This document describes the process for performing a total leukocyte count. It involves diluting a blood sample with Turk's fluid to lyse red blood cells while staining white blood cell nuclei. The cells are then counted in a calibrated chamber and reported as the number of leukocytes per cubic millimeter of undiluted blood. The Turk's fluid composition and dilution methods (microdilution vs bulk dilution) are provided along with the calculation steps and normal reference ranges. Clinical significance of leukocyte count results is also summarized.

1. TOTAL LEUCOCYTE COUNT
Mr Govardhan Joshi

2. Principle
• The sample of blood is diluted with a turk’s
fluid which destroy RBC and stain the nuclei of
leucocyte. The cell are then counted in a
calibrated chamber and their number in
undiluted blood reported as leucocyte/cumm.

3. Requirement
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Counting chamber
WBC count
Turk’s fluid
EDTA blood
Microscope
Timer

4. Turk’s fluid
Composition:
• Glacial acetic acid= 1.5ml→lyses RBC
• 1% gention violet= 1ml→ stain nuclei of WBC
• Distilled water= upto 100ml→ solvent
• Thymol= 1 pinch→prevent growth of fungus
The diluting fluid is not isotonic and the very
dilute acid solution lyses RBC but not WBC

5. Methods
• Microdilution method
• Bulk dilution method

6. Procedure microdilution method
• Draw the well mixed anticoagulated blood upto mark
0.5
• Clean the tip of pipette and adjust the level exactly
upto mark 0.5
• Draw diluting fluid upto mark 11
• Mix well by rotating pipette for 2-3 minutes
• Charge the neubauer chamber by discarding 1-2 drops
• Allow the cell to settle down for 2 minutes
• Count WBC under low power(10x) in four large corner
square

7. Procedure for bulk dilution method
• Mix 380µl of diluting fluid and 20µl of blood in
a clean test tube
• Other steps same as above

8. Calculation
WBC count=N x dil. Factor x depth factor/area
counted
WBC count= N x 50

9. In case of leucopenia
• Dilution→ 1:10
In case of leucocytosis
• Dilution→ 1:100

10. Normal values
Adult= 4,000- 11,000/cumm
Birth= 10,000- 25,000/cumm
1-3yrs= 6,000- 18,000/cumm
4-7yrs= 6,000- 15,000/cumm
8-12yrs= 4,500- 13,500/cumm

11. Clinical significance
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Leukocytosis
Leukaemia
Myeloproliferative disorder
Infection
Physiological: age, muscular
exercise etc
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Leucopenia
Typhoid
Aplastic anemia
Dengue
Influenza
Malaria

12. Corrected total WBC count for
nucleated RBC
• Done when nucleated RBC are seen in
peripheral blood smear
• Corr. WBC count=
100x Uncorrected WBC/100+NRBC
Uncorrected WBC= total nucleated cell counted
NRBC= nucleated RBC seen in PBS/ 100 WBC

13. or
Corr. WBC= Uncorr.WBC-(NRBCx Uncorr.WBC)
100+NRBC