Flow Cytometric Live Cell-Based Assay in Neuromyelitis Optica: Comparison with the Commercial Cell-Based Indirect Immunofluorescence Assay

Introduction: Neuromyelitis Optica (NMO) is an inflammatory disorder affecting the central nervous system, notably the optic nerve and spinal cord. Seropositive NMO is marked by serum IgG antibodies against aquaporin-4 (AQP4). The accurate identification of AQP4-IgG is crucial for distinguishing NMO from other demyelinating diseases of the central nervous system. However, traditional diagnostic assays have limitations in sensitivity and specificity. Here, we introduce our in-house flow cytometry live cell-based assay (FC-LCBA) for detecting AQP4 antibodies with enhanced sensitivity and specificity. Our objective is to report the accuracy and compare the efficacy of our newly developed in-house FC-LCBA against the commercial cell-based indirect immunofluorescence assay (IIFA) in detecting AQP4 antibodies. Methods: This single-blind study was approved by the ethical committee and involved 101 serum samples. Twenty-five samples (including retests) from 17 patients evaluated in the NMO spectrum who had at least one positive cell-based IIFA test during the diagnosis or follow-up are tested in parallel with our in-house FC-LCBA and cell-based IIFA. In addition, 36 serum samples from myelin oligodendrocyte glycoprotein-associated disease (MOGAD) patients and 40 serum samples from healthy subjects are also referred for specificity analysis. Results: Our in-house FC-LCBA displayed superior sensitivity, detecting positive results even when the cell-based IIFA yielded negative results in patients under immunosuppressive treatments. Additionally, FC-LCBA exhibited high specificity for NMO, showing negligible antibody levels in patients with MOGAD diagnosis and healthy individuals. The assay's stability was confirmed through consistent results in retests. Conclusion: Our in-house FC-LCBA emerges as a promising diagnostic tool for detecting AQP4 antibodies, offering improved sensitivity, specificity, and reliability, instilling confidence in its potential.