Name: A Simple Double Centrifugation Tube Method to Obtain Platelet-rich Plasma from Equine Blood (Video) | JoVE Journal
Uploaded: 2025-08-15T15:00:00.000+00:00
Duration: 6 min 10 s
Description: Learn a simple double centrifugation method to prepare platelet-rich plasma from equine blood for treating musculoskeletal disorders. This cost-effective protocol uses basic equipment and maintains sterility for routine clinical practice.

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5 minutes

We thank the Vicerrector&#237;a de Investigaciones y Postgrados of the Universidad de Caldas, Manizales, Colombia for their financial support to publish this article.

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
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
2. Aseptic blood collection
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Platelet-rich plasma procurement
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Statistical analysis
<ol>
	<li>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 &gt; 0.05), compare and analyze the cell data using the t-test. If platelet and leukocyte concentration data are not normally distributed (P &lt; 0.05), compare and analyze data using a Wilcoxon test.</li>
	<li>Determine the platelet and leukocyte efficiency concentration in P-PRP according to the following formulas25: 
	Platelet collection efficiency = <img alt="Platelet concentration calculation, formula: Platelet count/mL in PRP ÷ whole blood parameters." src="/files/ftp_upload/67985/67985eq01.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 1"> × 100 
	Leukocyte collection efficiency = <img alt="Leukocyte concentration ratio formula, blood analysis equation, clinical research calculation." src="/files/ftp_upload/67985/67985eq02.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 2"> × 100</li>
	<li>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.</li>
	<li>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.</li>
	<li>Consider p &lt; 0.05 values statistically significant for all the tests.</li>
</ol>

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<li>Argüelles, D., Carmona, J. U., Climent, F., Muñoz, E., Prades, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autologous+platelet+concentrates+as+a+treatment+for+musculoskeletal+lesions+in+five+horses%5BTitle%5D)+AND+%22Vet+Rec%22%5BJournal%5D)">Autologous platelet concentrates as a treatment for musculoskeletal lesions in five horses.</a> Vet Rec. 162 (7), 208-211 (2008).</li>
<li>Carmona, J. U., Argüelles, D., Climent, F., Prades, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autologous+platelet+concentrates+as+a+treatment+of+horses+with+osteoarthritis%3A+a+preliminary+pilot+clinical+study%5BTitle%5D)+AND+%22J+Equine+Vet+Sci%22%5BJournal%5D)">Autologous platelet concentrates as a treatment of horses with osteoarthritis: a preliminary pilot clinical study.</a> J Equine Vet Sci. 27 (4), 167-170 (2007).</li>
<li>Dejnek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Content+of+blood+cell+components%2C+inflammatory+cytokines+and+growth+factors+in+autologous+platelet-rich+plasma+obtained+by+various+methods%5BTitle%5D)+AND+%22World+J+Orthop%22%5BJournal%5D)">Content of blood cell components, inflammatory cytokines and growth factors in autologous platelet-rich plasma obtained by various methods.</a> World J Orthop. 13 (6), 587-602 (2022).</li>
<li>Giraldo, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+the+breed%2C+sex+and+age+on+cellular+content+and+growth+factor+release+from+equine+pure-platelet+rich+plasma+and+pure-platelet+rich+gel%5BTitle%5D)+AND+%22BMC+Vet+Res%22%5BJournal%5D)">Effects of the breed, sex and age on cellular content and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel.</a> BMC Vet Res. 9, 29(2013).</li>
<li>Zimmermann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sample+preparation+technique+and+white+cell+content+influence+the+detectable+levels+of+growth+factors+in+platelet+concentrates%5BTitle%5D)+AND+%22Vox+Sang%22%5BJournal%5D)">Sample preparation technique and white cell content influence the detectable levels of growth factors in platelet concentrates.</a> Vox Sang. 85 (4), 283-289 (2003).</li>
</ol>

The authors have no conflicts of interest to disclose.

The use of PRP in human and veterinary medicine for the treatment of chronic musculoskeletal disorders is increasing1,8,26,27. 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 ...

Platelet-rich plasma (PRP) is an orthobiologic product commonly used to treat chronic equine musculoskeletal conditions such as tendinopathies, desmopathies, osteoarthritis, and laminitis1,2,3. It is also effective in the treatment of limb wounds and ocular surface disease4,5. 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-&#946;1) 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 differentiation6,9. 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 horses6,11,12. One of the simplest and most common systems categorizes these hemocomponents based on leukocyte concentration11,12. 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&#39;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 horses6,15. 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 tissues1,2,20. 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-&#946;1 and PDGF-BB, which are integral to its therapeutic effects. By standardizing the process and demonstrating the product&#39;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 Arg&#252;elles 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.

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

a simple double centrifugation tube method to obtain platelet-rich plasma from equine blood

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

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 &#215; g for 5 min and 240 &#215; g for 5 min. The mean platelet concentration for PRP was 360.95 &#177; 56.31 platelets (PLT) &#215; 103/&#181;L, while the mean leukocyte concentration was 3.05 &#177; 1.53 cells &#215; 103/&#181;L. 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.

The mean platelet count (platelets × 103/µL) was significantly higher in PRP than in whole blood (p &lt; 0.001). In contrast, the mean leukocyte concentration (leukocytes × 103/µL) in PRP was significantly lower than that in whole blood (p &lt; 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 ...

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.

Platelet-rich plasma (PRP) is widely utilized as a routine treatment for chronic musculoskeletal conditions in horses, such as osteoarthritis, ...

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Research

JoVE Journal

Medicine

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Source:Sathish Muthu1,2,31Orthopaedic Research Group, Coimbatore.2Department of Biotechnology, Faculty of Engineering, Karpagam Academy of Higher Education.3Department of Orthopaedics, Government Medical College.This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and isolating platelets. This PRP is used in therapeutic applications for tissue regeneration and healing.

Preparation of Platelet-Rich Plasma from Human Peripheral Blood

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Purification of Platelets from Mouse Blood

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Platelet metabolism is of interest, particularly as it relates to the role of platelet hyper- and hypoactivity in bleeding and thrombotic disorders. Isolating platelets from plasma is necessary for some metabolic assays; presented here is a method for isolating of intracellular metabolites from washed platelets.

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Learn a simple double centrifugation method to prepare platelet-rich plasma from equine blood for treating musculoskeletal disorders. This cost-effective protocol uses basic equipment and maintains sterility for routine clinical practice.

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

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