● In a manual erythrocyte count, 10 µl of EDTA blood (collected with an
Eppendorf pipette) is diluted in 1990 µl of isotonic erythrocyte dilution solution.
● This results in a dilution of 1:200.
● This suspension must be well-mixed and be immediately placed into the
counting chamber.
● After approximately 3 minutes, the erythrocytes will have settled, and one may
begin counting the erythrocytes in 80 small squares.
● The calculation of the erythrocyte count is achieved by following the formula
below using these factors:
- Number of RBC counted in the small squares.
- Dilution of the cell solution.
- Number of counted small squares.
- Volume above one small square.
- Conversion factor is necessary in order to arrive at the volume of one liter. Since
we are dealing with one µl, this is equal to 106 (1 µl = 1 x 106 L or 1 L = 1 x 106
µl).
■ #Calculations:
RBC count / µl = {Number of RBC counted × Dilution (200)} ÷ {Number of
squares counted (80) ×volume of 1 small square (0.00025 µl)}
i.e.
RBC count / µl = (Number of RBC counted × 200) ÷ (80) ×0.00025 µl)
i.e.
RBC count / µl = (Number of RBC counted × 200) ÷ (0.02 µl)
● Example with 500 counted RBCs.
RBC count / µl = (Number of RBC counted × 200) ÷ (0.02 µl)
i.e. RBC count / µl = (500 × 200) ÷ (.02 µl) = 5000000 / µl