Ann Lab Med.  2012 Sep;32(5):331-338. 10.3343/alm.2012.32.5.331.

Clinical Usefulness of Cell-based Indirect Immunofluorescence Assay for the Detection of Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder

• Kang ES
• Min JH
• Lee KH
• Kim BJ

• KMID: 1387347
• DOI: http://doi.org/10.3343/alm.2012.32.5.331

Abstract

BACKGROUND
The presence of antibodies to aquaporin-4 (AQP4) has been identified as a key characteristic of neuromyelitis optica spectrum disorder (NMOSD), an autoimmune inflammatory demyelinating central nervous system (CNS) disorder. We evaluated the performance of a cell-based indirect immunofluorescence assay (CIIFA) for detecting AQP4 antibodies using antigen prepared with a recombinant AQP4 peptide transfection technique and assessed the usefulness of CIIFA for diagnosis of NMOSD in routine clinical practice.
METHODS
Forty-six serum samples from 36 patients as a comparison set and another 101 patients enrolled consecutively from a neurology clinic were included. CIIFA and fluorescence immunoprecipitation assays (FIPA) were performed. CIIFA was performed at 2 different institutions for comparison purposes.
RESULTS
CIIFA and FIPA sensitivity in the comparison set was 86% and 79% in neuromyelitis optica (NMO) patients and 55% and 36% in high-risk NMO patients, respectively. The semiquantitative titer measured by CIIFA correlated well with the arbitrary unit (fluorescence units [FU]) derived from FIPA (r=0.66). Titers measured by CIIFA and FIPA were elevated in NMO patients compared to high-risk NMO patients (1:240 vs. 1:180 and 8,390 vs. 4,059 FU, respectively). The frequency of AQP4 antibody detection by CIIFA in 101 consecutively enrolled patients was 100% in NMO and 23% in high-risk NMO patients, while only 4.6% in control patients, including those with multiple sclerosis.
CONCLUSIONS
Detection of AQP4 antibodies by CIIFA provides sensitive and highly specific diagnostic information for NMO and high-risk NMO patients, which can be used to differentiate these conditions from other demyelinating CNS diseases.

Keyword

Neuromyelitis optica; Aquaporin 4; Indirect immunofluorescence assay; Immunoprecipitation assay

MeSH Terms

Adult
Aged
Antibodies/*blood
Aquaporin 4/*immunology
Female
*Fluorescent Antibody Technique, Indirect
Humans
Male
Middle Aged
Neuromyelitis Optica/*diagnosis
Reagent Kits, Diagnostic

Figure

• 

  Fig. 1 Cell-based indirect immunofluorescence assay (CIIFA) for aquaporin-4 (AQP4) antibody detection with AQP4-transfected HEK 293 cell line as a substrate. Representative samples showing an intensity of w+ to 4+ are provided above. W+ was assigned when the intensity of fluorescence was less than 1+. Of note, fluorescence was also observed in round cells, which might not reflect healthy, viable HEK cells during slide preparation.

• 

  Fig. 2 Distribution of AQP4 antibody values in 46 comparison samples. (A) Correlation of the results between the semiquantitative titer of the cell-based indirect immunofluorescence assay (CIIFA) and the quantitative value derived from the fluorescence immunoprecipitation assay (FIPA) based on arbitrary fluorescence units (FU). (B) Titers of AQP4 antibody measured by CIIFA in sera of patients with various diseases. Abbreviations: MS, multiple sclerosis; NMO, neuromyelitis optica; OND, other neurological diseases.

• 

  Fig. 3 The fluorescence intensity of AQP4 antibodies measured by the cell-based indirect immunofluorescence assay (CIIFA) in sera of 101 patients with various diseases. There were only 3 AQP4 antibody-positive patients (*) among non-NMO and non-high-risk NMO patients. Weak positive intensity was not considered to be a significant positive result. Abbreviations: ADEM, acute disseminating encephalomyelitis; AND, autoimmune neurologic diseases; CIS, clinical isolated syndrome; LETM, longitudinal extensive transverse myelitis; MS, multiple sclerosis; NMO, neuromyelitis optica; OND, other neurological diseases; RON, recurrent optic neuritis.

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