Platelet-rich plasma: lyophilization as an alternative for conservation and transport

Abstract

Platelet-rich plasma (PRP) has been used in equine reproduction to improve pregnancy rates due to its immune-modulating effect. Platelet granules contain numerous proteins, such as growth factors, cytokines, and antimicrobial peptides. However, the shelf life of platelets is 5 days and used at a temperature above 20°C, where there is the concern of bacterial contamination. Also, preparation is a time-consuming procedure. Therefore, studies to find an effective method of PRP storage would be of great benefit, saving costs, and could also guarantee a faster and more effective treatment. This work compared the platelet levels between fresh and lyophilized PRP. Peripheral blood from 8 healthy equine males with a mean age of 4±2years was collected in 4.5-mL vacutainer tubes containing 3.2% sodium citrate. The PRP preparation was performed by double centrifugation in vacutainer tubes. Blood was homogenized and centrifuged at 120g for 10 minutes. After the first centrifugation, the upper 30% of the plasma was discarded. The remaining plasma (70%) was submitted to another centrifugation at 240g for 10 minutes. After the second centrifugation, 30% of plasma at the bottom of each tube was considered PRP. Platelet count was carried out with an automated hematology analyzer (HematoClin 2.6VET - Bioclin IL, USA). The lyophilization process was carried out at the Research Support Center of the Federal RuralUniversity of Pernambuco. Before being lyophilized, samples were frozen at -80°C for 24 h and then processed in an Alpha 1-4 LD Plus Christ apparatus, a drying chamber temperature of -27°C and vacuum of 0.52 mbar, for 24 hours. The lyophilized PRP obtained was stored in well-sealed microtubes and kept frozen at -4°C for further analysis. The results were subjected to analysis of variance (ANOVA) to determine the effects of lyophilization on the platelet concentrations. Significance was declared at P<0.05. The median platelet count in the fresh PRP was 108±35.2 × 103/uL. The platelet count after lyophilization was 46.6% lower (p<0.05) when compared with the initial count (56±17.7 × 103/uL). Although there was a significant reduction in platelet concentration after lyophilization, 53.4% of platelets were viable after the process. Our study showed the viability of lyophilizing PRP using a standard and easy system. Despite several published studies on PRP standardization, to the best of our knowledge, this is the first work on equine PRP lyophilization. In addition, lyophilized PRP appears to be a possible alternative to fresh PRP; with minor variability, a large-scale production with increased shelf life could be achieved following a single production process. Further studies are needed, mainly on growth factors after lyophilization and the use of PRP in vivo.