1. The number of cells counted.
2. The volume of fluid inside the chamber (i.e. the counting area ? depth).
3. Dilution of blood.
With the help of these known factors, the number of WBC/ cu mm of undiluted blood can be calculated.
For total WBC counting following equipments are required:
1. WBC diluting fluid
2. WBC pipette
5. Blood sample.
WBC Diluting Fluid:
WBCs are about 4000 to 10,000 per cubic mm of blood. Counting this much number is highly impossible. Therefore, the blood sample is diluted with the help of diluting fluid or Trucks fluid.
i. Glycial acetic acid – 3 ml
ii. Aqueous gention violet – 1%
iii. Distilled water – 97 ml.
Acetic acid of diluting fluid removes red cells, and gention violet stains the nucleus of WBC to facilitate the WBC counting.
WBC pipette is graduated to give blood dilution 1: 20. It has rubber tube, containing white bread. The bottom of pipette is marked with 0.5 and on top, it is 11. It has rubber tube attached for sucking.
It is a chamber used for cell count. Generally, improved Neubauers chamber is used. Other chambers like Burkers and Fusch-Rosenthal may also be used. Neubauer chamber consists of 9 square mm area marked and ruled. It has four squares, each with 1 mm. The outer four squares are divided into 16 small squares (Fig. 6.1).
16 ? 4 = 64 small squares.
The depth of Neubauer chamber is 0.1 mm. It has a special cover slip.
Capillary blood or EDTA anti-coagulated blood is used for WBC counting.
Assemble all the equipment. Draw the blood directly into WBC pipette upto 0.5 marks. Wipe off the tip of pipette to remove extra blood present at the tip. Then immediately draw up the WBC diluting fluid upto 11 marks. Now rotate the pipette gently, so that the fluid and the blood get properly mixed.
This will give dilution 1: 20. Place cover slip in position over the ruled area. Once again mix the solution thoroughly by rotating the pipette. Expel one drop of fluid from the pipette. Now the second drop is allowed to hang from the pipette.
This can be done by applying slight pressure on the rubber tube of the pipette. Immediately when the drop is in hanging position, touch the tip against the edge of cover slip.
The angle between the pipette and Neubauer chamber is about 45°. With this process, the fluid gets inside the chamber. This is known as charging of the chamber. The care should be taken during charging, so that there should not be any air bubble present inside the chamber and there is no over filling beyond the ruled area.
Leave the counting chamber as it is without disturbing for about 2 to 3 min. This will allow settling down the WBCs in the counting chamber. Place the counting chamber on the stage of microscope; adjust the light and the ruled area.
The difference between the two square cell counts should not be more than 10 WBC. Now, count the WBC in all four outer squares. Make the total and repeat the same procedure on the another side of the chamber. Take average of the two sides.
In case of marginal cells, count the cells on the margin of ‘L’ shape, i.e. either right and lower or left and upper side.
The known factors are:
1. The volume of fluid inside the chamber
= Area ? depth
= 4 ? 0.1
2. Dilution of blood = 20
3. The number of cells counted in four squares suppose, it is = N
TLC = N ? 20 / Area ? Depth
N ? 20 = N ? 20
= 4 ? 0.1 = 0.4
TLC = N ? 50
With the help of this formula, we can calculate the total number of leucocytes in blood.