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The diagnosis of systemic vasculitis (SV) is a major clinical challenge due to the very different forms of presentation and requires an interdisciplinary approach. Targeted laboratory diagnostics support making the diagnosis, differential diagnosis and classification and are also a key component in the detection of active organ manifestations and treatment complications. The basic laboratory tests include the erythrocyte sedimentation rate (ESR), C‑reactive protein (CRP), blood count, serum creatinine, urinalysis, specific autoantibodies, complement, immunoglobulins, cryoglobulins and hepatitis B and C serology. Antineutrophil cytoplasmic autoantibodies (ANCA), antiglomerular basement membrane antibodies (anti-GBM antibodies) and anti-C1q antibodies are valuable laboratory markers for the diagnosis of the various forms of small vessel vasculitis. There are no specific laboratory tests for the diagnosis of medium and large vessel vasculitis. Despite advances in our understanding of the pathogenesis of vasculitis, no biomarkers have yet been identified that can be reliably used to guide treatment or that are useful in distinguishing vasculitis from other inflammatory diseases such as infections or treatment complications., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Objectives: To determine the spectrum of anti-neutrophil cytoplasmic antibody (ANCA) antigen-specificities in eosinophilic granulomatosis with polyangiitis (EGPA), an ANCA-associated vasculitis (AAV) entity., Methods: We conducted a retrospective analysis of 73 EGPA patients from three German tertiary referral centres for vasculitis. In addition to in-house ANCA testing, pentraxin 3 (PTX3)- and olfactomedin 4 (OLM4)-ANCA were determined using a prototype cell-based assay for research (EUROIMMUN, Lübeck, Germany). Patient characteristics and clinical manifestations were evaluated and compared based on ANCA status., Results: Myeloperoxidase (MPO)-ANCA positive patients (n=8; 11%) significantly more frequently displayed peripheral nervous system (PNS) and pulmonary involvement and less frequently heart involvement compared to MPO-ANCA negative patients. PTX3-ANCA positive patients (n=5; 6.8%) had a significantly higher prevalence of ear, nose and throat, pulmonary, gastrointestinal and PNS involvement, and a lower prevalence of renal and central nervous system involvement compared to PTX3-ANCA negative patients. Proteinase 3 (PR3)-ANCA and OLM4-ANCA were detected in 2 patients (2.7%) each with multiorgan involvement. One PR3-ANCA positive patient was also positive for bactericidal permeability increasing protein (BPI)-ANCA., Conclusions: In addition to MPO, the spectrum of ANCA antigen specificities includes various other target antigens such as PR3, BPI, PTX3, and OLM4, potentially segregating further EGPA subgroups. A lower prevalence of MPO-ANCA was detected in this study compared with other studies. OLM4 is reported as novel ANCA antigen-specificity in EGPA, and thus AAV.

