Aim: To determine the platelet count of own blood.
Requirements: RBC pipette, Neubauer’s chamber, coverslip, cotton swabs, petri dish, platelet diluting fluid (1% formalin in 3% ammonium oxalate solution), microscope, disposable needle (26G), 70% alcohol, or any other suitable marketed antiseptic.
Principle: Platelet diluting fluid contains ammonium oxalate which reacts with blood and ruptures RBCs. It also acts as an anticoagulant which helps to prevent the aggregation of platelets and makes counting easy. Platelets are derived from the fragments of the plasma membrane of megakaryoblasts and are responsible for platelet plug formation that aids hemostasis.
- Place the coverslip on the counting grid of Neubauer’s chamber.
- Sterilize the tip of the ring finger with a suitable antiseptic solution.
- Gently prick the finger with the help of a pricking needle 26G and fill the pipette with blood by sucking accurately up to the 0.5 mark in the RBC pipette.
- Dip the tip of the pipette in the freshly prepared platelet diluting fluid kept in the watch glass and suck accurately up to the 101 mark on the pipette.
- Hold the pipette horizontally, and gently roll the pipette between the palms to mix the blood thoroughly with platelet diluting fluid.
- Wait for 10-15 minutes (During this RBCs rupture by the action of ammonium oxalate).
- Discard the first 3-5 drops and then hold the pipette at an angle of about 45 degrees and touch the tip of the pipette to the middle part of the upper edge of the coverslip till it spread completely.
- Allow the fluid to settle for the next 2 to 3 minutes.
- Count the platelets in all central 25 RBC squares under the higher objective of the microscope.
- Calculate platelet count using the formula.
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.
- Platelet count (per mm3) = Number of platelets count × Dilution/volume of fluid
- = Total number of platelet count in central 25 square × 200/0.1
- Normal Value: 1.5 to 4.0 lakhs/mm3.
Result: Platelets of given blood sample was found to be …….
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