Evaluation of a multiparametric immunofluorescence assay for standardization of neuromyelitis optica serology

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.

Figure 1. Recruitment of NMO patients, MS patients and HC.

OD: Other diseases, NMO: neuromyelitis optica, MS: multiple sclerosis, HC: healthy controls.

Figure 2. Fluorescence staining patterns as observed with positive and negative controls provided by the manufacturer (column 1 and 2, respectively), an anti-AQP4 antibody positive human serum sample (column 3), and an anti-AQP4 antibody negative human control sample (column 4).

The biochip mosaic consists of 5 substrates: HEK cells transfected with full length recombinant human AQP4 (row A), non-transfected HEK cells (row B) and cryosections of primate cerebellum (row C), cerebrum (row D) and optic nerve (row E). Bound IgG was visualized using secondary antibodies labeled with FITC (green). Cell nuclei, stained with DAPI, are shown in blue. Magnification 40×. GL: granular layer; WM: white matter; DAPI: 4,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate.

Figure 3. “Typical” (A) and “atypical” (B) white matter fluorescence staining as observed with anti-AQP4 positive NMO (A) and anti-AQP4 negative MS (B) human serum samples, respectively.

Bound IgG was visualized using secondary antibodies labeled with FITC (green). Cell nuclei, stained with DAPI, are shown in blue. Magnification 40×. NMO: neuromyelitis optica; MS: multiple sclerosis; DAPI: 4,6-diamidino-2-phenylindole; FITC: Fluorescein isothiocyanate.

Figure 4. Differential distribution of IIF staining patterns in five diagnostic substrates following incubation with serum samples from patients with NMO or controls.

The corresponding staining patterns (A, B, C, D; typical and atypical white matter staining), as defined in the results section, are indicated, together with the final evaluation of positivity or negativity for anti-AQP4 antibodies, and the healthy volunteers' clinical status. NMO: neuromyelitis optica, MS: multiple sclerosis, HC: healthy controls, CNT 1: commercial goat polyclonal anti-human AQP4 IgG (H19, Santa Cruz Biotechnology), CNT 2: human anti-AQP4 positive serum provided by the manufacturer, CNT 3: human anti-AQP4 negative serum provided by the manufacturer, TC = transfected cells, NTC = non-transfected cells, ON = optic nerve.