Use of Immunofluorescence and Confocal Laser Scanning Microscopy in Identifying Rare Cases of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) Showing Dual Specificities to Myeloperoxidase and Proteinase3

Abstract

Anti-neutrophil cytoplasmic antibodies (ANCA) are the immunodiagnostic markers for idiopathic necrotizing crescentic glomerulonephritis affecting mainly medium to small sized blood vessels. The diagnosis of ANCA associated vasculitis (AAV) is mainly based on clinical and histopathological characteristics along with the serological evidence. Immunofluorescence microscopy (IIF) is considered as the "gold standard" for ANCA detection, and ANCA showing two major patterns ie, cytoplasmic (c-ANCA) and perinuclear (p-ANCA) react with different antigenic targets of neutrophils like Proteinase3 (PR3) and Myeloperoxidase (MPO). A third unusual and rare immunofluorescence pattern called as "X- ANCA" or atypical ANCA is also sometimes seen. The difficulty in identification of ANCA immunofluorescence patterns is mainly seen due to the rare dual patterns seen in the same sera and also the additional nuclear immunofluorescence seen due to presence of anti-nuclear antibodies. ANCA testing by immunofluorescence and Confocal Laser scanning microscopy, as well as by specific ELISAs for detection of anti-PR3 and anti-MPO antibodies have helped in improving the diagnosis. Patients having dual specificities to MPO and PR3 in a patient is a rare finding. Among 425 clinically and histopathologically proven cases of AAV, eight patients (1.9%) had dual specificities, of which five patients showed mixed immunofluorescence pattern and 3 patients showed X-ANCA pattern which was confirmed by both immunofluorescence and Confocal Laser scanning microscopy and the dual specificities to MPO and PR3 were detected by individual ELISAs.