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

Detalhes bibliográficos
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
Resumo: 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.
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spelling Buffy Coat Vs blood collection tube mononuclear cells: a functional characterizationBlood processingBlood preservativesBuffy coatMonocyte functioniNKT cellT lymphocyte functionPeripheral 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.O sangue periférico é a amostra biológica mais prática de obter informações para fins clínicos e de investigação, servindo como fonte de células e de metabolitos. Sabe-se que diferentes fatores na colheita de sangue, como anticoagulantes e o tempo entre colheita e processamento, influenciam o fenótipo e a função dos leucócitos. Os buffy coats são produtos residuais do processamento de unidades de sangue-total de dadores saudáveis, comumente usados em investigação na área da Imunologia como fonte de leucócitos. A solução preservante presente nos buffy coats é o Citrato-Fosfato-Dextrose (CPD), em que o citrato atua como composto anticoagulante. Não existe conhecimento sobre a possível diferença na funcionalidade de leucócitos isolados de buffy coats quando comparados com leucócitos isolados de tubos de colheita de sangue com anticoagulantes. Este projeto teve como objetivo estudar a influência do tipo de colheita de sangue, nomeadamente, os anticoagulantes, na função, fenótipo e viabilidade das células mononucleares do sangue periférico (PBMCs). Os monócitos (células CD14+) e linfócitos T foram as PBMCs estudadas. As condições de colheita de sangue testadas foram buffy coats, que são gerados a partir de um saco de sangue colhido aproximadamente 20 h antes das experiências, e tubos contendo ácido etilenodiamino tetra-acético (EDTA), CPD suplementado com adenina (CPDA) ou citrato de sódio. A função dos monócitos foi investigada pela sua capacidade de apresentar antigénios e pela sua atividade lisossomal, uma vez que a ação deste organelo é importante na apresentação de antigénios externos. A função lisossomal foi analisada pela atividade da β-glicosidase e pela atividade lisossomal geral. A capacidade de apresentação dos monócitos foi analisada através da apresentação de antigénios lipídicos a células Natural Killer T invariantes (iNKT). Estas células são ativadas por lípidos ligados ao CD1d, uma molécula tipo complexo principal de histocompatibilidade (MHC) classe I não polimórfica, presente na superfície de células apresentadoras de antigénios. O facto destas células serem restritas para uma molécula não polimórfica torna-as mais práticas para ensaios com dadores diferentes do que outros tipos celulares, como linfócitos T convencionais. Este projeto usou duas abordagens diferentes, pois inicialmente o sangue foi colhido de dadores diferentes, que apresentavam elevada variabilidade inter-dador, logo, nas experiências seguintes, o sangue foi colhido do mesmo dador. Isto implicou um tempo de espera de 20 h entre a colheita e o isolamento das células, com tempo fixo para os buffy coats e os tubos de colheita de sangue. Os resultados mostraram que monócitos isolados de tubos de colheita de sangue com EDTA como anticoagulante têm menor capacidade de apresentar antigénios lipídicos às células iNKT do que monócitos isolados de buffy coats. Não foram encontradas diferenças na capacidade de apresentação entre monócitos isolados de citrato de sódio ou CPDA e os monócitos isolados de buffy coats. Isto foi acompanhado por uma diminuição na viabilidade e na eficiência de isolamento de células CD14+ colhidas com EDTA como anticoagulante. A análise por citometria de fluxo dos marcadores de superfície CD1d e CD86 revelou uma maior expressão destes marcadores em monócitos isolados com EDTA do que em células isoladas de buffy coats. Em contraste, não foram observadas diferenças na ativação de linfócitos T pela produção de citocinas após estimulação com PMA/ionomicina em células isoladas de buffy coats, e tubos de sangue com EDTA e citrato de sódio. Com estes resultados, pode-se concluir que tubos de sangue contendo EDTA não são a melhor escolha como anticoagulante para ensaios de apresentação de antigénios por monócitos. Os ensaios funcionais com linfócitos T parecem ser menos sensíveis às alterações nas condições de colheita. É aconselhável que as condições de colheita e o tempo entre a colheita e a análise da amostra biológica sejam cuidadosamente escolhidas ao definir desenhos experimentais no futuro.2024-12-05T00:00:00Z2022-11-25T00:00:00Z2022-11-25info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/35652engBorges, Sofia de Moraisinfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T12:08:44Zoai:ria.ua.pt:10773/35652Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:06:39.214494Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
title Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
spellingShingle Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
Borges, Sofia de Morais
Blood processing
Blood preservatives
Buffy coat
Monocyte function
iNKT cell
T lymphocyte function
title_short Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
title_full Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
title_fullStr Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
title_full_unstemmed Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
title_sort Buffy Coat Vs blood collection tube mononuclear cells: a functional characterization
author Borges, Sofia de Morais
author_facet Borges, Sofia de Morais
author_role author
dc.contributor.author.fl_str_mv Borges, Sofia de Morais
dc.subject.por.fl_str_mv Blood processing
Blood preservatives
Buffy coat
Monocyte function
iNKT cell
T lymphocyte function
topic Blood processing
Blood preservatives
Buffy coat
Monocyte function
iNKT cell
T lymphocyte function
description 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.
publishDate 2022
dc.date.none.fl_str_mv 2022-11-25T00:00:00Z
2022-11-25
2024-12-05T00:00:00Z
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