A Comparative Evaluation of Antigen-Specific Sandwich Immunoassay and Indirect Immunofluorescence Assay (IIF) in Detecting Antineutrophil Cytoplasmic Antibodies: Are We Ready to Replace IIF with ELISA as the Primary Screening Method?

Abstract

Background Antineutrophil cytoplasmic antibodies (ANCA) are important biomarkers in the diagnosis of ANCA-associated vasculitis, and indirect immunofluorescence (IIF) had been the method of choice for its detection from the very beginning. However, international consensus on ANCA testing (2017) advocates the use of high-quality immunoassays as the primary screening method. The purpose of this study was to evaluate the diagnostic performance of enzyme-linked immunosorbent assay (ELISA) compared to IIF in detecting ANCA. Methods One-hundred eighty-nine serum samples of suspected or known cases of systemic vasculitis were screened for ANCA by IIF and proteinase-3- and myeloperoxidase-ELISA. In IIF, positive results were further divided into cytoplasmic pattern of ANCA and perinuclear pattern of ANCA, depending upon the pattern of fluorescence. McNemar’s chi-squared test was applied to check the equality of proportions of positive results, and Kappa statistics was used to measure the agreement between the two methods. Diagnostic performance of ELISA was evaluated taking IIF as reference. Results IIF detected ANCA in 17.5% cases and ELISA detected it in 11.6% cases. A good agreement between the overall performance of ELISA and IIF was observed (K-value: 6.8, p-value: < 001). However, a significant difference in the proportion of positive results by the two methods was observed in McNemar’s test (two-sided p-value: 0.007). Taking IIF as standard, ELISA showed 60.6% sensitivity, 98.7% specificity, and predictive value of positive and negative results of 90.9 and 92.2%, respectively. Conclusion The new generation antigen-specific ELISAs had high specificity but the chances of missing cases in primary screening due to the low sensitivity and high false negativity (39.4%) need to be dealt with.

Interference of Antinuclear Antibody (ANA) in Indirect Immunofluorescence Assay (IIFA)-Based Perinuclear Antineutrophil Cytoplasmic Antibody (pANCA) Interpretation

“…All described procedures were part of our lab's routine testing flow, and data were analyzed in a retrospective manner [1].…”

“…Testing for ANCA in our lab: sera were subjected to IIFA-based ANCA testing (IIFT : Granulocyte Mosaic 13, Euroimmun, Lübeck, Germany, Catalog# FA1201-1005-13) and monospecific Sandwich third-generation ELISAs for anti-PR3 (anti-PR3-hn-hr-ELISA(IgG), Euroimmun, Lübeck, Germany, Catalog# EA1201-9601-2G) and anti-MPO (anti MPO-ELISA-IgG, Euroimmun, Lübeck, Germany, Catalog# EA1211-9601-G) to detect the presence of ANCA. [ 1 ].…”

“…The patient sera were diluted in 1/10 proportion, added to the substrate wells, and then incubated. In case sera containing ANCA (IgG, IgM, and IgA) were attached to antigens on fixed neutrophils [ 1 ]. FITC (green) labeled antihuman antibodies were allowed to react and observed under the fluorescence microscope.…”

“…In contrast, a smooth fluorescence wrapped around the granulocyte's cell nuclei was recorded as pANCA (perinuclear pattern). ELISA kits had reagent wells coated with a mixture of purified recombinant PR3 and native PR3 (in anti-PR3-hn-hrELISA, IgG) as well as purified-MPO (in anti-MPO-ELISA, IgG) antigens in two respective kits [ 1 ]. Prediluted samples were added to the wells.…”

“…Antineutrophil cytoplasmic antibodies (ANCAs) are crucial in diagnosis and pathogenesis of a group of conditions called ANCA-associated vasculitis [ 1 , 2 ]. ANCA-associated vasculitis is a rare disease associated with necrotizing inflammation of small/medium-sized blood vessels with and without granuloma formation in affected organs [ 2 , 3 ].…”