The detection of circulating autoantibodies against a variety of structural and functional molecules present in ubiquitous or tissue-specific cells is critical to the diagnosis of many autoimmune diseases (systemic - such as systemic rheumatic diseases - and organ-specific diseases). In particular, the determination of autoantibodies is one of the classification and/or diagnostic criteria for some autoimmune diseases and has a relevant predictive value, since many autoantibodies can be detected years before the clinical manifestation of a disease.The identification of these antigen-antibody systems and the simultaneous development of laboratory methods for detecting and measuring autoantibodies are considered one of the milestones in the history of clinical immunology over the last 60 years. Many different immunoassay methods have been developed and used in laboratory practice, from the early conventional (or monoplex) analytical methods that can detect single autoantibodies to the newer multiplex platforms that can quantify dozens of molecules. Various diagnostic immunoassays commonly used in current laboratory practice for the detection of autoantibodies are presented in this review., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Objective: This study was performed to evaluate the diagnostic accuracy of novel line and dot immunoassays for detection of MPO and PR3 ANCA., Methods: Sera from 50 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 45 disease controls were tested by IIF and for the presence of PR3-ANCA and MPO-ANCA by four different line or dot immunoassays, as well as by a chemiluminescence immunoassay., Results: The area under the curve of the receiver operating characteristic curve to discriminate AAV from controls was 0.858 (95% CI 0.785-0.931) for the IIF method. For the antigen-specific immunoassays, the area under the curve varied between 0.869 (95% CI 0.797-0.941) and 0.936 (95% 0.886-0.985)., Conclusions: Our comparison of various ANCA detection methods showed a high degree of diagnostic precision for all of the PR3- and MPO-ANCA line and dot immunoassays investigated. The performance was equal to or better than the performance of IIF. These results indicate that novel line and dot immunoassays can serve as a first-line test method in patients with the suspected diagnosis of AAV., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To evaluate the autoantibody profile in eosinophilic granulomatosis and polyangiitis (EGPA) patients., Methods: 33 EGPA patients were tested for anti-neutrophil cytoplasmic antibodies (ANCA), antinuclear antibodies (ANA), rheumatoid factor (RF), anti-alpha-enolase antibodies, and anti-eosinophil peroxidase (EPO) antibodies. Granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), hypereosinophilic syndrome (HES), rheumatoid arthritis (RA), primary biliary cirrhosis (PBC) patients and healthy subjects were tested as a control group., Results: Anti-alpha-enolase antibodies were positive in 82% of EGPA patients at high titers. Although a high sensitivity was shown for an anti-alpha-enolase antibody titer above 1/100 (82%), the specificity for EGPA remained low (44%) (AUC=0.653, p=0.008). Anti-alpha-enolase antibodies predominated in males with EGPA (p=0.048) and were associated with skin involvement (p=0.040). Most of the EGPA patients positive for anti-alpha enolase antibodies (20 out of 27) had a negative indirect immunofluorescence test (IFT) for ANCA. ANCA were positive in 8 EGPA patients (24%) with a perinuclear pattern in all but one patient. The ANCA-target antigen was myeloperoxidase (MPO) and/or alpha-enolase. A usually fine-speckled ANA pattern was observed in 42% of the EGPA patients. RF was positive in 1 (6%) of the 18 EGPA patients tested. There was no association between the presence and levels of autoantibodies and EGPA disease activity. None of the patients and controls was positive for anti-EPO antibodies., Conclusions: Alpha-enolase may be a target of autoimmunity in EGPA patients and shows usually negative ANCA IFT results.

The cornerstone of the laboratory diagnostics of small vessel vasculitis is the detection of antineutrophil cytoplasmic antibodies (ANCA). The current international consensus recommendations suggest that proteinase 3 (PR3) and myeloperoxidase (MPO) ANCA immunoassays should be used as a first-line test if there is a justified suspicion of ANCA-associated vasculitis (AAV). A second method is only recommendable when the immunoassay shows a negative or borderline result. The precise identification of all patients with active AAV and avoidance of misdiagnoses due to false positive ANCA measurements is achieved when the ANCA determination is limited to defined clinical situations, which are indicative for AAV. There is increasing evidence that the specificity of ANCA to define homogeneous groups of patients could be better with respect to the prognosis than the clinical subtype.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in eosinophilic granulomatosis with polyangiitis (EGPA) is presented. ANCA, specific for myeloperoxidase (MPO), can be detected in 30-35% of EGPA patients. 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. A positive MPO-ANCA result contributes to the diagnostic work‑up for EGPA. Patients with MPO-ANCA associated EGPA have more frequently vasculitis features, such as glomerulonephritis, neuropathy, and skin manifestations than patients with ANCA negative EGPA. However, the presence of MPO-ANCA is neither sensitive nor specific enough to identify whether a patient should be subclassified as having "vasculitic" or "eosinophilic" EGPA. At present, ANCA status cannot guide treatment decisions, that is, whether cyclophosphamide, rituximab or mepolizumab should be added to conventional glucocorticoid treatment. In EGPA, monitoring of ANCA is only useful when MPO-ANCA was tested positive at disease onset.

