Optimized preparation method of platelet-concentrated plasma and noncoagulating platelet-derived factor concentrates: maximization of platelet concentration and removal of fibrinogen

Abstract

Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230-270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.

Figures

FIG. 1.

Flow chart for the preparation process of platelet-rich plasma (PRP)-related products. Whole blood (WB) was drawn from donors by venipuncture and collected into conical tubes that contained an anticoagulant. The PRP or white blood cell-containing PRP (W-PRP) was isolated by the first spin for 10 min. PRP was centrifuged again for 10 min to spin down the platelets. The platelet-poor plasma (PPP) layer was partially removed to create platelet-concentrated plasma (PCP) or replaced with one-tenth volume of phosphate-buffered saline (PBS) for noncoagulating platelet-derived factor concentrate (PFC). After the platelets were resuspended, thrombin was added for platelet activation. Color images available online at www.liebertonline.com/tec

FIG. 2.

Differential centrifugations for isolating PRP or W-PRP. WB was centrifuged for 10 min at various centrifugal forces to isolate plasma. *p<0.05. (A) Collected plasma volume relative to WB volume. The collected plasma volume increased with increasing centrifugal force and peaked at up to 50% of the WB volume at 2330 g (n=20). (B) Platelet (PLT) and white blood cell (WBC) yield in collected plasma relative to that in WB. PLT yield peaked at between 190 and 320 g (n=15), whereas WBC yield decreased with as centrifugal force increased (n=16). The right figure shows the averaged data for PLT (red) and WBC (blue) yields. Values represent means±standard deviation. (C) Two-dimensional data of PLT and WBC collection rates at various centrifugal forces. PLT yield is relatively consistent, whereas WBC yield widely varies among donors. Yields (%) of PLT and WBC relative to WB were plotted on the x-axis and y-axis, respectively. Each centrifugal force is expressed in different colors (n=8). Color images available online at www.liebertonline.com/tec

FIG. 3.

Measurement of PLTs after centrifugation of PRP. PRP obtained by the first spin for 10 min at 270 g was centrifuged again to prepare PCP. *p<0.05. (A) PLT yield in PCP after the second spin for 10 min at various centrifugal forces relative to PRP (n=5). (B) PLT concentration in WB, PRP, and one-third and one-tenth volume of PCP (1/3v-PCP, 1/10v-PCP; n=11). PLTs in PRP were further concentrated by the second spin and the reduction of plasma volume in PCP. (C) Comparison of PLT yield (%) in PRP and 1/10v-PCP between two anticoagulants. PRP and 1/10v-PCP were prepared with the same protocol with either ethylene di-amine tetra-acetic acid (EDTA) or acid citrate dextrose (ACD) as the anticoagulant (n=10). EDTA showed significantly higher inhibitory effects on PLT aggregation than ACD.

FIG. 4.

Measurement of platelets and platelet-derived growth factor (PDGF)-BB in the preparation process of PCP and PFC. PCP and PFC were prepared using an optimized protocol. The number of PLTs was counted before PLT activation with thrombin, whereas the PDGF-BB protein level was measured after PLT activation. *p<0.05. (A) Comparison of PLT concentration in WB, PRP, 1/10v-PCP, and 1/10v-PFC obtained from a single sample (n=6). It was shown that PLTs were highly concentrated in 1/10v-PCP and 1/10v-PFC compared with PRP. (B) PLT yield relative to WB in PRP, 1/10v-PCP, and 1/10v-PFC obtained from a single sample (n=6). (C) Comparison of PDGF-BB concentration in WB, PRP, 1/10v-PCP, and 1/10v-PFC obtained from a single sample (n=6). PDGF-BB concentration differs between each product and was highest (nearly 50 times of WB) in 1/10v-PFC. (D) Total PDGF-BB content in WB, PRP, 1/10v-PCP, and 1/10v-PFC obtained from a single sample (n=6). Surprisingly, the total PDGF content in 1/10v-PFC was larger than the amount obtained from activated WB.

FIG. 5.

Measurement of PDGF-BB and fibrinogen in PFC. PFC was prepared by replacing supernatant PPP with PBS after the second spin. *p<0.05. (A) Comparison of two anticoagulants in regards to PDGF-BB concentration in 1/10v-PFC. Either EDTA or ACD was used as the anticoagulant for preparation of 1/10v-PFC with the rest of the protocol being the same (n=10). EDTA showed significantly higher inhibitory effects on platelet aggregation than ACD. (B) Comparison of fibrinogen concentration between 1/10v-PCP and 1/10v-PFC (n=3). Plasma replacement with PBS effectively reduced the fibrinogen concentration.

FIG. 6.

Conclusive preparation protocol for PRP-related products. Our optimized protocols for PRP-related products are as follows. WB collected using EDTA as an anticoagulant is centrifuged for 10 min at 230–270 g or 70 g in a 15-mL conical tube for the isolation of PRP and WBC-containing PRP, respectively. PRP is further centrifuged for 10 min at 2300 g (or higher) for the precipitation of platelets. For one-tenth volume of PCP (1/10v-PCP), nine-tenths volume of the supernatant PPP is removed, and the platelet pellet is resupended, followed by platelet activation with thrombin. For one-tenth volume of PFC (1/10v-PFC), all PPP is removed, one-tenth volume of PBS is added, and the platelet pellet is then resupended, followed by platelet activation with thrombin. Color images available online at www.liebertonline.com/tec