The Enumeration of Red Blood Cells (The Erythrocyte Count) The red cells count is the determination of the
number of erythrocyte in 1 mm3 of blood.
Apparatus and Reagent
1234Neubauer chamber with coverslip (Hemocytometer Chamber). Red cell pipette. Microscope. Diluting fluid.
Neubauer Counting Chambers The hemocytometer counting chamber (Neubauer) is used for cell counting . The surface of the chamber contains two ruled areas separated by an H- shaped moat . These are the primary squares. The primary squares consist of a total area of 9 mm2 (3 mm on each side, length* width), which is divided into nine large squares. Each of these large squares measures 1mm on each side, with the total area being 1mm2. The four corner large square are further divided in to 16 medium squares to aid in cell counting. They are used for counting leukocytes. The center large square in the surface of hemocytometer chamber has an area of 1 mm2 , that is divided into 25 medium squares. Each of the 25 medium squares is further divided into 16 small squares. So the total area of the center large square is divided into 400 small squares (25 * 16 ). The four corner and center medium squares of the center large square are used when counting erythrocytes.
The distance between the bottom of the coverslip and the surface of the counting area of the chamber is 1 mm. Red cell pipette consists of a distal capillary stem that is divided into 10 equal parts and a mixing bulb containing red bead (aid in mixing the diluting fluid and blood), which is continued as a tube to be attached to a small rubber tube. For RBC counts, the solution that is used is isotonic with erythrocytes. The diluting fluids used do not lyses the leukocytes. Normally, the leukocytes are too few to interfere with the RBC count.
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Diluting fluids
1- Hayems solution a- Mercuric chloride 0.5 gm b- Sodium chloride 1 gm c- Sodium sulphate 5 gm d- Distilled water to 200 ml 2- Normal saline (0.9 % Nacl) a- Sodium chloride 9 gm b- Distilled water 100 ml
Procedure
1- Draw blood to the 0.5 mark in the RBC pipette, without letting any bubbles into the pipette by holding the pipette almost horizontally . The pipette must be clean and dry. 2- Draw the diluting fluid up to the mark 101 (dilution 1 to 200), while filling the bulb the pipette should be gently rotated to obtain good mixing. 3- The coverslip is placed over the neubauers chamber so as to cover both the ruled platforms evently. 4- Now load the chamber. This is done in three steps: a- Mix the contents of pipette for 3 minutes. b- Expel 6 drops from the pipette to remove the fluid in the stem which has not been mixed with blood. c- By holding the pipette at an angle of 45 degree and touching the space between the coverslip and the chamber by the point of the pipette, an appropriate drop of the mixture is allowed to run under the coverslip by capillary action. 5- Allow two minutes for setting of the cells and then count. 6- The count is done as follows: In the erythrocyte count, the central double ruled square is used. Red cells lying in 80 very small squares have to be counted. These 80 small squares, comprise 5 medium sized squares, each of which is bound by a triple line. It is recommended that the five medium sized squares chosen for counting cells should consist of four corners and one central, this is to secure a never distribution of cells, In counting, cells which touch the left hand lines or the upper lines of the square are taken to be within that square and those which touch the lower or right hand lines are omitted as outside the square.
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Calculation
The total area of the whole large central square is 1mm2 The central large square divided into 25 medium squares Each of the 25 medium square is further divided into 16 small squares So the total number of the small squares is 16 * 25 = 400 The area of the each small square is 1/ 400 mm2 Since the depth is 1/ 10 mm So the volume of each small square is 1/ 400 mm2 * 1/10 mm = 1/4000mm3 Since the count was done in 80 small square (5 *16 = 80) So the total volume of 80 small square is 80 * 1/4000 = 1/50 mm3
The number of RBCs in 1mm3 The number of RBC in 1/50 mm3 Y *1 X= 1 / 50
x y 1 = y* 1/ 50
= y * 50 Dilution is 200 X = y* 50 * 200 So the number of RBCs in 1 mm3 = number of cell counted * 10000
