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

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.

A comparison of three column agglutination tests for red blood cell alloantibody identification.

OBJECTIVE: Commercial kits of column tests for pre-transfusion testing have progressively replaced conventional tube tests in most laboratories. Aim of this study was to compare three commercial test cell panels for the identification of irregular red blood cell (RBC) alloantibodies. Overall, 44 samples with a positive indirect antiglobulin test (IAT) by routine testing were used for comparison of following panels: Ortho RESOLVE® panelC (Ortho Clinical Diagnostics (OCD), Milan, Italy), ID-DiaPanel(-P) (Bio-Rad Laboratories, CA, USA) and Identisera Diana(P) (Grifols, Barcelona, Spain). Column agglutination techniques were used, with microtubes containing either microgel (Bio-Rad/Grifols) or glass bead microparticles (Ortho). RESULTS: Alloantibody identification was possible in 38 samples, of which identical identification was shown in 33 samples by all methods. The remaining samples showed differences between certain methods, with the gel card system being superior to the glass card system for analyzing stored samples Considering that not all samples were evaluated in all three methods, the concordance rate reached 100% between Bio-Rad and Grifols, 90.5% between Bio-Rad and OCD, 86.5% between OCD and Grifols and 90.5% between all methods. Although differences in sensitivities were seen for specific antibodies, the three methods showed comparable performance for the identification of RBC alloantibodies.