Vesículas extracelulares de concentrado de hemácias: tempo de armazenamento e controle de qualidade
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Data
2018-08-10
Autores
Risso, Mariane Aparecida
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Universidade Estadual Paulista (Unesp)
Resumo
As vesículas extracelulares (VEs) são formações membranárias heterogêneas de tamanhos submicrométricos, que possuem funções específicas dependentes de sua biogênese. Os Concentrados de hemácias (CH) sofrem inúmeras alterações bioquímicas e morfológicas durante a estocagem comprometendo o meio intracelular e extracelular. O controle de qualidade das unidade de CH avalia essa lesão de estoque das hemácias principalmente a partir da determinação do grau de hemólise através da dosagem de hemoglobina livre. Não se sabe ao certo a quantidade de vesículas extracelulares que um CH pode gerar durante seu armazenamento mas muitos estudos relacionam as VEs com complicações pós-transfusionais. O objetivo deste estudo foi analisar o controle de qualidade de concentrados de hemácias sob o aspecto da identificação de vesículas extracelulares, correlacionando o grau de hemólise e a identificação de VEs em CH de hemácias em diferentes dias de conservação. Métodos: CH foram obtidos de 20 doadores saudáveis, refrigerados armazenados por 35 dias, e avaliados para o perfil de doação de sangue, grau de hemólise e dosagem de VEs por citometria de fluxo nos dias 5, 15, 25 e 35 de armazenamento. Resultados: Não houve diferença estatística significante entre o perfil da doação e as dosagens de grau de hemólise e VEs. O número total de VEs aumentou significativamente com o armazenamento assim como o grau de hemólise, observando maior correlação entre os métodos em D25 (p=0,0002) e D35 (p<0,0001). O tamanho das VEs foi avaliado pela intensidade de fluorescência que demonstrou VEs < 3µm. O grau de hemólise demonstrou ser um parâmetro pouco confiável, já que mesmo nas unidades com baixo volume apresentou conformidade nos valores. Pesquisas em caracterização, quantificação e avaliação de danos provocados pelas VEs poderão nortear novos rumos para o esclarecimento das lesões de armazenamento e mostrar possíveis melhorias na avaliação de qualidade desses hemocomponentes.
The extracellular vesicles (EVs) are heterogeneous membrane formations of submicrometric sizes, which have specific functions depending on their biogenesis. Red blood cell (RBC) undergo numerous biochemical and morphological changes during storage age compromising the intracellular and extracellular environment. The quality control of these units evaluates this lesion RBCs inventory mainly from the haemolysis determination through the measurement of free haemoglobin. It is unclear how much EVs the RBCs can generate during its storage but many studies related the EVs with post-transfusion complications. The objective of this study was to analyze the RBCs quality control under the aspect of the identification of EVs, correlating the haemolysis and the identification of VEs on different days of conservation. Methods: RBC were obtained from 20 healthy donors, refrigerated and stored for 35 days, and evaluated for blood donation profile, haemolysis and EVs counting by flow cytometry on storage days 5, 15, 25 and 35. Results: There was no statistically significant difference between the donation profile and haemolysis percentual and EVs dosages. The total number of EVs increased significantly with storage as well as the haemolysis, observing a higher correlation between the methods in D25 (p = 0.0002) and D35 (p <0.0001). The size of the VEs was evaluated by the fluorescence intensity that demonstrated VEs < 3μm. The haemolysis proved to be an unreliable parameter, since even in the low volume units it showed compliance in the values. Research on characterization, quantification and evaluation of damage caused by EVs may guide new directions for the clarification of storage lesions and show possible improvements in the quality evaluation of these blood components.
The extracellular vesicles (EVs) are heterogeneous membrane formations of submicrometric sizes, which have specific functions depending on their biogenesis. Red blood cell (RBC) undergo numerous biochemical and morphological changes during storage age compromising the intracellular and extracellular environment. The quality control of these units evaluates this lesion RBCs inventory mainly from the haemolysis determination through the measurement of free haemoglobin. It is unclear how much EVs the RBCs can generate during its storage but many studies related the EVs with post-transfusion complications. The objective of this study was to analyze the RBCs quality control under the aspect of the identification of EVs, correlating the haemolysis and the identification of VEs on different days of conservation. Methods: RBC were obtained from 20 healthy donors, refrigerated and stored for 35 days, and evaluated for blood donation profile, haemolysis and EVs counting by flow cytometry on storage days 5, 15, 25 and 35. Results: There was no statistically significant difference between the donation profile and haemolysis percentual and EVs dosages. The total number of EVs increased significantly with storage as well as the haemolysis, observing a higher correlation between the methods in D25 (p = 0.0002) and D35 (p <0.0001). The size of the VEs was evaluated by the fluorescence intensity that demonstrated VEs < 3μm. The haemolysis proved to be an unreliable parameter, since even in the low volume units it showed compliance in the values. Research on characterization, quantification and evaluation of damage caused by EVs may guide new directions for the clarification of storage lesions and show possible improvements in the quality evaluation of these blood components.
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Palavras-chave
Vesículas extracelulares, concentrado de hemácias, controle de qualidade, citometria de fluxo, Extracellular vesicles, red blood cells, quality control, flow cytometry