritan tuberculosis and Mycobac- tertum leprae. Hematology 35 Normal Total Leukocyte Values: The total! leukocyte count varies normally with age, exercise, relationship to a meal, relationship to hot bath, high altitudes, and dehydration. There may also be physiological changes in the leuko- cyte count from day to day. Normal values for various age groups are as follows: Number per cu. num. Infants .. . . 10,000 to 22,500 Children : 8,300 to 10,800 Adults . 6,000 to 10,000 Leukocytosis: Leukocytosis is the term applied to an increase in the total leukocyte count above the upper limit of normal. Leukocytasis is the result of an absolute increase in number of one of the white cells; thus an increase in neutrophils (neutrophilia) in acute appen. dicitis or of eosinophils (eosinophilia) in trichinosis will result in an increase in the total white count. It is important ¢o do a differential count in addition to a total white count in order to detect an increase in number of one of the white cells, ‘his sometimes gives a clue to the cause of the under: lying condition as indicated in the following pages. Origin and Significance of Granulocytes: Polymorphonuclear Neutrophils: The youngest recognized forms of this series are the myeloblasts. Normally, these cells occur only in the bone marrow where they go through certain stages to reach maturity. In the myeloblast stage differentiation from lymphoblasts and monoblasts is difficult, The myeloblast next develops into the tmyelocyte, which is characterized by the presence of tiny, pale, red staining granules in the cytoplasm. Myeloblasts and myelocytes are Not in the peripheral blood except in conditions that stimulate great activity in the bone marrow, such as severe infections, myelog- cnous leukemia, and erythroblastosis foetalis. Nonsegmented Neutrophils (Band Forms): Normal values are as follows: adults from 2 to 5 per cent; children from three to ten years old, from 3 to 8 per cent. The myelocyte becomes a meta- 36 Body Fluids, Exereta, and Functional! Tests myelocyte by the indentation of the nucleus and later assumes the shape of a horseshoe. The fine pale-red granules remain in the cytoplasm. Metamyelocytes may be divided into young forms (with nucleoli and dark staining nuclei) and adult forms (without nucleoli). Metamyelacytes are the youngest forms that are found normally in the peripheral blood. They are also known as “band forms” and “nonsegmented" or “non-filamented” neutrophils. These forms are increased in acute infectious processes with or without an incease in the total leukocyte count. An increase in metamyelocytes constitutes a “shift 10 the left’ as described by Schilling. Segmented Polymorphonuclear Neutrophils: Normal values are as follows: adults from 54 to 62 per cent; children from three to ten years old, fram 16 to 60 per cent. The nucleus of the metamyel- ocyte becomes pinched off into from two to six or even ten lobes connected by threads of chromatin, Normally, the granutes remain pale red, but in severe infections they may take the basophilic stain and increase in size. These are known as toxic granules and are thought to be due to the effect of toxins in the cells. Taste 8; NormMat. VALUES OF LEUKOCYTES IN PERIPHERAL Boop Children. (8-10 years) Adults Absolute Count Absolute Count Per | Aver- Range Per | Aver- Range cent age cent age Granulocytes Nonseemented 1 trophils (band forms) ¥ ‘ 280 | 150-300 25 30g | 150-100 Segmented or mature reutropbils 16-60 | 3250 | 3000-8000 | 54-62 | 4000 | 3000-5500 Eosinophils... 13 300 50-700 1-3 200 50-250 Basophuls.. . 00.75, 35 0-50 0-0.75 25 15-50 te aioli “ 49-48 | 4000 | 3250-5000 | 25-33 | 2100 | 1500-3000 Monocytes. . 3-5 350 | 250-700 BT 375} 285-500 RS, Hematology 37 When an increase in white cells (leukocytosis) is due to an increase in neutrophils, it is called neutrophilia. In this instance leukocytosis and neutrophilia are synonymous. Usually the increase in neutrophils is “absolute”; that is, both the total number of neutrophils per cu. mm. and the percentage are increased. Neutro- penia is a reduction below normal of the total number of neutro- phils. An increase in neutrophils is found in the following; 1. Infectious processes, systemic: Septicemia, pyemia, - pneumonia, meningitis, gonorrhea, diphtheria, tularemia, actinomycosis, Weil's dis- ease, anterior poliomyelitis, rabies, typhus, herpes zoster, coccidioidomyco- sis, acute rheumatic fever, smallpox, chickenpox, scarlet fever, erysipelas, valvular heart disease, peritonitis, anthrax, plague, cholera, and tetanus, 2. Infectious processes, localized: Pyogenic abscess, carbuncle, furun- culosis, tonsillitis, mastoiditis, otitis media, sinusitis, ulcer, empyema, cholecystitis, pyelitis, pyelonephritis, salpingitis, appendicitis, 8. Metabolic disorders: Acidosis of diabetes mellitus, uremia, gout, and eclampsia. 4, Drugs and poisons: Digitalis, epinephrine, foreign proteins, venoms, mercury, lead, carbon monoxide, potassium chlorate, camphor, coal tar products, pyridine, pyrogallol, benzol compounds, turpentine. 5. Acute hemorzhage: Neutrophilia occurs particularly when the hemorrhage is into a body cavity, e.g., ruptured tubal pregnancy. 6. Diseases of the hemopoietic system: Myelogenous leukemia, ery- thremia, erythroblastosis foetalis, incompatible transfusions, sudden hemolysis. 7, Miscellancous conditions: Coronary occlusien, rapidly growing carcinoma, for 12 to 36 hours after a major operation, burns. 8. Physiological conditions: Strenuous exercise, pregnancy, labor, puerperium, during digestion, after a hot bath, fear, pain, dehydration, extreme sunlight and high altitude. Lupus Erythematosus Cells (“L.E.” Cells): These cells are usually neutrophils containing one or more masses of purplish- staining material which fills the cell and compresses the nucleus, These cells are thought to form as follows: When injured leukocytes come in contact with an abnormal globulin in the serum, their nuclei absorb protein, lose their normal chromatin structure and become homogencous masses that seem to be composed largely of 38 Body Fluids, Excreta, and Functional Tests desovyribanucleaprotcin, These masses became fiee by either extrusion or disintegration of the surrounding cytoplasm. They are then phagocytized by healthy neutrophils, forming “L.E." cells. These cells are found in stained bone marrow smears, and by appropriate incubation and concentration technic they may be found in the peripheral blood. “L.E.” cells are usually indicative of lupus erythematosus, but similar cells may be tound in rheumatoid arthritis and in toxic reactions to certain drugs. Eosinophils: Normal values of eosinophils in both adults and children are from 1 to 3 per cent. Eosinophils originate from myeloblasts; when they develop into myelocytes, large acid staining granules form in the cytoplasm thus differentiating them from neutrophilic myelocytes. The adult cells rarely have more than two lobes, but they retain the characteristic coarse acid-staining granules, Eosinophils are increased in the following conditions: 1. Blond diseases: Easinaphilic leukemia, myelogenous leukemia, polycythemia vera, and slightly in pernicious anemia. 