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Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based Automated Gating

  • Linskens, Eleni1
  • Diks, Annieck M.2
  • Neirinck, Jana3
  • Perez-Andres, Martín4, 5
  • De Maertelaere, Emilie1
  • Berkowska, Magdalena A.2
  • Kerre, Tessa6
  • Hofmans, Mattias1
  • Orfao, Alberto4, 5
  • van Dongen, Jacques J. M.2
  • Haerynck, Filomeen7
  • Philippé, Jan1, 3
  • Bonroy, Carolien1, 3
  • 1 Department of Laboratory Medicine, Ghent University Hospital, Ghent , (Belgium)
  • 2 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden , (Netherlands)
  • 3 Department of Diagnostic Science, Ghent University, Ghent , (Belgium)
  • 4 Cancer Research Centre (IBMCC, USAL-CSIC; CIBERONC CB16/12/00400), Department of Medicine and Cytometry Service (NUCLEUS Research Support Platform), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca , (Spain)
  • 5 Translational and Clinical Research Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca (USAL), Department of Medicine, IBSAL and CIBERONC, University of Salamanca, Salamanca , (Spain)
  • 6 Department of Hematology, Ghent University Hospital, Ghent , (Belgium)
  • 7 Department of Pediatric Pulmonology and Immunology and PID Research Laboratory, Ghent University Hospital, Ghent , (Belgium)
Published Article
Frontiers in Immunology
Frontiers Media SA
Publication Date
Nov 02, 2020
DOI: 10.3389/fimmu.2020.584646
  • Immunology
  • Original Research


Background Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles.

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