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To optimize aquaporin-4 (AQP4) antibody (Ab) detection and to assess the influence of the increased sensitivity of the assay on the demographic and disease-related characteristics of a group of AQP4-Ab–negative patients.Serum samples were obtained from patients included in the French NOMADMUS database with a definite diagnosis of neuromyelitis optica (NMO) (n = 87) and were compared with controls (n = 54). They were tested by indirect immunofluorescence and cell-based assays (CBAs) in various conditions and with several plasmids.We identified the CBA on live cells transfected with the untagged AQP4-M23 isoform as the best method, with a sensitivity of 74.4% and a specificity of 100%. We demonstrated a direct relationship between improvement of the sensitivity of the detection method and the distinctiveness and characteristics of the AQP4-Ab–negative NMO group. Whereas with the classic indirect immunofluorescence or current AQP4-M1 CBA we found only slight differences between the 2 populations, using the AQP4-M23 CBA, we demonstrated that patients with AQP4-Ab–negative NMO expressed specific demographic and disease-related features. They were characterized by an equal male/female ratio (p < 0.001), a Caucasian ethnicity (p = 0.029), and an overrepresentation of simultaneous optic neuritis and transverse myelitis at first episode (p = 0.015). In terms of disability, they experienced a better visual acuity at last follow-up compared with seropositive NMO (p = 0.007).This raises the question of a distinct physiopathology for patients with AQP4-Ab–negative NMO and of their place in the spectrum of the disease.