Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes.
J Immunol Methods
; 474: 112646, 2019 11.
Article
em En
| MEDLINE
| ID: mdl-31419409
ABSTRACT
INTRODUCTION:
Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting.METHODS:
Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65â¯years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20â¯min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation.RESULTS:
The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at Tâ¯=â¯0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1â¯h (Tâ¯=â¯1) depicted as means (95%Cl) CD35 2.2× (1.5×-2.5×) pâ¯=â¯.028, CD11b 2.5× (1.7×-3.1×) pâ¯=â¯.023, CD10 2.5× (2.1×-2.7×) pâ¯=â¯.009) and a further increase in activation markers was observed after 2 and 3â¯h. Monocytes also showed a increase in activation markers in 1â¯h (mean (95%Cl) CD35 1.8× (1.3×-2.2×) pâ¯=â¯.058, CD11b 2.13× (1.6×-2.4×) pâ¯=â¯.025) and also a further significant increase in 2 and 3â¯h was observed.CONCLUSION:
This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2â¯h to only 15-20â¯min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Palavras-chave
Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Neprilisina
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Imunofenotipagem
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Receptores de Complemento 3b
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Sistemas Automatizados de Assistência Junto ao Leito
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Antígeno CD11b
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Citometria de Fluxo
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Testes Imediatos
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Leucócitos
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Limite:
Adolescent
/
Adult
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Aged
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Holanda