In recent years, insight into immune pathogenesis and treatment of primary systemic vasculitides (PSV) has increased considerably, and has led to the development of many clinically relevant biomarkers. This review aims to provide an update on the main biomarkers discovered and their potential application to precision medicine in vasculitis. Genetic and molecular profiling of patients and promising biomarkers discoveries are very important for personalized medicine; however, there are very limited data in PSV. Genetic studies including mainly genome-wide association studies (GWAS) had led to important discoveries in disease pathogenesis of PSV while whole exome sequencing studies lead to discovery of monogenic vasculitides. Although there are numerous studies addressing novel biomarkers in PSV, few of these biomarkers are currently being used in routine clinical practice in the management of patients with PSV. Current studies indicate that ANCA types identify distinct prognostic subsets of ANCA vasculitis patients. Today, biomarkers-driven treatment algorithms are not available in PSV., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Considerable progress has been made in understanding the role of autoantibodies in systemic vasculitides (SV), and consequently testing for anti-neutrophil cytoplasmic antibodies (ANCA), anti-glomerular basement membrane antibodies (anti-GBM), and anti-C1q antibodies is helpful and necessary in the diagnosis, prognosis, and monitoring of small-vessel vasculitis. ANCA-directed proteinase 3 (PR3-) or myeloperoxidase (MPO-) are sensitive and specific serologic markers for ANCA-associated vasculitides (AAV), anti-GBM antibodies are highly specific for the patients with anti-GBM antibody disease (formerly Goodpasture's syndrome), and autoantibodies to C1q are characteristic of hypocomlementemic urticarial vasculitis syndrome (HUVS; anti-C1q vasculitis). The results of a current EUVAS study have led to changes in the established strategy for the ANCA testing in small-vessel vasculitis. The revised 2017 international consensus recommendations for ANCA detection support the primary use PR3- and MPO-ANCA immunoassays without the categorical need for additional indirect immunofluorescence (IIF). Interestingly, the presence of PR3- and MPO-ANCA have led to the differentiation of distinct disease phenotype of AAV: PR3-ANCA-associated vasculitis (PR3-AAV), MPO-ANCA-associated vasculitis (MPO-AAV), and ANCA-negative vasculitis. Further studies on the role of these autoantibodies are required to better categorize and manage appropriately the patients with small-vessel vasculitis and to develop more targeted therapy.

The current practice for detection of anti-neutrophil cytoplasm antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) has been screening by indirect immunofluorescence (IIF) followed by an antigen specific tests for PR3- and MPO-ANCA. However, ANCA diagnostics have undergone many technical developments that have affected the 1999 international consensus recommendations, and lead to a revision of the existing ANCA detection strategy. Recent European multicentre studies have compared the diagnostic performance of various ANCA detection methods and demonstrated that PR3- and MPO-ANCA immunoassays yielded the highest diagnostic accuracy. New guidelines for ANCA testing have been developed based on these data. According to the revised 2017 international consensus recommendations, testing for ANCA in small vessel vasculitis can be done by PR3- and MPO-ANCA immunoassays, without the categorical need for IIF. Thus, IIF can be discarded completely, or can be used as confirmation assays instead a screening test. Clearly, though, the new testing strategy for ANCA in vasculitis must identify the ANCA target antigen, as PR3- and MPO-ANCA serotype correlate well with disease expression. Furthermore, recent studies have shown that AAV can be classified based on ANCA serotype, since PR3- and MPO-ANCA- diseases are strongly associated with distinguishable genetic alleles, different clinical and histological features. ANCA presence and the antigen specificity also may have important value as a prognostic factor and may serve as a guide for immunosuppressive therapy. In the current review, we summarize the novelties in ANCA testing, present the 2017 revised international consensus on ANCA testing in vasculitis, evaluate the diagnostic significance of ANCA, and discuss the role of ANCA serotypes in the diagnostic work-up of patients with AAV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Anti-neutrophil cytoplasmic antibodies (ANCA) play a central role in the diagnostic and pathogenesis of patients with small vessel vasculitis, so called ANCA-associated vasculitis (AAV). ANCA in these diseases are almost always directed against proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA). Most laboratories worldwide use as standard the indirect immunofluorescence technique (IFT), on human neutrophils, to screen for ANCA, and then confirm positive IFT results with antigen specific immunoassyas for PR3- and MPO-ANCA. New guidelines for ANCA testing have been developed based on a recent European multicentre study, and according to the revised 2017 international consensus recommendations, testing for ANCA in small vessel vasculitis can be done by PR3- and MPO-ANCA immunoassays, without the categorical need for IIF. The clinical utility of ANCA depends on type of assay performed and the appropiate ordering of testing the right clinical setting. Accurate identification of all patients with AAV and the avoidance of misdiagnosis can be achieved using a "gating policy" based on clinical information given to the laboratory at the time of request., Competing Interests: EC has been a consultant for Inova Diagnostics and has received lecture fees from Bio-Rad, Inova Diagnostics, Medipan, Menarini, Orgentec and Thermo Fisher. None of the other authors declare a competing interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Anti-neutrophil cytoplasmic antibodies (ANCA) play a central role in the diagnosis and pathogenesis of patients with ANCA-associated vasculitis. ANCA-associated vasculitis is a rare disease characterized by necrotizing inflammation of small/medium-sized blood vessels with and without granuloma in different organs. The main syndromes are granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic GPA. ANCA in these diseases are almost always directed against proteinase 3 and myeloperoxidase. Most laboratories worldwide use as standard the indirect immunofluorescence technique to screen for ANCA and then confirm positive IFT results with antigen specific immunoassyas for PR3- and MPO-ANCA. New guidelines for ANCA testing have been developed based on a recent European multicentre study, and according to the revised 2017 international consensus recommendations, testing for ANCA in small vessel vasculitis can be done by PR3- and MPO-ANCA immunoassays, without the categorical need for IIF. The new testing strategy for ANCA in vasculitis directly identifies the ANCA target antigen and has a particular value for the AAV sub-classification. Recent studies have shown that AAV can be classified based on ANCA serotype. ANCA presence and the antigen specificity also may have important value as a prognostic factor and may serve as a guide for immunosuppressive therapy. The clinical utility of ANCA depends on the type of assay performed and the appropiate ordering of testing the right clinical setting. Accurate identification of all patients with AAV and the avoidance of misdiagnosis can be achieved using a "gating policy" based on clinical information given to the laboratory at the time of request., (© 2018 The Mediterranean Journal of Rheumatology (MJR).)

