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Evaluation of a Multiparametric Immunofluorescence Assay for Standardization of Neuromyelitis Optica Serology

Evaluation of a Multiparametric Immunofluorescence Assay for Standardization of Neuromyelitis Optica Serology

Letizia Granieri,#1,2,* Fabiana Marnetto,#1,2 Paola Valentino,1,2 Jessica Frau,3 Agata Katia Patanella,4 Petra Nytrova,5 Patrizia Sola,6 Marco Capobianco,1 Sven Jarius,7 and Antonio Bertolotto1,2

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Abstract.

Background

Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the central nervous system, which predominantly affects the optic nerves and spinal cord. In a majority of cases, NMO is associated with antibodies to aquaporin-4 (AQP4) (termed NMO-IgG).

Aims

In this study, we evaluated a new multiparametric indirect immunofluorescence (IIF) assay for NMO serology.

Methods

Sera from 20 patients with NMO, 41 patients with multiple sclerosis (MS), 30 healthy subjects, and a commercial anti-AQP4 IgG antibody were tested in a commercial composite immunofluorescence assay (“Neurology Mosaic 17”; Euroimmun, Germany), consisting of five different diagnostic substrates (HEK cells transfected with AQP4, non-transfected HEK cells, primate cerebellum, cerebrum, and optic nerve tissue sections).

Results

We identified AQP4 specific and non-specific fluorescence staining patterns and established positivity criteria. Based on these criteria, this kit yielded a high sensitivity (95%) and specificity (100%) for NMO and had a significant positive and negative likelihood ratio (LR+=?, LR?=0.05). Moreover, a 100% inter- and intra-laboratory reproducibility was found.

Conclusions

The biochip mosaic assay tested in this study is a powerful tool for NMO serology, fast to perform, highly sensitive and specific for NMO, reproducible, and suitable for inter-laboratory standardization as required for multi-centre clinical trials.

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Introduction.

Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the central nervous system, which mainly affects the optic nerves and spinal cord [1], [2]. In the majority of cases, NMO is associated with autoantibodies to the water channel aquaporin-4 (AQP4) (termed NMO-IgG) [3], [4]. Anti-AQP4 antibodies have also been found in patients with isolated longitudinally extensive transverse myelitis and in patients with isolated optic neuritis, conditions which are considered limited or inaugural forms of NMO [5]–[7]. In addition, anti-AQP4 antibodies have been found in a subset of patients with connective tissue disorders (CTD) such as lupus erythematosus (SLE), Sjogren’s syndrome and co-existing NMO spectrum disorders (NMOSD) [8]–[10].

Since the discovery of anti-AQP4 antibodies, several assays for the detection of NMO-IgG have been developed [11]. However, most of these assays are available only at few specialized laboratories. Moreover, most of them lack independent standardization and validation, and no generally accepted gold standard assay exists.

The present study aimed to evaluate a new commercially available multiparametric indirect immunofluorescence (IIF) assay in distinguishing NMO from MS patients. This assay consists of an array of five different diagnostic substrates including HEK cells transfected with AQP4, non-transfected HEK cells, and three monkey tissue sections (cerebellum, cerebrum, and optic nerve). The assay was evaluated through the following steps: 1. Characterization of distinct immunofluorescence staining patterns. 2. Correlation between staining patterns and the patients’ clinical diagnoses. 3. Evaluation of the diagnostic sensitivity, specificity, and clinical utility (as assessed by calculation of likelihood ratios) of each pattern. 4. Analysis of the assay’s inter- and intra-laboratory reproducibility.

Our results show that this IIF assay has high sensitivity and specificity and represents a powerful tool for NMO serology, permitting the identification of different AQP4 specific and non-specific patterns. Moreover this assay is fast to perform, highly reproducible and suitable for inter-laboratory standardization.

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