Adigrat University Department of Physiology in 2019 for medicine students Blood and Immunity physiology Laboratory session

1. Blood and Immunity Physiology
Laboratory Session
Day1
1. Total WBC count
2. RBC count
3. Differential WBC count

2. 1.Total white blood cell count
Significance of the test
• To investigate infections and unexplained
fever.
• To monitor treatments which can cause
leucopenia.
• To diagnose leukemia.

3. Principle of the test
• Whole blood is diluted 1:20 in weak acetic
acid solution which lyses red blood cells but
preserves leukocytes to be counted.
• The count is multiplied by dilution factor and
volume correction factor .
• The number of WBC is reported as number of
cells per microliter(µl) of whole blood.

4. Specimen
• EDTA anticoagulated blood or Capillary blood
can be used for counting WBC

5. Materials and Reagents
• Hemocytometer(improved neubauer chamber)
• WBc thoma pippet
• Diluting fluid(weak acetic acid)
• Cover glass
• Cotton
• Microscope
• alcohol

6. Cont’d….

7. WBC Thoma Pippet

8. Procedure
1.Mix the anticoagulated blood carefully
2.Suck the blood into WBC thoma pipette
exactly up to 0.5 mark.
3.Wipe the excess blood outside the pipete using
cotton
4.Take diluting fluid up to 11 mark
5.Gently rotate the pipette for mixing
6.Discard the first drops of unmixed solution
present at the stem of the pipette

9. Cont’d…..
7.Charge the counting chamber and allow 2
minutes for settling of cells
8.Count white cells in the four corner squares of
the haemocytometer using microscope.

10. About the haemocytometer
• The chamber has 9 large
squares
• Each square is 1mmx1mm
(area 1mm2
)and 0.1mm depth
(volume 0.1mm3
)
• WBC is count in the four
large Corner squares
• Volume used for counting
WBC is 4x0.1mm=0.4mm3

12. Calculation
• The dilution and the volume used must be corrected.
• The dilution is corrected by dilution correction factor(DCF)
• The volume is corrected by volume correction factor(VCF)
Total WBC Count = no of cell counted x DCF x VCF
DCF =20(1:20)
VCF =Volume desired/volume used
1mm3
/0.4mm3
= 2.5
WBC count =no of cell counted x20x2.5
= no of cell counted x50/µl
Normal range : 4,000-10,000/µl (4 - 10 x10 3
/µl)

13. Interpretation
• Increase in
Acute infection eg pneumonia,tonsilitis
 Inflammation and tissue necrosis
eg burn,Arthritis
Leukemia and myloproliferetive disorders
Stress
Strenuous exercise

14. Cont’d….
• Decrease in
Viral and parasitic infections
Drugs and reaction to chemicals
Hypresplenism
Aplastic anemia etc…

15. 2.Red blood cell count
Significant of the test
• Used to diagnose and classify anemia.
• To calculate red cell indices

16. Principle of the test
• Whole blood is diluted 1:200 in hayem’s
solution which maintains the disc like shape
of red cells and prevents agglutination.
• RBCs are counted under microscope
• The count is multiplied by dilution factor and
volume correction factor
• The number of RBC is reported as number of
cells per microliter(µl) of whole blood

17. Specimen
• EDTA anticoagulated blood or capillary blood
can be used for counting RBC

18. Material and reagents
• Haemocytometer
• RBC thoma pippet
• Cover glass
• Diluting fluid (hayem’s solution)
• Microscope
• Cotton
• alcohol

19. Procedure
1.Mix the anticoagulated blood carefully
2.Suck the blood into pipette exactly up to 0.5 mark.
3.Wipe the excess blood outside the pipete using
cotton
4.Take diluting fluid up to 101 mark
5.Gently rotate the pipette for mixing
6.Discard the first drops of unmixed hayem’s
solution present at the stem of the pipette

20. Cont’d….
7.Charge the counting chamber and allow 2
minutes for settling of cells
8.Count red cells in the central 5 small squares of
the haemocytometer using microscope

21. About the haemocytometer
• RBC counts in the central large
square(1mmx1mm)
• The central large square is divided
in to 25 smaller squares
• RBC is counted in the 5 out of these
25 smaller squares
• Each smaller square is
1/25(0.04mm3
)and its volume is
0.004mm3
• So volume used for RBC count is
5x0.004mm3
= 0.02mm3

