Evaluation of different platelet-rich plasma protocols for use in the equine reproductive clinic

Abstract

Platelet-rich plasma (PRP) is a biological product used in equine regenerative medicine., Several methods and protocols for obtaining the different forms of PRP exist, depending on the application. In reproductive medicine, there is a need to classify the product according to the number of leukocytes, erythrocytes, and platelets present in PRP. This evaluation allows the classification of pure plasma rich in platelets (P-PRP) or plasma rich in leukocytes and platelets (L-PRP) and not just with the generic name of PRP. The objective of this study was to evaluate the concentration of platelets, erythrocytes, leukocytes, and bacterial load present in PRP, produced by two protocols already used in the equine reproductive clinic and two new protocols that aimed for greater efficiency/simplicity in the separation process. Ten horses of different breeds were selected, and from each one, five total blood samples were collected by venipuncture: one for the complete blood count and four protocols to produce PRP using different protocols (P1, P2, P3, and P4). In P1 and P2, the blood was collected in a vacutainer (4 mL glass tubes) with sodium citrate at 3.2%. P3 and P4 blood collections were in an open system into 50 mL conical plastic tubes using sodium citrate at 3.2% and citrate-phosphate-dextrose adenine (CPDA-1) at 14%, respectively. P1 used 120g/ 10 min centrifugation, and P2 performed two centrifugations, one at 120g / 10min and the other at 240g / 10 min. In both protocols, P3 and P4 used two centrifugations (215g / 10 min and 863g / 10 min). Analysis of blood components and bacterial growth was performed. Protocols were considered independent factors, and platelet, erythrocyte, and leukocyte concentrations as dependent variables. Platelet concentration was analyzed with ANOVA and t-test; erythrocytes and leukocytes concentration with Kruskal Wallis and Mann Whitney test. Platelet concentration was higher in P3 and P4 than in P1 (P < 0.0009) and equal to P2. P1 presented a lower concentration of erythrocytes than P2, P3, and P4 (P = 0.0014). Leukocytes were observed in all groups. However, P1 had a lower number of leukocytes than P3 and P4 (P = 0.0120) but showed no difference from P2. All groups were identified as being P-PRP and without bacterial contamination. The characterization of quantities and proportions of the blood cells after each protocol will lead to a specific clinical use (intrauterine platelets will aid in persistent post-covering endometritis), since the presence of certain blood cells, can generate different results in tissue proliferation, differentiation, and immunity. Therefore, the P3 and P4 protocols may have advantages in treating reproductive diseases, as the larger volumes make the procedure faster and simpler.