2. Parasitic discases: Trichinosis, hydatid cyst, especially after rup- ture, schistosomiasis, infestation with Clonorchis sinensis, infestation with some intestinal parasites particularly in strongyloidiasis and ancylos tomiasis, infestation with Zasciola hefatica and in massive infestauon with Taena solium and amebiasis. 3. Skin diseases: Psoriasis, pemphigus, dermatitis herpetiformis, ery- thema multiforme, mycosis fungoides. and cutancous allergies such as urticaria and angioneurotic edema. . 4, Neoplasms: Some cases of malignant granuloma (Hodgkin's dis ease), certain ovarian and bone neoplasms, neoplasms impinging upon se1ous surfaces, and neoplasms showing extensive nectosis. 5. Allergic diseases of the respirator tract: Bronchial asthma, hay fever, and pollinosis. 6 Poisons: Phosphorus, copper sulfate, camphor, and pilocarpme 7. Disease of unknown or doubtful etiology: Loefller’s syndrome some cases Of periarteritis nodosa, chorea, scarlet fever, tropical eosino philia, and familial eosinophilia. ; 8. Specific infections: Brucellosis, tuberculosis, coccidioidomycosis. Eosinophils are decreased (cosinopenia), or disappear entirely in severe infections and reappear upon recovery. ‘They are decreased Hematology 39 also in shock, major surgical operations, severe alarm reactions, Cushing’s disease, and following injection of adrenal cortica! hor- mones, or substances that increase the output of these hormones, e.g., ACTH and epinephrine. For more accurate enumeration of the eosinophils to determine the percentage of decrease in the Thorn test, they are counted in a counting chamber and the total per cu. mm. is reported. (See page 635). The normal range is usually 150 to 250 per cu. mm. Basophils: Normal values of basophils in both adults and chil- dren are from 0 to 0.75 per cent. Basophils originate from myelo- blasts, but the mature nuclei do not become lobulated. The cyto- plasm is characterized by coarse basic-staining granules. Basophils are increased in the following conditions: 1. Blood diseases: Basophilic leukemia, myelogenous leukemia, polycythemia vera and chronic hemolytic anemia. 2. Miscellaneous conditions: Sometimes in Hodgkin's disease, chickenpox, smallpox, chronic infections of the bony sinuses, follow- ing splenectomy, and sometimes after administration of nonspecific proteins. Origin and Significance of Nongranulocytes: Lymphocytes: Normal values of lymphocytes are as follows: Adults, from 25 to 33 per cent; children from three to ten years old, from 42 to 48 per cent. Lymphocytes supposedly originate from lymphoblasts in the spleen, lymph glands, tonsils, thymus, and possibly the bone marrow. Lymphoblasts are not found normally in the peripheral blood, but they are found in blood smears of acute lymphatic leukemia. Lymphocytes are sometimes classified as “large” and “small,” the large form representing the more immature stage. Some authorities assert, however, that the large forms originate in the spleen, while the small forms originate in the lymph glands. Abnormal lymphocytes, sometimes called “atypical lymphocytes” are found characteristically in blood smears of infectious mononu- cleosis. These cells are usually large, have an oval, kidney-shaped or 40 Body Fluids, Excreta, and Functional Tests lobulated nucleus and a vacuolated or foamy nongranular cyto- plasm. The nucleus is a coarse network of strands and masses of chromatin. These are thought to be well differentiated, mature lymphocytes by most hematclogists. Lymphocytes are increased in the following conditions with an increased white cell count: Blood diseases: Lymphocytic leukemia and sometimes in pur- pura hemorthagica. Acute infections: Infectious mononucleosis, acute infectious lymphocytosis, pertussis, and during convalescence from acute pyo- genic infections. Chronic infections: Tuberculosis in persons with good resistance, congenital syphilis, and secondary syphilis. Metabolic diseases: Rickets, thyrotoxicosis, and malnutrition. Normal increase in infants and children. Lymphocytes are increased in the following conditions with a normal or decreased white cell count: Blood diseases: Pernicious anemia and familial splenic anemia {Gaucher's disease). Acute infections: Typhoid fever, influenza, undulant fever (brucellosis), infections hepatitis, German measles, and mumps. Monocytes {Large Mononuclear Cells): Normal values are as follows: Adults from 8 to 7 per cent; children from three to ten years old, from 3 to 5 per cent. Monocytes are believed to originate in the bone marrow from monoblasts. These develop into pre- monocytes, which in tarn become adult monocytes. Monocytes are increased in the following conditions: Blood diseases: Monocytie leukemia. Acute infections: Brucellosis, subacute bacterial endocarditis, typhus fever, Rocky Mountain spotted fever. Certain parasitic infestations: Kala-azar, cutaneous leishmaniasis, malaria, and trypanosomiasis. Miscellaneous conditions: Sometimes in Hodgkin's disease, familial splenic anemia (Gaucher's disease), Niemann-Pick’s dis- ease, Hand-Schiller-Christian’s disease, and poisoning by tetra- chlorethane. Hematology 4t Plasma Cells: Plasma cells are not found normally in the per- ipheral blood. They are thought to originate from lymphocytes or primitive connective tissue cells.6 They are round cells with deep blue cytoplasm and an eccentric nucleus containing deep-staining chromatin arranged like the spokes of a wheel. Plasma cells are found in the peripheral blood in the following conditions: Blood diseases: Plasma cell leukemia. Exanthematous diseases: Rubella, slight increase in measles, scarlet fever, and chickenpox. Miscellaneous conditions: They may be present in multiple myeloma and serum reactions. Leukopenia: Leukopenia is a reduction in the total number of leukocytes below the lower limit of normal. It is usually due to a decrease in granulocytes, particularly neutrophils. Causes of Leukopenia: The important causes of leukopenia may be grouped as follows: 1. Certain acute and chronic infections: Typhoid and paratyphoid fever, brucellosis, miliary tuberculosis, overwhelming pyogenic infections. 