1. White Blood Cell (WBCs) Count
Practical Hematology Lab
- LAB 2 -
White Blood Cell (WBCs) Count

2. White blood cells Characters of WBCs:
Whenever a germ or infection enters the body the white blood cells have a variety of ways by which they can attack. Some will produce protective antibodies that will overpower the germ. Others will surround and devour the bacteria.
The white blood cells have a rather short life cycle, living from a few days to a few weeks.
Several different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a hematopoietic stem cell.

3. Characters of WBCs
Leukocytes are found throughout the body, including the blood and lymphatic system.
The name "White Blood Cell" derives from the fact that after cenrifugation of a blood sample, the white cells are found in the Buffy coat, a thin layer of nucleated cells between the sedimented red blood cells and the blood plasma, which is typically white in color. The scientific term leukocyte directly reflects this description, derived from Greek leuko - white, and cyte - cell.

4. Principle of WBCs count test
Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent) at a specific volume in the thoma pipette.
The diluent lyses the erythrocytes but preserves leukocytes and platelets.
The diluted blood is added to the hemacytometer chamber.

5. Specimen: Reagents, supplies and equipment:
EDTA- anticoagulated blood or capillary blood is preferred.
Reagents, supplies and equipment:
White blood cells count diluting fluid which may be one of the following:
Acetic acid 2% (v/v) in distilled water.
HCL 1% (v/v) in distilled water.
Turks' solution which is formed of:
Glacial acetic acid 3 ml
Crystal violet 1 ml
100 ml distilled water.

6. Equipment White blood cells count diluting fluid Thoma white pipette
Hemacytometer and coverslip
Microscope
Lint-free wipe
Alcohol pads

7. haemocytometer chamber
Thoma white pipette
Rubber sucking tube

8. Hemacytometer
The hemacytometer counting chamber is used for cell counting.
It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm.
The surface of the chamber contains two square ruled areas separated by an H-shaped moat.

9. Hemacytometer

10. Procedure Draw the blood up to 0.5 mark in the thoma pipette.
Wipe the outside of the capillary pipette to remove excess blood that would interfere with the dilution factor.
Holding the pipette almost vertical place into the fluid. Draw the diluting fluid into the pipette slowly until the mixture reaches the 11 mark, while gently rotating the pipette to ensure a proper amount of mixing.
Place the pipette in a horizontal position and firmly hold the index finger of either hand over the opening in the tip of the pipette, detach the aspirator from the other end of the pipette now the dilution of the blood is completed

11. Procedure
Mix the sample for at least 3 minutes to facilitate hemolysis of RBCs.
Clean the hemacytometer and its coverslip with an alcohol pad and then dry with a wipe.
Before filling the chamber, discard the first four to five drops of the mixture on apiece of gauze to expel the diluent from the stem.

12. Procedure
Carefully charge hemacytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.

13. Procedure for counting WBC’s
Under 10 x magnifications, scan to ensure even distribution. Leukocytes are counted in all nine large squares of counting chamber.
Count cells starting in the upper left large corner square. Move to the upper right corner square, bottom right corner square, bottom left corner square and end in the middle square.
Count all cells that touch any of the upper and left lines, do not count any cell that touches a lower or right line.

15. Calculations Depth= 0.1 Correction for dilution: Correction of volume:
The thoma pipette is 1:20
Dilution factor 20
Correction of volume:
Volume of 1small square = 1x1x0.1= 0.1mm3
Volume of 4 large squares = 4x0.1= 0.4 mm3 or μL

16. Suppose that you count 50 cells in 4 squares (0
Suppose that you count 50 cells in 4 squares (0.4mm3), found the count in 1mm3?
o.4 mm3
X mm3
X = 50 x 1\ 0.4
Volume correction = 1\ 0.4
Total count \ 1mm3 =
No. of cells x volume correction x dilution =
no. of cells x ( 1\0.4 ) x =

17. Discussion
A highly elevated leukocyte count (leukocytosis) may make accurate counting difficult. In either instance, a secondary dilution should be made. When calculating the total count, adjust the formula to allow for secondary dilution. Or a red pipette can be used to make 1:100 dilution.
If count is less than 3000 cell/mm3, a smaller dilution of blood should be used to ensure a more accurate count. This can be accomplished by drawing the blood up to 1.0 mark and the diluting fluid to the 11 mark. The dilution will then be 1 : 10, and the dilution factor in the calculation will be 10

18. Discussion
If more than 5 nucleated RBC’s are seen on the differential, the total leukocyte count should be corrected using the following calculation:
Corrected WBC =
(Uncorrected leukocyte count x 100)
(100 + # of NRBC’s/100 WBC’s on differential)

19. Sources of errors
Common Sources of Error: a) Failure to have required blood volume. b) Failure to mix well. c) Failure to discard the first 4 drops. d) Failure to properly charge the counting chamber.

20. The least Frequent Sources of Error are: a) Inaccurate pipet or counting chamber. b) Moist or unclean pipet. c) Excessive pressure in finger when obtaining the blood. d) Too little or too much diluting fluid. e) Slowness in manipulation, thus allowing the blood to clot. f) Air bubbles in the pipet. g) Air bubbles in the counting chamber. h) Presence of yeast or other contaminants in the diluting fluid. i) Presence of many nucleated red cells causing a high white cell count. j) Mistakes in counting or calculations.