Background:

Platelet rich plasma (PRP) is a blood-derived source of growth factors and several cytokines, which are essential for tissue regeneration and important for wound healing due to their angiogenic, mitogenic, and chemotactic activities. To date no protocol has been established for PRP production. Standardization of this technique should consider fundamental factors such as experimental model used, blood collection method, anticoagulant choice, rotation and amount of centrifugations, elapsed time between sample activation and its clinical use in order to ensure quality and biological effects of the product. This study aimed to compare three protocols for PRP achievement in order to evaluate platelet enrichment ability and method reproducibility for further use in clinical investigations regarding PRP therapeutic properties. Materials, Methods &

Results:

New Zealand higid rabbit's whole blood was collected in tubes containing sodium citrate. Samples were obtained through exsanguination, via abdominal aortic puncture, and separated in four aliquots designed for PRP processing and basal platelet count. The count was conducted at the time blood was collected and after every concentration protocol. Methods were tested in triplicates, and three different individuals repeated each technique for three times, reaching 27 repetitions. Selected methodologies consisted in two centrifugations protocols protocol A used 250 g for 10 min for the first separation, and another 10 min at 2000 g during the second centrifugation; protocol B proposed that first centrifugation would last 20 min at 160 g, and the second would last 15 min at 400 g, and protocol C consisted of 10 min at 400 g for the first separation, and 10 min at 800 g during second separation. Protocols were performed at the same time in three similar centrifuges, in order to standardize the variables (operator, time, environment, equipments), and also to diminish biases. Comparison objects in this study include ability of raising platelet concentration, time required for preparing the final product, reproduction handiness, and need for equipment for proper hemoconcentrated production. Achieved platelet count in each protocol and basal value were analyzed following randomized complete. Kurskal-Wallis test was used for independent samples comparison, considering a 5% significance level. For each tested sample, elapsed time for product preparation was evaluated. Subjective analyzes comprehended execution easiness and the need for special material, and were evaluated through questionnaire after each protocol. Protocol A showed a 25-fold increase in platelet count, whereas protocols B and C had 13 and 7-fold, respectively. Results indicate all protocols were efficient in concentrating the samples at least 3 times more than basal count. Elapsed time for product preparation in each protocol was 35, 52, and 41 min for A, B, and C methods, respectively. Subjective analyzes considered protocols A and C as low complexity, and protocol B was defined as medium complexity in regards to execution. With reference to material accessibility for protocols, all were considered of easy reproducibility.

Discussion:

Besides analyzing experimental model and most proper way to access blood collection, this study was limited to verify in a quantitative manner the platelet concentration in specific protocols, without evaluating their biological effects. Therefore, in regards to proposed objectives - relation between platelet concentration increase, spent time, and easiness of protocol - we conclude that protocol A, formulated by Nagae et al. (2007), was the method that most fitted the work needs, and greatly suited the challenges posed.