Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole Blood Samples after Extended Storage

Abstract

The platelet-rich fibrin-like matrix (PRFM) is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB) samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl₂ in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB) levels were examined by scanning electron microscopy (SEM), flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA), respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared-without evident loss of quality-from WB samples stored for up to 7 days by our previously developed method.

Keywords: fibrin fiber; platelet-derived growth factor; platelet-rich fibrin; platelets; storage.

Figure 1

(a–c) Stable counts of platelets, red blood cells (RBCs), and white blood cells (WBCs) in stored whole blood samples (n = 8); (d) A comparison of WBC components between fresh and 7-day-stored WB samples. The data were calculated from an average of 8 samples. W-SCR: WBC small cell ratio, W-MCR: WBC middle cell ratio, W-LCR: WBC large cell ratio.

Figure 2

Immunofluorescent staining of CD41 and CD62P expressed in platelets isolated from 2-day- or 7-day-stored WB samples. (a,d) Control resting platelets; (b,e) platelets stimulated by 0.1% CaCl₂ for 15 min; and (c,f) platelets stimulated by 10 mM ADP for 15 min. The platelets were derived from the same donor and were distributed with almost the same density in all the dishes (views).

Figure 3

Flow-Cytometric (FCM) analysis of CD41- and CD62P-double-positive platelets in platelet fractions that were prepared from fresh or stored WB samples and stimulated with 10 mM ADP or 0.1% CaCl₂ for 15 min (n = 4). * p < 0.05 as compared with control platelets at the same time points. No significant differences were observed in time-course changes.

Figure 4

Stable Ca²⁺ (a) and glucose levels (b) and pH (c) of fresh and stored WB samples. Because stored WB samples contained ACD-A as an anticoagulant, CaCl₂ was added to the samples for PRF clot formation. Ca²⁺ levels were determined before and after the addition of CaCl₂. Glucose levels were determined in WB samples before the addition of CaCl₂. * p < 0.05 as compared with the individual control levels on day 1 (n = 8).

Figure 5

Visual appearance of platelet-rich fibrin–like matrixs (PRFMs) prepared from WB samples stored for the indicated periods. WB samples were simultaneously collected from the same donor. Similar PRFM samples were obtained from three other experiments.

Figure 6

Scanning electron microscopy (SEM) images of fibrin fibers formed in PRFMs prepared from WB samples stored for the indicated periods. WB samples were simultaneously collected from the same donor. Similar findings were obtained in three other experiments.

Figure 7

Time-dependent changes in the concentration of PDGF-BB extracted from PRFM samples that were prepared from stored WB samples and compressed to squeeze out PRFM exudates. * p < 0.05 as compared with fresh WB samples as controls (n = 8).