Evaluation of Platelet and Leukocyte Counts in Canine Platelet-Rich Plasma Obtained After Successive Blood Collections From the Same Patient
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Published: Frontiers in Veterinary Science, 2022
Keyword: Canine, Platelet-Rich Plasma, PRP, Freezing, Growth Factors, Platelet, Leukocyte, Storage
Author(s): V. DeMello, G. Chen, J. Wakshlag, and D. Mason
Overview
This study evaluated whether the timing of collection influences platelet and leukocyte counts of PRP samples and evaluated growth factor concentrations in canine PRP after freezing and storage without a preservation agent for 6 months of time. Whole blood was collected from 15 canines three times over 4 weeks and processed for PRP then stored. CBC analysis was performed immediately on all whole blood and PRP samples. The platelet and leukocyte counts were not statistically significant between the timing of blood draws over the course of 4 weeks. The PRP samples were frozen without a preservation agent for the remainder of the 6 months. The samples were thawed for growth factor concentration assessment. All three growth factors were present in measurable quantities.
Materials/Methods
Whole blood collection from 15 dogs was performed three times over four weeks: initial blood draw, 2 weeks post-initial blood draw, and 4 weeks post-initial blood draw (time 2). The blood was processed for PRP using the Companion Regenerative Therapies Pure-PRP II kit and manufacturer’s instructions were followed. CBC analysis was immediately performed on all whole blood and PRP samples with a benchtop hematology analyzer. The PRP samples were then frozen and stored without a preservation agent using a -20°C freezer for the duration of the 4 week study. Baseline growth factor concentrations were not assessed prior to freezing. The samples were then transferred to the laboratory of analysis and continued to be stored at -20°C for the remainder of the 6 months. The samples were thawed on ice and subjected to centrifugation at 10,000 g for 10min. Supernatants were then utilized for ELISA testing for VEGF, TGF-β1, and PDGF-BB.
Data was assessed for normality using a Shapiro-Wilk test for all cellular counts in the PRP and for growth factor data across time. One-way analysis of variance was used to assess differences over time for lymphocytes and platelets. A Friedman’s test was utilized to assess differences across time for red blood cell and neutrophil counts as well as VEGF. A one-way ANOVA was used to assess PDGF and TGF-B. A P < 0.05 was set. A Pearson correlation test was used to identify the correlations between platelet recovery and growth factor concentration and a P <0.05 was set. Correlation strength was based on R values obtained from the testing.
The sample consisted of 15 healthy adult canines (8 spayed females and 7 neutered males) with an average age of 5 years (range 15 months – 12 years) and average weight of 36.9Kg (range 17-70Kg).
Results
The platelet, leukocyte, and red blood cell counts were not statistically significant between the timing of blood draws over the course of 4 weeks. All three growth factors (PDGF, VEGF, and TGF-β1) were present in measurable quantities after freezing and storage for 6 months without a preservation agent. PDGF and TGF-β1 were correlated with platelet count of the final PRP.
Conclusions
This study showed that PDGF and TGF-β1 are directly correlated with the platelet count of the final PRP and that growth factor concentrations are present and measurable in canine PRP frozen and stored without a preservation agent for 6 months which is encouraging from a clinical perspective regarding storage for future use. It also concluded that PRP preparation is unlikely to be influenced by time or the kits utility on a daily basis showing consistency in product delivered.