A white blood cell (WBC) count is used to investigate HIV/AIDS,

infections and unexplained fever, and to monitor treatments which can

cause leukopenia.
:
Whole blood is diluted 1 in 20 in an acid reagent which haemolyzes
the red cells (not the nucleus of nucleated red cells), leaving the white
cells to be counted. White cells are counted microscopically using an
Improved Neubauer ruled counting chamber (haemocytometer) and
the number of WBCs per litre of blood calculated.
1- Blood sample: EDTA anticoagulated blood or capillary
blood can be used for counting white cells.
2- Counting chamber (hemocytometer)
The counting chamber recommended for cell counts is a
metallized surface (‘Bright-line’) double cell Improved
Neubauer ruled chamber.
3- Counting chamber cover glasses
Special optically plane cover glasses of defined thickness
(designed for use with haemocytometers) are required. Other
cover glasses must not be used. Manufacturers of counting
chambers provide two cover glasses with each chamber.
4- Pipettes/calibrated capillaries and safe filling device
A 20 ul (0.02 ml) micropipette e.g. white shellback type, or calibrated
capillary is required to measure blood samples. A safe pipette/capillary
filler should be used to aspirate and dispense the blood.
5- Hand counter
To count white cells accurately, a simple inexpensive mechanical hand
tally counter is required. Turning the knob on the side returns the
counter to zero after each count.
6- WBC diluting fluid
7- Microscope
8- Test Tubes
9- Tips
To make 100 ml:
Acetic acid, glacial . . . . . . . . . . . . . . . . . . . . . . 2 ml
Distilled water. . . . . . . . . . . . . . . . . . . . . . . . . 96 ml
Gentian violet, 1% w/v* . . . . . . . . . . . . . . . . . 2 ml
*Prepare by dissolving 0.1 g gentian violet in 10 ml distilled water and filter.
1- Fill a 100 ml cylinder to the 98 ml mark with distilled water.
2- Add 2 ml concentrated (glacial) acetic acid and mix.
Caution: Glacial acetic acid is a corrosive chemical with an irritating vapour, therefore
handle it with care and in a well ventilated room. Do not mouth-pipette (use a pipette
filler).
3- Add the gentian violet solution and mix. Transfer to a storage bottle and label.
Store in
the dark at room temperature.
1- Measure 0.38 ml of diluting fluid and dispense it into a small
container or tube.
2- Add 20 µl of well-mixed EDTA anticoagulated venous blood or
free- flowing capillary blood and mix. Important: The volume of blood
used in the test must be correct.
3- Assemble the counting chamber:
– Make sure the central grid areas of the chamber and the special
haemocytometer cover glass are completely clean and dry.
– Slide the cover glass into position over the grid areas and press
down on each side until rainbow colours (Newton’s rings) are seen.
Prior moistening of the chamber surface on each side of the grid areas
will help the cover glass to adhere to the chamber.
4- Re-mix the diluted blood sample. Using a capillary, Pasteur pipette, or plastic bulb
pastette held at an angle of about 45o, fill one of the grids of the chamber with the
sample, taking care not to overfill the area.
Important: The chamber must be refilled if the sample overfills into the channel beyond
the gridor an air bubble forms in the grid area.
5- Leave the chamber undisturbed for 2 minutes to allow time for the white cells to settle.
Note: To prevent drying of the fluid, place the chamber in a petri dish or plastic container
on dampened tissue or blotting paper and cover with a lid.
6- Dry the underside of the chamber and place it on the microscope stage. Using the 10x
objective with the condenser iris closed sufficiently to give good contrast, focus the rulings
of the chamber and white cells. Focus the cells until they appear as small black dots.
7- Count the cells in the four large corner squares of the chamber marked W1, W2, W3,
W4 in (total area of 4 mm2). Include in the count the cells lying on the lines of two sides
of each large square.
8- Report the number of white cells per litre of blood using the following
simple calculation:
– Divide the total number of cells counted by 2.
– Divide the number obtained by 10.
The number obtained ×109 is the white cell count.
Example
Cells counted in 4 squares = 84
84 ÷ 2 = 42
42 ÷ 10 = 4.2
WBC count = 4.2 cells × 109/l
WBC calculation details
WBC count (per litre) =Cells counted × 20 ×106 / 4× 0.1
where 20= 1 in 20 dilution of blood, 4= 4 mm2 area counted, 0.1= mm depth
of chamber.
For Example if cell count is 50 on hemocytometer;
WBC count (per litre) =Cells counted × 20 ×106 / 4× 0.1
WBC count (per litre) =50 × 20 ×106 / 4× 0.1
WBC count (per litre) = 1000 × 106 / 0.4
WBC count (per litre) = 2500 × 106
WBC count (per litre) = 2.5 × 109/L
Counting chamber in petri dish to prevent drying of the preparation.
Whenever possible perform WBC counts in-duplicate. The difference between the two counts (as a percentage of the
mean) should not be more than 20%.
How to calculate the % difference between two counts
1 - Record the number of cells counted in Count 1 and Count 2.
2 - Calculate:
– the difference in the number of cells counted between the two counts.
– the mean of the two counts
3 -Calculate the difference of the two counts as a percentage of the mean.
Example; Cells counted in Count 1 = 88, Count 2 = 76
– Difference in numbers of cells between the two counts: 88–76 = 12
– Mean of Count 1 and Count 2: = 82
– Difference of the two counts as a % of the mean: 12×100/82=14%
Check that the diluting fluid is free from particles which could be mistaken for WBCs. To do this, fill a counting chamber
with a sample of the diluting fluid and examine the grid areas microscopically using the 10x objective with greatly
reduced condenser iris. If the fluid contains particles resembling WBCs, filter it and recheck or discard the fluid and
prepare fresh diluting fluid.
When examining the blood film, check that there is no major discrepancy between the total white cell count and white
cells seen in the blood film.
 ● Incorrect measurement of blood due to poor technique (see Text Method ) or
using a wet or chipped pipette.
● When using anticoagulated blood, not mixing the blood sufficiently or not
checking the sample for clots.
● Inadequate mixing of blood with diluting fluid.
● Not checking whether the chamber and cover glass are completely clean.
● Not using a haemocytometer cover glass.
● Over-filling a counting chamber or counting cells when the sample contains air-
bubbles.
● Not allowing sufficient time (2 minutes) for the cells to settle in the chamber.
● Using too intense a light source or not reducing the iris diaphragm sufficiently to
give good contrast.
● Not completing counting of the cells before the sample begins to dry in the
chamber.
● Counting too few cells.Precision increases with the number of cells counted.
Children at 1 y . . . . . . . . . . . . . . . . 6.0–18.0 × 109/L
Children 4–7 y. . . . . . . . . . . . . . . . 5.0–15.0 × 109/L
Adults . . . . . . . . . . . . . . . . . . . . . . . 4.0–10.0 × 109/L
Pregnant women . . . . . . . . . . . . . . Up to 15 × 109/L
The main causes of a raised WBC count are:
● Acute infections
● Inflammation and tissue necrosis
● Metabolic disorders
● Poisoning
● Acute haemorrhage
● Leukaemias and myeloproliferative disorders
● Stress, menstruation, strenuous exercise.
The main causes of a reduced WBC count are:
● Viral, bacterial, parasitic infections
● Drugs (e.g. cytotoxic) and reactions to chemicals
● Hypersplenism
● Aplastic anaemia
● Folate and vitamin B12 deficiencies (megaloblastic anaemia)
● Anaphylactic shock
● Ionizing radiation