RBC Count Procedure PDF

## **To perform Total RBC Count** ### **Red Blood Cells** - Red blood cells, or erythrocytes, are the most abundant cells in the bloodstream and contains hemoglobin, the compound that carries oxygen through the body. - Any disruption of the red blood cells, its quantity, shape, size, structure or l...

## **To perform Total RBC Count** ### **Red Blood Cells** - Red blood cells, or erythrocytes, are the most abundant cells in the bloodstream and contains hemoglobin, the compound that carries oxygen through the body. - Any disruption of the red blood cells, its quantity, shape, size, structure or life cycle can therefore affect the oxygen-carrying capacity of the blood. ### **Shape and Size** - Normally disk shaped & bi-concave. - Central portion thinner and periphery thicker. - **Size**: Diameter = 7.2µ (6.9-7.5 μ) - **Thickness**: - At periphery= 2.2 μ - At center = 1 μ ### **Methods of RBC Count** 1. Visual haemocytometer( manual method) 2. Automated method ### **Manual Method** - NEUBAUER CHAMBER - RBC PIPETTE - RBC DILUTING FLUID - EDTA SAMPLE ### **RBC Diluting Fluid** - RBCs are around only 5 millions/cumm 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 RBCs. - It is isotonic to RBCs. ### **Neubauer Chamber** - The most frequently used haemocytometer is the Neubauer (or 'Improved Neubauer') chamber. - The ruled area is 3mm2 divided into 9 large squares each with a 1 mm2 area. - The large central square (which can be seen in its entirely with the 10X objective), is divided into 25 medium squares with double or triple lines. - Each of these 25 squares are again divided into 16 small squares with single lines. - So that each of the smallest squares has an area of 1/400 mm2. - The large center square is used for RBC counts. - This area is subdivided into 25 medium squares, which in turn are each divided into 16 squares. - Of the 25 medium squares, only the four corner squares and the center square within the large center square are used for counting RBCs. ### **Procedure** - Assemble all the equipment. Draw the blood directly from the finger or collected sample into the RBC pipette up to a 0.5 mark. - Wipe off the tip of the pipette to remove any extra blood. - Then immediately draw up the diluting fluid up to the 101 mark. - Now rotate the pipette gently for 2-3 mins so that the diluting fluid gets mixed properly. - This will give a dilution of 1:200. - Place the cover slip in position over the ruled area of the chamber. Once again mix the solution thoroughly by rotating the pipette. - Discard the first 1-2 drops of blood from the pipette. - Now apply slight pressure on the rubber tube of the pipette, so that the third drop of the fluid is in a hanging position. - Touch the tip of the pipette (hanging drop.) against the edge of the cover slip. - The angle between the pipette and the cover slip is 45 degrees. - With this process, the chamber gets filled. - This is known as charging the chamber. - Make sure no air bubble is present inside the chamber and that there is no overfilling beyond the ruled area. - Now count the RBC using 40x in the central square. - The central square is subdivided into 25 squares. - Out of the 25 squares, count the four at each corner and one at the center. - Each of these five squares is subdivided into 16 small squares. - RBC's are counted in 16 x 5 = 80 small squares. - In case of marginal cells, count the cells on the 'L' line. - Either the right and lower or the left and the upper margin. ### **Diluting Fluid** - Two types of diluting fluids are used for RBC counting. 1. Hayem's fluid 2. Dacie's fluid ### **Composition of Hayem's Fluid** - Sodium chloride – 0.5gm - Sodium sulfate – 2.5gm - Mercuric chloride – 0.2.5gm - Distilled water – 100ml ### **Composition of Dacie's Fluid** - Trisodium citrate – 3 gm. - Formalin – 1ml. - Distilled water – 99 ml. - This diluting fluid is cheap and commonly used. ### **Errors of RBC Count** - **Technical Errors** - Defective apparatus/defective technique - Technical errors can be minimized by the use of accurately calibrated apparatus and by careful technique. - **Inherent Error** - The distribution of RBCs is of an irregular pattern in the counting chamber. - This affects the accuracy of visual count which can be minimized by counting a large number of RBCs. ### **Reference Range** - Adult male - 4.5 to 5.5 million/cumm - Adult female - 3.8 to 5.2 million /cumm - At birth - 6 to 8 million/cumm