Box 4.

Red flags to be considered before making a diagnosis of NMOSD (modified from Wingerchuk et al. 2015 [228])

Clinical and laboratory features Progressive overall clinical course (neurologic deterioration unrelated to attacks; consider MS) Time to attack nadir < 4 h (consider ischemia/infarction) or continual worsening for > 4 weeks from attack onset (consider sarcoidosis or neoplasm) Partial TM, especially when not associated with LETM MRI lesion (consider MS) AQP4-IgG positivity only in the CSF, not in the serum (can be true positive in very rare cases; e.g., if serum testing is hampered by strong background staining, or shortly after PLEX; always consider retesting of serum and CSF in an alternative assay) AQP4-IgM and/or AQP4-IgA positive, but AQP4-IgG negative (clinical significance unknown; not sufficient for making a diagnosis of seropositive NMOSD) “Double positivity” for AQP4-IgG and MOG-IgG (extremely rare/implausible; repetition of both tests recommended in all cases) Presence of CSF-restricted OCB (present in ≤ 20% of cases of NMO vs > 90% of MS [lower in Asian and other populations]) Presence of a bi- or trispecific MRZ reaction (present in around 67% of MS patients, virtually absent in NMOSD)

Neuroimaging findings Brain imaging features (T2-weighted MRI) suggestive of MS (MS-typical): Lesions with orientation perpendicular to a lateral ventricular surface (Dawson fingers), or lesions adjacent to lateral ventricle in the inferior temporal lobe, or juxtacortical lesions involving subcortical U-fibers, or cortical lesions Spinal cord characteristics more suggestive of MS than NMOSD: Lesions < 3 complete vertebral segments (sagittal T2), or lesions located predominantly (> 70%) in the peripheral cord (axial T2), or diffuse, indistinct signal change (T2, seen with longstanding or progressive MS) Lesions with persistent (> 3 months) Gd enhancement (suggestive neither of NMOSD nor MS) or persistent Gd enhancement despite immunotherapy (consider tumor/lymphoma or vascular malformation) Brain linear perivascular radial Gd enhancement (consider GFAP-IgG-associated astrocytopathy and, possibly, neurosarcoidosis, vasculitis, lymphoma)

Comorbidities Sarcoidosis, established or suggestive findings thereof (e.g., mediastinal lymphadenopathy, fever and night sweats, elevated serum ACE or soluble IL2R levels [153, 163], leptomeningeal enhancement) Cancer, established or with suggestive clinical, radiologic, or laboratory findings thereof; consider also lymphoma or paraneoplastic disease (e.g., CV2/CRMP5-associated optic neuropathy [88] and myelopathy, or anti-Ma-associated diencephalic syndrome) Chronic infection, established or with suggestive findings thereof (e.g., HIV, syphilis, Tb)

ACE angiotensin-converting enzyme, CSF cerebrospinal fluid, IL2R interleukin-2 receptor, LETM longitudinally extensive transverse myelitis, MRZ measles/rubella/zoster virus reaction, i.e., intrathecally produced antibodies (positive antibody index) against at least two of these three viral agents, MS multiple sclerosis, OCB oligoclonal bands, PLEX plasma exchange, Tb tuberculosis, TM transverse myelitis