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An international consensus on antineutrophil cytoplasm antibodies (ANCA) testing in eosinophilic granulomatosiswith polyangiitis (EGPA) is presented.ANCA, specific formyeloperoxidase (MPO), can be detected in 30-35% of patients with EGPA. MPO-ANCA should be tested with antigen-specific immunoassays in any patient with eosinophilic asthma and clinical features suggesting EGPA, including constitutional symptoms; purpura; polyneuropathy; unexplained heart, gastrointestinal, or kidney disease; and/or pulmonary infiltrates or hemorrhage.Apositive MPO-ANCA result contributes to the diagnostic workup for EGPA. Patients with MPO-ANCA-associated EGPA have vasculitis features, such as glomerulonephritis, neuropathy, and skin manifestations, more frequently than patients with ANCA-negative EGPA. However, the presence of MPO-ANCA is neither sensitive nor specific enough to identifywhether a patient should be subclassified as having "vasculitic"or "eosinophilic"EGPA. At present, ANCA status cannot guide treatment decisions, that is,whether cyclophosphamide, rituximab, ormepolizumab should be added to conventional glucocorticoid treatment. In EGPA, monitoring of ANCA is only useful when MPO-ANCA was tested positive at disease onset. © 2020 by the American Thoracic Society.

This document follows up on a 2017 revised international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in granulomatosis with polyangiitis and microscopic polyangiitis and focuses on the clinical and diagnostic value of ANCA detection in patients with connective tissue diseases, idiopathic interstitial pneumonia, autoimmune liver diseases, inflammatory bowel diseases, anti-glomerular basement membrane (GBM) disease, infections, malignancy, and during drug treatment. Current evidence suggests that in certain settings beyond systemic vasculitis, ANCA may have clinical, pathogenic and/or diagnostic relevance. Antigen-specific ANCA targeting proteinase-3 and myeloperoxidase should be tested by solid phase immunoassays in any patient with clinical features suggesting ANCA-associated vasculitis and in all patients with anti-GBM disease, idiopathic interstitial pneumonia, and infective endocarditis associated with nephritis, whereas in patients with other aforementioned disorders routine ANCA testing is not recommended. Among patients with autoimmune liver diseases or inflammatory bowel diseases, ANCA testing may be justified in patients with suspected autoimmune hepatitis type 1 who do not have conventional autoantibodies or in case of diagnostic uncertainty to discriminate ulcerative colitis from Crohn's disease. In these cases, ANCA should be tested by indirect immunofluorescence as the target antigens are not yet well characterized. Many questions concerning the optimal use of ANCA testing in patients without ANCA-associated vasculitis remain to be answered. © 2020 Elsevier B.V.

Anti-neutrophil cytoplasmic antibodies (ANCAs) are valuable laboratory markers used for the diagnosis of well-defined types of small-vessel vasculitis, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). According to the 1999 international consensus on ANCA testing, indirect immunofluorescence (IIF) should be used to screen for ANCAs, and samples containing ANCAs should then be tested by immunoassays for proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. The distinction between PR3-ANCAs and MPO-ANCAs has important clinical and pathogenic implications. As dependable immunoassays for PR3-ANCAs and MPO-ANCAs have become broadly available, there is increasing international agreement that high-quality immunoassays are the preferred screening method for the diagnosis of ANCA-associated vasculitis. The present Consensus Statement proposes that high-quality immunoassays can be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the categorical need for IIF, and presents and discusses evidence to support this recommendation.

Objective: To evaluate the performance characteristics of direct and capture ELISA for the detection of PR3-ANCA in Wegener's granulomatosis (WG) in international ANCA reference laboratories. Methods: Serum samples were derived from patients with histological and clinical diagnosis of WG (n = 60), rheumatoid arthritis (RA) (n = 30) and healthy controls (n = 30). Each of them was tested for the presence of ANCA by indirect immunofluorescence technique (IFT), direct and capture ELISA in six international reference laboratories (Massachusetts General Hospital, Boston; Wieslab AB, Lund; University of Maastricht; University Hospital Groningen; Mayo Clinic, Rochester; Rheumaklinik Bad Bramstedt/University of Schleswig-Holstein Campus Lubeck). Each centre tested the sera according to their house protocols of IFT and ELISA. The diagnostic performance of each test was estimated by receiver operating characteristic curve analysis and sensitivity and specificity in detection of ANCA/PR3-ANCA were calculated for the respective methods. Results: In patients histologically and clinically known as WG, the detection of ANCA by IFT varied between 52 and 83% among the participating centres. PR3-ANCA positivity with the different ELISAs ranged from 53 to 80% in direct ELISA and from 72 to 76% in capture ELISA. While most capture ELISAs successfully detected PR3-ANCA, there were significant differences between IFT and direct ELISA results between laboratories. ROC curve analysis demonstrated that in five of six laboratories the overall diagnostic performance of capture ELISA was superior to IFT and direct ELISA, respectively. Conclusion: Capture ELISA is a highly sensitive assay for detection of PR3-ANCA in WG and should be used in conjunction with compatible clinical picture and histological evidence.

