Aim: Introduction to haemocytometry.
Theory: Haemocytometry is the study of counting several cells in a blood sample. Since the whole blood contains a large number of blood cells, it is difficult to count them under a microscope. This difficulty can be overcome by diluting blood with suitable diluting fluid and then counting them. The whole process of counting blood cells involves:
- Collection of a blood sample from the finger prick.
- Filling the blood in the pipette.
- Dilution of blood with suitable diluting fluid.
- Filling counting chamber with diluted blood i.e. charging the chamber.
- Counting cells and reporting results in terms of the number of cells per cubic millimeter.
Blood cell counting is performed using specific apparatus called a hemocytometer which consists of:
- The diluting pipettes.
- The counting chamber/ Neubauer’s chamber.
1. The diluting pipettes: Two different glass capillary pipettes having a bulb are provided for diluting the blood for RBCs and WBCs counting. The pipette has a long narrow stem with a capillary bore and a conical tip. It is divided into ten equal parts and only two markings are numbered; i.e. 0.5 in the middle of the stem, and 1.0 at the junction of the stem and the bulb.
The stem widens into a bulb containing a free-rolling bead which helps in mixing blood with diluting fluid. The color of the bead is red in the RBC pipette and white in the WBC pipette.
The bulb narrows again into a short stem to which a long rubber tube is attached wearing a mouthpiece (Red mouthpiece for RBC pipette and white for WBC pipette). Just above the bulb, a marking of 101 is present on the RBC pipette and 11 on the WBC pipette.
Points to remember for differentiation of RBC and WBC pipettes are shown in the following table.
|RBC pipette||WBC pipette|
|1. Calibrations are 0.5 and 1.0 below the bulb and 101 above the bulb.||1. Calibrations are 0.5 and 1.0 below the bulb and 11 above the bulb.|
|2. Capillary bore is narrow, thus it is a slow-speed pipette.||2. The capillary bore is wider, hence it is a fast-speed pipette.|
|3. Bulb is larger and has a red bead.||3. Bulb is smaller and has a white bead.|
|4. The volume of the bulb is 100 times the volume contained in the stem.||4. The volume of the bulb is 10 times the volume contained in the stem.|
|5. The dilution can be 1: 100 or 1: 200.||5. The dilution can be 1: 10 or 1: 20.|
2. Neubauer’s chamber or counting chamber: The counting chamber is a single solid heavy glass slide. There are three parallel platforms or pillars separated from each other by shallow trenches extending across the middle third portion of the slide. The central platform is wider and exactly 0.1 mm lower than the two lateral platforms. It is divided into two equal parts by a short transverse trench in its middle. Thus, there is H shaped trench enclosing the two platforms. The lateral platforms support the coverslip and the central platform contains a counting grid.
The counting grid: Each counting grid measures 9 mm2 (3 mm × 3 mm) and is divided into 9 squares each of 1 mm2 (1 mm × 1 mm).
Out of these 9 squares, four large corner squares are used for counting WBCs. Each large square is divided by single lines into 16 medium-sized squares. Each of these medium squares has a side of ¼ mm and an area of 1/16 mm2 (1/4 mm × 1/4 mm).
The central large square is divided into 25 medium-sized squares each is having a side of 1/5 mm. These medium squares are separated by double or triple lines. These lines extend in all directions beyond the boundaries of 9 mm square i.e. in between all the WBCs squares around the central RBCs squares. Each of the 25 medium squares is further divided into 16 smallest squares by single lines. These smallest squares are having a side of 1/20 mm and an area of 1/400 mm2. RBCs are counted in four corners and one central medium square each containing 16 smallest squares i.e. in a total of 80 smallest squares.
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