2. Ceitain viral and rickettsial diseases: Measles, rubella, smallpox up to about the fourth day, influenza after third or fourth day, infectious hepatitis, dengue, psittacosis, scrub typhus {tsutsugamushi fever), sand fly fever (pappataci fever), sometimes in infectious mononucleosis. 3. Certain protozoa! infestations: Systemic leishmaniasis, cutaneous leishmaniasis, malaria except during a paroxysm. 4. Diseases of the hemopoietic s}stem: Aplastic anemia, agranulocyto- sis, aleukemic leukemia, relapse of pernicious anemia, chronic hypo- chromic anemia, Banti's syndrome, familial splenic anemia (Gaucher's disease). 5. Certain drugs and poisons: Sulfonamides, barbiturates, amido pyrine, benzol, dinitrophenol, thiouracil compounds, tridione, pyri benzamine, arsenic, plasmochin, quinine, Chloromycetin, salol and thio- glycolic acid, nitrogen mustards. 6. Radiation: X-rays, radium, and radiation from atomic disintegra- tion. 7. Miscellaneous conditions: Portal cirrhosis, anaphylactoid shock, nonspecific protein therapy, rheumatoid arthritis, splenic neutropenia, Felty’s syndrome, cachexia, and debilitated states. 42 Body Fluids, Exereta, ond Functional Tests - PLATELETS The Life Cycle of Platelets: Platelets are thought to originate from the megakaryocytes in the bone marrow during adult life. The mechanism of their forma tion is not clear, but apparently the cytoplasm of the megakaryo- cytes undergoes fragmentation to form platelets. Wright®* came to this conclusion in 1906 and his work has been confirmed by Runting®? and Downey.s Platelets have a short span of life of eight to nine days. In fact, all of the platelets in the peripheral blood can be replaced in three to five days, forming at the rate of approximately 100,000 per day.s? Function of Platelets: Platelets are concerned in the clotting of blood and also in clot retraction (syneresis) . Physical Function: By virtue of their adhesiveness, platelets tend to stick to such surfaces as injured endothelium and form numerous pseudopods. Thus, a mass of platelets in a strictly physical way can plug openings in capillaries. In connection with clot retraction, it has been observed that when the platclet count is low, the clot is less firm, less rigid and less adhesive than normal. Tocantins*® demonstrated experimentally that clot retraction is absent when the platelet count is 70,000 per cu. mm. or lower. It is felt, therefore, that platelets play an important role in clot retraction probably by contraction of the pseudopods thrown out by the platelets into the fibrin network.7% Capillary Fragility: Those conditions in which there is a marked dmombocytopenia are associated with increased capillary fragility resulting in capillary hemorrhages when the tourniquet test is applied. At the present time the mechanism whereby platelets maintain the integrity of capillary endothelium is not clear. Chemical Function. Platelets contain a number of active amines, among which is serotonin. This was originally discovered as the vasopressor agent released from clotted blood?9 and is now thought to reinforce local vasoconstrictive mechamusms following trauma. Hematology . 43 Platelet Coagulation Factors: The following platelet factors have been partially purified: 7 . Factor 1, a factor which resembles plasma proaccelerin also known as Factor V; Factor 2, which accelerates the formation of fibrin by the action of thrombin on fibrinogen; Factor 3, the lipoid platelet thromboplastic factor; Factor 4, an antiheparin. Factor 3, the thromboplastic factor, appears to be most important in blood coagulation. Normal Platelet Values: The counting of platelets is not satisfactory because of their small size, agglutination potential and tendency to break up Normal adults are said to have 250,000 to 500,000 per cu. mm. Figures vary depending on the technic used as indicated below: Olef?? 437,000 to 586,000 per cu. mm. Dameshek?t 500,000 to 900,000 per cu. mm, Tocantins® cutaneous blood 250,000 + 7458, standard deviation 58,500 venous blood 310,000 = 11,937, standard deviation 110,750 arterial blood 350,000 + 13,889, standard deviation 128,000 Thrombocytosis: ‘Thrombocytosis is an increase in platelets above the upper limits of normal. Due consideration must be given for the variation in different methods as shown above. Causes of Thrombocytosis: Thrombocytosis is noted in the fol- lowing: PHYSIOLOGICAL CONDITIONS: High altitudes, severe exercise, cachexia, and malnutrition. . DISEASES OF THE HEMOPOIETIG SYSTEM: Polycythemia vera, hemo- lytic anemias, acute hemorrhage, chronic myelogenous leukemia, and. chlorosis. INFECTIONS: Suppurative processes and acute rheumatic fever. MISCELLANEOUS: After surgical operations, particularly splenec- tomy, after fractures, particularly of the neck of the femur, and during asphyxia. 44 Body Fluids, Exereta, and Functional Tests Thrombocytopenit A decrease in the total platelet count below the lower limit of normal is called thrombocytopenia and is generally associated with protonged bleeding time, normal coagulation time, increased capillary fragility and reduced prothrombin consumption. This condition is found mostly in primary and secondary purpura hemorrhagica as follows: 1, Idiopathic thrombocytopenic purpura. 2. Symptomatic thrombocytopenic purpura caused by the following: Chemical and physical agents, acute and chronic leukemias; hypo chromic, aplastic, pernicious, and myclophthisic anemias; certain infec tions, such as septicemia, typhoid fever, early pneumonia, diphtheria and bacterial endocarditis; hemolytic jaundice; Banti’s disease; familial splenic anemia (Gaucher's disease); and on the first day of menstruation COAGULATION OF BLOOD ‘The clotting of blood appears as a very simple reactton, but it is in reality a very complex process. The most widely accepted concept is that it @kes place in three steps or stages: Stage 1 Interaction of platelet fac- tor, antihemophilic factor Calcium and Plasma (AHF), plasma thrombo + accesiory > thromboplastin platin component (PTC) factors Stage 2 Prothrombin -+ Thromboplastin, calcium and ~ Thrombin accessory Jactors Stage 3 Fibrinogen’ + Thrombin 2 Fibrin {blood dot) Clotting Factors: Confusion in terminology is one of the major problems in studying blood coagulation. Dilferent investigators have given different names to the same factor. Following is a list of international numbers with synonyms: Hematology 45 Factor F Fibrinogen Factor 1 Prothrombin ° Factor HI = Thromboplastin (tissue) ‘Thrombokinase Factor IV) Calcium Factor V Labile factor Proaccelerin Plasma Ac globulin Factor VE Serum Ac globulin Accelerin Factor VIL Stable factor Proconyertin