A1 Refereed original research article in a scientific journal
Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood
Authors: van der Pan Kyra, de Bruin-Versteeg Sandra, Damasceno Daniela, Hernandez-Delgado Alejandro, van der Sluijs-Gelling Alita J, van den Bossche Wouter BL, de Laat Inge F, Diez Paula, Naber Birgitta AE, Diks Annieck M, Berkowska Magdalena A, de Mooij Bas, Groenland Rick J, de Bie Fenna J, Khatri Indu, Kassem Sara, de Jager Anniek L, Louis Alesha, Almeida Julia, van Gaans-van den Brink Jacqueline AM, Barkoff Alex-Mikael, He Qiushui, Ferwerda Gerben, Versteegen Pauline, Berbers Guy AM, Orfao Alberto, van Dongen Jacques JM, Teodosio Cristina
Publisher: Frontiers Media SA
Publication year: 2022
Journal: Frontiers in Immunology
Journal name in source: FRONTIERS IN IMMUNOLOGY
Journal acronym: FRONT IMMUNOL
Article number: 935879
Volume: 13
Number of pages: 26
ISSN: 1664-3224
eISSN: 1664-3224
DOI: https://doi.org/10.3389/fimmu.2022.935879
Web address : https://doi.org/10.3389/fimmu.2022.935879
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/176905984
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.
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