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

Summary

Platelet-rich plasma (PRP) is used in the treatment of equine musculoskeletal disorders. Different techniques yield PRP with varying platelet and leukocyte concentrations. This article describes a method for obtaining PRP from horse blood.

Abstract

Platelet-rich plasma (PRP) is widely utilized as a routine treatment for chronic musculoskeletal conditions in horses, such as osteoarthritis, tendinopathies, and desmopathies. This effectiveness stems from the high concentration of growth factors and anti-inflammatory cytokines that are released upon activation of this orthobiologic agent, whether activated endogenously or exogenously. Despite its growing popularity, there is a notable absence of instructional videos that demonstrate the techniques for obtaining PRP in horses.

This study examines a double centrifugation tube method for obtaining PRP in horses. Whole blood from six horses was collected in sodium citrate tubes and centrifuged twice at 120 × g for 5 min and 240 × g for 5 min. The mean platelet concentration for PRP was 360.95 ± 56.31 platelets (PLT) × 10³/µL, while the mean leukocyte concentration was 3.05 ± 1.53 cells × 10³/µL. The platelet count in PRP was 2.41x higher than in whole blood, while the leukocyte concentration in PRP was 0.44x lower. The collection efficiency for PLT in PRP was 17%, while the collection efficiency for leukocytes in PRP was 3.1 %. The mean concentrations of transforming growth factor beta 1 and platelet-derived growth factor BB in PRP were 2601.82 (95% confidence interval (CI): 2358.88-2844.76) pg/mL and 1208.05 (95% CI: 1051.09-1365.02) pg/mL, respectively. These results indicate that PRP can be efficiently and reproducibly obtained through a simple, cost-effective method suitable for use by equine practitioners.

Introduction

Platelet-rich plasma (PRP) is an orthobiologic product commonly used to treat chronic equine musculoskeletal conditions such as tendinopathies, desmopathies, osteoarthritis, and laminitis^1,2,3. It is also effective in the treatment of limb wounds and ocular surface disease^4,5. The rationale for its use is based on the high concentrations of growth factors (GFs) released primarily by platelets⁶. Leukocytes also contribute by releasing regulatory and anti-inflammatory cytokines⁷. GFs such as transforming growth factor beta 1 (TGF-β₁) and platelet-derived growth factor BB (PDGF-BB) exert potent anabolic, proliferative, angiogenic, and anti-inflammatory effects⁸. Upon exogenous or endogenous activation, PRP polymerizes into a platelet-rich gel, facilitating cell trafficking, anchoring, and differentiation^6,9. In this way, the PRP serves as a complex living scaffold and not just as a container for GFs and cytokines¹⁰.

Several systems classify PRP products used in both humans and horses^6,11,12. One of the simplest and most common systems categorizes these hemocomponents based on leukocyte concentration^11,12. According to this classification, PRP products with low to negligible leukocyte concentrations and low to moderate platelet concentrations -- relative to the levels in the horse's whole blood -- are referred to as pure PRP (P-PRP)^6,11,12. In contrast, PRP products with detectable to high leukocyte concentrations and moderate to high platelet concentrations are referred to as leukocyte- and platelet-rich plasma (L-PRP)^6,11,12.

P-PRP products are typically obtained by tube-to-tube centrifugation in which whole blood is collected from horses in tubes containing anticoagulants such as sodium citrate and citrate, phosphate, and dextrose¹³. The blood is centrifuged twice to separate platelets from other components¹⁴. In contrast, L-PRP products are primarily prepared using semi-automated kits designed for human blood, some of which have been validated for use in horses^6,15. These semi-automated kits generally require specialized centrifuges and come with a higher cost¹⁶, as well as potential concerns about aseptic handling, which can complicate their practical use in clinical settings¹⁷. Additionally, these kits may result in the undesirable concentration of excessive leukocytes or red blood cells, which could be detrimental in certain clinical situations¹⁸. For example, elevated leukocyte counts may provoke excessive inflammation, and an overabundance of red blood cells could interfere with the healing process or lead to complications such as tissue irritation or rejection¹⁵; however, a commonly used method for producing equine L-PRP involves a gravitational process that does not require specialized equipment¹⁹.