The diagnosis of systemic vasculitis is challenging. Laboratory testing may provide useful information. Routine laboratory tests include erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), blood count, serum creatinine, urinalysis, specific autoantibodies, complement, immunoglobulin, cryoglobulin, and Hepatitis B and C serology. Although ESR and CRP are often helpful for the diagnosis of vasculitis, they are nonspecific and do not help in distinguishing between vasculitis disease activity and a concomitant infection or another source of inflammation. A few autoantibodies are helpful for diagnosis, such as anti-neutrophil cytoplasmic antibodies (ANCAs) (in ANCA-associated small-vessel vasculitis), anti-glomerular basement membrane (GBM) antibodies (in anti-GBM antibody disease), and anti-C1q antibodies (in immune complex-associated small-vessel vasculitis). The 2017 revised consensus recommendations on ANCA testing state that high-quality antigen-specific immunoassays are the preferred screening methodology for the diagnosis of ANCA-associated vasculitis. ANCA subtypes (proteinase-3-ANCA and myeloperoxidase-ANCA) are associated with different epidemiological, genetic, and clinical features., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

The 2016 European Vasculitis Society (EUVAS) meeting, held in Leiden, the Netherlands, was centered around phenotypic subtyping in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). There were parallel meetings of the EUVAS petals, which here report on disease assessment; database; and long-term follow-up, registries, genetics, histology, biomarker studies, and clinical trials. Studies currently conducted will improve our ability to discriminate between different forms of vasculitis. In a project that involves the 10-year follow-up of AAV patients, we are working on retrieving data on patient and renal survival, relapse rate, the cumulative incidence of malignancies, and comorbidities. Across Europe, several vasculitis registries were developed covering over 10,000 registered patients. In the near future, these registries will facilitate clinical research in AAV on a scale hitherto unknown. Current studies on the genetic background of AAV will explore the potential prognostic significance of genetic markers and further refine genetic associations with distinct disease subsets. The histopathological classification of ANCA-associated glomerulonephritis is currently evaluated in light of data coming out of a large international validation study. In our continuous search for biomarkers to predict clinical outcome, promising new markers are important subjects of current research. Over the last 2 decades, a host of clinical trials have provided evidence for refinement of therapeutic regimens. We give an overview of clinical trials currently under development, and consider refractory vasculitis in detail. The goal of EUVAS is to stimulate ongoing research in clinical, serological, and histological management and techniques for patients with systemic vasculitis, with an outlook on the applicability for clinical trials.

