A Simple Double Centrifugation Tube Method to Obtain Platelet-rich Plasma from Equine Blood.

Overview

• This study presents and validates a simple, double centrifugation tube method to obtain platelet-rich plasma (PRP) from horse blood, demonstrating its efficiency and reproducibility for clinical use in treating chronic musculoskeletal conditions in horses.

Introduction and Background

• Platelet-rich plasma (PRP) is an orthobiologic treatment commonly used in equine medicine to address chronic musculoskeletal disorders such as osteoarthritis, tendinopathies, and desmopathies.
• PRP is effective due to its high concentration of growth factors and anti-inflammatory cytokines, which are released upon activation and promote tissue healing.
• Despite its frequent clinical use, there is a lack of instructional resources demonstrating how to prepare PRP from equine blood, limiting wider practical adoption among veterinarians.

Research Objective

• The study aimed to evaluate a straightforward, cost-effective double centrifugation technique to isolate PRP from equine blood, focusing on its platelet and leukocyte concentrations and growth factor content.
• The goal was to determine whether this method could reliably produce PRP suitable for clinical applications in horses.

Methodology

• Blood Collection: Whole blood samples were taken from six horses using tubes containing sodium citrate as an anticoagulant.
• Double Centrifugation Process:
  • First centrifugation at 120 × g for 5 minutes to separate blood components.
  • Second centrifugation at 240 × g for 5 minutes to concentrate the platelets in the plasma fraction.
• The centrifugation speeds and durations were selected to optimize platelet recovery while minimizing leukocyte inclusion.

Key Results

• Platelet Concentration:
  • PRP had a mean platelet count of approximately 360.95 × 10³ platelets/µL.
  • This concentration was 2.41 times higher than that found in whole blood, indicating effective platelet enrichment.
• Leukocyte Concentration:
  • PRP had a mean leukocyte count of 3.05 × 10³ cells/µL.
  • This was only 44% of the leukocyte concentration in whole blood, showing that the method reduced leukocyte presence.
• Collection Efficiency:
  • Platelet collection efficiency was 17%, meaning that 17% of the total platelets present in whole blood were recovered in the PRP.
  • Leukocyte collection efficiency was only 3.1%, indicating that leukocytes were largely excluded from the PRP.
• Growth Factor Quantification:
  • Transforming Growth Factor Beta 1 (TGF-β1) mean concentration was measured at 2601.82 pg/mL (95% CI: 2358.88-2844.76).
  • Platelet-Derived Growth Factor BB (PDGF-BB) was found at a mean concentration of 1208.05 pg/mL (95% CI: 1051.09-1365.02).
  • These growth factors are key to PRP’s regenerative properties.

Interpretation and Significance

• The provided double centrifugation technique is simple, uses common lab equipment, and does not require expensive commercial kits, making it accessible for equine practitioners.
• By enriching platelets while simultaneously reducing leukocytes, the method creates a PRP product that is likely to maximize therapeutic benefits and minimize inflammatory responses.
• The measured levels of important growth factors confirm that the PRP obtained is biologically active and suitable for promoting tissue healing.
• This method offers a reproducible and cost-effective option for veterinary clinicians to prepare PRP, potentially improving the management of musculoskeletal diseases in horses through regenerative therapies.

Conclusion

• The study successfully demonstrated that PRP with clinically relevant platelet concentrations and desirable leukocyte depletion can be prepared using a simple double centrifugation tube method.
• This cost-effective approach is recommended for use by equine practitioners to enhance the treatment of chronic musculoskeletal conditions, helping to standardize PRP preparation in veterinary practice.