Serum prothrombin conversion accelerator (SPCA) Co-thromboplastin Autoprothrombin I +Factor VIL Antihemophilic factor (AHF) Thromboplastinogen Platelet co-factor I Plasma thromboplastic factor A Factor IX Plasma thromboplastin component (PTC) Christmas factor Platelet co-factor J] Plasma thromboplastic factor B Autoprothrombin JI Factor X Stuart-Prower factor Factor XI Plasma thromboplastin antecedent (PTA) Factor XII Hageman factor Following is a description of the nature of the various factors: FACTOR I. FI8RINOGEN: This is a molecule of large molecular weight, in plasma but not in serum. Fibrinogen is changed to fibrin by the proteo- lytic action of thrombin. FACTOR 11. PROTHROWBIN: This is a stable plasma glycoprotein which is converted to thrombin by thromboplastin, calcium and certain other factors. It is virtually absent from normal serum. . FACTOR Il. TISSUE THROMBOPLASTIN: Tissues which show the greatest thromboplastic activity are brain, ]ung and placenta. Tissue thrombo- plastin can be fractionated into a heat-stable lipoid substance which seems closely related chemically to platelet factor 3, and a heat labile protein which seems to resemble the product of the interaction of AHF, PTC, PTA and other plasma factors.75 FAcToR Iv. CALGIUM: Calcium secms to be required in many phases of the clotting process. Calcium and accessory factors are necessary for 46 Body Fluids, Excreto, ond Funetional Tests the completion of stages 1 and 2 of blood clotting. Details of the inter- actions which take place must await further research. FAGIOR V. PROACCELERIN: This has the character of 2 globulin which is necessary for the formation of thrombin from prothrombin by throm. boplastin and calcium. One group in ute United States has called it plasma accelerator globulin,?® while another has called it labile factor.?7 Ta Australia, Fantl and Nance’® designated it accelerator substance, and in Norway, Owren?? used the term Factor V. It disappears rapidly during coagulation, hence fresh serum contains virtually none. FACTOR vi, AccILIRIN: This facto. was named aceelerin because it was considered to be the active form of proaccelerin, Factor V. TAGTOR VII, PROCONVERTIN: This is a stable B globulin which is found in both plasma and serum. Owren?? described it as Factor VII, Ware and Scegers*? as serum Ac-globulin and Alexander®? as prothrombin con- version accelerator, It can correct the prolonged prothrombin times of certain patients treated with coumarin, of infants during the neonatal period, of those with liver cell damage, and of those rare patients who have a congenital Factor VII deficiency. FACTOR Vill. ANTIHEMOPHILIG rAGroR (Ar): This factor is 2 glebulin which corrects the clotting in true hemophiliacs. It is normally present in plasma and is used up in the process of clotting, hence is not present in serum. FACTOR 1X. PLASMA THROMBOPLASTIN COMFONENT: This factor is 2 betas globulin found sn normal serum and plasma. It corrects the clotiing defect in Christmas disease and is also known as Christmas Tactor FACTOR X. STUART-PROWER 1 AcToR‘®} This is an alpha globulin which ip found in both serum and plasma. Ie is necessary for the formation of thromboplastin and the action of tissue thromboplastin in converting prothrombin to thrombin. FACTOR XI. PLASMA ‘THROMBOPLASTIN ANTECEDENT (FTA): This is a globulin which is present in normal serum and plasma. The lack of this globulin causes a hemophiha-like disease, but it oceurs in esther sex FACTOR Xit M1AGEMAN FACTOR:64 This 15 a2 heatlabile globulin witch is present in both serum and plasma. The lack of this factor causes no clinical symptoms. Thrombin: Thrombin is formed from prothrombin by the acuon of thromboplastin, calcium and certain other factors. [t appears that plasma thromboplastin reacts with calcium and Factor V (proac- celerin) to form an unstable product, prothrombinase, which can carry aut the final change of prothrombin to thrombin in the Hematology 47 presence of calcium. From the reaction of tissue thromboplastin, calcium, Factor V, Factor VII and Stuart Factor an apparently similar prothrombinase is formed. Thrombin not only forms fibrin from fibrinogen, but it speeds up the disruption of platelets and activates Factor V, thus catalyzing the first two stages of blood coagulation. Fibrin: Fibrin is formed from fibrinogen which acts as the substrate for the proteolytic action of thrombin. Fibrin strands are then formed by polymerization.85 TESTS FOR HEMORRHAGIC DISORDERS Platelet Count: The platelet count yields information which may be of help in evaluating a hemorrhagic condition, In addition to counting platelets, they should be evaluated qualitatively, ¢.g., for the presence of giant forms. Both thrombocytosis and thrombocytopenia may be associated with a hemorrhagic disorder. If the platelet count is sufficiently reduced, there 1s usually an associated prolonged bleeding time, increased capillary fragility and poor clot retraction. However, the clotting time is within normal limits, and prothrombin con- sumption is reduced. Coagulation Time: Coagulation time is the measurement of the clotting of blood in vitro without tissue juices. This may be done in several ways, but the same technic and the same caliber glassware should be used each time. Capillary-Tube Method: A common technic is the capillary-tube method. Five pieces of capillary tubing are filled with fresh blood from a deep cut. A section of tube is broken off at each one-half. minute interval. The end-point is the appearance of a thin thread of fibrin between the fragments as they are pulled apart. The nor- mal coagulation time by this method is from three to seven minutes. Lee and White Method: Another popular method is that of Lee and White, in which 1 ec. of blood is placed in a tube with a di- ameter of 8 mm. (%o inch). The tube should be absolutely clean 48 Body Fluids, Excreta, and Functional Tests and should be rinsed with isotonic saline solution, It is advisable to use at least three tubes so chat one tube is undisturbed while the others are being examined. The test is best carried out at 98.6° F, (37° C) The normal range by this method is from six to 15 minutes. A normal coagulation time does not exclude a clotting defect and does not prove adequacy of plasma thromboplastin formation. The coagulation time may be shortened by the accidental inuo duction of tissue fluids, rapid passage of blood through the needle, introduction of air bubbles, or unclean or rough glassware. It may be prolonged by very low or high pH, very low or high temperature, the use of ordinary plastic, silicone, or paraffin-lined tubes, or after administration of hepain. In a silicone tube the normal range 1s 19 to 60 minutes, The coagulation time is prolonged under the following condi- tions: Hypoprothrombinemia in which there is a deficiency of Factors Il, V, VII or X, vitamin K deficiency or inability to absorb it, Hemophilia, Factor VIII deficiency. Plasma ‘thromboplastin component deficiency, Factor XI deficiency. Hageman wait, Factor XII deficiency. Fibrinogenopenia or afibrinogenemia. Hyperheparinemia in heparin therapy, peptone and anaphy lactic shack. 