Total Erythrocyte Count by Hemocytometer

The manual method of RBC count is time consuming and imprecise. Introduction The red blood cell count is important in diagnostic hematology. It permits the MCV and MCH values to be calculated. The manual method of RBC count is time consuming and imprecise. The reference method preferred For RBC count is an automated method. Clinical Significance At birth the total erythrocyte count varies from 6.5 millions to 7.25 millions per cubic mm (cu mm). There is steady decline after a few hours and at the end of 15 days to one month there is a small rise to normal adult levels. An increase in total erythrocyte count is observed in conditions such as 1. hemoconcentration due to burns, cholera etc. 2. in central cyanotic states as seen in chronic heart disease, conditions of decreased lung function such as emphysema and 3. in polycuthemia. Decrease in erythrocyte count is observed in 1. old age 2. in pregnancy and in 3. group of diseases classified under anemias Normal values Male: 4.5 to 6.0 x 106 cells/cu mm (l). Female: 4.0 to 4.5 X 106 cells/cu mm (l). Specimens 1. Double oxalated or EDTA blood or 2. Capillary blood (The specimen need not be a fasting sample) Principle The blood specimen is diluted 1:200 with the RBC diluting fluid and cells are counted under high power (40 X objective) by using a counting chamber. The

number of cells in undiluted blood are calculated and reported as the number of red cells per cu mm (l) of whole blood. Requirements Microscope Improved Neubauer Chamber RBC pipette RBC diluting fluid. It is prepared as follows: sodium citrate : 3.0 g formalin : 1.0 ml distilled water to : 100 ml This solution is table at room temperature (25oC5oC) for at least one year. Note 1. RBC diluting fluid is isotonic with blood hence hemolysis does not take place. Normal saline also can be used, but it causes slight creation of red blood cells and allows rouleaux formation. 2. Formalin acts (as a preservative and checks bacterial and fungal growth. 3. Sodium citrate prevents coagulation of blood and provider correct osmotic pressure. Procedure 1. Mix the anticoagulated blood carefully by swirling the bulb. 2. In the case of capillary blood the lancet stab should be sufficiently deep to allow free flow of blood. It is drawn quickly in the RBC Pipette 3. Draw blood up to 0.5 mark. 4. Carefully wipe the excess blood outside the pipette by using cotton or a gauze 5. Draw diluting fluid up to 101 mark. 6. The pipette is rotated rapidly by keeping it horizontal during mixing. 7. After five minutes, by discarding few drops from the pipette and holding it slightly inclined small volume of the fluid is introduced under the cover s which is placed on the counting chamber. 8. Allow the cells to settle for 2 to 3 minutes. 9. Place the counting chamber on the stage of the microscope. 10. Switch to low power (10 x ) objective. Adjust light and locate the large square in the center with 25 small squares. 11. Now switch to high power (40 x ) objective. 1. 2. 3. 4.

12. The red blood cells in the four corner squares and in the center square (marked in the diagrams as R) are counted. 13. Use following formulae for the calculation of red blood cells, 6 Total red blood cells per liter of blood = RBCs / cu mm (l) x 10 Or use following formula Red cell count (per liter) = [(no. of cell counted) / (volume counted (l))] x dilution x 106 Total red blood cells/cu mm (l) = [number of red cells counted x dilution] / [area counted x depth of fluid] Where (1) Dilution = 1:200 (i.e., 200) (2) Area counted = 80 / 400 = 1/5 sq.mm Since cell are counted in 5 bigger squares and such square is further divided into 16 small squares. Each small square = 1/400 sq.mm Hence area of (5 * 16) = 80 such areas = 80/400 sq.mm = 1/5 sq.mm (3) Depth of fluid = 1/10 mm (4) Number of red cells counted = N Hence total red blood cells, cu mm = [(N * 200)] / [(1/5) * (1/10)] = N * 200 *50 = N * 10,000 Additional information For dilution of blood, instead of using an RBC pipette following methodology is preferred. - By using a 20 l (standardized) pipette, 20 l of blood is mixed with 4 ml of RBC diluting fluid in a glass test tube (10 x 75 mm). It is mixed well and then small volume of this fluid is introduced into a counting chamber. - The errors of blood counting by visual means are very considerable due to the random distribution of the cells in the counting chamber. The movement of the cells in the chamber during the filling process causes them to collide and this influences their distribution. The inherent distribution error can be reduced (a) by counting more cells. - To confirm specific results, it is better to re peat a count using a second chamber and pipette than to count double the number of cells in single filling of the counting chamber. Sources of Error

Falsely high counts 1. Collection of blood from the area where there is hemoconcentration. 2. Inadequate wiping of the pipette. 3. Improper pipetting of blood as well as the fluid. 4. Improper mixing. 5. Uneven distribution in the counting chamber. 6. Errors in calculations. Falsely low counts 1. Blood dilution with tissue fluid due to edema or squeezing. 2. Improper pipetting and dilution (when blood drawn is less and if dilution is above the mark). 3. Dilution of the contents in the pipette by saliva. 4. Uneven distribution of the cells in the counting chamber. 5. Error in calculations. 6. Use of improperly standardized counting chamber and bad adjustment of cover slip