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 -80ºC. 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) x10 3 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±65.5x10 3 µL -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±66.1x10 3 µL -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 o C for 14 days, but studies on platelet function of thawed PRP are still needed. Key words: Platelets. Storage. Equine. Dmso. ResumoNeste estudo preliminar, um novo protocolo de criopreservação de plasma rico em plaquetas (PRP) de equinos foi avaliado. O PRP foi obtido através de dupla centrifugação do sangue total coletado de oito pôneis clinicamente saudáveis. Uma amostra de PRP fresco foi analisada quanto ao número total de plaquetas, volume plaquetário médio (VPM) e morfologia plaquetária. Na avaliação morfológica 200 plaquetas foram contadas utilizando um microscópio com contraste de fase com objetiva de 40x e classificadas como ativadas (com pseudópodes), inativas (forma discóide normal) ou estado incerto (forma esférica, mas sem peseudópodes). Duas outras amostras de PRP foram armazenadas em um freezer mecânico a -80 o C, uma contendo dimetil sulfóxido (DMSO) como crioprotetor e outra sem 3 µL -1 ), maior VPM (5,7±0,08fL) e mais plaquetas ativadas (13,9%) do que os outros grupos (p<0,05). DMSO a 6% foi capaz de preservar a morfologia plaquetária no PRP armazenado a -80 o C durante 14 dias, mas estudos a respeito da função plaquetária ainda não necessários. Palavras-chave: Plaquetas. Armazenamento. Equino. Dmso.

Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study

“…Shelf life of the remaining product may be prolonged by freezing, as carried is out in human medicine [75,76], though studies demonstrate that freezing is detrimental to platelet morphology and function and continuous synthesis of growth factors [36]. In veterinary medicine, there are few studies considering extension of the shelf life by freezing of platelet concentrates [54,[77][78][79][80] and in the dog there are no literature reports that confirm the best preservation method. In fact, there is only one research study focused on growth factors evaluation in which a small number of canine PRP samples was frozen at −80 • C and subsequently analyzed, demonstrating an increase in the growth factors following freezing similar to that obtained with calcium chloride activation [36] and one clinical study reporting PRP preservation (at −76 • C) before application to skin wounds in the dog, with a subsequent excellent clinical response [15].…”