Practical Hematology Lab

- LAB 2 -

White Blood Cell (WBCs)

Count
 White blood cells
• Characters of WBCs:
1. 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.
2. The white blood cells have a rather short life cycle,
living from a few days to a few weeks.
3. 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.
 Characters of WBCs
4. Leukocytes are found throughout the body,
including the blood and lymphatic system.
5. 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.
 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.
• Specimen:
• 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.
 Equipment
1. White blood cells count diluting
fluid
2. Thoma white pipette
3. Hemacytometer and coverslip
4. Microscope
5. Lint-free wipe
6. Alcohol pads
 haemocytometer chamber

Thoma white pipette

Rubber sucking tube

 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.
Hemacytometer
 Procedure
1. Draw the blood up to 0.5 mark in the thoma pipette.
2. Wipe the outside of the capillary pipette to remove
excess blood that would interfere with the dilution
factor.
3. 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.
4. 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
 Procedure
5. Mix the sample for at least 3 minutes to facilitate
hemolysis of RBCs.
6. Clean the hemacytometer and its coverslip with an
alcohol pad and then dry with a wipe.
7. 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.
 Procedure
8. Carefully charge hemacytometer with diluted blood
by gently squeezing sides of reservoir to expel
contents until chamber is properly filled.
 Procedure for counting WBC’s

1. Under 10 x magnifications, scan to ensure even

distribution. Leukocytes are counted in all nine large
squares of counting chamber.
2. 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.
3. Count all cells that touch any of the upper and left
lines, do not count any cell that touches a lower or right
line.
 Calculations
• Depth= 0.1
• Correction for dilution:
• 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
• Suppose that you count 50 cells in 4 squares (0.4mm3),
found the count in 1mm3?

 50 o.4 mm3
 X 1mm3
 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 20 =
 Discussion
1. 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.

2. 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
 Discussion

3. 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(

)of NRBC’s/100 WBC’s on differential # + 100(

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.
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.