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

Our research focuses on developing a simple cost-effective method to produce platelet-rich plasma PRP from horse blood for treating musculoskeletal disorders like osteoarthritis and tendon injuries. Key challenges include maintaining sterility during manual processing and achieving consistent leukocyte reduction, which vary more than platelet concentration.

Our protocol fills the gap of standards. We demonstrate techniques for equine PRP preparation, especially for clinics without access to advanced kits. Our method is affordable, uses basic lab equipment, and avoids costly kits, making PRP accessible for routine equine practice while maintaining therapeutic efficacy.

We'll explore PRP's long-term clinical outcomes and refine protocols to improve platelet and consistency for diverse equine muscular skeletal conditions.

[Narrator] To begin, restrain the horse securely using either a clinical examination chute or a bridle depending on its behavior. Clip a 5 by 5 centimeter area of hair using an electric shaver in the mid-third of one side of the neck over the jugular vein. Using non-sterile gloves and gauze, perform a preliminary non-sterile skin preparation over a 10-centimeter length of the jugular area for approximately three minutes. Apply several pieces of gauze soaked in antiseptic foam in a circular motion. Now, using sterile gloves, scrub the skin with gauze soaked in disinfectant in a circular motion for approximately five minutes. Then, clean the area with sterile gauze soaked in 95% ethyl alcohol. Place 16 4.5 milliliter sodium citrate tubes previously wiped with gauze soaked in 95% ethyl alcohol in a clean plastic rack. Disinfect the rubber caps of the vacuum tubes, especially at the needle coupling recess with 95% ethyl alcohol. Now, sterilely insert a butterfly needle into the prepared jugular vein site and attach a 4.5 milliliter sodium citrate tube to the needle holder to collect blood. Then, gently shake the filled tube and place it back in the rack. Place the tubes containing whole blood in a four-place swing out rotor at a 90 degree angle in a benchtop centrifuge. Centrifuge the tubes at 120 G for five minutes at room temperature. After centrifugation, place a sterile impervious drape in a class 2 laminar flow hood. Wearing a face mask and sterile gloves, place a sterile 10-milliliter luer syringe with a 21-gauge 5 by 8 inch sterile needle on the dominant hand side. Insert the sterile needle three millimeters above the buffy coat in each centrifuge citrated blood tube and gently aspirate 50% of the plasma without disturbing the buffy coat. Continue aspirating the plasma from the tubes and distribute it into 5 to 10 milliliter sterile plain tubes. Close the tubes with rubber stoppers and place them in a four-place swing out rotor in the centrifuge. Centrifuge at 240 G for five minutes at room temperature to obtain small cellular buttons. After placing the tubes back on a rack inside the laminar flow hood, place two sterile 10 milliliter luer syringes, a sterile spinal needle, and a sterile plastic cap inside the hood. Open them and use the non-dominant hand to remove the rubber caps. With the sterile dominant hand, insert an 18-gauge 90 millimeter spinal needle attached to a 10 milliliter sterile luer syringe into the tube and aspirate the top 75% of the plasma. Then, aspirate the remaining 25% of the plasma using the same needle attached to a fresh 10 milliliter sterile syringe. Finally, wrap the sterile luer syringe containing PRP in a small, sterile impermeable drape and cap with a sterile needle or closure to prevent contamination. The mean platelet count was 2.41x higher in PRP than in whole blood. In contrast, the mean leukocyte concentration in PRP was significantly lower than that in whole blood. TGF-Beta1 concentrations were significantly different between plasma, PRP-supernatants and PRP-lysate with the highest concentrations for PRP-lysate. On the other hand, PDGF-BB concentrations were significantly higher in PRP-supernatants and PRP-lysate compared to plasma.