How to count Platelets by the manual method using Improved Neubauer Counting Chamber?

We are living in the era of modernization, in terms of using fully automatic Hematology analyzer in Laboratory but still today, the old gold standard manual method of counting platelets by the manual method using Improved Neubauer Counting Chamber is very important.

Manual counts are still necessary, If there is a significant proportion of giant platelets. Every Medical Lab Technician should know how to do it ?

What are Platelets ?

Platelets are irregular in outline with fine red granules that may be scattered or centralized in the cell.

Size : –  1. Normal platelets are 1-3 µm in diameter.

              2. A small number of larger platelets are up to 5 µm in diameter.

Normal Count :- 1.5 lakh to 4.5 lakhs/cumm

Reagent required for doing manual Platelet Count.

1%  aqueous Ammonium Oxalate

Weigh 1 gm Ammonium Oxalate and dissolve in 100 ml of distilled water/deionized water. Mix it properly.

Use clean glassware to prepare the solution.

To avoid contamination, make less quantity not more than 500 ml at a time.

Store it at 4 degree C.Labeled the bottle of reagent with date of preparing the reagent.

How to do manual Platelet Count ?

Dilution : –  Generally we make 1 : 200 dilution

Blood sample : 20 µl

1% Ammonium Oxalate solution : – 3.980 µl

Blood sample required :- EDTA Blood Sample

Mix it well and stand it for 10-15 minutes.  In 1%  aqueous Ammonium Oxalate solution, red cells are get lysed. Fill the neubauer chamber with the help of micropipette or Pasteur pipette.Place the counting chamber in a moist petridish for at least 20 minutes.

Points to remember

1. We can use wet gauge piece or tissue paper in petridish to make it moist. It would not dry filled suspension in counting chamber.
2. We keep it in  moist petridish for at least 20 minutes as platelets get settled.

Counting of Platelets in neubauer chamber :- first focus in 10 x then change in 40 x and count in centre square which is divided into 25 small squares.

How to calculate Platelets ?

Calculation:-

Platelet  count ( per litre) =

No. of cells counted/ volume counted(µl) x Dilution x 106( 10 raise to the power 6)

Let’s understand

N = number of platelets counted in an area of 1 square mm ( 0.1 µl in volume)

 , the number of platelets per litre of blood is : – N x 10 x 200(dilution) x 106( 10 raise to the power 6) =  N x 2000 x 106( 10 raise to the power 6)

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