2 Circulating anticoagulants against factors V, VIII and 1X Clot-Retraction Time: Determinalion: Determination of clot-retraction time may be made by placing I cc. of venous blood in each of three small rubes (8 mm. [3%o inch] in diameter) and § cc, in a fourth, All tubes are placed in a 98.6° F. (87° C.) incubator and inspected (not handled) after one-half, one, two, four, and 24 hours for evidence of retrac- tion from the side, There is retraction of normal blood in from 30 to GO minutes. Hematology . 49 Clot retraction is prolonged and is not satisfactory in the follow- ing conditions: Primary thrombocytopenia. . Thrombocytopenia secondary to aplastic anemia, pernicious anemia, hemorrhagic disease of the newborn, acute leukemias, multiple mycloma and malignant granuloma (Hodgkin's disease) of the bone marrow. Bt Fig. 4: Clot retraction. A: Normal retraction of clot, B: No retraction of lot, Thrombasthenia in which the platelet count is normal, but there is imparred platelet aggregation. Development of fibrinolysin in shock may confuse the result by dissolution of the clot. $0 Body Fluids, Excreta, and Functional Tests Factors Influencing Glot Retraction: (1) A decrease in the amount of fibrinogen results in the formation of a small clot which gives the impression of normal clot retraction. (2) A decrease in the number of red cells gives the impression of normal clot retraction. (3) An increase in the number of red cells (polycythemia) results in an apparently poor clot retraction, since the clot is large and the amount of clear serum is small. Dissolution of Clot: Fibrinolysins may be detected in whole blood or im plasma by observing shrinkage of a clot kept at 37° C. for 24 hours. A normal clot docs not change in this time. Fibrinolysins da not usually produce a severe bleeding disorder unless they can cause lysis of a clot in two hours or less, An increase in fibrinolysins may be found in major surgery of the lung, prostate or uterus, shock, premature separation of the placenta, death of fetus in utero, advanced hver disease and carcinomatosis, Fibrinogen Level: ‘The normal plasma fibrinogen level is 150 to 300 mg. per 100 cc. Blood congulation is not usually defective unless the plasma fibrinogen level is less than 100 mg. per 100 cc. Clinically significant reduction of fibrinogen levels fs seen in abruptio placentae, retention of a dead fetus, amniotic fluid embolism, congenital afibrino genemia, liver discases, severe malnutrition, typhoid fever, marked anemia and for a short time after extensive hemorrhage. Bleeding Time: ‘The normal bleeding time is one to three minutes. The bleeding time is protonged in the following conditions: Primary thrombocytopenia. Thrombocytopenia secondary to pernicious anemia, aplastic anemia, hemorrhagic disease of the newborn, acute lerhemias, chronic lymphocytic leukemia, multiple myeloma and Hodghin’s disease of the bone marrow. Vascular hemophilia. Thrombocytopathia, a condition in which there is a decrease in platelet factor 3. Hematology 51 Value of Bleeding and Clotting Time: The concept of the value of bleeding and clotting time has undergone a change. With the availability of more specific tests, bleeding and clotting times as screening tests have little to offer except in severe disorders of blood clotting. Capillary Fragility Test: Determination: The capillary fragility test is also Known as the “tourniquet test.” A blood-pressure cuff is applied above the elbow, and the pressure is maintained at a point just above the diastolic pressure. In a positive test, a large number of tiny hemorrhages develop within a few minutes. The test is positive in idiopathic thrombocytopenic purpura and in some cases of secondary thrombocytopenic purpura, scurvy, scarlet fever, measles, influenza, achylia gastrica, vitamin-K defi- ciency, and chronic nephritis. Prothrombin Determination: Schofield’s8* discovery of the part played by spoiled sweet clover in hemorrhagic disease of cattle was followed by isolation and synthesis of the active principle, dicoumarin. Dicoumarin under various trade names, such as Dicumarol, now has an important role in the prevention and treatment of coronary thrombosis, phlebothrombosis and thrombophlebitis. In such cases, the blood- clotting mechanism is too efficient. The use of Dicumarol is an attempt to reduce the efficiency of this mechanism to the lower limit of safety. This is done by giving an initial dose of from 200 to 300 mg. (3 to 414 grains) of Dicumarol, which does not become effective for 48 hours. A prothrombin time or prothrombin activity (Fig. 5) is determined at this time to establish a base line, or normal, for the patient. To eliminate confusion, the prothrombin time only is re- ported in seconds, The complete report should inclide the pro- thrombin time of the control as well as that of the patient. The range of the normal prothrombin time is usually from 12 to 14 seconds. In view of the fact that a difference of a few seconds in pro- thrombin time is indicative of wide differences in prothrombin 52 Body Fluids, Excreto, ond Functional Tests concentration, procedures have been developed in which the plasma is diluted, thus creating a longer coagulation time and making it easier to detect minor variations in prothrombin concentration. The optimal dilution appears to be 12.5 per cent plasma in isotonic saline solution, and this is said to increase the sensitivity and relia- bility of the test. The normal range using diluted plasma according to the method ot Shapiro4? is fiom 37 to 42 seconds ® | | TT coe 1 je -—— 4 i Fig. 5: Prothrombin activity curve for plasma with prothrombin time of 12 seconds, Curve is determined by tests on serial dilutions of plasma. (Quiek, A. Ja Am, J. Clin. Path., 10:222, 1940.) The prothrombin time is prolonged in the following conditions: Treatment with anticoagulants which reduce prothrombin Factors Vil and X. Parahemophitia in which there isa deficiency of Factor V. Serum prothrombin conversion accelerator (SPCA), Tactor VII deficiency. Hemorrhagic disease of the newborn, Liver disease in which there 1s suficient reduction of Factors V, VI and X and fibrinogen. Circulating antiprothrombin and anuthromboplastin Hematology 53 Sources of Error: The physician as well as the technician should be aware of the sources of error in determining the prothrombin time. I. Fresh plasma should be used. Plasma allowed to stand at room or refrigerator temperature for more than two or three hours may give clotting times slightly higher than fresh plasma. 