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Improved Standardization of Flow Cytometry Diagnostic Screening of Primary Immunodeficiency by Software-Based
Eleni Linskens1, Annieck M Diks2, Jana Neirinck3
1Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.
Insights
Automated Gating and Identification (AG&I) tools improve the reproducibility of flow cytometry (FC) for diagnosing primary immunodeficiencies (PIDs). While AG&I offers greater robustness than manual gating, expert review remains crucial for complex cases.
Area of Science:
- Immunology
- Clinical Diagnostics
- Biotechnology
Background:
- Multiparameter flow cytometry (FC) is crucial for diagnosing primary immunodeficiencies (PIDs).
- The EuroFlow PID Orientation tube (PIDOT) facilitates lymphocyte subpopulation identification.
- Standardization of FC data analysis is needed for accurate PID diagnosis.
Purpose of the Study:
- To evaluate the contribution of Automated Gating and Identification (AG&I) tools to FC standardization in PID diagnostics.
- To compare AG&I with expert-based Manual Gating (MG) in terms of reproducibility and clinical interpretation.
- To assess the impact of AG&I on the diagnostic work-up of PIDs.
Main Methods:
- FC data from 44 PID patients and 26 healthy donors stained with PIDOT were analyzed using both MG and AG&I.
- Percentage differences in absolute lymphocyte counts/µL were calculated between MG and AG&I.
- Intra- and inter-observer reproducibility of MG vs. AG&I was evaluated on a subset of 12 samples.
Main Results:
- AG&I identified >99% of lymphoid events with significantly higher reproducibility than MG.
- High agreement (<20% difference) between MG and AG&I was observed in most samples (83% HD, 68% patients).
- AG&I had minimal impact on clinical interpretation, with expert review needed for specific aberrant cases.
Conclusions:
- Replacing MG with AG&I enhances reproducibility and robustness in PID diagnostics.
- Expert revision of AG&I results is still necessary for samples with numerical alterations or aberrant cell maturation/marker expression.
- AG&I tools represent a significant advancement for standardized PID diagnosis via flow cytometry.
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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