Cryopreservation protocol for equine platelet-rich plasma

Abstract

In this preliminary study, a new equine platelet-rich plasma (PRP) cryopreservation protocol was evaluated. PRP was obtained by a double centrifugation technique of whole blood collected from 8 adult healthy ponies. A fresh sample of PRP was analyzed for total platelet count, mean platelet volume (MPV), and platelet morphology. Upon morphological evaluation, 200 platelets were counted using a differential interference contrast microscope with a 40x phase objective and classified as activated (with pseudopodia), inactivated (normal discoid shape), or uncertain state (spherical shape, without pseudopodia). Two other PRP samples, one containing DMSO as a cryoprotectant and the other without DMSO, were stored in a mechanical freezer at-80°C. After 14 days, the frozen samples were thawed and submitted to the same analysis as described above. The fresh PRP showed a platelet count of 830 (±95.3) ×103 μL-1, an MPV of 5.2 (±0.07) fL, and composed of 4% activated platelets. There was no significant difference in platelet count, MPV, and activated platelets between fresh and 6% DMSO frozen PRP samples (617.9±65.5×103μL-1; 5.3±0.06fL; 9.5%) (p>0.05). On the other hand, samples frozen without DMSO showed a significantly lower platelet count (519.6±66.1×103μL-1), higher MPV (5.7±0.08fL), and more activated platelets (13.9%) than the other groups (p<0.05). The 6% DMSO was able to preserve platelet morphology in PRP stored at-80°C for 14 days, but studies on platelet function of thawed PRP are still needed.