2. Hemolysis of the specimen of blood. 3. Inadequate amount of blood resulting in an excess of the antico- agulant. 4. The use of old thromboplastin extract. It should be used within four hours after preparation. Prolonged aging results in a loss of potency and prolonged clotting time. 5. Metallic fons, such as copper, in the distilled water used in mak- ing the saline solution for extraction of the thromboplastin may lead to prolonged clotting time, 6. Failure to recognize the end-point. 7. Glassware must be chemically clean. Soaps and other detergents used in cleaning glassware, if net properly rinsed off, will tend to give prolonged clotting time. 8. The calcium chloride, sodium chloride, sodium oxalate, potassium oxalate, and ammonium oxalate must be anhydrous and of reagent grade. Prolonged clotting times have been traced frequently to faulty calcium chloride. {: should be used in an anhydrous state. 9. Failure to mix the reagents thoroughly with the plasma at the time of addition may result in a prolonged clotting time. 10, The storage of thromboplastin at room temperature may result in slight loss of potency in one to two months. It is, therefore, advisable to store it in the refrigerator. IL. The use of improper technic, such as improper use of the pipet, neglecting to blow into it after each transfer, and neglecting to wipe the outside of the pipet at each transfer. Prothrombin Consumption Test:* The prothrombin consumption test is really a measure of the available plasma thromboplastin. By determining the prothrombin before and after coagulation is complete one obtains a measure of the thromboplastin that reacts with prothrombin. 54 Body Fluids, Excreta, and Functional Tests Prothrombin consumption and thromboplastin generation are reduced in the following conditions: Hemophilia in which there is a deficiency of Factor VIE. Deficiency of Factors V, IX, X, XL or XL, Thrombocytopenia with impaired thromboplastin formation, Thrombocytopathia, a condition in which thee is a deficiency of platelet factor 3. Circulating anuithromboplastin Thromboplastin Generation Test (TGT);**™ This test is of value in differentiating blood coagulation abnormalities in which there is deficient formation of thambo. plastin. Such abnormalities are found in hemophiha with deficiency of Factor VIII (AHG), deficiency of Factor IX (PTC) and in abnormality of platelet function. It is based upon the fact that normal plasma treated with aluminum hydroxide which contains Factor VHI (AHG), Factor V (proaccelerin), Factor XI (PTA), Factor XII (Hageman Factor), platelets plus normal serum which supphes Factors IX (PFC) and X (Stuart-Prower Factor) as well as Factor XI (PTA) and XII (Hageman Factor) react to form thromboplastin in the presence of calaum chloride. A Jack of any ol these seven factors will cause abnormal thromboplastin formation 1, When patient's adsorbed plasma, normal serum and normal plate- lets are incubated, then deficiency of Factor V or VIII is present. They are differentrated by the prothrombin time. I{ prothrombin time 1s pro- longed, Factor V deficiency exists, but not Factor VIII deficiency 2. When patient's serum and normal adsorbed plasma and normal platelets are incubated, then deficiency of Factor IX or X is present. Differentiate by prothrombin time which is normal in Factor IX defi- erency and prolonged in Factor X deficiency. 3. Patient's adsorbed plasma plus patient's serum: and patient's or normal platelets are incubated, then deficiency of Factor NJ or XII is present . 4, When patent's platelets plus normal adsorbed plasma and normal serum are incubated, thrombocytopenia or thrombocytopathia is present. Hf the platelet count 1s normal, the cause 1s thrambocytopathia. Hematology 55 Partial Thromboplastin Time (PTT): This is an overall screening test of the total coagulation mechanism except for platelet factor deficiency and Factor VII deficiency. It is based on the observation that with whole thrombo- plastin (saline extract of rabbit brain) the results obtained with hemophilic plasma are the same as with normal plasma, while with partial thromboplastin (cephalin) the results with hemophilic plasma are much longer than those with normal plasma. In carrying out the test, a standardized amount of platelet-like reagent is added to plasma. The reaction is initiated by the addition of calcium chloride, and a stopwatch is started simultaneously. The tube is incubated in a 37° C. waterbath for 30 seconds and is then checked periodically for clot formation. The normal range is 40 to 100 seconds. NEW TERMINOLOGY OF BLOOD CELLS The first two reports*!. 92 of the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood- Forming Organs recommend certain changes in names and defini- tions of blood cells. It seems advisable to give a summary of these recommendations here with the hope that it will help to promote a universal adoption of some form of standard nomenclature. The following terms are recommended for the cells of the differ- ent series and for diseases affecting any cell of that series: Lympho- cytic, granulocytic, monocytic, plasmacytic, thrombocytic, and erythrocytic. Table 9 gives recommended terms for each of the white cells and thrombocytes. The definitions decided on are as follows: Lymphoblast: Any cell of the lymphocytic series having fine chromatin struc- ture in the nucleus. Cells of blast morphology associated with pro- lymphocytes should be tentatively classified as lymphoblasts. Prolymphocyte: Any cell of the lymphocytic series intermediate in morphology 56 Body Fluids, Exereta, and Functional Tesis Taste 9: RecoMMENDED Terms AnD TERMS TO BE AVOIDED WHEN REFERRING TO SreciFic CELLs oF BLoop AND Bioop-Forminc OrcANS eee Nase of Series| Term to be Used Terms to be Avoided Lymphocytic. .} Lymphoblast Myeloblast, hemocytoblast, Iymphoidocyte, stem cell, lymphocyte Prolymphocyte Large lymphocyte, pathologie large lympho- eyte, atypical leukocytoic lymphocyte, monocyte, immature lymphocyte Lymphocyte Small, medium, or large lymphocyte, normal lymphocyte, smalt, medium, or large mononuclear Monocytic. ...