Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells.

Autor:	Delgado AH; Cytognos SL, Salamanca, Spain.; Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain., Fluxa R; Cytognos SL, Salamanca, Spain., Perez-Andres M; Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain.; Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.; Biomedical Research Networking Centre Consortium of Oncology (CIBERONC), Instituto de Salud Carlos III, Madrid, Spain., Diks AM; Department of Immunology (IMMU), Leiden University Medical Center (LUMC), Leiden, Netherlands., 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, University of Turku (UTU), Turku, Finland., Blanco E; Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain.; Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain., Torres-Valle A; Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain.; Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain., Berkowska MA; Department of Immunology (IMMU), Leiden University Medical Center (LUMC), Leiden, Netherlands., Grigore G; Cytognos SL, Salamanca, Spain., van Dongen JJM; Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain.; Department of Immunology (IMMU), Leiden University Medical Center (LUMC), Leiden, Netherlands., Orfao A; Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain.; Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.; Biomedical Research Networking Centre Consortium of Oncology (CIBERONC), Instituto de Salud Carlos III, Madrid, Spain.
Jazyk:	angličtina
Zdroj:	Frontiers in immunology [Front Immunol] 2023 Oct 17; Vol. 14, pp. 1268686. Date of Electronic Publication: 2023 Oct 17 (Print Publication: 2023).
DOI:	10.3389/fimmu.2023.1268686
Abstrakt:	Background: Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Objective: Here, we designed, constructed and validated an automated database-guided gating and identification (AGI) approach for fast and standardized in-depth dissection of B-lymphocyte and PC populations in human blood. Methods: For this purpose, 213 FC standard (FCS) datafiles corresponding to umbilical cord and peripheral blood samples from healthy and patient volunteers, stained with the 14-color 18-antibody EuroFlow BIgH-IMM panel, were used. Results: The BIgH-IMM antibody panel allowed identification of 117 different B-lymphocyte and PC subsets. Samples from 36 healthy donors were stained and 14 of the datafiles that fulfilled strict inclusion criteria were analysed by an expert flow cytometrist to build the EuroFlow BIgH-IMM database. Data contained in the datafiles was then merged into a reference database that was uploaded in the Infinicyt software (Cytognos, Salamanca, Spain). Subsequently, we compared the results of manual gating (MG) with the performance of two classification algorithms -hierarchical algorithm vs two-step algorithm- for AGI of the cell populations present in 5 randomly selected FCS datafiles. The hierarchical AGI algorithm showed higher correlation values vs conventional MG (r 2 of 0.94 vs. 0.88 for the two-step AGI algorithm) and was further validated in a set of 177 FCS datafiles against conventional expert-based MG. For virtually all identifiable cell populations a highly significant correlation was observed between the two approaches (r 2 >0.81 for 79% of all B-cell populations identified), with a significantly lower median time of analysis per sample (6 vs. 40 min, p=0.001) for the AGI tool vs. MG, respectively and both intra-sample (median CV of 1.7% vs . 10.4% by MG, p<0.001) and inter-expert (median CV of 3.9% vs . 17.3% by MG by 2 experts, p<0.001) variability. Conclusion: Our results show that compared to conventional FC data analysis strategies, the here proposed AGI tool is a faster, more robust, reproducible, and standardized approach for in-depth analysis of B-lymphocyte and PC subsets circulating in human blood. Competing Interests: JvD, AO, AMB, MB, EB, MP and AH-D, are listed as co inventors on the EuroFlow 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 panel used in this study, and RF and AO are listed as co inventors on the patent “Method of digital information classification” US 10,133,962 B2, filing date 9 June 2017 owned by Cytognos S.L. JvD 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 Consortium. These royalties are exclusively used for continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium. JvD and AO report an Educational Services Agreement and a Scientific Advisor Agreement from BD Biosciences San Joseé, CA; all related fees and honoraria are for the involved University Departments at University of Salamanca. AH-D, GG and RF are employees of Cytognos S.L. Salamanca Spain. 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. (Copyright © 2023 Delgado, Fluxa, Perez-Andres, Diks, van Gaans-van den Brink, Barkoff, Blanco, Torres-Valle, Berkowska, Grigore, van Dongen and Orfao.)
Databáze:	MEDLINE
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