In light of these limitations, simpler and more cost-effective alternatives for PRP preparation are highly desirable. Various in vitro studies, experimental models of musculoskeletal disease, and observations in equines have shown that both P-PRP and L-PRP products have beneficial effects on treated cells and tissues^1,2,20. These products promote healing, reduce inflammation, and enhance tissue regeneration⁶. However, the decision to use P-PRP or L-PRP to treat an equine could be influenced by technical and economic factors.

As per the authors, there are no published studies describing and fully demonstrating a widely validated step-by-step procedure for obtaining a PRP product from equine blood. This article outlines the procedures necessary to safely collect whole blood from horses and produce a PRP product suitable for both experimental and clinical use. It also includes validation of this orthobiologic by quantification of platelets and leukocytes and determination of the concentration of TGF-β₁ and PDGF-BB, which are integral to its therapeutic effects. By standardizing the process and demonstrating the product's efficacy, this study seeks to provide a reliable and accessible method for equine practitioners to prepare PRP with confidence and precision.

Although the double centrifugation method for producing equine PRP was first described by Argüelles et al.¹⁴ in 2006, this is the first time the protocol has been fully presented in a video. This video is intended to serve as a practical resource for equine practitioners who face technical limitations and budgetary constraints that prevent them from using more sophisticated and expensive PRP kits for horses with musculoskeletal disorders.

Protocol

This study was conducted in accordance with the internal protocols for the preparation and use of PRP in horses at the Veterinary Teaching Hospital of the Universidad de Caldas, Manizales, Colombia. This study did not require Internal Animal Care Committee approval as the data were obtained from lame horses treated clinically at our institution. Data from six Colombian Creole horses, three mares and three geldings, with an average age of 7.4 (± 2.6) years were used to develop this protocol. The owners of the horses were informed of the nature of the study and signed an informed consent form. All horses included in the study were clinically healthy except for the musculoskeletal problem being treated. Only animals with a basal platelet count greater than 100 × 10³/mL of whole blood were included. It is important to note that horses with lower platelet concentrations may be considered thrombocytopenic¹⁴. In addition, these animals exhibited normal blood counts, liver and kidney clinical biochemistry parameters, and urine analysis values within the normal range for the equine species.

1. Horse restriction and skin disinfection

1. For the safety of both the horse and the veterinarian, restrain the horse (Eqqus caballus) securely in a clinical examination chute or with a bridle (Figure 1A), depending on the behavior of the animal. Next, clip a 5 cm x 5 cm area of hair with an electric shaver in the mid-third of one side of the neck, over the jugular vein.
   NOTE: Some owners prefer that the horse not be clipped. A study indicates that hair clipping at the venipuncture site is not necessary to prevent bacterial contamination of P-PRP²¹. Therefore, this procedure is at the discretion of the veterinarian.
2. Carry out a preliminary non-sterile skin preparation over the clipped area of the jugular vein for at least 10 cm using non-sterile gauze and gloves for approximately 3 min. Apply several pieces of gauze soaked in antiseptic foam in a circular pattern (Figure 1B).
3. Prepare the venipuncture site aseptically using both sterile gloves and gauze soaked in disinfectant. Scrub the skin in the neck area in a circular motion for approximately 5 min. Clean the area with sterile gauze soaked in 95% ethyl alcohol.

