This document describes the procedure for performing a total white blood cell count. It involves diluting a blood sample with a solution that lyses red blood cells and stains white blood cell nuclei. The diluted sample is then placed in a counting chamber and the number of white blood cells in a specific area are counted under a microscope. The total white blood cell count is then calculated based on the dilution factor and area counted. Normal ranges provided are 4-11 x 103 cells/μL or 4-11 x 109 cells/L. Abnormal values above or below this range would indicate leukocytosis or leukopenia, respectively. Potential sources of error are also noted but not described.

1. Total white blood cell
count
Ebtihal Ahmed Babekir

2. Principle:
Whole blood is diluted by mixing with a fluid that lyse the red cells,
platelets, dilute the white cells and stains their nuclei deep violet-black.

3. WBCs diluting fluid(2% glacial acetic acid)
• consists of:
Glacial acetic acid 2ml
Gentian violet 1-2 drop
Distilled water 98ml

4. Test sample:
• EDTA anticoagulated venous blood. Alternatively, freely flowing
capillary blood.

5. Method:
• Add 0.02ml of blood to 0.38ml of diluting fluid in a 75
X 12mm glass or plastic tube (dilution 1 in 20).
• Mix well for at least 2 min.
• Prepare the counting chamber (Clean, dry and fix the
cover slip).
• Smoothly fill the chamber.

6. Method(con)
• Leave the chamber undisturbed on a bench for at least 2 min for the
cell to settle.
• Using X 10 objective, count the white cells in the specific area in the
chamber.

8. Calculation:
T.WBCs = No. of cells counted X dilution factor
Volume counted

9. Reference range:
4 - 11 X 10 3 c/µL
4 - 11 X 10 9 c/ L

10. Comment on the result:
• Leukocytosis :( if the result is more than the upper limit)
• Leukopenia :( if the result is less than the lower limit)

11. Source of errors??

12. platelet count

13. Uses
Platelets count is one of the first line investigations of
coagulation problems.

14. principle
Whole blood is diluted with a 1% ammonium oxalate solution. The
isotonic balance of the diluent is such that all erythrocytes are lysed
while the leukocytes and platelets remain intact.

15. Requirements:
-Platelet diluting fluid.(1% Ammonium oxalate)
-Counting chamber and cover glass.
-1ml pipette.
-0.02ml pipette.
-Test tubes.
-Blood sample.
-Gauze.
-Moist cotton on a Petri dish.
-Microscope.

16. Reagent preparation
1% Ammonium oxalate:
Ammonium oxalate 1g
D.W 1L

17. Sample:
EDTA anticoagulated blood is preferable, as platelet count appears to
be higher in venous than in capillary blood, this may be due to
adhesion of platelets to the site of the skin puncture.

18. Method:
• Add 0.02ml of blood to 0.38ml of diluting fluid (dilution 1in 20).
• Mix well for at least 2 min.
• Prepare the Haemocytometer chamber (Clean, dry and adhere the cover glass).
• Using a Pasteur pipette or capillary, fill the counting chamber. Avoid air bubbles
and over flowing.
• Place the chamber on a moistened Petri dish for at least 20 min to allow platelets
to settle.
• Using X 40 dry objective, count the platelets in the specific area in the chamber.

19. Calculation:
Platelets count = No. of cells counted X dilution factor
Volume counted

20. Reference range:
150 - 400 X 103 c/µL
150 - 400 X 109 c/ L

21. Comment on the result:
• thrombocytosis :( if the result is more than the upper limit)
• thrombocytopenia :( if the result is less than the lower limit)

22. General comments
• Platelet counts should be performed within three hours after the
dilution has been prepared.
• If blood is collected by a skin puncture, carefully remove the first
drop of blood and collect free-flowing blood for the platelet count.

23. • If clumps of platelets are seen in the Haemocytometer, the
procedure should be repeated.
• Clumps may be due to inadequate mixing of blood or to poor
technique in obtaining the blood specimen.
• A Wright's/leishman's-stained peripheral blood smear should be
examined and the platelet estimate determined to confirm the
Haemocytometer platelet count for each sample.

24. • Platelet satellitism will result in falsely decreased platelet counts.
With platelet satellitism, platelets adhere to neutrophils.

25. • It is very important that all platelet counts be confirmed by slide
examination so that a false increase of platelets (due to the counting
of fragments) is not reported.

26. Source of errors in platelet count
???