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 cytokines^7....

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 muscu....

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.