The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months

Abstract

Background: Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our objectives were two-fold. First, to determine the effect of a single freeze-thaw cycle on PRP protein concentrations and establish their baseline levels. Second, to evaluate the effect of storage in -20 °C automatic defrost freezer, - 20 °C manual defrost freezer, - 80 °C manual defrost freezer, and liquid nitrogen for 1, 3, and 6 months on PRP protein concentrations, compared to the established baseline concentrations. Results: Fold-change between fresh activated and snap frozen PRP were analyzed using paired t-test. A snap frozen-thaw cycle resulted in increased MMP-9 (p = 0.0021), and a small significant decrease in TGF-β1 (p = 0.0162), while IGF-1 and PDGF-BB were unchanged compared to fresh activated PRP. Fold-change over time within storage method were analyzed using repeated measures ANOVA and Tukey post-hoc test. IGF-1 decreased in all conditions (p < 0.0001). At all time-points at -20 °C (p < 0.0001), and at 3 and 6 months at -80 °C (p < 0.0070), PDGF-BB decreased. TGF- β1 was unchanged or increased after 6 months (p < 0.0085). MMP-9 decreased at 3-months at -20 °C, and at all times at -80 °C and in liquid nitrogen compared to snap frozen (p < 0.0001). Conclusions: The protein profile of equine frozen-stored PRP differs from fresh PRP. For clinical applications equine PRP can be stored at -80 °C for 1 month or in liquid nitrogen for 6 months to maintain PDGF-BB and TGF-β1 concentration, but IGF-1 concentrations will be reduced. The storage temperature and duration should be reported in studies measuring protein concentrations in PRP. To accurately measure IGF-1 concentrations, PRP samples should be analyzed immediately.