Standardization of canine platelet-rich plasma preparation and its functional characterization

Platelet-rich plasma (PRP) is widely used in clinical and research applications due to its abundant growth factors and cytokines. However, the lack of a standardized method for PRP preparation results in variability in platelet yield, leukocyte carryover, and function.

To identify centrifugation parameters that maximize platelet recovery while preserving function in canine PRP.

Whole blood from 10 healthy dogs was processed by single-spin (10 min at 800, 1,200, 1,500, or 2,000 rpm) or dual-spin (two-time immediate sequential repetition of the same centrifugation speed for 5 min) using a fixed-angle rotor (radius = 15 cm; 1,500 rpm = 364 × g equivalent). Platelet recovery and white blood cell (WBC) contamination were quantified, and platelet aggregation and dense-granule secretion (2-MeSADP, thrombin), thromboxane (Tx) A₂ generation (TxB₂ enzyme-linked immunosorbent assay [ELISA]), protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) phosphorylation (immunoblot), and platelet-derived growth factor (PDGF)-BB release (ELISA) were measured.

Among single-spin conditions, PRP obtained at 1,500 rpm/10 min yielded the highest platelet recovery (≈25.1%) with < 1% WBC carryover. This PRP exhibited maximum 2-MeSADP- and thrombin-induced platelet aggregation and secretion, TxA₂ generation, AKT/ERK phosphorylation, and PDGF-BB release, suggesting that optimizing the centrifugal speed is crucial for achieving the optimal PRP collection with the utmost platelet function. Compared with the optimal single-spin condition, dual-spin at 1,500 rpm/5+5 min further increased platelet recovery (≈48.7%) and enhanced functional readouts.

A dual-spin at 1,500 rpm/5+5 min protocol produces leukocyte-poor PRP with superior platelet recovery and function in dogs, supporting more effective and reproducible clinical and research applications.