Total leucocyte count

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 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 Leukocytosis Leukaemia Myeloproliferative disorder Infection Physiological: age, muscular exercise etc 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= 100 x 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