Proteinase 3 was identified as the target antigen of anticytoplasmic antibodies (ACPA/c-ANCA) in Wegener's granulomatosis (WG). Using the murine monoclonal antibody (MAb) WGM2, the authors localized proteinase 3 on normal and pathologic tissue and in various human cell lines. Proteinase 3 was found in all granulocytes, mast cells, and a subset of monocytes (2-10% of peripheral blood monocytes). No crossreactivity of the MAb with other cells or tissue components was observed. Comparing various vasculitic and granulomatous tissues, WG granulomas contained more granulocytes. In tissue of WG, no crossreactivity of the MAb with tissue components such as blood vessel wall or endothelium was seen. The monocytic line THP-1 and the promyelocytic line HL 60 contained proteinase 3. Since proteinase 3 was only found in neutrophil granulocytes, mast cells, and a subset of monocytes, the presented data provide further evidence that granulocytes play a central role in the pathogenesis of WG.

To investigate the distribution of proteinase 3, the target antigen of anti-cytoplasmic antibodies (ACPA or C-ANCA), within the organelles of resting normal human polymorphonuclear leukocytes and monocytes, the authors used immunocytochemical techniques on thin frozen sections. To obtain valuable tools for immunolabeling, two murine monoclonal antibodies (MAbs) directed against the ACPA antigen were produced. In neutrophils, the authors observed immunogold label for the ACPA antigen, predominantly in myeloperoxidase-positive azurophil granules, and in smaller amounts on the plasma membrane. In monocytes, the ACPA antigen could be detected in small granules, which occasionally also contained myeloperoxidase, and rare labeling was found on the monocyte membrane. The finding that the ACPA antigen is expressed on the plasma membrane of neutrophils and monocytes, thereby becoming accessible to circulating autoantibodies, supports the supposition that ACPA are not only markers of disease activity, but also are involved in the pathogenesis of Wegener's granulomatosis.

This document follows up on a 2017 revised international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in granulomatosis with polyangiitis and microscopic polyangiitis and focuses on the clinical and diagnostic value of ANCA detection in patients with connective tissue diseases, idiopathic interstitial pneumonia, autoimmune liver diseases, inflammatory bowel diseases, anti-glomerular basement membrane (GBM) disease, infections, malignancy, and during drug treatment. Current evidence suggests that in certain settings beyond systemic vasculitis, ANCA may have clinical, pathogenic and/or diagnostic relevance. Antigen-specific ANCA targeting proteinase-3 and myeloperoxidase should be tested by solid phase immunoassays in any patient with clinical features suggesting ANCA-associated vasculitis and in all patients with anti-GBM disease, idiopathic interstitial pneumonia, and infective endocarditis associated with nephritis, whereas in patients with other aforementioned disorders routine ANCA testing is not recommended. Among patients with autoimmune liver diseases or inflammatory bowel diseases, ANCA testing may be justified in patients with suspected autoimmune hepatitis type 1 who do not have conventional autoantibodies or in case of diagnostic uncertainty to discriminate ulcerative colitis from Crohn's disease. In these cases, ANCA should be tested by indirect immunofluorescence as the target antigens are not yet well characterized. Many questions concerning the optimal use of ANCA testing in patients without ANCA-associated vasculitis remain to be answered.

Antineutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3-ANCA) are valuable serological markers for entities termed "ANCA-associated vasculitides" (AAV: granulomatosis with polyangiitis "Wegener" (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, formerly known as Churg Strauss syndrome) and, in particular, for GPA. Consensus guidelines currently recommend performing indirect immunofluorescence (IIF) testing together with an enzyme-linked immunosorbent assay (ELISA) (or other immunometric assays) to detect PR3-ANCA. Generally, antibodies to PR3 give a cytoplasmic staining (C-ANCA) on ethanol-fixed neutrophils and the specific antigen is usually detected by different ELISAs (. direct, capture, and anchor). The prevalence of PR3-ANCA is between 60% and 97% in GPA, and is much lower in MPA and EGPA. However, PR3-ANCA have also been observed in other disorders, such as ulcerative colitis, infectious disorders (i.e., subacute bacterial endocarditis), drug-induced syndrome (i.e., cocaine-induced midline destructive lesions), and other disorders not associated with vascular inflammation. In general, PR3-ANCA levels are useful to monitor disease activity but should not be used by themselves to guide treatments. A genome-wide association study (GWAS) in AAV confirms a genetic component to AAV pathogenesis, demonstrates genetic distinctions between GPA and MPA that are associated with ANCA specificity, and suggests that the response against the autoantigen PR3 is a central pathogenic feature of PR3-ANCA-associated vasculitis. © 2014 Elsevier B.V. All rights reserved.