2. Acknowledgements
Addisa Ababa University
Jimma University
Haramaya University
Hawassa University
University of Gondar
American Society for Clinical Pathology
Center for Disease Control and Prevention-
Ethiopia
2
4. General introduction of the course
Discussion of course syllabus
Review of course chapters
Discussion of course expectations, attendance policies,
evaluation criteria
5. Course Objective
At the end of this course students will be able to:
Compare and contrast normal and abnormal red blood
morphology
Define anemia and its ways of classification
Describe the lab investigations for different types of
anemia
Define leukemia and explain its origin
Discuss the different types of leukemias and other
proliferative disorders
Describe hemostasis
Explain the clinical implication of different screening and
confirmatory tests for bleeding and clotting disorders
6. Course objective
At the end of this course students will be able to:
Describe techniques for the examination of L.E cells,
bone marrow, CSF, other body fluids and osmotic
fragility of red cells
Explain the principle of automated hematology analysis
Indicate important reference ranges in hematology
Recognize the importance and application of quality
assurance in hematology
Perform safety rules
Apply quality control for hematological tests
Recognize sources of error and possible remedies in
hematologic tests
8. Objectives
Upon completion of this chapter the student will be able to:
Discuss the overview of hemopoiesis
Discuss basic hematolological investigations (cell count
and differential, Red cell indices)
Discuss the significance of well prepared well stained
peripheral blood smear
9. Outline
Overview of haematopoiesis
Haematological investigation
cell counts
Leukocyte differential count
Red cell indices
Quality of peripheral blood film
14. RBC maturation
Pronormoblast
Basophilic normoblast
Polychromatophilic normoblast
Orthochromatic normoblast
Polychromatophilic erythrocyte
(Reticulocytes)
Erythrocyte
A. Cell Size and cytoplasm color
B. Nuclear chromatin structure
C. Composite (appearance
under the microscope):
A
B
C
Immature RBCs Mature RBCs
14
15. Review of a well prepared well
stained blood smear
Check With Coworkers
When You Are Uncertain
of What You See On a
Blood Smear!
15
16. Identification of cells (Wright’s stained
smear)
Size of the cell
Nuclear-cytoplasmic ratio
Nuclear characteristics
Chromatin pattern
Nuclear shape
Presence of nucleoli
Cytoplasmic characteristics
Color
Granulation
Vacuoles
Shape
16
17. Identification of cells (cont’d)
With maturation:
Size of the cell decreases
Nuclear:cytoplasmic ratio decreases from 4:1 or 3:1 to 2:1
or 1:1 in most cases except for erythrocytes and
thrombocytes (no nuclei) and lymphocytes which
frequently retain the original ratio.
Nuclear characteristics
Chromatin pattern becomes more coarse and dense
Nuclear shape changes to many lobes or segments
(in Granulocytes)
Nucleoli disappear
17
18. With maturation:
Cytoplasmic characteristics
Color changes from deep blue to lighter blue,
blue-gray or pink
Granulation: no granules to non-specific
granules to specific granules
Vacuoles: increase with age (except for
monocytes which frequently have vacoules
throughout their life cycle)
Shape: megakaryocyte has more irregular
outline
In identification of cells,
examine more systematically
by assessing various
maturational features
18
19. Segmented neutrophils (“segs,” also
called polymorphonuclear neutrophil
leukocytes [PMNs or “polys”])
Note that the size of the erythrocytes is
about the same as the nucleus of the
small resting lymphocyte.
Band neutrophil. Note the
unsegmented horseshoe-shaped
nucleus
19
20. Eosinophil. Note the bilobed nucleus
and the large granules.
Basophil. Note the dark granules that
obscure the nucleus.
Neutro
20
21. Resting lymphocyte. Note the small
round nucleus and scant cytoplasm.
Reactive lymphocyte. Note the
abundant cytoplasm that “hugs”
erythrocytes
Large granular lymphocyte.
21
22. Monocyte. Note the large size, folded
nucleus, and cytoplasmic vacuoles
Platelets (arrow).
Retics
22
23. Red Blood Cell Indices
Calculation
MCV (fl) = PCV(l/l) X 1000
RBC (cells/l)X 10-12
MCH(pg) = Hb (g/dl) X 10
RBC (cells/l) X 10-12
MCHC (g/dl) = Hb (g/dl)
PCV(l/l)
23
24. Case Study 1: Red cell Indices
Calculations: MCV
2. Calculate the MCV if a patient has:
RBC 2.72 x 1012/L
Hgb 10.1 g/dL
Hct 30.0%
HCT % X 10
= fL
Healthy individual
(range) = 80-100 fL
RBC 1012/L
5 minutes!
