[Laboratory diagnostics for vasculitis beyond antineutrophil cytoplasmatic autoantibodies]. / Labordiagnostik bei Vaskulitiden jenseits von antineutrophilen zytoplasmatischen Autoantikörpern.

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), C­reactive 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.

Spectrum of ANCA-specificities in eosinophilic granulomatosis with polyangiitis. A retrospective multicentre study.

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.

[Methods of autoantibody diagnostics - when immunofluorescence test, when ELISA?] / Methoden der Autoantikörperdiagnostik ­ wann Immunfluoreszenztest, wann ELISA?

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.

Autoantibody profile in eosinophilic granulomatosis and polyangiitis: predominance of anti-alpha-enolase antibodies.

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.

Evaluation of PR3- and MPO-ANCA line and dot immunoassays in ANCA-associated vasculitis.

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.

2020 international consensus on ANCA testing beyond systemic vasculitis.

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.

International Consensus on ANCA Testing in Eosinophilic Granulomatosis with Polyangiitis.

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 work­up 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.

Usefulness of vasculitis biomarkers in the era of the personalized medicine.

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.

The Diagnostic and Clinical Utility of Autoantibodies in Systemic Vasculitis.

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.

Investigations in systemic vasculitis. The role of the laboratory.

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.

[ANCA-associated vasculitis: recent methodological advances for ANCA detection]. / Neues zur Labordiagnostik bei ANCA-assoziierten Vaskulitiden.

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.

Anti-neutrophil cytoplasm antibodies (ANCA): Recent methodological advances-Lead to new consensus recommendations for ANCA detection.

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.

New concepts in ANCA detection and disease classification in small vessel vasculitis: the role of ANCA antigen specificity.

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.

Position paper: Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis.

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.

A multicentre study to improve clinical interpretation of proteinase-3 and myeloperoxidase anti-neutrophil cytoplasmic antibodies.

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.

Neutrophil Extracellular Traps Contain Selected Antigens of Anti-Neutrophil Cytoplasmic Antibodies.

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.