| Monoblast Myeloblast, hemocytoblast, lymphoidocyte, lymphocyte, stem cell, immature monocyte Promenocyte Premonocyte, hemohistioblast, immature monocyte, Ferrata cell Monocyte Large mononuelest, transitional, plasmato- oyte, endothelial leukocyte, histtocyte, resting wandering cell Granulocytic. .| Myeloblast Granuloblast, hemocytoblast, lymphoidocyte, lymphocyte, stem cell Progranulocyte Promyclocyte Il, levkoblast, myeloblast, premyelocyte, promyelocyte, progranulo- eyteA Myelocyte Granulocyte, myelocyte B, nonfilament, class I eS Hematology 57 TABLE 9: (Continued) Name of Series| Term to be Used Terms to be Avoided Granulocytic. | Metamyelocyte Metagranulocyte, juvenile, myelocyte C, Continued nonfilament, ¢elass I Band cell Staff cell, stab cell, nonfilament, class I, rod nuclear, polymorphonuclear, stabkernige, thabdocyte, nonsegmented Segmented Polymorphonuclear, filamented, class IT, Ii, IV, or V, loboeyte Plasmacytic. ..]| Plasmablast Myeloblast, hemocytoblast, lymphoidocyte, lymphocyte, stem cell, lymphoblastic plasma cell, myeloma cell Proplasmacyte Tork cell, Turk irritation form, lympho- blastic or myeloblastic plasma cell, myeloma cell Plasmacyte Plasma cell, Unna’s plasma cell, Marschalko’s plasma cell, plasmacytoic lymphocyte, myeloma cell Thromboeytic | Megakaryoblast | Megalokaryoblast Promegakaryocyte | Premegalokaryocyte Megakaryocyte Megalokaryocyte Thrombocyte Platelet, thromboplastid Disintegrated cell Senile cell, smudge, basket cell, smear cell, degenerated cell 58 Body Fluids, Exereta, ond Functional Tests between the lymphoblast and the lymphocyte. It will always have toa coarse a chromatin structure to fit the criteria for a blast and to0 fine a chromatin structure or too large a cell diameter to be classed as a lymphocyte. Usually, but not always, prolymphocytes are larger than 15 microns in diameter, which is the upper limit for the lymphocyte. Lymphocyte: Any cell of the lymphocytic series having the morphology of those commonly found in the blood of healthy adults. Monoblast: Any cell of the monocytic series having fine chromatin structure, Usually nucleoli are visible. Cells of blast morphology found in association with promonocytes should be tentatively classed as monoblasts. Promonocyte: Any cell intermediate in morphology between the monoblast and the monocyte. It is differentiated from the monoblast by having an irregularly shaped nucleus and somewhat coarser chromatin struc- ttre, and from the monocyte by the presence of one or more nucleoli. Monocyte: Any cell of the monocytic series having the morphology of these commonly found in the blood of healthy adults. Ic is differentiated from the promonocyte by the absence of nucleoli, Myeloblast: Any cell of the granulocytic series having fine chromatin stru ture and containing no specific granules. Usually nucleoli are vi ble. Cells of blast morphology found in association with progranu- locytes should tentatively be classed as myeloblasts. Progranulocyte: Any cell of the granulocytic series which has a nuclear structure too coarse for that of a blast cell and which has not yet developed Hematology 59 discernible, specific granules. This term was selected rather than “promyelocyte” because of its clear relationship to the definition of granulocyte, given below, and because the term “promyelocyte” has been in wide use for cells which do contain specific granules. The reason that the terms granuloblast, granulocyte, and meta granulocyte were not chosen was that the terms myeloblast and mye- locyte were already in general use with essentially the definitions here given. This is true also for the term granulocyte, which would otherwise have to be synonymous with the term myelocyte. Specific Granules: Neutrophilic, eosinophilic, or basophilic granules. This term does not include azurophilic granules. Granulocyte: An inclusive term to apply to any cell containing specific gran- ules. The plural form “granulocytes” would therefore include all myelocytes, metamyelocytes, band cells, and segmented cells whether neutrophils, eosinophils, or basophils. Myelocyte: Any cell containing specific granules, with a round or oval nu- cleus. It is distinguished from the progranulocyte by the presence of specific granules and from the metamyelocyte by the absence of indentation in the nucleus, It may be further subdivided, at the option of the user, into early and Jate stages, but the definition of early or late should be clearly stated in any publication. This and all subsequent cells of the granulocytic series should be additionally characterized as neutrophil, eosinophil, or basophil. Metamyelocyte: Any cell of the granulocytic series having specific granules in the cytoplasm and a nucleus intermediate in shape between that of the myelocyte and the band cell. The nucleus usually has an in- dented oval shape, resembling 2 bean or kidney. Band Cell: Any cell of the granulocytic series which has a nucleus that could be described as a curved or coiled band, no matter how 60 Body Fluids, Exereta, one Functional Tests marked the indentation, if it does not completely segment the nucleus into lobes connected by a filament, It is differentiated from the metamyelocyte by an appreciable length of the nucleus having parallel sides, and from the segmented neutrophil by having no indentation which could be described as a filament. Segmented Cell: Any cell containing specific granules in which the lobes of the nucleus are connected by a filament. A filament is defined as a thread-like structure, Since at times, in viewing a three-dimensional object from one direction, it is impossible to be certain whether two parts of the nucleus are connected by 2 filament or band, it is suggested chat such cells always be placed in the segmented category since this is the more differentiated and more common cell. The term “toxic neutrophils,” followed by 2 1 to 4-4 designa tion, is recommended for the grading of toxic granules, basophilia of the cytoplasm, vacuoles, and condensation of nuclear chromatin in the neutrophils, since its meaning is clear, although it is recog- nized that it is not an adequately descriptive term. The grading should depend more on the degree of change than on the percenage of the cells involved and should be recorded in the report whenever the degree of change exceeds 2--. Plasmablast: Any cell of the plasmacytic series having fine chromatin structure in the nucleus. Cells of blast morphology found in association with proplasmacytes are usually seen only in plasmacytic leukemia or plasmacytic sarcoma. The cytoplasm tends to be more opaque in staining than in the other Icukocytic blast cells. Proplasmacyte: Any cell of the plasmacytic series with a nuclear structure too coarse for that of a blast cell but with one or more nucleoli