Formulation and storage of platelet-rich plasma homemade product

Abstract

The platelet-rich plasma (PRP) is an autologous biotherapy based on platelet-healing properties. Here, we developed a simple and reproducible PRP purification protocol based on two successive centrifugations. We evaluated different centrifugation speeds and time-storage durations on the platelet quantity and quality. Sterility and stability of our PRP homemade product were also performed. We prepared PRP from 54 healthy volunteers. We tested activation state, reactivity, and stability of platelets by flow cytometry using basal and adenosine diphosphate (ADP)-induced P-selectin expression markers; growth factor release after platelet activation by an enzyme-linked immunosorbent assay (ELISA); platelet aggregation capacity by aggregrometry assays; clot formation and retraction by thromboelastography; and platelet morphology by ultrastructural analysis. About 130 and 250 g successive speed centrifugations further concentrated platelets while preserving their bioactivity during 6 h (after that, platelet functions were significantly altered). In these conditions, we obtained a highly concentrated pure PRP product (with a low leukocyte count) suitable to study platelet properties. To avoid the loss of efficacy, we recommend injecting PRP under 3 h after preparation.

FIG. 1.

(A) Platelet counting in whole blood and in four conditions of PRP preparation. Data obtained for different donors (130 g, n=11; 250 g, n=10; 400 g, n=10; 1000 g, n=6). ***p<0.0003 comparing with whole blood. (B) Platelet concentration factor versus speed of second centrifugation. Data obtained for different donors (130 g, n=10; 250 g, n=8; 400 g, n=8; 1000 g, n=6). *p=0.02. PRP, platelet-rich plasma.

FIG. 2.

Aggregation rate versus speed of the second centrifugation. Data obtained for different donors (130 g, n=10; 250 g, n=8; 400 g, n=8; 1000 g, n=6).

FIG. 3.

Ultrastructure of platelet issued of one PRP preparation and analyzed by transmission electron microscopy. Centrifugation protocol (A) 130 g; (B) 250 g; (C) 400 g, and (D) positive control induce by ADP. (A) Typical section of a resting platelet, discoid forms without pseudopodia, with well-preserved granular structure (panel left). Note the formation of loose aggregates in (A) and (B) (right panel) with largely preserved granular structure and nonactivated platelets. (D) Typical section corresponding to morphological signs of activation: spherical forms with pseudopodia, a partial centralization of a-granules, and some large aggregates composed of mostly degranulated platelets. ADP, adenosine diphosphate.

FIG. 4.

Thromboelastrography results. Time to clot formation (k) and maximum amplitude (MA). Results from three independent PRP preparations.

FIG. 5.

P-selectin expression versus speed of the second centrifugation. Data obtained for different five donors. **p<0.008 and *p<0.016. Signal/isotypic=P-selectin expression (MFI) of basal or ADP-induced condition/MFI of isotypic control in arbitrary units. MFI, mean fluorescence intensity.

FIG. 6.

(A) Concentrations of growth factors in two different plasma preparations. PPP and PRP (enriched in platelets 3.47-fold over peripheral blood). Data obtained for different 14 donors. ***p<0.0001. (B) Scatter plot of the PRP growth factor levels versus platelet count. Data obtained for different 14 donors. *Statistically significant. PPP, platelet-poor plasma; rS, Spearman's rank correlation coefficient.

FIG. 7.

(A) The spontaneous binding of FITC-conjugated CD62P (basal) to platelets. T0, unstored PRP preparation; T3, storage period of 3 h; T6, 6 h; and T24, storage period of 24 h. The binding of CD62P-FITC to platelets induced by ADP (reactivity). Data obtained for different five donors. *p<0.008. (B) Representative flow cytometry histograms. FITC, fluorescein isothiocyanate.

FIG. 8.

Growth factor levels in PRP and PPP during storage. T0, unstored PRP preparation; T3, storage period of 3 h; T6, 6 h; and T24, storage period of 24 h. Data obtained for different three donors. ***p<0.0001.