Antineutrophil Cytoplasmic Antibodies Testing Research Articles

Determination of Subset-Restricted Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Immunofluorescence Cytochemistry.

Neutrophils have long been considered a homogeneous cell type where all circulating cells of a particular individual express the same proteins. Lately, however, this view is changing and distinct neutrophil subsets, defined by the presence or absence of different proteins, are being increasingly recognized. At least two separate protein markers, CD177 and Olfactomedin-4 (OLFM4) are known to be expressed by some, but not all, circulating neutrophils of a given individual. We recently described the existence of subset-restricted serum autoantibodies targeting OLFM4; these were discovered during clinical testing for anti-neutrophil cytoplasmic antibodies (ANCAs). ANCA testing is part of the clinical examinations routinely carried out to support diagnosis of suspected autoimmune conditions, especially vasculitis. Positive sera typically react with all neutrophils from a single donor, whereas subset-restricted ANCA sera (such as those containing anti-OLFM4 antibodies) only react with a fraction of neutrophils. Described in this chapter is an indirect immunofluorescence (IIF) approach to test human sera for the presence of subset-restricted ANCA as well as instructions for costaining experiments using sera and purified antibodies directed against established subset markers.

Detection of Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Indirect Immunofluorescence.

The eventual presence of anti-neutrophil cytoplasmic antibodies (ANCA) can initially be screened with indirect immunofluorescence (IIF). The majority of laboratories that facilitate ANCA testing use commercial kits. Although in-house assays are not encouraged in routine clinical laboratories, knowledge on the methodological aspects of the assay remains of importance. These aspects include choice of substrate, choice of fixative, staining procedure, and interpretation procedure. In this paper details on the methodology are provided and discussed in the context of the clinical application.

Pulmonary disorders that are potentially associated with anti- neutrophilic cytoplasmic antibodies: A brief review.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis frequently manifests with involvement of the lungs and kidneys, and it also may affect other anatomic sites. This article presents the patterns of pulmonary injury in which ANCA-associated vasculitis is included in the differential diagnosis, with a discussion of antineutrophil cytoplasmic antibody testing methods. The histologic features of non-vasculitic, potentially-ANCA-associated pulmonary lesions are reviewed briefly as well.

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.

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.

Borderline positive antineutrophil cytoplasmic antibodies (ANCA)-PR3/MPO detection in a large cohort tertiary center: lessons learnt from a real-life experience.

Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) are the best strategies for antineutrophil cytoplasmic antibodies (ANCA) detection. In a minority of subjects, ELISA-based ANCA testing may result in a borderline positive titre. Therefore, we assessed the clinical significance of such a result. This is a retrospective study, which included all subjects screened for ANCA subtypes (myeloperoxidase (MPO) or proteinase-3 (PR3)) with subsequent identification of borderline positive results, as determined by ELISA and retested using IIF. The demographic, clinical and laboratory data of subjects with borderline positive ANCA test results were extracted from their medical records. A total of 14,555 PR3/MPO-ANCA tests were performed with ELISA during the study period (2006-2016). Of the 14,555 PR3-ANCA antibody tests that were performed, 94 were borderline positive (titre 0.9-1.1), and of 14,555 MPO-ANCA antibody tests, 43 were borderline positive (titre 0.9-1.1). The male-to-female ratio was 1:1.08 and the mean age was 50.95±21.79 years. Four MPO-ANCA (9.30%) and 11 PR3-ANCA (11.70%) antibody borderline samples resulted positive on IIF testing. Subjects with borderline positive MPO-ANCA were found to have a poorer outcome in terms of renal failure and the requirement of dialysis. Subjects with borderline positive MPO-ANCA and positive p-ANCA (IIF) seem to have a less favorable outcome. Physicians should be aware of these findings and possibly perform a closer follow-up and routine screening for these subjects.

Carbimazole-induced agranulocytosis – A rare case report

A 30-year-old lady with hyperthyroidism for the past 1 year was initially on carbimazole 30 mg twice orally and then changed to 30 mg once daily. She presented with intermittent fever since 3 weeks, h/o oral ulcers, difficulty in swallowing, and palpitations. Routine investigations showed anemia, neutropenia, leucopenia, and C-reactive protein elevation. Peripheral smear showed normocytic normochromic anemia with leucopenia. Widal test, RA factor test, immunoglobulin M (Ig M) dengue, Ig M Lepto, antinuclear antibody screen, cytoplasmic antineutrophil cytoplasmic antibodies, and perinuclear anti-neutrophil cytoplasmic antibodies tests were negative. Viral markers are negative.

Diagnostic outcome and indications for testing in patients with positive ANCA at a Canadian tertiary care centre.