24
25. Case Study 1 Answer: Indices
Calculations: MCV
2. Calculate the MCV:
RBC 2.72 x 1012/L
Hgb 10.1 g/dL
Hct 30.0%
Answer:
MCV = 110.3 fL
fL
/L
12
10
in
RBC
10
x
HCT%
25
26. Case Study 1: Indices
Calculations: MCV
3. Calculate the MCV:
RBC 7.30 x 1012/L
Hgb 13.9 g/dL
Hct 49.0%
Do you remember the formula?
5 minutes!
26
27. Case Study 1 Answer:
Indices Calculations: MCV
3. Calculate the MCV:
RBC 7.30 x 1012/L
Hgb 13.9 g/dL
Hct 49.0%
Answer:
MCV = 67.1 fL Healthy individual
(range) = 80-100 fL
fL
/L
12
10
in
RBC
10
x
HCT%
27
28. Case Study 1: Red cell Indices
Calculations: MCH
4. Calculate the MCH if a patient has:
RBC 3.79 x 1012/L
Hgb 11.6 g/dL
Hct 32.0%
Do you remember the formula?
5 minutes!
28
29. 4. Calculate the MCH:
RBC 3.79 x 1012/L
Hgb 11.6 g/dL
Hct 32.0%
Answer:
MCH = 30.6 pg
Case study 1 Answer: Red cell
Indices Calculations: MCH
Hgb in g/dl X 10
= pg
Healthy individual
(range) = 27-33 pg
RBC in 1012/L
29
30. Case study 1: Red cell Indices
Calculations: MCHC
1. Calculate the MCHC if a patient has:
Erythrocytes 4.50 x 1012/L
Hgb 13.5 g/dL
Hct 40.0%
5 minutes!
Hgb in g/dL x 100
= %
Healthy individual
(range) = 32-36%
Hct %
30
31. Case Study 1 Answer: Indices
Calculations: MCHC
1. Calculate the MCHC:
Erythrocytes 4.50 x 1012/L
Hgb 13.5 g/dL
Hct 40.0%
Answer:
MCHC = 33.8%
31
32. Platelet Procedures Calculation
Formula
Number of cells X dilution factor x10*
Area counted
Dilution factor = invert dilution used (if 1:20, then use x20)
Area counted = 2 sq mm (if two center squares counted
use divided by 2)
*invert 0.1 depth of chamber
32
33. Case Study 2: WBC Calculation Formula
Question:
Using the formula, calculate the
WBC for the following:
WBC dilution using traditional
pipette (1:20)
Diluent 380 to Sample 20
WBC counting area
(4 corner squares on each side)
Counted 100 cells on one side of
Hemacytometer, 110 cells on
other side
Number of cells X
dilution factor X 10
Area counted
2 minutes!
33
34. Case Study 2 Answer:
WBC Calculation Formula
Question:
Using the formula, calculate
the WBC for the following:
WBC dilution using
traditional pipette (1:20)
WBC counting area (4
corner squares)
Counted 100 cells on
one side of
Haemacytometer, 110
cells on other side
Number of cells X
dilution factor X 10
Area counted
Answer: 210 X 20 X 10
8
= 5.3 X 109/L
34
35. Case Study 2: WBC
Calculation Formula
Question:
Using the formula, calculate
the WBC for the following:
WBC dilution: 1:20
Cells counted WBC area
(4 corner squares) =160
total of both sides
Number of cells X dilution
factor X 10
Area counted
2 minutes!
35
36. Case Study 2 Answer: WBC
Calculation Formula
Question:
Using the formula, calculate
the WBC for the following:
WBC dilution: 1:20
Cells counted WBC area
(4 corner squares) =160
total of both sides
Number of cells X
dilution factor X 10
Area counted
Answer:
160 X 20 X 10
8
= 4.0 X 109/L
36
37. Case study 2: Platelet count
If 204 platelets were counted in a volume of 0.02 cu mm
of a 1 in 20 diluted blood, what would be the total
number of platelets in 1cumm or 1L of undiluted blood ?
a. 204x109/L
b. 10,200/cumm
c. 204x1012/L
d. none
37
38. Case study 2: CSF Cell count
If a technician counted 81 WBC from undiluted CSF in
an area of 9 sq mm using the Improved Neaubaeur
counting chamber, the total No. of cells per cu mm of
CSF would be
a. 100
b. 90
c. 1,800
d. 4,050
38
39. Bibliography
MA Lichtman, E Beutler, U Seligsohn, K Kaushansky,
TO Kipps (Editors). William’s Hematology. 7th Ed.
McGraw-Hill Co. Inc. 2008.
Dacie, John V and Lewis, S.M. Practical Hematology
10th Edition Churchill-Livingstone 2006.
Wintrobe, Maxwell M. Clinical Hematology 11th Edition
Lea and Febiger, Philadelphia 2003.
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