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

Kyra van der Pan, Sandra de Bruin-Versteeg, the TiMaScan, IMI2, PERISCOPE, EuroFlow Consortia, Daniela Damasceno, Alejandro Hernández-Delgado, Alita J. van der Sluijs-Gelling, Wouter B.L. van den Bossche, Inge F. de Laat, Paula Díez, Brigitta A.E. Naber, Annieck M. Diks, Magdalena A. Berkowska, Bas de Mooij, Rick J. Groenland, Fenna J. de Bie, Indu Khatri, Sara Kassem, Anniek L. de Jager, Alesha Louis, Julia AlmeidaJacqueline A.M. van Gaans-van den Brink, Alex Mikael Barkoff, Qiushui He, Gerben Ferwerda, Pauline Versteegen, Guy A.M. Berbers, Alberto Orfao, Jacques J.M. van Dongen*, Cristina Teodosio

*Corresponding author for this work

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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.

Original languageEnglish
Article number935879
JournalFrontiers in Immunology
Publication statusPublished - 14 Sep 2022

Bibliographical note

Funding Information:
The presented work was funded by the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme with an ERC Advanced Grant (ERC-2015-AdG 695655, TiMaScan) and the IMI2 PERISCOPE project, financed by the Innovative Medicines Initiative 2 Joint Undertaking (grant number 115910). This joint undertaking receives support from the European Union’s Horizon 2020 Research and Innovation Programme, the European Federation of Pharmaceutical Industries and Associations (EFPIA), and The Bill and Melinda Gates Foundation (BMGF). This manuscript only reflects the author’s views. The Joint Undertaking is not responsible for any use that may be made of the information this manuscript contains. The coordination and innovation processes of this study were supported by the EuroFlow Consortium. The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP).

Publisher Copyright:
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.


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