2. Aseptic blood collection

1. Place 16 4.5 mL sodium citrate tubes, previously cleaned with gauze soaked in 95% ethyl alcohol, in a clean plastic rack. Impregnate the rubber caps of the vacuum tubes with 95% ethyl alcohol, especially in the recess for the needle coupling.
   NOTE: In general, eight 4.5 mL sodium citrate tubes are required to obtain 2.5 mL of PRP. The number of tubes used in a particular case will depend on the soft tissues or specific joint being treated.
2. Sterilely insert a butterfly needle into the disinfected jugular site (Figure 2A) and attach 4.5 mL sodium citrate tubes to the holder (Figure 2B). Gently shake the tubes and place them in a plastic rack.
   NOTE: A 4.5 mL sodium citrate tube is used for automated hemograms and to prepare plasma after hard spin centrifugation. This plasma is then used as a negative control for growth factor and cytokine enrichment comparisons with PRP.

3. Platelet-rich plasma procurement

1. Place the 4.5 mL sodium citrate tubes containing whole blood into a four-place swing-out rotor at an angle of 90° in a bench centrifuge. Centrifuge the tubes at 120 × g for 5 min at room temperature. After centrifugation, position the 4.5 mL tubes containing citrated blood and two additional 10 mL plain tubes in a rack and place the rack on the non-dominant hand side of the operator inside a class II laminar flow hood over a sterile, impervious drape.
   NOTE: One study suggests that a Class II laminar flow hood is not essential to prevent bacterial contamination during PRP procurement²¹. Instead, PRP can be prepared in a clean room free of air currents and dust. In this scenario, the blood tube rack can be placed on a table covered with a sterile, impervious drape.
2. First centrifugation: Wearing a face mask and sterile gloves, place a 10 mL sterile luer syringe coupled with a sterile needle (21 G 5/8") at the dominant hand side in the inside of the laminar flow hood.
   NOTE: From this moment onward, the dominant gloved hand always remains sterile, while the rubber caps of the sodium citrate tubes should be removed using the gloved non-dominant hand.
3. Insert the sterile needle in each centrifuged citrated blood tube 3 mm over the buffy coat (Figure 3A) and gently aspirate 50% of the plasma fraction, avoiding fluid turbulence and blood aspiration. Once 10 mL of plasma from four 4.5 mL tubes are aspirated, distribute it in 5-10 mL sterile plain tubes (Figure 3B).
   NOTE: Remove the rubber caps of these tubes with the non-dominant hand and place them over a sterile site of the drape or hold them to avoid bacterial contamination.
4. Second centrifugation: close the tubes again with the rubber stoppers and place them in an equilibrated state in a four-place swing-out rotor at an angle of 90° in the bench-top centrifuge and spin these tubes at 240 × g for 5 min at room temperature.
   NOTE: The choice of the relative centrifugation force (RCF or g) is based on a seminal study¹⁴ that first described this method for obtaining PRP from equine blood. Furthermore, this method has been evaluated for reproducibility by other authors²².
5. Place the tubes in a rack and position them on the non-dominant side inside the class II laminar flow hood, on top of the previously laid, sterile, impermeable drape.
6. Use the non-dominant hand to remove the rubber caps and the sterile dominant hand to slowly aspirate the first 75% of plasma using an 18 G, 90 mm spinal needle attached to a 10 mL sterile luer syringe (Figure 3C). Discard this syringe or reserve it for other experimental or clinical uses as it contains platelet-poor plasma (PPP). Next, attach the previously used sterile spinal needle to a 5 or 10 mL sterile luer syringe to aspirate the remaining plasma (25%) from the 5-10 mL sterile plain tube. This fraction is known as P-PRP (Figure 3D).
   NOTE: Under certain clinical and experimental conditions, an aliquot of 0.5-1 mL of PRP is used for automated hemograms followed by growth factor (TGF-β₁ and PDGF-BB) analysis by enzyme-linked immunosorbent assay. Measurement of these mediators may be performed 3 h after activation of P-PRP with calcium salts or thrombin²³ or after inducing cell damage by temperature change or the addition of nonionic detergents²⁴.
7. Wrap the sterile luer syringe containing PRP in a small impermeable sterile drape with an attached sterile needle or cap to prevent bacterial contamination of the orthobiologic product (Figure 4A).
   NOTE: The anatomical structure of the horse to be treated should be prepared for aseptic injection as described in section 1 of this protocol. The horse should be adequately restrained and sedated to minimize discomfort and ensure the safety of both the horse and the veterinarian. The PRP product should be handled using sterile gloves and needles and may or may not be activated based on the practitioner's preference (Figure 4B).

