OBJECTIVE: Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). RESULTS: Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. CONCLUSIONS: The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology.

Clinical Department of Neurology Medical University of Innsbruck Innsbruck Austria

Clinical Neurobiology Unit Neuroscience Institute Cavalieri Ottolenghi University Hospital San Luigi Gonzaga Regional Referring Multiple Sclerosis Centre Orbassano Italy

Department of Clinical Immunology Odense University Hospital Odense Denmark

Department of Clinical Medicine University of Bergen Bergen Norway Department of Neurology Haukeland University Hospital Bergen Norway

Department of Immunology and Biotechnology University of Pécs Pécs Hungary

Department of Immunology Istanbul University Institute of Experimental Medicine Istanbul Turkey

Department of Neurology Azienda ULSS 9 Treviso Treviso Italy

Department of Neurology Center of Clinical Neuroscience 1st Faculty of Medicine General University Hospital and 1st Faculty of Medicine Charles University Prague Prague Czech Republic

Department of Neurology MS Centre ErasMS Erasmus MC Rotterdam The Netherlands

Department of Neurology University Hospital of Schleswig Holstein Campus Lübeck Lübeck Germany

Euroimmun AG Lübeck Germany

Faculty of Medecine RTH Laennec Lyon Neurosciences Research Centre Neuro inflammation and Neuro oncology Team Lyon France

Institute for Quality Assurance Lübeck Germany

Institute of Clinical Research University of Southern Denmark Odense Denmark Department of Neurology University of Pécs Pécs Hungary

Klinikum rechts der Isar der TU München Klinik für Neurologie Munich Germany

Laboratory for CSF and Neuroimmunology Topelex Ltd Prague Czech Republic

Medical Faculty Department of Neurology Heinrich Heine University Düsseldorf Düsseldorf Germany

Molecular Neuroimmunology Group Department of Neurology University of Heidelberg Germany

NeuroCure Clinical Research Center Charité Universitätsmedizin Berlin Berlin Germany

Neuroimmunology Program Hospital Clinic and Institut d'Investigació Biomèdica August Pi i Sunyer Universitat de Barcelona Barcelona Spain

Neuroimmunology Program Hospital Clinic and Institut d'Investigació Biomèdica August Pi i Sunyer Universitat de Barcelona Barcelona Spain Institute of Neurology Medical University of Vienna Vienna Austria

Neurology Department Istanbul University Cerrahpasa Medical School Istanbul Turkey

Nuffield Department of Clinical Neurosciences University of Oxford Oxford UK

Sanquin Diagnostic Services Department of Immunopathology and Blood Coagulation Amsterdam The Netherlands

Servei de Neurologia Neuroimmunologia Centre d'Esclerosi Múltiple de Catalunya Vall d'Hebron Institut de Recerca Hospital Universitari Vall d'Hebron Universitat Autònoma de Barcelona Barcelona Spain

Zdravotni ustav se sidlem v Usti nad Labem Centrum imunologie a mikrobiologie Usti nad Labem Czech Republic

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