PRP method
Obtain WB by venipuncture in acid citrate dextrose (ACD) tubes
Do not chill the blood at any time before or during platelet separation.
Centrifuge the blood using a ‘soft’ spin.
Transfer the supernatant plasma containing platelets into another sterile tube (without anticoagulant).
Centrifuge tube at a higher speed (a hard spin) to obtain a platelet concentrate.
The lower 1/3rd is PRP and upper 2/3rd is platelet-poor plasma (PPP). At the bottom of the tube, platelet pellets are formed.
Remove PPP and suspend the platelet pellets in a minimum quantity of plasma (2-4 mL) by gently shaking the tube.
With regard to the type of anticoagulant for use, most authors agree on not using EDTA because it could damage the platelet membrane. Therefore, anticoagulants with citrate and dextrose of sodium citrate are recommended.[38]
The authors compared the effects of sodium citrate and ACD-A on platelet aggregation, pH and extracellular iCa concentration. The anticoagulant ACD-A is the choice for collection of platelets by apheresis, whereas trisodium citrate (3.2% or 3.8%) is the anticoagulant most commonly used for diagnostic evaluations of platelets. Trisodium citrate and ACD-A solutions differ markedly in pH, with ACD-A having a pH of 4.9 and 3.8% sodium citrate having a pH of 7.8. In addition, the citrate ion concentration in ACD-A is 15.6 mg/mL, whereas 3.8% sodium citrate contains 24.4 mg of citrate ion/mL. It has been reported. In several species that alterations in the pH and extracellular iCa concentration of PRP can affect platelet aggregation in vitro, with aggregation typically impaired at acidic pH and lower extracellular iCa concentrations.[39,40]
the authors found that at 900g x 5 mins for 1st centrifugation and 1000g x 10 mins for 2nd centrifugation at 16° C in a refrigerated centrifuge