The counting of blood cells, manually with the help of microscope, is not possible. Therefore, to count the cells, blood is diluted and placed in a special type of counting chamber. This technique is called haemocytometeric counting. The cells often counted by this method are red cells, white cells, platelets, and eosinophills.
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For total red cell counts following equipment are required:
1. RBC diluting fluid
2. RBC pipette
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3. Haemocytometer
4. Blood sample.
1. RBC Diluting Fluid:
RBCs are about 5 millions/cu mm of blood. Counting this much number is highly impossible. Therefore, the blood sample is diluted with the help of RBC diluting fluid. It fixes and preserves RBC. It is isotonic to red cells.
Following two types of RBC diluting fluid are commonly used:
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Formalin Citrate Diluting Fluid:
Composition:
i. Trisodium citrate – 3 gm.
ii. Formalin – 1 ml.
iii. Distill water – 99 ml.
This diluting fluid is cheap and commonly used.
Haym’s Diluting Fluid:
Composition:
a. Sodium chloride -0.5 gm
b. Sodium sulfate -2.5 gm
c. Mercuric chloride -0.25 gm
d. Distilled water – 100 ml.
The RBC diluting fluid prevents haemolysis and removes unwanted blood cells.
2. RBC Pipette:
RBC pipette is graduated to give dilution 1: 100 or 1: 200. It has bottom marked with 0.5 and 1 and the top is marked with 101. It has a round bulb containing red bead. A rubber tube is attached to the top for sucking.
Blood sucked upto 0.5 mark gives dilution 1: 200. In case of anemic patient, it is sucked upto 1 mark to give dilution 1: 100. After blood, RBC diluting fluid is sucked upto 101 marks and mixed gently.
3. Haemocytometer:
This is a chamber used for cell count. Generally improved Neubauer chamber is used. Other chambers like Burkers and Fusches Rosenthal may also be used. The Neubauer chamber has ruled area of total 9 sq. mm and the depth is 0.1 mm. The central one square is highly ruled.
The central square is divided into 25 squares. Each square is further subdivided in 16 small squares. For RBC count, count the cells in each corner and one central square, i.e. total 5 squares. As each square is further divided into 16 small squares, the area to be counted is, 16 x 5 = 80 small squares.
4. Blood Sample:
Capillary blood or EDTA anticoagulated blood may be used.
Principle:
The blood specimen is diluted (usually 200 times) with red cell diluting fluid. This diluted blood is placed in haemocytometer. The cells are counted under proper magnification over the specified area of haemocytometer.
The known factors are:
1. Number of cells counted
2. Volume of fluid inside the counting chamber
3. Dilution of blood
With the help of these known factors, the number of RBC / cu. mm of undiluted blood can be calculated.
Procedure:
Assemble all the equipment; draw the blood directly from finger or collected sample into RBC pipette upto 0.5 mark. Wipe off the tip of pipette to remove extra blood, if present. Then immediately draw up the diluting fluid upto 101 mark.
Now rotate the pipette gently so that the diluting fluid gets mixed properly. This will give dilution 1: 200. Place the cover slip in position over the ruled area of chamber. Once again mix the solution thoroughly by rotating the pipette.
Now apply slight pressure on the rubber tube of pipette, so that the second drop of fluid is in hanging position. Touch the tip of pipette (hanging drop) against the edge of cover slip. The angle between pipette and cover slip is 45°. With this process, chamber gets filled with the fluid. This is known as charging of the chamber.
The care should be taken that, no air bubble is present inside the chamber and there is no over filling beyond the ruled area.
Leave the counting chamber as it, without disturbing for about 3 min. This will allow the settling down of RBCs. Place the chamber on stage of microscope. Adjust the light and ruled area. Make sure that, the distribution of RBC over the chamber is uniform.
Now count the RBC in Central Square. The central square is subdivided into 25 squares. Out of 25 squares, count four at each corner and one at center. Each of these five squares is subdivided into 16 small squares. Thus, RBCs are counted in 16 x 5 = 80 small squares.
In case of marginal cells, count the cells on ‘L’ line, i.e. either right and lower or left and upper margin. Make the total of cells counted in 5 squares and repeat the procedure to another side of the chamber.
Calculations:
Thus, the known things are:
1. Number of cells counted:
Suppose the number of cells counted is ‘N’.
2. The volume of fluid inside the chamber,
i.e. Area × depth
Area = Central 1 sq. mm
= 25 sq.
Out of which, 5 are counted.
25 = 1. Sq. mm.
5 =?
5/ 25 =
Total RBC count = N × Dilution / Area × Depth
N × 200 / 1/ 5 × 0.1
Total RBC count = N × 10,000.
Thus, with the help of this formula, we can calculate the total number of RBC present in blood.