Objective: The objective of this multicentre study was to improve the clinical interpretation of PR3- and MPO-ANCAs as an adjunct for the diagnosis of ANCA-associated vasculitis (AAV) by defining thresholds and test result intervals based on predefined specificities and by calculating test result interval-specific likelihood ratios (LRs)., Methods: Eight different PR3- and MPO-ANCA immunoassays from seven companies were evaluated using 251 diagnostic samples from AAV patients and 924 diseased controls., Results: Thresholds for antibody levels were determined based on predefined specificities (95, 97.5, 99 and 100%) and used to delimit test result intervals. Test result interval-specific LRs were determined. For all assays, the LR for AAV increased with increasing antibody level. For all but one immunoassay, high antibodies levels (associated with LR >55) were found in a substantial fraction (>65%) of patients. The area under the curve (AUC) of receiver operating characteristics analysis of a diagnostic approach in which positive results were confirmed by IIF or another immunoassay was not substantially higher than the AUC of performing immunoassay only. The highest AUC was found when immunoassay was combined with another immunoassay or with IIF., Conclusion: To diagnose AAV based on PR3- and MPO-ANCA, it is useful to define thresholds for antibody levels and to assign test result interval-specific LRs. Higher antibody levels are associated with a higher likelihood for disease. Such information improves clinical interpretation., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Neutrophil extracellular traps (NETs) are chromatin filaments decorated with enzymes from neutrophil cytoplasmic granules. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from neutrophil cytoplasmic granules and are biomarkers for the diagnosis of systemic vasculitides. ANCA diagnostics are based on indirect immunofluorescence (IIF) of ethanol-fixed neutrophils. IIF shows a cytoplasmic staining pattern (C-ANCA) due to autoantibodies against proteinase 3 (PR3) or a perinuclear staining pattern (P-ANCA) due to autoantibodies against myeloperoxidase (MPO). The distinct ANCA-staining patterns are an artifact of ethanol fixation. Here, we tested NETs as a substrate for the detection of ANCAs in human sera. We observed that P-ANCAs specifically stained NETs, while C-ANCAs targeted the cell bodies of netting neutrophils. The distinct ANCA-staining patterns were caused by the presence of MPO, but not PR3, in NETs. Using NETs as a substrate for IIF, we characterized ANCAs in sera of patients with ANCA-associated vasculitis (AAV). Furthermore, we inhibited serine proteases by diisopropylfluorophosphate to prevent chromatin unfolding and the release of NETs and thus generated neutrophils with MPO-positive nuclei and PR3-positive cytoplasm, which resembled the appearance of ethanol-fixed neutrophils. In conclusion, our data suggest that NETs are selectively loaded with antigens recognized by P-ANCAs, and netting neutrophils provide a physiological substrate for ANCA detection in patients with AAV.

