Laboratory

session
Light
microscopy of
WBC and
Erythroid cells

Eldad J.Dann MD
WBC Differential Cytochemistry
Comparison of Wright Stain and Peroxidase Stain

E M
L
L = Lymphocyte
N B = Basophil
N
M M = Monocyte
B
N
N = Neutrophil
N
L N E = Eosinophil
L

Wright Stain Peroxidase Stain

 Intracellular Peroxidase
Flow Cytometric Analysis

1
5
SCATTER (Size)

3
4

ABSORBANCE
 eos

Monocytes
lymphocytes
Neutrophils
MN PMN
MCV mean corpuscular volume 80-94 fl (10-15( L
MCH mean corpuscular hemoglobin 27-31 pg (10-12(gm
MCHC mean corpuscular hemoglobin concentration
32-36 gm/dl
% RDW- 11.5-14.5
Example: Hct 45% RBC 5x106/ul Hb 15 gm/DL
MCV= 450(L)x1000/ 5 X1012(L)= 90 fL
MCH= 150gm/L /5 X1012(L)= 30 pg
MCHC= MCH /MCV =(15 gm/DL)/ 0.45= 33g/DL
 RBC Analysis
High angle detector
(5o - 15o)

670nm
Laser
Diode

Low angle detector

(2o-3o)
RBC Cytogram and Histograms

Macro 0 60 120 200 fL

RBC Volume (V)
RBC Volume (fL)

120fL

60fL
28g/dL 41g/dL 0 28 41 50 g/dL
Micro Hgb Concentration (HC)

Hypo Hyper
Hgb Concentration (g/dL)
0 100 pg
Hgb Content (CH)
Normal Sample: MCV > 80 fL
 Sickle Cell Anemia

• MCV < 80 (78.5) and M/H Ratio > 0.9 (1.5)

• Broad distribution width for RBC Volume and HC histograms
• Presense of Hyperchromia suggestive of sickle cell anemia
Criteria for performance of
manual differential count
Lymphocyte > 50%

Large Unstained Cells (LUC) > 7%

WBC Flags > 3

Left Shift > +

Making Blood Film on Slide
Schematic drawing of blood film made on a slide

The film has been spread from left to right.

An indication is given of the way the white blood cells are distributed
Different Qualities of Blood Films Made on Slides

A B C D E
 ‫צביעת משטחים של תאי דם במעבדה ההמטולוגית‬
‫מרכז רפואי רמב"ם‬
‫שימוש במכשיר‪:‬‬

‫‪AUTOMATED SLIDE STAINER HEMA-TEK‬‬

‫צביעת המשטחים מבוססת על עקרונות צביעת ‪ Ramnovsky‬הכוללת‬
‫‪ :Azure B (trimethylthionin‬חומר צבע המגיב עם מרכיבים‬
‫חומציים‪ ,‬בעיקר עם חומצות הגרעין וכן עם מרכיבים חומציים‬
‫בציטופלסמה‪.‬‬
‫‪ :Eozine (tetrabromo fluorescein‬חומר צבע המגיב עם מרכיבים‬
‫בסיסיים‪ ,‬בעיקר עם ההמוגלובין שבתאים האדומים‪.‬‬
‫תערובת הצבעים מומסת במתנול הגורם לפיקסציה של התאים‬
‫למשטח ושמירה על צורתם‪.‬‬
Chronic
Myelogenous
Leukemia
WBC
100,000/UL
Leukocytosis
Lymphocytosis
REACTIVE LYMPHOCYTES
Leukopenia & Neutropenia
Thrombocytopenia
Anemia
Increased No of Large
Unstained Cells
 High nuclear to
Blasts cytoplasmic ratio
very fine chromatin lace
like appearance
nucleoli are seen
cytoplasmic granules
when blast are from
myeloid
Cytoplasm has typically
gray ground glass
appearance with fine
azurophilic granules.
Rather prominent
cytoplasmic vacuoles
AML on the ADVIA 120
AML M3

Manual 90% Blasts (Promyelocytes)

ADVIA 120 66% Blasts
3+ BLASTS Flag, 3+ IG Flag
WBC 40,000 cells/µL
Iron deficiency anaemia
B12 deficiency anaemia
Megaloblastic
Anemia
 Autoimmune Hemolytic
Increased
Anemia
reticulocyte count
High MCV
High serum lactic
dehydrogenase
((LDH
High billirubin
Haemolytic anaemia
Haemolytic anaemia – red cell fragmentation
 Sickle cell anaemia and refractory anaemia and excess of blast

Blood 2007;109:858

Copyright ©2007 American Society of Hematology. Copyright restrictions may apply.

