Platelet-rich Plasma Preparation
Platelet-rich plasma is defined as the plasma concentrate of autologous blood with a platelet quantity above normal physiologic level.[24] Normal platelet concentrations in blood range from 150,000 to 450,000 platelets/mm3.[25] Some studies report baseline platelet counts to standardize platelet-rich plasma concentrations. Clinically, this is not practical, as it adds unnecessary costs, and it is unclear whether this translates to higher efficacy.
Various forms of platelet-rich plasma exist as detailed in Table 2. These are classified based on the contents of the concentrate (i.e., leukocytes and/or platelets), and are also classified based on the density of the fibrin network that supports this leukocyte and platelet concentrate. Such variation in the final platelet-rich plasma product is attributable to a wide range of preparation protocols, differing in the number, force, and time of centrifugation.[26,27] For the single-spin technique, a sample of whole blood is centrifuged, and three layers are formed because of different densities (Figure 1). The bottom layer consists of red blood cells; the middle layer, known as the buffy coat, consists of platelets and leukocytes; and the top layer consists of plasma.[18] Although the bottom layer is always discarded, the use of variable quantities of the buffy coat and plasma layer can yield different platelet concentrations. A second spin can further separate the solution, potentially creating a solution with a higher platelet concentration that either includes or excludes leukocytes, depending on the amount of buffy coat transferred to the second tube.[9,26,28] For pure platelet-rich fibrin and leukocyte- and platelet-rich fibrin, a high-density fibrin-platelet clot is produced during the centrifugation process. It allows for slower release of growth factors over time and has been shown to dissolve more slowly than the low-density fibrin matrix of pure platelet-rich plasma or leukocyte- and platelet-rich plasma.[27] In the literature, differing opinions exist on the role of leukocytes in platelet-rich plasma solution. Proponents of platelet-rich plasma high in leukocyte concentration discuss the importance of these leukocytes in immune regulation, infection prevention, and promotion of angiogenesis through release of vascular endothelial growth factor.[26,29,30] Alternatively, studies that advocate for the exclusion of leukocytes argue that the inflammatory response produced by cytokine release from these leukocytes causes negative effects on the treated tissue.[31,32]
Figure 1.
Three layers of centrifuged whole blood.
Three factors can be addressed when making platelet-rich plasma: anticoagulation, centrifugation, and activation. A generalized platelet-rich plasma preparation guide is listed in Figure 2.
Figure 2.
Platelet-rich plasma preparation overview.
Anticoagulation
Anticoagulation is used during the processing of whole blood to prevent clotting. Platelet-rich plasma is stable in the anticoagulated state for approximately 8 hours after its preparation.[10,24,33] Coagulation can be halted by disabling calcium ions, which are necessary for clot formation in the coagulation cascade. This is done with the addition of citrate ion, which binds and deactivates calcium, forming calcium citrate. Many anticoagulants are used and include sodium citrate, acid citrate dextrose, citrate phosphate dextrose adenine, trisodium citrate, trisodium phosphate, and citrate. Most commercial platelet-rich plasma device venipuncture collection tubes are coated with an anticoagulant.
Centrifugation
Variation exists in the literature with regard to the number, force, and time of centrifugations performed.[34,35] A standard laboratory centrifuge or commercial collecting system may be used for platelet-rich plasma preparation. These systems differ in the platelet concentration produced, with variable ranges based on the device used.[18,24,36–38] Some collection systems also use a separator gel during the centrifugation process that provides a physical gradient for blood to pass through.[39–41]
During processing, platelet activation should be avoided as much as possible. Platelet fragmentation should also be avoided, as this can result in premature release of activated proteins, with poor bioactivity.[36] To help prevent such fragmentation, low gravity forces during centrifugation can be implemented along with acid citrate dextrose anticoagulant, which preserves platelet membrane integrity.[24,36]
Activation
An exogenous activator can be added to the platelet-rich plasma to facilitate release of biologically active proteins. This is done with a calcium-based activator alone (e.g., calcium chloride), calcium gluconate, or thrombin. Calcium that was previously bound by the anticoagulant is replenished with the addition of calcium activator.[15,39,42–45] Alternatively, thrombin functions by directly activating platelets.
Biologically active proteins are secreted from platelets within 10 minutes of clot initiation, and more than 95 percent of these presynthesized growth factors would have been secreted within the first hour.[10,46] For this reason, delivery of platelet-rich plasma within 10 minutes is advised. Some studies do not activate platelets, arguing that this step is unnecessary and that platelets are ready to exert their function once injected.[35,47–50] [See Video 1 (online), which demonstrates the preparation process of platelet-rich plasma.]
Plast Reconstr Surg. 2021;147(1):219-230. © 2021 Lippincott Williams & Wilkins
