Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood.

Autor:	van der Pan K; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., de Bruin-Versteeg S; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Damasceno D; 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., Hernández-Delgado A; 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., van der Sluijs-Gelling AJ; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., van den Bossche WBL; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands.; Department of Immunology, Department of Neurosurgery, Brain Tumor Center, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, Netherlands., de Laat IF; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Díez P; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Naber BAE; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Diks AM; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Berkowska MA; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., de Mooij B; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Groenland RJ; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., de Bie FJ; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Khatri I; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Kassem S; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., de Jager AL; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Louis A; Department of Immunology, Leiden University Medical Center, Leiden, Netherlands., Almeida J; 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., van Gaans-van den Brink JAM; Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands., Barkoff AM; Institute of Biomedicine, Research Center for Infections and Immunity, University of Turku (UTU), Turku, Finland., He Q; Institute of Biomedicine, Research Center for Infections and Immunity, University of Turku (UTU), Turku, Finland., Ferwerda G; Section of Paediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands., Versteegen P; Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands., Berbers GAM; Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands., Orfao A; 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., van Dongen JJM; 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., Teodosio C; 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.
Jazyk:	angličtina
Zdroj:	Frontiers in immunology [Front Immunol] 2022 Sep 14; Vol. 13, pp. 935879. Date of Electronic Publication: 2022 Sep 14 (Print Publication: 2022).
DOI:	10.3389/fimmu.2022.935879
Abstrakt:	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. Competing Interests: JD and AO report to be chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow Foundation. These royalties are exclusively used for continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium. JD and AO report an Educational Services Agreement from BD Biosciences (San José, CA) and a Scientific Advisor Agreement with Cytognos; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. AH-D is an employee of Cytognos (Salamanca, Spain). Lastly, JD, CT, AO, JA, WB, KP, MB, AD, DD, and AH-D, are listed as (co)inventors on the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” (NL2844751, filing date 5 November 2019), owned by the EuroFlow scientific consortium, which describes the flow cytometry panels developed in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2022 van der Pan, de Bruin-Versteeg, Damasceno, Hernández-Delgado, van der Sluijs-Gelling, van den Bossche, de Laat, Díez, Naber, Diks, Berkowska, de Mooij, Groenland, de Bie, Khatri, Kassem, de Jager, Louis, Almeida, van Gaans-van den Brink, Barkoff, He, Ferwerda, Versteegen, Berbers, Orfao, van Dongen and Teodosio.)
Databáze:	MEDLINE
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