present. Plasmacyte: A cell characterized by extremely coarse chromatin structure, Hematology 61 with the deeply staining chromatin of the nucleus aggregated into large, sharply demarcated clumps. It is differentiated from the pro- plasmacyte by the absence of nucleoli. The cytoplasm of all cells of the plasmacytic series tends to be deeply basophilic and opaque in appearance. Azurophilic granules may be present or absent, but are more commonly absent. Megokaryoblast: Any cell of the thrombocytic series having a nucleus with fine chromatin structure. Usually these are larger than the other blast cells. Promegakaryocyte: Any cell of the thrombocytic series with a nucleus containing nucleoli but having a chromatin structure too coarse for a blast cell. The nucleus is usually similar in shape to that of the megakaryocyte. Fine azurophilic granules are usually diffusely scattered through the cytoplasm. Megakaryocyte: Any nucleated cell of the thrombocytic series in which nucleoli are not discernible, The azurophilic granules are often aggregated into clumps. Megakaryocytes and promegakaryocytes are typically much larger than other cells found in the marrow. Thrombocyte: Any cell of the thrombocytic series containing no nucleus; in other words, any non-nucleated fragment of megakaryocytic cyto- plasm containing azurophilic granules similar to those of the mature megakaryocyte. The term “thromboplastid” was recognized as being anatomi- cally correct, but it was felt that to be consistent with the use of the term “erythrocytic” and to permit the use of “thrombocytic” and “erythrocytic” in describing these cell series, the suffix “cyte” was preferable for these two non-nucleated forms. 62 Body Fluids, Excreta, and Functional Tests Disintegrated Cell: Any cell of any series in which the cytoplasmic outline has been disrupted or the nuclear chromatin is no longer surrounded by a membrane, excluding the changes in the nucleus that occur in mitotic division. Disintegrated cells should be recorded as such in the differential report, even though they could be identified by dispersed granules. They should be counted even if only shreds of nuclear material are discernible, since they are undoubtedly in- cluded in the total leukocyte count. Table 10 gives the recommended terms for cells of the erythro- cytic series. ‘The definitions of cells of the erythrocytic series are as follows: Rubriblast: Any cell of the erythrocytic series having fine chromatin struc- ture in the nucleus. Nucleoli are usually discernible. A stippled chromatin pattern is more common than the lace-net pattern usually seen in other blast cells. Tate 10; RecomMenpen Terms anp Terms To BE Avoipep Wuen Rererring To Speciric Cetrs oF EryrHrocytic Series Name of Series | Term ta be Used Terms to be Avorded Erythroeytic | Rubriblast Crythroblast, megaloblast, pronormoblast, promegaloblast, normoblast, hemocyto- blast, stem cell, myeloblast, lymphordo- eyte, karyoblast. Prorubricyte Erythroblast, megaloblast, pronormoblast, normoblast, macronermoblast, macrablast, prokaryocyte Normoblast, pronormoblast, macronormo- blast, erythroblast, polychtomatophilic wormoblast, karyooyte Metarubrieyte Normoblast, erythroblast, metakaryocyte Reticulocyte* Erythrocyte Red blood cell, erythroplastid, normocyte, akaryocyte "Tt is recommended that the reticulocyte stage be considered a subdivision of the erythrocyte stage. Hemotclogy 63 Prorubricyte: Any cell of the erythrocytic series in which one or more nucleoli are discernible in the nucleus and which has a chromatin structure tao coarse to be classified as a rubriblast. Rubricyte: Any cell of the erythrocytic series having definite structure of the nuclear chromatin, but containing no discernible nucleoli. This stage is differentiated from the prorubricyte by the absence of nucleoli in the nucleus, and from the metarubricyte by net having a pyknotic, fragmented, or partially extruded nucleus. Some may wish to subdivide and qualify this stage—or other stages—further into basophilic, polychromatic, or normochromatic rubricytes, ac- cording to the amount of hemoglobin present in the cytoplasm. Metarubricyte: Any nucleated cell of the erythrocytic series having a pyknotic, fragmented, partially extruded, or partially autolyzed nucleus. Pyk- notic describes a dense, solid, structureless nuclear mass. The phe- nomenon of karyorrhexis, or fragmentation of uuclei, should be clearly distinguished from the occurrence of double, well-formed nuclei, which are occasionally seen in prorubricytes and rubricytes, as well as in other cells which may divide amitotically. Reticulacyte: Any non-nucleated cell of the erythrocytic series in which, when supravitally stained—usually with brillant cresyl blue—one or more granules or a diffuse network of fibrils are discernible. All reticulo- cytes are included under the term “erythrocytes” since, without a special stain, reticulocytes are indistinguishable from erythrocytes. Erythrocyte: Any non-nucleated cell of the erythrocytic series. Pemicious-Anemia Type: The qualifying adjective phrase to be applied to any cell of the erythrocytic or granulocytic series, and to the marrow and blood 64 Body Fluids, Exereta, and Functional Tests pictures as a whole, to indicate the presence of the morphologic changes characteristically seen in pernicious anemia and other macrocytic anemias which respond to liver extract or folicacid therapy. In the nucleated cells of the erythrocytic series, the major feature of this change is a relative increase in the palestaining para chromatin with a corresponding decrease in the deep-staining basi- chromatin. In the cells of the granulocytic series, the characteristic change is the presence of giant forms having very bizarre nuclei; and in the segmented neutrophils, the occurrence of many cells with more than five lobes, REFERENCES 1, Dacie, J. V. and White, J. C.: Exythropoiesis with Particular Refer- ence to Its Study by Biopsy of Human Marrow. J. Clin. Path, 2:1, 1949. 2. Brannan, D.: Extramedullary Hematopoiesis in Anemias. Bull Johns Hopkins Hosp., 41:104, 1927. 3. Lyall, A.: Massive Extramedullary Bone Marrow Formation in a Case of Pemicious Anemia. J. Path. & Bact., 41:469, 1935. 4. Meyer, E. and Heineke, A. Uber Blutbildung in Milz und Leber bei schweren Anamien. Verhandl. d. deutsch pat. Gesellsch, 9:224, 1905. 5. Wintrobe, M, M.: Relation of Discasc of the Liver to Anemia. Arch Int. Med., 57:289, 1936, G. Jordan, H. E.: Extramedullary Blood Production. Physiol. Rev., 22:375, 1942. 7. Blaisdell, J. L.: Extramedullary Hematopoiesis in a Retroperitoneal Tumor. 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