Manual platelet count test
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Platelets are a component of blood , that help your body form clots , in case of bleeding.
 Platelets are fragments of cytoplasm that are genarate from the megakaryocytes of the bone marrow.
A healthy adult individual , normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood.
Having more than normal range platelets , called thrombocytosis & having less than normal range is known as thrombocytopenia. So platelet count is really good clue to , diagnose hidden diseases , that may be affect the process of blood clotting , leading to bleeding .
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Therefore , Manual platelet count is really important test for get idea about some critical disease conditions.
Principle
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Blood is diluted in a filtered solution of 1% Ammonium oxalate at a ratio of 1:20.
It lyses RBCs and remains WBCs and platelets. Then platelets are counted microscopically using improved Neubauer counting chamber and number of platelets per liter of blood is calculated.
Requirements
- EDTA anticoagulated fresh veneous blood
- Improved Neubauer counting chamber
- Pipettes
- Filtered 1% Ammonium oxalate – dilution fluid ( 1mL of Ammonium oxalate and 100mL of Distilled water)
- Humid chamber
- Microscope
Method
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- Â Mix the blood sample carefully and add 0.38mL of filtered 1% Ammonium oxalate in to a clean glass tube.
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2)  Add 20μL of blood and mix it well. Leave it for 3-4 min to lyse RBC.
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3)Â Assemble and clean the counting chamber
4) Then charge it carefully with above suspension.
5) To prevent drying of charged fluid, keep the chamber in humid container for 15-20 min , allowing   platelet’s to the settle down.
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After that focus the ruling area of counting chamber under 10X objective and count the platelets in the middle large square( all 25 small squares) using 40X objective and lower the condenser to reduce the light intensity.
In this case platelets appear as highly retractile particles.
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WBC count is low , so it does not interfere with platelets count …
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Platelet count per liter of blood= cells counted X dilution factor (20) X106
                              volume (1X1X0.1mm3)
Normal range = 150-400 X 106/ L
Sources of errors
- Unmixed blood sample
- Blood collected from capillaries (heal/finger prick)- results falsely low platelet count – however if we remove first blood drop we can minimize the error
- If clumps of platelts present in the , hemocytometer the procedure should be repeat.
- Pipetting errors
- Improper mixing after dilution
- Dirty counting chamber or cover glass
- Uneven distribution of platelets
- Over filling or under filling of counting chamber
- Wrong counting techniques
- Intense illumination
- Wrong calculation
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