Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood
Frontiers in Immunology, vol. 13, pp. 935879
Abstract
Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual “expert-based”) gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.
Authors 28
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Universidad de Salamanca · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca
Affiliation as printed
Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
Translational and Clinical Research Program, Cancer Research Center (IBMCC
University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
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Universidad de Salamanca · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca
Affiliation as printed
Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
Translational and Clinical Research Program, Cancer Research Center (IBMCC
University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center · Erasmus MC
Affiliation as printed
Department of Immunology, Department of Neurosurgery, Brain Tumor Center, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, Netherlands
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Leiden University Medical Center
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
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Universidad de Salamanca · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca
Affiliation as printed
Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
Translational and Clinical Research Program, Cancer Research Center (IBMCC
University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
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National Institute for Public Health and the Environment
Affiliation as printed
Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands
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Affiliation as printed
Institute of Biomedicine, Research Center for Infections and Immunity, University of Turku (UTU), Turku, Finland
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Affiliation as printed
Institute of Biomedicine, Research Center for Infections and Immunity, University of Turku (UTU), Turku, Finland
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Radboud Institute for Molecular Life Sciences
Affiliation as printed
Section of Paediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands
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National Institute for Public Health and the Environment
Affiliation as printed
Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands
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National Institute for Public Health and the Environment
Affiliation as printed
Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands
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Universidad de Salamanca · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca
Affiliation as printed
Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
Translational and Clinical Research Program, Cancer Research Center (IBMCC
University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
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Jacques J. M. van Dongen corresponding Aachen Department of Immunology, Leiden University Medical Center
Universidad de Salamanca · Leiden University Medical Center · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
Translational and Clinical Research Program, Cancer Research Center (IBMCC
University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
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Universidad de Salamanca · Leiden University Medical Center · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca
Affiliation as printed
Department of Immunology, Leiden University Medical Center, Leiden, Netherlands
Translational and Clinical Research Program, Cancer Research Center (IBMCC; University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
Translational and Clinical Research Program, Cancer Research Center (IBMCC
University of Salamanca - CSIC), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (Universidad de Salamanca, and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
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