4. Statistical analysis

1. Collect data on platelet and leukocyte counts in whole blood and PRP. Evaluate data for normality using the Kolmogorov-Smirnov test. If the data are normally distributed (P > 0.05), compare and analyze the cell data using the t-test. If platelet and leukocyte concentration data are not normally distributed (P < 0.05), compare and analyze data using a Wilcoxon test.
2. Determine the platelet and leukocyte efficiency concentration in P-PRP according to the following formulas²⁵:
   Platelet collection efficiency = × 100
   Leukocyte collection efficiency = × 100
3. Collect data from TGF-β₁ and PDGF-BB concentrations in plasma (negative control) PRP supernatants (after 3 h of activation with calcium gluconate), and PRP lysates obtained by the addition of a nonionic detergent solution.
4. Use a generalized linear mixed model to compare growth factor concentrations between, plasma, PRP supernatants, and PRP lysate. In case of significant statistical differences, apply a Tukey's post-hoc test.
5. Consider p < 0.05 values statistically significant for all the tests.

Results

The mean platelet count (platelets × 10³/µL) was significantly higher in PRP than in whole blood (p < 0.001). In contrast, the mean leukocyte concentration (leukocytes × 10³/µL) in PRP was significantly lower than that in whole blood (p < 0.001) (Table 1). Specifically, the platelet count in PRP was 2.41x higher than in whole blood, while the leukocyte concentration in PRP was 0.44x lower. The collection efficiency for PLT in PRP was 17%, while leukocyte collection efficiency ...

Discussion

The use of PRP in human and veterinary medicine for the treatment of chronic musculoskeletal disorders is increasing^1,8,26,27. However, a major criticism of PRP applications in experimental and clinical settings is the lack of basic data on the techniques and protocols used to prepare these hemocomponents²⁸. In addition, there is insufficient information on the cellular ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL plain venous blood collection tubes	BD Vacutainer, Franklin Lakes, NJ, USA	Ref 366430	This is only an example, tubes of other brands can be used.
4.5 mL sodium citrate tubes (105 M/3. 2%)	BD Vacutainer, Franklin Lakes, NJ, USA	107451	This is only an example, tubes of other brands can be used.
Bench Centrifuge	Rotofix 32A, Hettich GmbH, Tuttlingen, Germany		Other bench centrifuges can be used.
Butterfly needle (21 G × 0.75 in.) with 12 in. tubing and pre-attached holder	Safety-Lok Blood Collection Set, BD Vacutainer, Franklin Lakes, NJ, USA	Ref 367296	This is only an example, butterfly needles of other brands can be used.
Chlorhexidine foam solution at 2.3-4%			The brand or trade names of these products may vary from country to country.
Class II laminar flow hood	Thermo Fischer Scientific, Waltham, MA, USA		Other class II laminar flow hoods can be used
ELISA reader	Thermo Fischer Scientific, Waltham, MA, USA
Ethyl alcohol at 95%			The brand or trade names of these products may vary from country to country.
Human PDGF-BB DuoSet ELISA	R&D Systems, Inc, McKinley Place, MN, USA	DY220
Human TGF-beta 1 DouSet ELISA	R&D Systems, Inc, McKinley Place, MN, USA	DY240
Povidone iodine foam solution at 4-10%			The brand or trade names of these products may vary from country to country.
SPSS 25.0 software	IBM Corp, SPSS, NY, USA
Sterile luer syringes of various volumes	Discardit Syringe, BD Vacutainer, Franklin Lakes, NJ, USA		This is only an example, syringes of other brands can be used.
Sterile spinal needle (18 G × 90 mm)	BD Quincke Spinel Needle, Franklin Lakes, NJ, USA		This is only an example, spinal needles of other brands can be used.