Influence of Manual Double-Spin Preparation Protocols on the Cellular and Growth Factor Composition of Equine Platelet-Rich Plasma.

Influence of Manual Double-Spin Preparation Protocols on the Cellular and Growth Factor Composition of Equine Platelet-Rich Plasma

Overview

• This study examined how different manual double-spin centrifugation methods affect the cellular and growth factor makeup of platelet-rich plasma (PRP) derived from horses.
• The researchers aimed to identify which preparation protocols optimize platelet concentration and growth factor levels, important for therapeutic use in veterinary medicine.

Study Design and Phases

• The study was prospective, randomized, and conducted ex vivo using blood samples from 20 healthy horses.
• The research was divided into two phases:
  • Phase A: Tested nine different first-spin centrifugation protocols using samples from 10 horses to select the best based on platelet enrichment and leukocyte reduction.
  • Phase B: Utilized the two best first-spin protocols combined with nine different second-spin conditions on samples from the remaining 10 horses, creating 18 PRP preparations.

Measurements

• Cellular components measured included:
  • Platelets (PLT)
  • Leukocytes (white blood cells, WBC)
  • Erythrocytes (red blood cells, RBC)
• Concentrations of key growth factors associated with healing and regeneration were quantified:
  • Platelet-derived growth factor-BB (PDGF-BB)
  • Transforming growth factor-beta (TGF-β)

Key Findings – Phase A

• No statistically significant differences were found among the nine first-spin protocols regarding the cellular composition (p > 0.05).
• Two protocols were selected for Phase B based on their balance of platelet enrichment and leukocyte reduction.

Key Findings – Phase B

• All 18 final PRP preparations achieved:
  • Platelet enrichment—higher platelet concentration than whole blood.
  • Leukocyte depletion—lower white blood cell count compared to whole blood.
  • Negligible contamination with red blood cells.
• The force applied during the second spin significantly influenced growth factor concentrations (p < 0.001):
  • PDGF-BB increased from 2629 pg/mL at 200×g centrifugal force to 3467 pg/mL at 1000×g.
  • TGF-β increased from 13,364 pg/mL to 16,998 pg/mL over the same centrifugation force increase.
• Longer second-spin duration was associated with increased TGF-β levels (p = 0.01).

Correlations and Associations

• Platelet concentration was positively correlated with both PDGF-BB (correlation coefficient ρ = 0.604) and TGF-β (ρ = 0.691), with both correlations highly significant (p < 0.001).
• Leukocyte concentration did not show a significant correlation with either growth factor.

Interpretation and Implications

• The manual double-spin centrifugation method effectively produces platelet-rich plasma with enriched platelets and reduced leukocytes, which are critical for therapeutic efficacy.
• Increasing second-spin centrifugal force and spin duration can enhance growth factor content, potentially improving the biological activity of PRP.
• Variability among donors (individual horses) influences PRP composition, indicating that even with standardized protocols, biological variability must be considered.
• Standardizing PRP preparation procedures alone may not be enough to guarantee consistent biologically active products, which has implications for clinical use and research reproducibility.

Conclusions

• Both preparation technique parameters (centrifugation force and time) and donor factors impact the cellular and growth factor composition of equine PRP.
• Optimizing and possibly individualizing PRP preparation protocols could improve therapeutic outcomes in equine regenerative medicine.
• Future work should focus on reducing biological variability and further refining protocols for consistent clinical use.