 Microangiopathic
Anemia

Helmet cells due

to intra vascular
microangiophatic
dammage
High LDH
Low platelet
count
Sepsis
Chronic Myeloid Leukemia - CML
Acute Lymphoblastic Leukemia - ALL
Acute Promyelocytic Leukemia - APL
Acute Myeloid Leukemia - AML
Myeloperoxidas Staining positive reaction in blast cells - AML
Myeloperoxidas Staining negative reaction in blast cells - ALL
Reticulocyte staining
Peripheral-Blood Smear Showing Abnormalities Characteristic of Thrombotic Thrombocytopenic
Purpura.

George, J. N. N Engl J Med 2006;354:1927-1935

 Miscellaneous Conditions in Which the Blood Smear Can Be Diagnostically Important.

Bain, B. J. N Engl J Med 2005;353:498-507

A 24-year-old man presented with a three-day history of a blurred patch
near the center of his left visual field. He reported no fatigue or weight
loss. Ophthalmoscopy showed venous dilatation and multiple Roth's
spots in both retinas (arrows in Panel A, showing the fundus of the left
eye). Physical examination revealed splenomegaly extending to the right
iliac fossa. No fever and no cardiac murmurs were noted. When blood
was examined to measure the erythrocyte sedimentation rate, an
elongated buffy-coat zone was observed (Panel B, bracket); a similar
examination of blood from a healthy person is shown on the left. The
patient's leukocyte count was 303,000 per cubic millimeter. The blood
smear (Panel C) and the morphologic features of the bone marrow were
consistent with chronic myeloid leukemia in the chronic phase. The
patient underwent urgent leukapheresis for the symptoms of
hyperviscosity and was treated with hydroxyurea. His ophthalmic
symptoms resolved, and there was a reduction in the leukocyte count. He
subsequently underwent allogeneic hematopoietic stem-cell
transplantation and is doing well. Roth's spots, or white-centered retinal
hemorrhages, may be seen in a variety of conditions, including leukemia,
subacute bacterial endocarditis, ischemic events associated with elevated
venous pressure, and systemic vascular conditions with capillary
fragility.
 Roth
spots

Varga, Z. et al. N Engl J Med 2005;353:1041

 Spirochetes on a Blood Smear.

Sill, H. et al. N Engl J Med 2005;353:1862

A 31-year-old woman, 33 weeks pregnant, presented with a fever
(40{degrees}C) and chills 2 months after returning from a trip to
Brazil. A mixed infection of Plasmodium malariae and P. falciparum
was diagnosed and treated with quinine. She had a relapse one
month after the diagnosis, and labor was induced because of fetal
distress. A review of both sets of smears led to a revised diagnosis of
P. vivax, confirmed by polymerase-chain-reaction testing. In the
current smear, a ring form is seen within a red cell with basophilic
stippling, or what are known as Schuffner's dots (arrow), and also
several serpentine forms, which were identified as P. vivax
microgametes (arrowhead). Because parasites were seen in cord
blood obtained at delivery, both mother and infant were treated with
chloroquine. Subsequently, the mother was treated with primaquine
for terminal prophylaxis, which the infant did not require since he
was not exposed to the sporozoite and thus would not have had the
dormant hepatic hypnozoite. One year later, both mother and child
are doingKarplus,
well,R. etwith no
al. N Engl further
J Med relapses.
2006;354:1064
 Hemolytic Anemias, Characterized by Different Types of Poikilocytes.

Bain, B. J. N Engl J Med 2005;353:498-507

 Red-Cell Changes in Various Types of Hemolytic Anemia.

Bain, B. J. N Engl J Med 2005;353:498-507

 Red-Cell Changes in Various Types of Macrocytic Anemia.

Bain, B. J. N Engl J Med 2005;353:498-507

 Red-Cell Changes in Various Types of Hemolytic Anemia.

Bain, B. J. N Engl J Med 2005;353:498-507

Blood-Smear Features Associated with Thrombocytopenia and Errors in the Platelet Count.

Bain, B. J. N Engl J Med 2005;353:498-507

Fetal hemoglobin
Foetalstaining
haemoglobin staining