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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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.
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4.	中國醫療保健國際交流促進會骨科分會. 關節腔注射富血小板血漿治療膝骨關節炎的臨床實踐指南 (2018 年版). 中華關節外科雜志 (電子版), 2018, 12(4): 1-5.
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8.	袁霆, 張長青. 骨組織及軟組織修復作用中富血小板血漿的制作及其原理. 中國臨床康復, 2004, 8(35): 7939-7941.
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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. 富血小板血漿治療膝關節軟骨退行性病變的臨床實驗研究. 上海: 上海交通大學, 2011.
19.	李明, 章軍輝, 狄正林, 等. 應用自體富血小板血漿治療慢性難愈性創面的實驗研究. 中華關節外科雜志 (電子版), 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. 中國醫療保健國際交流促進會骨科分會. 關節腔注射富血小板血漿治療膝骨關節炎的臨床實踐指南 (2018 年版). 中華關節外科雜志 (電子版), 2018, 12(4): 1-5.
5. 中國醫療保健國際交流促進會骨科分會. 富血小板血漿在骨關節外科臨床應用專家共識 (2018 年版). 中華關節外科雜志 (電子版), 2018, 12(5): 596-600.
6. 位付濤, 王振. 去白細胞富血小板血漿對距骨骨軟骨損傷的療效及機制研究. 中國修復重建外科雜志, 2019, 33(5): 555-562.
7. 蘇文珍, 林永杰, 王國偉, 等. 體外沖擊波聯合富血小板血漿注射治療膝關節骨關節炎的前瞻性臨床對比研究. 中國修復重建外科雜志, 2019, 33(12): 1527-1531.
8. 袁霆, 張長青. 骨組織及軟組織修復作用中富血小板血漿的制作及其原理. 中國臨床康復, 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. 富血小板血漿治療膝關節軟骨退行性病變的臨床實驗研究. 上海: 上海交通大學, 2011.
19. 李明, 章軍輝, 狄正林, 等. 應用自體富血小板血漿治療慢性難愈性創面的實驗研究. 中華關節外科雜志 (電子版), 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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