Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization

• Autor(a) principal: Borges, Sofia de Morais
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/35652
• topic: Blood processing; Blood preservatives; Buffy coat; Monocyte function; iNKT cell; T lymphocyte function

Peripheral blood is the most practical biospecimen to obtain information for clinical and research purposes, as a source of cells and metabolites. It is widely known that different blood collection conditions, including anticoagulants and the time between blood collection and processing, influence leukocyte phenotype and function. Buffy coats are commonly used in immunological research as a source of leukocytes. They are residual products of healthy donor whole blood units processing. The preservative solution present in buffy coats is Citrate- Phosphate-Dextrose (CPD), in which citrate is the anticoagulant. There is a lack of information on the possible difference in the functionality of leukocytes isolated from buffy coats compared to leukocytes isolated from blood collection tubes with anticoagulants. This project aimed to study the influence of the blood collection condition, namely the anticoagulants, on peripheral blood mononuclear cells (PBMCs) function, phenotype, and viability. Monocytes (CD14+ cells) and T lymphocytes were the PBMCs studied. The blood collection conditions tested were buffy coats, which are generated from a blood bag collected approximately 20 h before the experiments, ethylenediaminetetraacetic acid (EDTA), CPD supplemented with adenine (CPDA), and sodium citrate-containing tubes. Monocyte function was investigated by the capacity to present antigens and the lysosomal activity since this organelle is important for antigen presentation. The lysosomal function was analyzed by the b-glucosidase activity and the general lysosomal activity. The monocyte capacity to present antigens was analyzed through lipid antigen presentation to invariant Natural Killer T (iNKT) cells. iNKT cells are activated by lipids bound to CD1d, a non-polymorphic MHC-class I-like molecule, present on the surface of antigen-presenting cells. The fact that these cells are restricted to a non-polymorphic molecule makes them more practical to use in assays with different donors than conventional T lymphocytes. This work was conducted using two different approaches, initially with blood collected from different donors, that had high inter-donor variability, therefore in the consecutive experiments blood was collected from the same donor. This implied that there was a 20 h wait after blood collection, before cell isolation, with a fixed time for the buffy coat and the collection of blood from tubes. The results showed that monocytes isolated from EDTA blood tubes have a lower capacity to present lipid antigens to iNKT cells than monocytes isolated from buffy coats. No differences were found between monocytes isolated from sodium citrate or CPDA and the ones isolated from buffy coats. This was accompanied by a decrease in the viability and in the efficiency of isolation of CD14+ cells of the EDTA-isolated monocytes. Flow cytometry analysis of the expression of the surface markers CD1d and CD86 showed a higher expression of these markers for monocytes isolated from EDTA than those isolated from buffy coats. In contrast, no difference in cytokine production upon T lymphocyte activation was observed with PMA/ionomycin stimulation, in cells isolated from buffy coats, EDTA, and sodium citrate-containing tubes. In conclusion, EDTA-containing blood tubes are not the ideal choice of anticoagulant for monocyte antigen presentation assays. T lymphocyte functional assays seem to be less sensitive to the alterations in blood collection conditions. We advise that the blood collection condition and the time between biospecimen collection and analysis should be carefully considered when designing experimental procedures.