Optimized preparation method of leukocytes-rich platelet-rich plasma by varying conditions during centrifugation_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Zhaoyuan ¹ , WEI Yuxuan ² ^Δ ,  ZHANG Changqing ¹ ,  YUAN Ting ¹

1. Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, P.R.China;
2. Department of Orthopaedics, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen Guangdong, 518116, P.R.China;

Corresponding author：

ZHANG Changqing, Email: zhangcq@sjtu.edu.cn; YUAN Ting, Email: terrenceyuan@gmail.com

Keywords：

Leukocytes-platelet-rich plasma; preparation; optimization; high tolerance rate; generalization

DOI：

10.7507/1002-1892.201911054

Video：

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Objective To identify a more popularized preparation protocol of leukocytes-rich platelet-rich plasma (L-PRP) for higher tolerance rate.Methods The peripheral blood samples of 76 volunteers (45.0 mL/case) were mixed with 5 mL sodium citrate injection for blood transfusion, and L-PRP was prepared by twice centrifugations. All blood samples were divided into three groups according to the parameters of twice centrifugation: experimental group A (12 cases, 400×g, 10 minutes for the first time and 1 100×g, 10 minutes for the second time), experimental group B (27 cases, 800×g, 10 minutes for the first time and 1 100×g, 10 minutes for the second time), and control group (37 cases, 1 360×g, 10 minutes for the first time and 1 360×g, 10 minutes for the second time). The platelet recovery rate and platelet and leukocyte enrichment coefficient of L-PRP in each group were calculated and compared.Results After removal of abnormal blood samples (platelet recovery rate was more than 100% or white thrombus), the remaining 55 cases were included in the statistical analysis, including 10 cases in experimental group A, 21 cases in experimental group B, and 24 cases in control group. The platelet enrichment coefficient and platelet recovery rate of experimental group B were significantly higher than those of experimental group A and control group (P<0.05); there was no significant difference between experimental group A and control group (P>0.05). There was no significant difference in leukocyte enrichment coefficient between experimental groups A, B, and control group (P>0.05).Conclusion The preparation quality of PRP is affected by various factors, including centrifugal force, centrifugal time, temperature, and operation process, etc. Twice centrifugation (800×g, 10 minutes for the first time and 1 100×g, 10 minutes for the second time) is an ideal and feasible centrifugation scheme, which can obtain satisfactory platelet recovery rate and enrichment coefficient with thicker buffy coat, which can reduce the fine operation requirements for operators, improve the fault tolerance rate and generalization.

Citation： ZHANG Zhaoyuan, WEI Yuxuan, ZHANG Changqing, YUAN Ting. Optimized preparation method of leukocytes-rich platelet-rich plasma by varying conditions during centrifugation. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(8): 1025-1030. doi: 10.7507/1002-1892.201911054 Copy

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3.	Yuan T, Zhang CQ, Tang MJ, et al. Autologous platelet-rich plasma enhances healing of chronic wounds. Wounds, 2009, 21(10): 280-285.
4.	中国医疗保健国际交流促进会骨科分会. 关节腔注射富血小板血浆治疗膝骨关节炎的临床实践指南 (2018 年版). 中华关节外科杂志 (电子版), 2018, 12(4): 1-5.
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13.	Jo CH, Roh YH, Kim JE, et al. Optimizing platelet-rich plasma gel formation by varying time and gravitational forces during centrifugation. J Oral Implantol, 2013, 39(5): 525-532.
14.	Araki J, Jona M, Eto H, et al. Optimized preparation method of platelet-concentrated plasma and noncoagulating platelet-derived factor concentrates: maximization of platelet concentration and removal of fibrinogen. Tissue Eng Part C Methods, 2012, 18(3): 176-185.
15.	Du L, Miao Y, Li X, et al. A Novel and convenient method for the preparation and activation of PRP without any additives: Temperature controlled PRP. Biomed Res Int, 2018, 2018: 1761865.
16.	Yin W, Xu Z, Sheng J, et al. Erythrocyte sedimentation rate and fibrinogen concentration of whole blood influences the cellular composition of platelet-rich plasma obtained from centrifugation methods. Exp Ther Med, 2017, 14(3): 1909-1918.
17.	Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg, 2004, 62(4): 489-496.
18.	李明. 富血小板血浆治疗膝关节软骨退行性病变的临床实验研究. 上海: 上海交通大学, 2011.
19.	李明, 章军辉, 狄正林, 等. 应用自体富血小板血浆治疗慢性难愈性创面的实验研究. 中华关节外科杂志 (电子版), 2016, 10(6): 624-629.
20.	Weibrich G, Kleis WK, Hafner G, et al. Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg, 2002, 30(2): 97-102.
21.	Piao L, Park H, Jo CH. Theoretical prediction and validation of cell recovery rates in preparing platelet-rich plasma through a centrifugation. PLoS One, 2017, 12(11): e0187509.
22.	Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte-and platelet-rich fibrin (L-PRF). Trends Biotechnol, 2009, 27(3): 158-167.
23.	Bausset O, Giraudo L, Veran J, et al. Formulation and storage of platelet-rich plasma homemade product. Biores Open Access, 2012, 1(3): 115-123.

1. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent, 2001, 10(4): 225-228.
2. Anitua E. Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants, 1999, 14(4): 529-535.
3. Yuan T, Zhang CQ, Tang MJ, et al. Autologous platelet-rich plasma enhances healing of chronic wounds. Wounds, 2009, 21(10): 280-285.
4. 中国医疗保健国际交流促进会骨科分会. 关节腔注射富血小板血浆治疗膝骨关节炎的临床实践指南 (2018 年版). 中华关节外科杂志 (电子版), 2018, 12(4): 1-5.
5. 中国医疗保健国际交流促进会骨科分会. 富血小板血浆在骨关节外科临床应用专家共识 (2018 年版). 中华关节外科杂志 (电子版), 2018, 12(5): 596-600.
6. 位付涛, 王振. 去白细胞富血小板血浆对距骨骨软骨损伤的疗效及机制研究. 中国修复重建外科杂志, 2019, 33(5): 555-562.
7. 苏文珍, 林永杰, 王国伟, 等. 体外冲击波联合富血小板血浆注射治疗膝关节骨关节炎的前瞻性临床对比研究. 中国修复重建外科杂志, 2019, 33(12): 1527-1531.
8. 袁霆, 张长青. 骨组织及软组织修复作用中富血小板血浆的制作及其原理. 中国临床康复, 2004, 8(35): 7939-7941.
9. Petrungaro PS. Using platelet-rich plasma to accelerate soft tissue maturation in esthetic periodontal surgery. Compend Contin Educ Dent, 2001, 22(9): 729-732.
10. Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg, 2000, 58(3): 297-300.
11. Aghaloo TL, Moy PK, Freymiller EG. Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study. J Oral Maxillofac Surg, 2002, 60(10): 1176-1181.
12. Amable PR, Carias RB, Teixeira MV, et al. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther, 2013, 4(3): 67.
13. Jo CH, Roh YH, Kim JE, et al. Optimizing platelet-rich plasma gel formation by varying time and gravitational forces during centrifugation. J Oral Implantol, 2013, 39(5): 525-532.
14. Araki J, Jona M, Eto H, et al. Optimized preparation method of platelet-concentrated plasma and noncoagulating platelet-derived factor concentrates: maximization of platelet concentration and removal of fibrinogen. Tissue Eng Part C Methods, 2012, 18(3): 176-185.
15. Du L, Miao Y, Li X, et al. A Novel and convenient method for the preparation and activation of PRP without any additives: Temperature controlled PRP. Biomed Res Int, 2018, 2018: 1761865.
16. Yin W, Xu Z, Sheng J, et al. Erythrocyte sedimentation rate and fibrinogen concentration of whole blood influences the cellular composition of platelet-rich plasma obtained from centrifugation methods. Exp Ther Med, 2017, 14(3): 1909-1918.
17. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg, 2004, 62(4): 489-496.
18. 李明. 富血小板血浆治疗膝关节软骨退行性病变的临床实验研究. 上海: 上海交通大学, 2011.
19. 李明, 章军辉, 狄正林, 等. 应用自体富血小板血浆治疗慢性难愈性创面的实验研究. 中华关节外科杂志 (电子版), 2016, 10(6): 624-629.
20. Weibrich G, Kleis WK, Hafner G, et al. Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg, 2002, 30(2): 97-102.
21. Piao L, Park H, Jo CH. Theoretical prediction and validation of cell recovery rates in preparing platelet-rich plasma through a centrifugation. PLoS One, 2017, 12(11): e0187509.
22. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte-and platelet-rich fibrin (L-PRF). Trends Biotechnol, 2009, 27(3): 158-167.
23. Bausset O, Giraudo L, Veran J, et al. Formulation and storage of platelet-rich plasma homemade product. Biores Open Access, 2012, 1(3): 115-123.

Chinese Journal of Reparative and Reconstructive Surgery

Optimized preparation method of leukocytes-rich platelet-rich plasma by varying conditions during centrifugation

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