Objectives:

1.To fill the hemocytometer correctly

2. To count the WBCs accurately in blood and body fluids
3.To know how to calculate the WBCs count in blood and body fluids
4.To know the sources of error in manual WBCs count
5.To know how to make a rough estimate for WBCs count from a stained PB film
6.To know physiological causes of WBCs count variation
 Requirements
Sample Reagents
●Venous blood collected on EDTA
●Diluent
or heparin or capillary blood
2% acetic acid
●Counts are stable for samples
stored at room temperature for (minute amount of
48hrs and at 4°C for 72 hrs. Leishman or brilliant cresyl
blue powder is added to
give a blue tinge to the
solution.)
Equipments
●Pipette 20µL volume
- Hemoglobin micropipette
- Automatic pipette
● Tubes
- Glass or plastic
● Haemocytometer
- Neubauer chamber
- Improved Neubauer
chamber
● Clean gauze
● Microscope
• The hemocytometer is a glass chamber with raised sides that will hold a glass
cover slip exactly 0.1mm above the chamber floor.

• The counting chamber contains a ruled area composed of 9 large squares of

equal size.

• The side length of each large square is 1mm and its depth is 0.1mm.

• The 4 large corner squares are used for counting WBCs, each of which is
divided into 16 smaller squares.

• The central square is used for counting platelets and is divided into 25 smaller
squares.
Principle:
Whole blood is mixed with a weak acid solution to
dilute the blood and lyse the RBCs
Procedure:
1- Mix the blood sample by gentle shaking to allow for homogenous distribution of the
cells in the plasma.
2- Withdraw 20µL of whole blood by the pipette.
3- Wipe excess blood carefully from the outside of the pipette.
4- Evacuate the blood into a test tube containing 380µL diluent (dilution 1:19; dilution
factor 20).
Procedure Cont.
5- If hemoglobin pipette is used, blood is rinsed from the pipette by withdrawing
and releasing the diluent into the test tube several times.

6- Cover the test tube and invert it several times (for 1 min.) to mix thoroughly.

7- Allow the preparation to stand for 2 minutes for complete haemolysis of red cells
to occur.

8-Clean the counting chamber (haemocytometer) and the glass cover with a clean
gauze (95% ethanol will facilitate the cleaning process).
Procedure Cont.
9- Wet the raised edges of the counting chamber and put the glass cover on top of the ruled area
of the chamber.

10- Fill the pipette with the prepared mixture and load the 2 chambers of the haemacytometer by
allowing the mixture to sweep under the glass cover.

11- The counting chamber should be filled in one action and no fluid should flow into the
surrounding area.

12- Leave the counting chamber undisturbed for few minutes to allow the cells to settle down, but
not much longer to avoid dryness at the edges of the preparation.
Procedure Cont.
13- Put the counting chamber on the microscope stage, lower down the
condenser and use the low power objective lens (10x) for counting the
cells. The high power objective lens (40x) can be used for more accurate
discrimination of cells.

14- Count the cells in the 4 large corner squares of each side of the
hemacytometer. Counts from each side of the chamber should match
within 25%.
Derivation of Formula for
Calculating WBCs Count:
This is done by using the number of cells counted and then a correction for volume and a
correction for dilution are made.
1. Volume of 1 WBC square = Area for square x depth
=1mm2 x 0.1 mm = 0.1mm3
2. Volume of squares counted = Volume of 1 WBC square x no. of counted squares
e.g. 0.1mm3 x 4 = 0.4mm3
3. Volume correction to get cell count in 1mm3(1µL) = Nx1/0.4 = N x 2.5

4. For dilution correction, multiply by dilution factor (20)

5. No. of WBCs counted in 1mm3 (1µL)

= No. of WBCs in 4 chambers x volume correction x dilution factor
= No. of WBCs x 2.5 x 20
= No. of WBCs x 50

OR No. of WBCs counted in 1mm3 (1µL) = No. of cells x dilution factor

Volume (area X depth)
 Sources
of errors

Apparatus Personal technique Inherent error

1.Not a well calibrated

not thoroughly mixed blood
pipette
inadequate shaking
• With best perfection,
improper loading of chamber
there is inherent error
(overfilling, trapped air bubbles) approx. 15%.
2. dirty glassware
counting cells inaccurately • Correlation with WBC
count made by rough
(skipping cells, counting cells twice,
estimation from a PB film
counting on wrong borders)
3. non-optically
plane cover glasses calculation error
WBC estimate from blood smear
Rough estimate of WBCs is used to overcome manual error (inherent)
and automated errors (large platelets, platelet clumps and nucleated
RBCs)
Procedure
• Use the low power objective of the microscope
• Look at 5-6 fields and determine the average number of WBC per
field
• Divide the average number by 4 and multiply by 1000 to get the
estimated WBC count
Example
• WBCs seen: 5 4 6 4 5
• Average : 4.8
• Calculation :4.8/4 x 1000 = 1.200
 Physiological variation of WBCs count:
- Age: highest at birth, elderly receiving influenza vaccine have decreased count due to decrease in
lymphocytes
- Sex: slightly higher in females, but tend to fall after menopause to become lower than males of similar age
- Pregnancy: common in 2nd trimester, may be up to 15x109/L
- Some contraceptive pills
- Cyclic variation: some individuals have variation every 14-21 days, which may be related to the menstrual
cycle in females
- Diurnal variation: minimum counts in the morning at rest
- Exercise: random activity causes slight increase, vigorous exercise causes rise up to 30x109/L (decreases
splenic blood flow and hence pooling of neutrophils)
- Adrenaline injection: release of mature neutrophils from BM, spleen and margination pool in blood vessels
- Ethnic variation: Africans tend to have lower counts
 WBCs Count in Body Fluids:
● Manual count of WBCs is specially important for counting cells in body fluids as ascitic, pleural, pericardial, cerebrospinal and
synovial fluids, specially when count is less than 1000/ mm3 (µL).
● Cells are counted directly without adding a diluent, or diluted 1:1 with acetic acid to lyse the small no. of RBCs which may be
present as a result of the trauma during sample collection.
● No. of WBCs counted in 1mm3 (1µL) without dilution
= No. of WBCs x volume correction
= No. of WBCs x 1/0.4
= No. of WBCs x 10/4
= No. of WBCs x 5/2
● No. of WBCs counted in 1mm3 (1µL) with 1:1 dilution
= No. of WBCs x volume correction x dilution factor
= No. of WBCs x 1/0.4 x 2
= No. of WBCs x 10/4 x 2
= No. of WBCs x 5/2 x 2
= No. of WBCs x 5
WBCs Count in Body Fluids
● Contamination of a body fluid sample with many RBCs does not
guarantee an accurate WBCs count; hence another sample should be
asked for. If this is not possible, the counted WBCs can be
approximately corrected by counting the no. of RBCs in this sample
and then calculating the WBCs using the ratio between both elements
in the peripheral blood.