Live MOG-IgG cell-based assay: Comparison across flow cytometers and diagnostic validation on high-sensitivity full spectrum flow cytometry

MOG antibody-associated disease (MOGAD) diagnosis rests on seropositivity for MOG antibody (MOG-IgG). Live cell-based assays (CBA) are gold standards. Although flow cytometry live CBAs have high real-world sensitivity, their global implementation and diagnostic deployment have been challenged by perceptions of "in-house" design and custom optimization. Herein, we compared the analytical robustness of flow live MOG-IgG CBA across various cytometers in both research and diagnostic laboratories. Flow live CBAs were performed on three conventional (Fortessa, BDLSRII, Gallios), and two spectral cytometers (Aurora, ID7000). MOG-IgG titers were calculated by median fluorescence intensity (MFI), and intra- and inter assay precisions (CV%) and serostatuses were determined. The MFI detection range on Fortessa, currently used for testing, was significantly lower than spectral ID7000 (4.75-fold, p = 0.04) and Aurora (12-fold, p = 0.0001), albeit all MFIs correlated ( p < 0.0001; R 2 = 0.99). Interestingly, the high detection range was attributed to technology, not laboratory environments ( p > 0.05). Intra- and inter-assay precisions were similar across cytometers. ID7000 and Fortessa had the lowest variation, with 4.6 % and 6.8 % intra-CV, respectively, and 15.7 % inter-CV. FACS ratio, currently reported as MOG-IgG titre, and MFIs were comparable and correlated for all cytometers ( p < 0.001; R 2 ≤ 0.99), regardless of the analysis software ( p < 0.0001, R 2 = 0.98). All serostatuses were highly concordant (κ = 1). Our results demonstrate that flow live CBAs can be validated in diagnostic laboratories across a range of flow cytometers with high reproducibility and repeatability. In particular, excellent assay performance on spectral flow cytometry strongly supports the proof-of-concept use of this technology for diagnostic purposes.