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. 2017 Jun 26;13(1):198.
doi: 10.1186/s12917-017-1123-3.

Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma

Hyeok-Soo Shin  1 , Heung-Myong Woo  1 , Byung-Jae Kang  2
Affiliations

Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma

Hyeok-Soo Shin et al. BMC Vet Res. .

Abstract

Background: Platelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method.

Methods: Whole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl2-activated platelet fractions.

Results: The highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions.

Conclusions: We have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

Keywords: Dog; Double centrifugation; Platelet recovery; Platelet-derived growth factor-BB; Platelet-rich plasma.

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Conflict of interest statement

Authors’ information

This manuscript is part of a MS thesis submitted by Dr. Shin to the College of Veterinary Medicine, Kangwon National University.

Ethics approval and consent to participate

All experimental dogs were housed at Kangwon National University (Chuncheon, Korea) in compliance with guidelines outlined by the Kangwon National University Animal Care Committee. The study protocol was approved by the Institutional Animal Care and Use Committee of Kangwon National University (Korea), Approval no.: KNU-160511-1.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Scatter plots for platelet recovery and yield of selected 4 conditions after the second centrifugation step. a Percent platelet recovery relative to PRP1 and (b) yield expressed as fold increase relative to whole blood. Data are presented by groups, the line represents the median of the group; error bars represent the interquartile range. Asterisk (*) indicates a recovery or yield significantly different (P < .05) between the two groups. Condition 5, 1000 g, 15 min; condition 8, 1500 g. 15 min; condition 9, 1500 g, 20 min; condition 10, 3000 g, 10 min
Fig. 2
Fig. 2
Scatter plots for PDGF-BB concentrations in plasma, PRP1 and PRP2 * Indicates a statistically significant difference (P < 0.05) compared with plasma. # Indicates a statistically significant difference (P < 0.05) compared with PRP1. Data are presented by groups, the line represents the median of the group; error bars represent the interquartile range. Condition 5, 1000 g, 15 min; condition 8, 1500 g. 15 min; condition 9, 1500 g, 20 min; condition 10, 3000 g, 10 min
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