Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns

Abstract

Objective:: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods.

Methods:: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1).

Results:: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1β. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-β1 in relation to other systems and low FGF-2 levels.

Conclusion:: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used.

Objetivo:: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação.

Métodos:: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-β1).

Resultados:: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1β. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-β1 em relação aos demais sistemas e reduzidos níveis de FGF-2.

Conclusão:: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.

Figure 1. Growth factors concentration. (A) transforming growth factor beta 1 and (B) fibroblast growth factor 2 analyzed in platelet-rich plasma obtained by Magellan (n=6), GPSIII (n=6), and open (n=4) methods

*Comparison of open system versus GPSIII, p <0.05. TGF-β1: transforming growth factor-beta1; FGF-2:fibroblast growth factors-2.

Figure 2. Interleukin levels in platelet-rich plasma measured by Magellan (n=6), GPSIII (n=6), and open (n=4) methods. (A) IL-8; (B) IL-1β; (C) IL-6; (D) IL-10; (E) tumor necrosis factor (TNF-α); (F) IL-12p70

* p value <0.05; ** p value <0.01. TNF-α: tumor necrosis factor alpha; IL: Interleukin levels.

Figure 3. Chemokine levels measured in platelet-rich plasma samples by Magellan (n=6), GPSIII (n=6), and open (n=4) methods

* p value <0.05. MIG: monokine induced by gamma; MCP: monocyte chemotactic protein; IP: inducible protein; RANTES: regulated on activation, normal T cell expressed and secreted.