Aim: To determine the red blood corpuscles (RBC) count of own blood using a haemocytometer.
Requirements: Neubauer’s chamber, RBC pipette, coverslips, compound microscope, sterile disposable needle (26G), cotton swab, 70% alcohol or any other suitable marketed antiseptic, RBC diluting fluid i.e. Hayem’s fluid.
Composition of RBC Diluting Fluid
|Sr. No.||Name of ingredient||Quantity||Role|
|1.||Sodium chloride||0.5 g||Provides isotonicity|
|2.||Sodium sulphate||2.5 g||Provides isotonicity, an anticoagulant|
|3.||Mercuric chloride||0.25 g||An antimicrobial agent|
|4.||Distilled water||Up to 100 ml||Vehicle|
Principle: RBCs (also called erythrocytes) are major blood cells that carry oxygen and carbon dioxide. Millions of RBCs are present in one mm3 of blood, it is difficult to count them in the whole blood under a microscope. Hence, the blood is diluted 100 or 200 times and then cells are counted using Neubauer’s chamber. Their number is again calculated in undiluted blood by knowing the dilution factor.
Sodium chloride and sodium sulphate present in the RBC diluting fluid provide isotonicity so that RBCs remain suspended in diluted blood without changing shape and size. Sodium sulphate also acts as an anticoagulant and mercuric chloride act as an antimicrobial agent and prevents contamination and growth of micro-organisms.
- Examine the Neubauer’s chamber with coverslip under low magnification (10 X) and focus the central 1 mm RBC counting square.
- Get a finger prick under aseptic conditions using a sterile disposable needle. Discard the first two drops of blood and fill the RBC pipette up to the mark of 0.5 without any air gap.
- Immediately, suck the RBC diluting fluid to the mark of 101 and mix the content of the bulb gently by rolling the bulb between the palms for 3-4 minutes.
- Discard the first two drops from the pipette and fill the chamber with two drops of diluted blood.
- Switch over to high magnification (45 X) and count the number of cells in four corner squares and one central square of the RBC counting area (i.e. in 80 smallest squares).
Rules for counting:
- Cells lying within a square are to be counted with that square.
- Cells lying on or touching the lower horizontal line and left vertical line are to be counted in that square (‘LL’ rule of counting; L-lower, L- left) to avoid counting errors.
- First, count the cells in the upper four horizontal smallest squares from left to right and come down to the next row. Then count from right to left, come down to the next row and so-on.
Note: The normal RBC counts in undiluted blood are:
- Males: 5 million/mm3 (4.5-6.0 million/mm3)
- Females: 4.5 million/mm3 (4.0-5.5 million/mm3)
- A number of cells in 80 smallest squares = y.
- Area of one smallest square = 1/20 × 1/20 = 1/400 mm2.
- Depth = 1/10 mm i.e. distance between slide and counting chamber.
- Volume of one smallest square = 1/400 × 1/10 = 1/4000 mm3.
- Volume of 80 smallest square = 1/4000 × 80 = 1/50 mm3.
- A number of cells in 1/50 mm3 volume of diluted blood = y.
- In 1 mm3 volume, undiluted blood number of cells will be = 50 × y × dilution factor. = y × 10000
Result: The RBC count of own blood was found to be ……/mm3.
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