Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays

Abstract

Objectives: Neuromyelitis optica (NMO) immunoglobulin G (IgG) (aquaporin-4 [AQP4] IgG) is highly specific for NMO and related disorders, and autoantibody detection has become an essential investigation in patients with demyelinating disease. However, although different techniques are now used, no multicenter comparisons have been performed. This study compares the sensitivity and specificity of different assays, including an in-house flow cytometric assay and 2 commercial assays (ELISA and transfected cell-based assay [CBA]).

Methods: Six assay methods (in-house or commercial) were performed in 2 international centers using coded serum from patients with NMO (35 patients), NMO spectrum disorders (25 patients), relapsing-remitting multiple sclerosis (39 patients), miscellaneous autoimmune diseases (25 patients), and healthy subjects (22 subjects).

Results: The highest sensitivities were yielded by assays detecting IgG binding to cells expressing recombinant AQP4 with quantitative flow cytometry (77; 46 of 60) or visual observation (CBA, 73%; 44 of 60). The fluorescence immunoprecipitation assay and tissue-based immunofluorescence assay were least sensitive (48%-53%). The CBA and ELISA commercial assays (100% specific) yielded sensitivities of 68% (41 of 60) and 60% (36 of 60), respectively, and sensitivity of 72% (43 of 60) when used in combination.

Conclusions: The greater sensitivity and excellent specificity of second-generation recombinant antigen-based assays for detection of NMO-IgG in a clinical setting should enable earlier diagnosis of NMO spectrum disorders and prompt initiation of disease-appropriate therapies.

Figure 1. Distribution of neuromyelitis optica (NMO)/aquaporin-4 (AQP4)-immunoglobulin G (IgG) titers by 6 assays in serum samples of patients and controls

Scatterplots of the 6 assays with cutoffs shown as dotted lines. The ELISA-R has 2 cutoffs, the recommended cutoff (5 U/mL) and a modified cutoff (1.6 U/mL). CBA = cell-based assay; DC = disease control; FACS = fluorescence-activated cell sorting; FIPA = fluorescence immunoprecipitation assay; FU = fluorescence units; HC = healthy controls; IIF = indirect immunofluorescence; M = Mayo Clinic; ΔMGFI = difference in median green fluorescence intensity; NMOSD = neuromyelitis optica spectrum disorder; O = Oxford.

Figure 2. Concordance of assays in neuromyelitis optica (NMO) and NMO spectrum disorder (NMOSD) cohort

Positive samples are marked + with an orange background. Negative samples are marked – with a white background. Longitudinally extensive transverse myelitis (LETM, TM) samples are highlighted in blue; recurrent optic neuritis (rON, ON) in pink. (B) The results from the fluorescence immunoprecipitation assays (FIPAs), performed in both centers on 9 samples from a single patient plotted against time, demonstrate the consistency of this simple assay to monitor serial samples. AUC = area under the curve; CBA = cell-based assay; E = EUROIMMUN; FACS = fluorescence-activated cell sorting; FU = fluorescence units; IF = immunofluorescence; M = Mayo Clinic; O = Oxford; ROC = receiver operating characteristic curve.