Interference of anti-nuclear antibodies on determination of anti-neutrophil cytoplasmic antibodies in patients suspected of vasculitis

Abstract

Anti-neutrophil cytoplasmic antibodies (ANCA) are mainly associated with medium and small vessel vasculitis. Two main methodologies currently available for detection of these antibodies are indirect immunofluorescence (IIF) and monospecific proteinase 3 (PR3) and myeloperoxidase (MPO) based immunoassays. However, well-defined guidelines regarding mode of testing for ANCA in laboratories still don’t exist, leading to problems in diagnosis and further patient management. Anti-neutrophil cytoplasmic antibodies testing by IIF and enzyme linked immunosorbent assay (ELISA) often pose a significant challenge in diseases other than vasculitis and in overlapping autoimmune conditions. Anti-neutrophil cytoplasmic antibodies reporting by IIF can be challenging in certain circumstances. This case series aims to discuss four cases with probable interference of anti-nuclear antibodies (ANA) during ANCA testing by IIF resulting in ANCA false positivity. All four cases on subsequent reflex testing by line immunoassay (LIA) for PR3, MPO and glomerular basement membrane (GBM) antigens proved otherwise. While analysing for the presence of ANCA by IIF, the possible interference of ANA leading to a false positive ANCA result should be kept in mind and alternative methods of testing like ELISA, extended granulocyte based IIF assays with MPO and PR3 coated beads, etc., should also be advised. Probability of atypical ANCA in diseases other than vasculitis should also be considered in case of ambiguous results.

Effect of antinuclear antibody positivity on antineutrophil cytoplasmic antibody results by indirect immunofluorescence assay

“…Detection of ANCA is important in many inflammatory diseases that are based on autoimmune mechanisms. [12,13] Indirect immunofluorescence, utilizing ethanol-fixed neutrophils as a substrate, is still widely utilized as the primary screening technique for ANCA detection in many clinical laboratories. [14] In line with the 1999 International Consensus on ANCA testing, the practice was to utilize IIFA for ANCA screening, followed by immunoassays for PR3 in cases of positive ANCA results.…”

“…The distinction between a true p-ANCA and ANA is crucial, as these autoantibodies are associated with different diseases, each having distinct pathogenic mechanisms, clinical presentations, and treatment strategies. [12]…”