ENUMERATION OF RBCS
- 1. ENUMERATION OF RBCs The red blood cell count is the determination of the number of erythrocytes in 1 mm3 of blood. Apparatus and Reagent - Neubauer s chamber with cover slip - Red cell pipette - Microscope - Diluting fluid
- 2. Protocol The center secondary square in the surface of hemocytomer chamber has an area of 1 square mm, which is divided into 25 tertiary squares. Each of the 25 squares is further divided into 16 smaller quaternary squares (as described above). So the total area of the center secondary square is divided into 400 very small squares (25 x 16). -The RBC pipette like WBC pipette except that contain a red bead and making dilutions of 1:200. -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. (In cases of leukocytosis, however, the leukocytes are easily identified and are not counted).
- 3. Protocol Diluting fluids a) Hayem s fluid - Mercuric chloride 0.5 gm -Sodium chloride 1.0 gm Sodium sulphate 5.0gm Distilled water to 200 ml. Procedure 1) Draw blood to the o.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) Wipe tip clean and draw in the diluting fluid up to the mark 101 (dilution 1 in 200). While filling the bulb the pipette should be gently rotated to obtain good mixing. 3) The coverslip is placed over the neubauer s chamber so as to cover both the ruled platforms evenly.
- 4. Protocol • 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; it must be sufficiently large to cover the whole ruled plate- form yet not large enough to fill the moat .Also there must be no air bubbles. (5)Allow two minutes for setting of the cells and then count.
- 5. Protocol • (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.
- 6. Calculations • The total area of the whole large central square is 1 mm2. The area of the smallest square is 1/400 mm2 and since the depth is 1/10 mm, its volume is 1/4000 mm3. Total volume of 80 small squares is therefore 80/4000 mm3--=1 / 50 mm3. Dilution is 1 in 200. RBC count =Dilution x I/Volume x number of cells counted (N) =200 x50xN =10,000 x No of cells/cu.mm. **************** Normal range RBC = 4.7-5.2 million cell/ mm3 of blood in female and 5.3-5.7 cell/ mm3 of blood in male.
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