22. Calculation
RBC Count = no of cell counted x DCFxVCF
DCF = 200(1:200)
VCF=volume desired/volume used
= 1cumm/0.02cumm=50
RBC count = no of cell counted x200x50
= no of cell counted x10,000/µl
Normal range
Adult male 5-6x10^6/µl
adult female 4- 5x10^6/µl

23. Interpretation
 RBC count increases in
 Polycythemia Vera
 Conditions that result in low oxygen(heart
failure,COPD,pulmonary fibrosis, high altitude)
 Kidney cancers
 Dehydration
RBC count decreases in
Anemia
 Sever falciparum parasitic infection
 Blood loss

24. 3 Differential WBC count
Significance of the test
 To help determine the cause of abnormal results on a WBC
count.
 To diagnose or monitor an illness affecting the immune
system.
Principle
• Blood film is prepared then allow to air dry.
• The dried smear stains with Romanowsky stains (giemsa
or wright stain).
• Cells will be counted
• Then the percent distribution of each cell is calculated

25. Morphology of different leukocytes
• Leukocytes differ in
Their size
Shape of nucleus
Color of cytoplasm
Presence/absence of granules in cytoplasm
Color of granules

26. cont’d
Segmented neutrophil
• Size: 12-15µm
• Nucleus: segmented
with 3-5 lobes
• Cytoplasm : pale
staining with small
neutrophilic granules
• Segmented neutrophil

27. Cont’d….
 lymphocyte
Small lymphocyte
• Size :10-12µm
• Nucleus: dark round
• cytoplasm:blue
Large lymphocyte
• Size : 12-16µm
• Nucleus : round , oval
or indented
• Cytoplasm :blue

28. Cont’d…….
Monocyte
• Size :15-20 µm
• Nucleus: round
,indented or folded
monocyte

29. Eosinophil
• Size: 12-17µm
• Nucleus : bilobed
• Cytoplasm :pale staining
with orange red granules
Eosinophil

30. Contd…..
 Basophil
• Size: 10-12µm
• Nucleus: bilobed but usually
obscured by granules
• large blue-violet granules in
the cytoplasm
Basophil

31. • The are two types of counting
1 Relative count: is the simple percentage distribution
of white blood cells.
2 Absolute count: is the exact count of leukocytes in
the peripheral blood.
Absolute count = relative count X total leukocyte
count.

32. Cont’d…..
Differential WBC reference range
Adults
Neutrophils …………… 2.0 – 7.5 × 10^9/l (40 – 75%)
Lymphocytes ………….1.2 – 4.0 × 10^9/l (21 – 40%)
Monocytes …………….0.2 – 1.0 × 10^9/l (2-10%)
Eosinophils …..………. 0.02 - 0.6 × 10^9/l (1-6%)
Basophils …..………… 0.01 – 0.1 × 10^9/l (0-1%)

33. Interpretation
Neutrophilia
• It is caused by:
 Acute bacterial infections
Physical and emotional stress
 Tissue destruction or necrosis
Myeloid leukomia

34. Cont’d…..
Neutropenia:
It is caused by:
Bone marrow failure
 Myeloid hypoplasia
 Drugs (chloramphenicol, phenylbutazone)
Ionizing radiation

35. Cont’d…..
Eosinophilia:
it Occurs during:
 Allergic diseases: bronchial asthma
 Intestinal parasitic infections
Basophilia:
Some allergic reactions
Chronic myelogenous leukemia(CML)

36. Cont’d…..
Monocytosis
• Chronic bacterial infections, e.g. tuberculosis,
typhoid, bacterial endocarditis
• Chronic myelomonocytic leukaemia

37. Cont’d…..
Lymphocytosis:
occurs during
• Infections in children, e.g. whooping cough,
mumps, measles, chicken pox
• viral infections: Epstein-Barr virus
,cytomegalovirus,
• Acute and chronic lymphocytic leukemia

38. Cont’d
Lymphocytopenia
 Immune deficiency disorders: HIV/AIDS
 Drugs, radiation therapy