Method Article
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January 15th, 2026
Source:
Sathish Muthu1,2,3
1Orthopaedic Research Group, Coimbatore.
2Department of Biotechnology, Faculty of Engineering, Karpagam Academy of Higher Education.
3Department of Orthopaedics, Government Medical College.
This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and isolating platelets. This PRP is used in therapeutic applications for tissue regeneration and healing.
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Sample collection
Screen the patients using preliminary blood counts to rule out thrombocytopenia and establish baseline cell counts. To do this, use a clinical-grade hemocytometer for cell counting. Ensure the platelet counts are above 150,000 cells/µL.
Ensure the patient is hydrated well before sample collection. This could be ensured by visual inspection of the tongue, skin turgor, and blood pressure measurement.
Collect 100 mL of peripheral venous blood from the antecubital vein of the patient using 2 x 50 mL syringes filled with 5 mL of Acid-Citrate-Dextrose-Adenine (ACDA) anticoagulant in a ratio of 1:10. Tilt the collected syringes for proper mixing of the anticoagulant to avoid clotting of the blood sample before proceeding to next step.
NOTE: Slow blood draw may result in inadequate mixing of the collected blood with the anticoagulant and clotting; hence, use a minimum 18G cannula for blood collection.
Transfer 100 mL of blood in the syringes to 15 mL conical bottom centrifugation tubes with 10 mL in each tube.
2. Centrifugation and isolation
Place the centrifugation tubes in a clinical-grade tabletop centrifugation machine maintained at room temperature in a sterile environment and run the initial soft spin at the speed of 100 x g for 15 min. The anticoagulated blood separates into three layers, i.e., red blood cells at the bottom, plasma on the top, and buffy coat in the middle.
Pipette the top layer of the tri-layer sample in a sterile fashion using an 18G sterile spinal needle and collect it in a separate conical bottom centrifugation tube. Ensure individual tubes for every 10 mL of plasma collected.
NOTE: The decision to include the buffy coat depends on the nature of the platelet-rich plasma we are trying to isolate, which in turn depends on the nature of its clinical application. If leucocyte-poor PRP is the desired product, refrain from including the buffy coat layer, whereas if leucocyte-rich PRP is the desired product, then the top plasma layer along with the buffy coat is pipetted out for the second round of centrifugation.
Subject the collected plasma in 15 mL centrifugation tubes with 10 mL of plasma each to hard spin centrifugation at 1600 x g for 20 min. The platelets present in the plasma pellet at the bottom of the test tube. Collect the supernatant in a separate tube.
Pipette the platelet pellet into a 5 mL syringe with the help of an 18 G sterile spinal needle. Collect all the test tubes with pellets into a single 5 mL syringe for application.
Reconstitute the collected pellet with sufficient volumes for clinical use with the supernatant plasma in the centrifugation tube following the second round of centrifugation.
3. Validation
Subject the final PRP prepared to cell counting using clinical grade hemacytometer to calculate the platelet count and calculate the absolute platelet count in the PRP, platelet concentration gradient achieved over baseline, and platelet recovery efficacy of the protocol using the following formula.
Absolute Platelet Count = platelet count of the PRP in µL x 1000 x volume of PRP in mL
Platelet concentration gradient = 
Platelet recovery efficiency = 
For example, 100 mL of blood with a baseline platelet count of 200,000 / µL is used for PRP preparation, and 5 mL of PRP with a final platelet count of 3,000,000 / µL is obtained. The above-mentioned parameters are calculated as follows:
Absolute Platelet Count = 3,000,000 x 1000 x 5 = 15 billion platelets
Platelet concentration gradient = 3,000,000 / 200,000 = 15
Platelet recovery efficiency = 3,000,000 x 1000 x 5 / 200,000 x 1000 x 100 = 75%
Ensure the recovery efficiency of the protocol through repeated cycles. An effective protocol utilized delivers a concentration gradient above 5 times the baseline with >80% recovery efficiency. Based on the desired usage of the volume of PRP, platelet counts that need to be delivered can be titrated accordingly.
