Delgado, Alejandro HFluxa, RafaelPerez-Andres, MartinDiks, Annieck Mvan Gaans-van den Brink, Jacqueline A MBarkoff, Alex-MikaelBlanco, ElenaTorres-Valle, AlbaBerkowska, Magdalena AGrigore, Georgianavan Dongen, J J MOrfao, Alberto2023-11-092023-11-092023-10-173791556910.3389/fimmu.2023.1268686http://hdl.handle.net/10029/627048The 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 (r2 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 (r2>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.enCopyright © 2023 Delgado, Fluxa, Perez-Andres, Diks, van Gaans-van den Brink, Barkoff, Blanco, Torres-Valle, Berkowska, Grigore, van Dongen and Orfao.B-cellEuroFlowautomated gating and identification approachmultiparameter flow cytometryplasma cellstandardizationArticle1664-3224Front Immunol 2023;14:1268686