Objective: This multicentre study was performed to evaluate the diagnostic accuracy of a wide spectrum of novel technologies nowadays available for detection of myeloperoxidase (MPO) and proteinase 3 (PR3)-antineutrophil cytoplasmic antibodies (ANCAs)., Methods: Sera (obtained at the time of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF) (at two sites) and for the presence of PR3-ANCA and MPO-ANCA by eight different immunoassays., Results: The area under the curve (AUC) of the receiver operating characteristic curve to discriminate AAV from controls was 0.923 (95% CI 0.902 to 0.944) and 0.843 (95% CI 0.814 to 0.871) for the two IIF methods. For the antigen-specific immunoassays, the AUC varied between 0.936 (95% CI 0.912 to 0.960) and 0.959 (95% CI 0.941 to 0.976), except for one immunoassay for which the AUC was 0.919 (95% CI 0.892 to 0.945)., Conclusions: Our comparison of various ANCA detection methods showed (i) large variability between the two IIF methods tested and (ii) a high diagnostic performance of PR3-ANCA and MPO-ANCA by immunoassay to discriminate AAV from disease controls. Consequently, dual IIF/antigen-specific immunoassay testing of each sample is not necessary for maximal diagnostic accuracy. These results indicate that the current international consensus on ANCA testing for AAV needs revision., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: Up to now indirect immunofluorescence (IIF) followed by an antigen-specific assay specific for proteinase 3 (PR3) or myeloperoxidase (MPO) has been the standard method for the detection of antineutrophil cytoplasmic antibodies (ANCA). The development of more sensitive and highly specific PR3-ANCA and MPO-ANCA immunoassays for the diagnosis of ANCA-associated vasculitis (AAV) has raised doubts about the two-stage diagnostic strategy currently recommended for ANCA detection., Objective: Presentation and discussion of the new international consensus recommendations on ANCA testing in AAV., Methods: This article presents the new guidelines for ANCA testing that have been developed based on the results of a recent large multicenter study by the European Vasculitis Society (EUVAS). The draft of the author committee was revised by each contributor and subsequently distributed to 12 experts on 4 continents. After further revision the final document was returned for ratification and submitted for publication., Results/conclusion: The current study results confirm the superiority of the diagnostic value of antigen-specific immunoassays compared to IIF. The current consensus recommendations support the primary use of PR3-ANCA and MPO-ANCA immunoassays for diagnostic evaluation of patients with AAV without the categorical need for additional IIF.

Objectives: The aim of this study was to analyse the role of netting neutrophils in the pathogenesis of granulomatosis with polyangiitis (GPA), especially their interplay with peripheral blood mononuclear cells (PBMCs)., Methods: The amount of cell-free DNA (cfDNA) was determined in sera from GPA patients (pairs active/inactive state of disease, n=18) and from healthy controls (HCs, n=10). Furthermore, we performed in vitro incubation experiments using PBMCs and NETs from patients and HCs for accessing the effect of NETs on PBMC behaviour. We determined proliferation of T- and B-cells (CSFE assay), B-cell maturation (CD38 staining and flow cytometry), production of IgG (ELISpot, ELISA), and secretion of the cytokines IFN-γ, IL-4, IL-10, IL-17A (ELISA)., Results: We detected a significant increase in serum cfDNA levels of GPA patients compared to HCs. The concentration of cfDNA was associated with disease activity. NETs of patients and HCs induced proliferation of CD4+ T- cells and CD19+ B-cells and maturation of B-cells. Furthermore, we detected an increase in IL-17A secretion after stimulating PBMCs with NETs. A significant difference between PBMCs from GPA patients and HCs was not detectable., Conclusions: NETs activate PBMCs of HCs and GPA patients. Our findings give supportive evidence that NETosis plays a role in the pathogenesis of GPA.

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: The aim of this multicenter EUVAS study was to evaluate the diagnostic performance of multi-parametric indirect immunofluorescence (IIF) assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA)., Patients and Methods: The study included 912 samples from diseased controls and 249 diagnostic samples from GPA (n=183) and MPA (n=66) patients. The performance of two automated multi-parametric assays [Aklides (Medipan/Generic Assays) and EuroPattern (Euroimmun)] combining IIF on cellular and purified antigen substrates was compared with two manual IIF analyses and with commercially available ELISAs for MPO- and PR3-ANCA (Euroimmun)., Results: The area under the curve (AUC) of the receiver operating characteristics (ROC) curve to discriminate AAV from controls was 0.925, 0.848, 0.855 and 0.904 for, respectively, the two manual analyses, Aklides and EuroPattern, and 0.959, 0.921 and 0.886 for, respectively, antigen-specific ELISA, antigen-coated beads, and microdot, respectively. Variation in pattern assignment between IIF methods was observed., Conclusion: The performance of IIF depends on the substrate used and the definition of IIF patterns. The performance of automated IIF is improved by multi-parameter testing (combined IIF and antigen-specific testing). Given the variability between IIF methods, the diagnostic importance of this technique is questioned., (Copyright © 2016. Published by Elsevier B.V.)

Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are a defining feature of ANCA-associated vasculitides (AAV). They play a pivotal role in disease pathophysiology and have strongly improved early diagnosis and treatment of these infrequent, but potentially fatal diseases. Neutrophils and their products are major players in initiating the autoimmune response and tissue destruction in vasculitic as well as granulomatous inflammation. This review highlights recent findings on old and novel players (ANCA, neutrophils, neutrophil extracellular traps, fibroblasts, immune cells and complement) and puts them into context with the current understanding of disease mechanisms in AAV., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.

Detection of antineutrophil cytoplasmic antibodies (ANCAs) is a well-established diagnostic test used to evaluate suspected necrotizing vasculitis of small blood vessels. Conditions associated with these antibodies, collectively referred to as ANCA-associated vasculitides, include granulomatosis with polyangiitis (formerly known as Wegener granulomatosis), microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis (formerly known as Churg-Strauss syndrome). The diagnostic utility of ANCA testing depends on the type of assay performed and on the clinical setting. Most laboratories worldwide use standard indirect immunofluorescence tests (IFT) to screen for ANCA and then confirm positive IFT results with antigen-specific tests for proteinase 3 (PR3) and myeloperoxidase (MPO). Developments such as automated image analysis of immunofluorescence patterns, so-called third-generation PR3-ANCA and MPO-ANCA ELISA, and multiplex technology have improved the detection of ANCAs. However, challenges in routine clinical practice remain, including methodological aspects of IFT performance, the diverse antigen-specific assays available, the diagnostic value of testing in clinical settings and the prognostic value of serial ANCA monitoring in the prediction of disease relapse. This Review summarizes the available data on ANCA testing, discusses the usefulness of the various ANCA assays and advises on the clinical indications for the use of ANCA testing.

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity., (© FASEB.)

Introduction: Plasma cells residing in inflamed tissues produce antibodies in chronic inflammatory and systemic autoimmune diseases. This study examined if plasma cells, located within inflamed nasal tissue in granulomatosis with polyangiitis (GPA), express features potentially associated with the autoimmune and destructive character of this disease., Methods: Ig gene mutation patterns of individual tissue-derived plasma cells from GPA (n = 5) were analyzed, by using laser-assisted microdissection followed by semi-nested polymerase chain reaction (PCR). Signs of B-lymphocyte maturation (ectopic lymphoid structures, ELS) and survival (a proliferation-inducing ligand, APRIL; B-cell maturation antigen, BCMA; transmembrane-activator and calcium modulator and cyclophilin interactor, TACI; receptor activator of nuclear factor κB ligand, RANKL) were examined in nasal tissues or serum, respectively, by using immunohistochemistry/fluorescence and enzyme-linked immunosorbent assay, ELISA., Results: Plasma-cell derived Ig genes (light- and heavy-chain pairs, n = 4; heavy chains, n = 33) resembled mutation patterns seen in other autoimmune diseases, predominantly displaying selection against replacement mutations within the framework region of Ig genes (10 of 15), which is responsible for structural integrity. Ectopic lymphoid structures were similar between GPA and a disease control (that is, unspecific chronic rhinosinusitis. However, histomorphologic features distinguishing GPA from rhinosinusitis (that is, neutrophilic microabscess and granuloma) expressed considerable amounts of membrane-associated and secreted APRIL, respectively. The latter was co-localized with CD138 and found in close proximity to cells expressing IgG, TACI, and BCMA. Interestingly, plasma cells strongly expressed receptor activator of nuclear factor κB ligand (RANKL), apart from fibroblast-like cells., Conclusions: Plasma cells within granulomatous inflammation appear to display features that might be required for autoreactivity and, possibly, RANKL-mediated destruction in GPA.

During the past 30 years, remarkable progress has been achieved in understanding the role of the antineutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) in so-called "ANCA-associated vasculitides". In the right clinical context, ANCAs are a good seromarker for these diseases, have improved nomenclature of systemic vasculitides and have contributed to new pathogenic concepts. However, problems with the clinical applications of ANCA testing in daily practice remain. They can be summarised as follows: assay standardisation and performance; the use of ANCA testing in a clinical setting with a low pretest probability; the relationship between ANCA titres and disease activity remains unclear. The solution to problems regarding the ANCA diagnosis is focussed on the fundamental methods, i.e., correct implementation of IFT and ELISA, the cautious use of commercial assays and restricting the use of the tests to clinical situations with a rather high pretest probability of AAV.

