Neubauer counting chamber’s calculations for RBC; Platelets and WBC:-
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● The counting grid is composed of 9 big squares, measuring 1 x 1 mm.
● From these squares, the central square contains 25 medium sized squares each measuring 0.2 x 0.2 mm.
● These are further divided into 16 small squares each measuring 0.05 x 0.05 mm.
● The large central square is also called the erythrocyte grid.
● The squares highlighted in red correspond to 80 small squares, that are used to establish the erythrocyte and platelet counts.
● The large squares marked in blue are used to establish the leukocyte count.
A) Erythrocyte (RBC ) Counting:
● 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
B ) Platelet Counting:
● In a platelet count, 50 µl EDTA blood (collected with an Eppendorf pipette) is mixed in 950 µl dilution solution (collected with an Eppendorf pipette).
● This results in a dilution of 1:20.
● The mixture must stand approximately 5 minutes so that the erythrocytes are completely lysed.
● Then the suspension is mixed and put into the counting chamber.
● The chamber is left in a moist environment for 20-30 minutes so the platelets can settle without the chamber drying.
● Like the erythrocyte count, 80 small squares are counted.
● Calculation of the platelet count is achieved by using the formula below using these factors:
- Number of platelets counted in the small squares.
- Dilution of the cell solution.
- Number of counted squares.
- Volume above a square.
- Conversion factor which is necessary in order to come to the volume of one liter. Since we are moving in the µl area, this is equal to 106 (1 µl = 1 x 106 L or 1 L = 1 x 106 µl).
■ #Calculation:
Platelet counts / µl = Number of platelets counted × Dilution (20) ÷Number of squares counted (80) ×volume of 1 small square (0.00025 µl)
i.e.
Platelet count / µl = (Number of Platelets counted × 20 ) ÷ (80 × 0.00025 µl)
i.e.
Platelet count / µl = (Number of Platelets counted × 20) ÷ (0.02 µl)
● Example with 200 counted platelets.
Platelet count / µl = (Number of platelets counted × 20) ÷ (.02 µl)
i.e. Platelets count / µl = (200 × 200) ÷ (.02 µl) = 200000 / µl
C) Leukocyte (WBC ) Counting :
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● In the manual WBC count, 50 µl of EDTA-blood (collected with an Eppendorf pipette) is mixed together with 950 µl dilution solution (e.g. Türk’s solution, collected with an Eppendorf pipette).
● This constitutes a dilution of 1:20. The erythrocytes will be lysed, and the leukocyte nucleus will be stained.
● The counting chamber is immediately filled after mixing. After 2 minutes, one may begin counting the leukocytes in the 4 large squares.
● Calculation of the leukocyte count is achieved by following the formula below using these factors:
- Number of leukocytes counted in the big squares.
- Dilution of the cell solution
- Number of counted big squares.
- Volume above a big square.
- Conversion factor which is necessary in order to come to the volume of one liter. Since we are moving in the µl area, this is equal to 106 (1 µl = 1 x 106 L or 1 L = 1 x 106 µl)
■ #Calculations:
WBC count / µl = Number of WBC counted × Dilution (20) ÷Number of squares counted (4) ×volume of 1 big square (0.1 µl)
i.e.
WBC count / µl = (Number of WBC counted × 20) ÷ (4 × 0.1 µl)
i.e.
WBC count / µl = (Number of WBC counted × 20) ÷ (0.4 µl)
■ Example with 200 counted WBCs.
WBC count / µl = (Number of WBC counted × 20) ÷ (0.4 µl)
= (200 × 20) ÷ (0.4 µl) =10000 / µl