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The ability to estimate platelet counts is an essential skill when working in a laboratory. Platelets are fragments of cytoplasm that help blood to clot. Platelet counts are used to determine thrombocytopenia (low platelet counts) and thrombocytosis (high platelet counts). According to the National Institute of Health, normal platelet counts are between 150,000 to 400,000 platelets per microliter of blood. Thrombocytopenia is associated with aplastic anemia, folate deficiency, vitamin B12 deficiency and acute leukemias. Thrombocytosis is associated with hemolytic anemia and polycythemia vera (excessive red blood cell production).
Dilute the blood sample in a 1:100 ratio of blood to diluent (ammonium oxalate). Remove the pipette from its shield aseptically by pushing the tip of the pipette shield through the diaphragm in the neck of the reservoir. Touch the pipette lip to blood and allow bore of pipette to fill with blood. Cover opening of pipette with index finger and place pipette in reservoir neck. Release pressure on the reservoir and remove your index finger from the pipette. Let stand for 10 minutes to allow red blood cells to hemolyze.
Fill the hemocytometer, which is a glass side with a counting chamber containing measured lines of known depth, with blood by squeezing the sides of the reservoir; then place the hemocytometer in a covered Petri dish and let stand for 10 minutes. Place the hemocytometer on the stage (the flat platform used for mounting slides) of a bright light or phase microscope and bring the ruled area of the hemocytometer into focus using the low power objective.
Switch to the 43X objective to increase your magnification and bring the large center square of the hemocytometer into focus. Count the plateletes in all 25 squares within the one large square of the hemocytometer, counting from left to right in the first row, then left to right in the second row until all rows are complete.
Multiply the number of cells counted by 1,000 to arrive at the total platelet count; for example, if the number of cells counted = 250, then 250 x 1,000 = 250,000 platelets per microliter.
You should do your estimate in an area of the smear in which the red blood cells barely touch one another. Repeat the procedure if you see clumps of platelets.
Perform platelet counts within 3 hours of preparing the dilution.
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