This article outlines the procedure for isolating platelet-rich plasma (PRP) from human peripheral blood. The method involves centrifugation to separate blood components and reconstitution of platelets for clinical applications.
Begin with human peripheral blood in a conical tube.
Centrifuge the tube at a low speed to separate the blood into three layers: red blood cells at the bottom, the buffy coat in the middle, and platelet-containing plasma at the top.
Transfer the plasma layer to a fresh tube and centrifuge it at a high speed to pellet the platelets.
Transfer the plasma supernatant to a new tube.
Collect the platelet pellet and reconstitute it with an appropriate volume of the plasma supernatant to obtain platelet-rich plasma or PRP, which contains a high concentration of platelets.
To begin, place the centrifugation tubes containing the anticoagulated blood in a clinical grade tabletop centrifugation machine set to room temperature within a sterile environment. Run the initial soft spin at 100 g for 15 minutes to separate the anticoagulated blood into three layers: red blood cells at the bottom, plasma on top, and the buffy coat in the middle.
Using an 18-gauge sterile spinal needle, pipette the top layer of plasma from the trilayer sample in a sterile manner. Collect the plasma into separate conical bottom centrifugation tubes, ensuring each tube holds 10 milliliters of plasma.
Then, place the 15-milliliter centrifugation tubes containing 10 milliliters of plasma each into the centrifuge. Perform a hard spin centrifugation at 1,600 g for 20 minutes to pellet the platelets at the bottom of the tubes. Collect the supernatant into separate tubes.
Next, using an 18-gauge sterile spinal needle, pipette the platelet pellet into a 5-milliliter syringe. Collect all platelet pellets from different tubes into a single 5-milliliter syringe for application.
Afterward, reconstitute the collected pellet with the required volume of supernatant plasma from the second centrifugation for clinical use. Using a clinical grade hemocytometer or automated hematology analyzer, count the platelets in the final PRP sample. The absolute platelet count, platelet concentration gradient, and platelet recovery efficiency are calculated using these formulas.
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