With widespread availability of anti-neutrophil cytoplasmic antibody (ANCA) testing, interpreting positive results has become increasingly challenging. Here, we conducted a retrospective study to evaluate indications for testing and diagnosis of patients with positive ANCA. Positive ANCA tests (immunofluorescence or immunoassay) performed between April 2014 and March 2015 were identified using the Ottawa Hospital (TOH) laboratory information system. TOH electronic records of subjects with positive ANCA were reviewed. 96 patients had first-time positive ANCA in the study year. The indications for testing were suspicion for: AAV in 22 patients (23%), unspecified vasculitis in 24(25%), an inflammatory condition in 46(48%), and unknown in 4(4%). Twenty-eight patients (29% of first-time positives) were diagnosed with AAV, corresponding to 16(72%), 8(33%), 4(9%), and 0 patients tested for these indications, respectively; 49(51%) of patients had other inflammatory or infectious etiologies, and non-inflammatory diagnoses accounted for the remaining 19(20%). One hundred and forty-four repeat ANCAs were performed with life-time mean of 4.4 re-tests per patient (range 0-44). Routine monitoring accounted for 86(72%) of all repeat tests. Management was changed following 34% of all re-tests performed for changed clinical status and 1% of re-tests conducted routinely. Few patients who start with low clinical suspicion for AAV and have positive ANCA are subsequently diagnosed with AAV. Serial ANCA testing is common but is not supported by clear evidence, and rarely leads to change in management. Clarification of guidelines on effective ANCA ordering may reduce hospital laboratory costs.

Angina in granulomatosis with polyangiitis: A case report.

Granulomatosis with polyangiitis (GPA) often causes small blood vessel lesions in multiple organs, with less impact on large vessels. We report a case involving not only large blood vessels, but also angina. The computed tomographic angiography of large vessels and vascular ultrasonography of the extremity also suggested great arteries of the body were involved. Further cytoplasmic antineutrophil cytoplasmic antibody tests, pathological examination and dynamic chest computed tomography scan confirmed the diagnosis of GPA. Anti-inflammatory therapy and coronary secondary prevention were given after diagnosis by pathological examination and laboratory evaluation. The symptoms of angina pectoris were relieved. This rare case report reminds us that among the well-known risk factors of atherosclerotic diseases, GPA is also an important risk factor for acute coronary syndrome. Early foresight for this rare risk factor can improve the early diagnosis and treatment of the disease, which will improve the prognosis of the patients.

An international survey on anti-neutrophil cytoplasmic antibodies (ANCA) testing in daily clinical practice.

Abstract Background: Detection of anti-neutrophil cytoplasmic antibodies (ANCA) is important for the diagnosis of the ANCA-associated vasculitides (AAV). For AAV, especially ANCA directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are most relevant. ANCA with less well-defined specificities may, however, also be detected in other inflammatory and non-inflammatory conditions. Methods: A questionnaire, initiated by the European Autoimmunity Standardisation Initiative (EASI), was used to gather information on methods and testing algorithms used for ANCA in clinical laboratories of 12 European countries (EASI survey). Results: Four hundred and twenty-nine responses were included in the EASI survey analysis which revealed differences within countries and between countries. Laboratories overall were poor in adherence to international consensus on ANCA testing. Substantial variation was observed with respect to the use of ANCA indirect immunofluorescence (IIF) in the algorithm, application of distinct methods for MPO- and PR3-ANCA, the daily availability of new ANCA results, and interpretation of test results. Conclusions: Awareness of these differences may stimulate further harmonization and standardization of ANCA testing. This may be promoted by an update of the international ANCA consensus and the introduction of international standards.

Évaluation de la pertinence des demandes d’anticorps anticytoplasme des polynucléaires neutrophiles : étude rétrospective menée au sein du centre hospitalier universitaire de Bordeaux

IntroductionDuring year 2013, 5943 tests for antineutrophil cytoplasmic antibodies (ANCA) detection were performed in Bordeaux hospital, France. This seemed disproportionate, with regard to the low prevalence of ANCA-associated vasculitis (AAV). Our purpose was to evaluate the relevance of these requests. MethodsRequests for detection of ANCA during 2013 were recorded, with their results. A sample of 501 requests was secondarily established. Relevance of requests was assessed independently by two reviewers. During year 2014, we developed strategies of information, in order to reduce the number of requests and increase their relevance. ResultsOnly 17.8 % of the 5943 requests for detection of ANCA resulted in a positive test using indirect immunofluorescence (including 10.6 % of the requests with titles above 1/50). Using Luminex©, 9.7 % of the test of detection against antimyeloperoxidase or antiproteinase 3 antibodies were positive. Within the sample of 501 patients, only 28.7 % of the requests were relevant. A percentage of 40.2 of them weren’t justified by a clinical affection typically associated with AAV. Exactly 15.9 of the requests were performed during systematic autoimmune screening. None of these requests could lead to the diagnosis of AAV. Combination of information procedures and use of a request form enabled a 19 % decrease of the number of requests. The percentage of requests without clinical justification also reduced from 40.2 % to 17.1 %. The reduction of the number of requests led to a 46,865 € saving. ConclusionThe majority of the requests for detection of ANCA was not relevant and could not lead to the diagnosis of AAV. Simple solutions enabled a partial but significant improvement of their relevance.

