HAEMOCYTOMETRY (COUNTING OF CELLS IN BLOOD):- Counting of blood cells, such as red blood cells (RBCs), white blood cells (WBCs) and platelets is a fundamental measurement in a haematology laboratory. These cells can be counted either manually by microscopy or with the help of automated electronic devices.

MANUAL CELL COUNT:-The manual method of counting involves:

1. Dilution of blood in an appropriate diluting fluid

2. Use of a haemocytometer to count the number of cells per unit volume of blood.e.g. One cubic millimetre (mm'.ul) or one litre (L)

Note:-One cubic millimetre is equal to 1 microlitre (14). One litre is equal to 10° microlitres.

Dilution of Blood for Cell Counts Using Thoma Pipettes:- A Thoma pipette (Fig. 3.2) consists of a graduated capillary tube divided into 10 parts marked 0.5 at the fifth mark and 1.0 at the tenth mark. The capillary tube opens into a mixing bulb containing a glass bead which facilitate mixing of the blood and the diluent. There is another short capillary tube

above the bulb marked 11.0 on the white cell pipette and 101.0 on the red cell pipette. In the Thoma white cell pipette, if blood is collected up to the 0.5 mark and diluted up to the 11.0 mark, the resulting dilution within the bulbis 1:20. The red cell pipette allows a ten times greater dilution than the white cell pipette and when 0.5 units is measured and diluted up to 101.0 mark, the dilution is 1:200. Each Thoma pipette is provided with an aspirating tube.

Note:-While making dilutions, it must be remembered that 1.0 unit in the stem of the pipette contains only the diluent and therefore, must not be used for counting cells. Discarding first 3-5 drops removes this fluid.

Techniques :-

1. Using an appropriate (RBC or WBC) Thoma pipette, aspirate well mixed blood into the capillary tube of the pipette to slightly above the 0.5 mark.

2. Wipe off the outside of the pipette with a gauze and adjust the blood level exactly to 0.5 mark by tapping the tip of the pipette with a gloved finger or using any other non absorbent material. Use of absorbent material such as guaze should be avoided as it will absorb only the liquid portion of blood, thus increasing actual concentration of cells in blood.

Hold the pipette in a horizontal position to maintain the blood level at 0.5 mark and place the tip of the pipette into the diluting fluid. Using a constant suction, draw the fluid into the pipette while twirling the pipette between the fingers. Continue aspirating slowly till the mixture reaches the top mark (11.0 in WBC pipette or 101.0 in RBC pipette) above the bulb.

 Remove the pipette from the diluting fluid and still holding in horizontally, remove the aspiration tube. Hold the pipette with the thumb and third finger at either end and shake vigorously at right angles to the long axis of the pipette. The glass bead in the pipette helps in mixing the suspension

Dilution of Blood for Cell Counts Using a Self Filling, Self Measuring Dilution Pipette (Unoppette)

The unoppette cell diluting system consists of a straight, thin-walled capillary tube, fitted into a plastic holder. It is also provided with a plastic reservoir containing a premeasured volume of diluent. The capillary tube is enclosed in a plastic shield . Special unoppette systems are available for leucocyte counts, red cell counts, platelet counts and eosinophil counts. Each uses the respective diluting fluid to obtain an appropriate dilution.

Technique :-

1. Open the diluent reservoir by forcing the blunt end of the plastic shield into the reservoir cap.

2. Draw the blood into the capillary pipette either from an EDTA-anticoagulated bloodor from a finger prick. The blood will fill the pipette by capillary action to a fixed volume.

3. Wipe the exterior of the pipette with a guaze and introduce the blood into the reservoir. Squeezing the reservoir helps to release the blood into the diluting fluid.

4. Mix the diluted blood in the reservoir.

5. Before charging the counting chamber remove the capillary tube from the reservoir and reattach it in a reversed position. Squeeze the reservoir gently to force a few drops of diluted blood out through the capillary to fill the counting chamber.

USE OF COUNTING CHAMBER