Background: We determined if PR3-ANCA is a biomarker that differentiates ulcerative colitis (UC) from Crohn's disease (CrD)., Methods: A total of 946 sera were tested, including 86 granulomatosis with polyangiitis (GPA) and 491 inflammatory bowel disease (IBD) patients (283 UC and 208 CrD), 264 pathological controls (various diseases) and 105 healthy individuals. All samples were tested for PR3-ANCA by ELISA (QUANTA Flash Lite®, INOVA Diagnostics) and chemiluminescent immunoassays (CIA QUANTA Flash PR3). Conventional anti-neutrophil cytoplasmic antibody (ANCA) indirect immunofluorescence assays (IIF) was performed with NOVA Lite™ (INOVA Diagnostics)., Results: PR3-ANCA by CIA were detected in 31.1% UC vs. 1.9% CrD sera (p=2.2E-16), and by ELISA in 6% UC and 0% CrD (p=0.0003). In GPA patients, PR3-ANCA were detected in 75.6% by CIA and 61.6% by ELISA (p<0.05). PR3-ANCA by CIA were more prevalent in E3-UC compared to E1/2-UC (p<0.05), and in patients with shorter disease duration (p<0.0001). PR3-ANCA showed similar sensitivity, but significantly higher specificity (p<0.05), compared to atypical pANCA by IIF., Conclusion: The novel PR3 CIA may prove helpful in the differentiation of CrD from UC, as well as in the identification of UC patients with more extensive disease., (© 2013 Elsevier B.V. All rights reserved.)

Granulomatosis with polyangiitis (Wegener's) (GPA) is a multisystem disease of unknown etiology, characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Antineutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3) are a defining feature of this disease. GPA usually starts as a granulomatous disease of the respiratory tract and, in the majority of patients, progresses to systemic disease with PR3-ANCA-associated vasculitis. Today, epidemiological evidence indicates that GPA develops as a result of complex gene-environment interactions. The nature of these risk factors and pathogenic mechanisms involved, however, are only just beginning to be understood. Clinical data and in vitro experimental results point to the pathogenic pathways involved in tissue lesion development, in which ANCA, cellular immunity, neutrophils extracellular traps, fibroblasts, vascular endothelial cells and inflammatory mediators play a major role. Today, the pathophysiological significance of PR3-ANCA is still unclear and the pathogenic pathways leading to granuloma formation are not explained. New data unexpectedly suggest that the destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids.

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: Indirect immunofluorescence (IIF) employing ethanol-fixed neutrophils (ethN) is still the method of choice for assessing antineutrophil cytoplasmic antibodies (ANCA) in ANCA-associated vasculitides (AAV). However, conventional fluorescence microscopy is subjective and prone to high variability. The objective of this study was to evaluate novel pattern recognition algorithms for the standardized automated interpretation of ANCA patterns., Methods: Seventy ANCA-positive samples (20 antimyeloperoxidase ANCA, 50 antiproteinase3 ANCA) and 100 controls from healthy individuals analyzed on ethN and formalin-fixed neutrophils (formN) by IIF were used as a 'training set' for the development of pattern recognition algorithms. Sera from 342 patients ('test set') with AAV and other systemic rheumatic and infectious diseases were tested for ANCA patterns using the novel pattern recognition algorithms and conventional fluorescence microscopy., Results: Interpretation software employing pattern recognition algorithms was developed enabling positive/negative discrimination and classification of cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA). Comparison of visual reading of the 'test set' samples with automated interpretation revealed Cohen's kappa (κ) values of 0.955 on ethN and 0.929 on formN for positive/negative discrimination. Analysis of the 'test set' with regard to the discrimination between C-ANCA and P-ANCA patterns showed a high agreement for ethN (κ = 0.746) and formN (κ = 0.847). There was no significant difference between visual and automated interpretation regarding positive/negative discrimination on ethN and formN, as well as ANCA pattern recognition (P > 0.05, respectively)., Conclusions: Pattern recognition algorithms can assist in the automated interpretation of ANCA IIF. Automated reading of ethN and formN IIF patterns demonstrated high consistency with visual ANCA assessment.