Total Erythrocyte Count by Haemocytometer

The total erythrocyte count is an important diagnostic tool for haematology. It permits the MCV and MCH values to be calculated. At birth the total erythrocyte count varies from 6.5 million to 7.25 million/cu.mm. There is steady decline after a few hours and at the end of two weeks to one month there is a slow rise to normal adult level.
An increase of total erythrocyte count above normal level is called erythrocytosis. Erythrocytosis is observed in condition such as (1) haemoconcentration due to burn, cholera, vomiting, dehydration etc.  (2) Central cyanotic states that is chronic lung diseases or congenital cyanotic heat diseases and (3) Polycythemia Vera etc. Decrease in erythrocyte count is observed in (1) Old age, (2) In pregnancy due to haemodilution, (3) Leukemia and in (4) Group of diseases classified under anaemia.

Normal values :

• Male        : 4.5 – 6.0 million/cu.mm
• Female     : 4.0 – 5.0 million/cu.mm

Principle :

The blood specimen is diluted 1 in 200 with the RBC diluting fluid and the red cells are counted under high power objective (40X) by using a counting chamber. The number of cells in undiluted blood is reported as per cu.mm (µl) of whole blood.

Specimen :

• Double Oxalated or EDTA anticoagulated venous blood.
• Capillary blood (specimen not be a fasting sample).

Requirements :

1. Test tube with test tube rack.
2. RBC pipette or Micropipette with microtips.
3. RBC diluting fluid :
   • Sodium Citrate            : 3.0 gm
   • Formalin                      : 1.0 ml
   • Distilled water             : 99.0 ml
   • This solution is stable at room temperature (25°C ± 5°C). A pinch of thymol may be added as a preservative.
4. Microscope.
5. Improved Neubauer counting chamber.
6. 5.0 ml graduated pipette.

Procedure :

1. Taken a clean and dry test tube in the test tube rack.
2. Pipettes 3.98 ml (3980 µl) of RBC diluting fluid in the test tube by using a 5.0 ml graduated pipette.
3. Added 0.02 ml (20 µl) of well mixed blood by using Micropipette with microtips.
4. Mixed will and keep for five minutes.
5. Mixed well and small amount of solution is introduced under the coverslip which is placed on the Neubauer counting chamber.
6. Allow the cells to settle for 2 – 3 minutes.
7. Placed the Neubauer chamber on the stage of the microscope. Observed first under low power objective (10X) and afterwards switch to high power objective (40X) and count the number of red cells in the 4 corner squire and in the centre squire in the middle chamber of the Neubauer chamber.

[Note : For dilution, We also used RBC pipette their blood
drawn upto 0.5 mark and afterwards RBC diluting fluid is drawn upto 101 mark
and mixed the solution.]