Discrepancies between two immunoassays for the determination of MPO and PR3 autoantibodies

BackgroundTesting for autoantibodies to myeloperoxidase (MPO) and proteinase 3 (PR3) is part of anti-neutrophil cytoplasmic antibodies (ANCA) test that aids the diagnosis of a number of autoimmune diseases including small-vessel vasculitis. We characterized the differences between two automated immunoassays at three facilities for measuring MPO- and PR3-ANCA autoantibodies. Methods117 serum samples were analyzed for MPO and PR3 autoantibodies. The INOVA QUANTA Lite® IgG assay (INOVA Diagnostics) were performed at two facilities and the Bio-Plex® 2200 Vasculitis Panel (Bio-Rad) were performed at a third reference lab. The results were compared both qualitatively (between INOVA QUANTA Lite® and Bio-Plex® methods) and quantitatively (between two sites performing INOVA QUANTA Lite® assays). ResultsComparison of the INOVA QUNATA Lite® assays at two different facilities (n=36) demonstrated high concordance (97.2% for MPO and 94.4% for PR3) and quantitative correlation (R2=0.973 for MPO and R2=0.935 for PR3). Conversely, INOVA QUNATA Lite® and Bio-Plex® methods showed poor concordance at 70.4% for MPO (n=81; 95% CI: 59.7% to 79.2%) and at 76.5% for PR3 (n=81; 95% CI: 66.2% to 84.4%). ConclusionThis study demonstrated low concordance between two methods for MPO-ANCA and PR3-ANCA measurements. Given the discrepancies, the performance of different autoantibody immunoassay methods should be taken into consideration when evaluating MPO-ANCA and PR3-ANCA results.

CS_1 Revised international consensus recommendation on testing of anti-neutrophil cytoplasm antibodies (ANCA) in small vessel vasculitis: evaluation of ANCA testing by antigen specific assays

CS_1 Revised international consensus recommendation on testing of anti-neutrophil cytoplasm antibodies (ANCA) in small vessel vasculitis: evaluation of ANCA testing by antigen specific assays

Pathology of Antineutrophil Cytoplasmic Antibody-Associated Pulmonary and Renal Disease.

-Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis commonly presents with pulmonary and renal involvement that may present diagnostic challenges. -To highlight the updates in the classification of small vessel vasculitis, present the patterns of pulmonary and renal pathology in which ANCA-associated vasculitis is included in the differential diagnosis, analyze the screening and specific antineutrophil cytoplasmic antibody testing methods in the clinical laboratory, compare and contrast the 3 major ANCA-associated vasculitis diseases, and review the pathophysiologic mechanisms of tissue injury in this setting. -Data are derived from published literature and clinical experience. -Although rare, ANCA-associated vasculitis diseases are often considered in the differential diagnosis of many pathologic patterns of pulmonary and renal disease. Histopathologic diagnosis of specific entities in this context nearly always requires correlation of the pathology with clinical and serologic data.

Incidence of anti-neutrophil cytoplasmic antibody-associated vasculitis before and after the February 2011 Christchurch Earthquake.

It has been suggested that environmental pollution from an earthquake might be associated with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). To determine the incidence of AAV during the 3-year period before (period 1), and the 3 years following (period 2), the earthquake that occurred on 22 February 2011 in Christchurch, New Zealand. All ANCA tests performed in the Canterbury region for 3 years before the earthquake (period 1, 2007-2010), and for 3 years after the earthquake (period 2, 2011-2014) were examined. AAV was defined according to The European Medicines Agency classification algorithm. Medical records were reviewed and cases were included if they were newly diagnosed within the study period. Incidence was calculated using population data from the 2013 New Zealand census. A total of 52 new cases of AAV was identified. The incidence in period 1 was 1.87/100 000/annum (95% C.I. 1.23-2.72), and for period 2 was 1.73/100 000/annum (95% C.I. 1.12-2.55). There was no statistically significant difference in incidence between the two study periods. There was no difference when analysing by myeloperoxidase (MPO) or proteinase-3 status, or restricting the analyses to those residing in an urban environment. The mean age at diagnosis for MPO AAV was significantly younger in period 2 than period 1 (61 years vs 71 years, P = 0.05). There were no other clinically important differences between the two groups. This study does not support the hypothesis that an environmental agent, caused by dust pollution related to earthquake damage, has a causative role in the pathogenesis of AAV.

Detection of antineutrophil cytoplasmic antibodies (ANCAs): a multicentre European Vasculitis Study Group (EUVAS) evaluation of the value of indirect immunofluorescence (IIF) versus antigen-specific immunoassays

ObjectiveThis 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...

The clinical presentation and therapy of diseases related to anti-neutrophil cytoplasmic antibodies (ANCA).

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies that react with proteins predominantly expressed in cytoplasmic granules of polymorphonuclear neutrophil granulocytes (PMNs). ANCA was initially detected using indirect immunofluorescence, allowing for different patterns such as p-ANCA (perinuclear) and c-ANCA (cytoplasmic) to be distinguished. Today it is common to detect the antibodies by immunochemical assays such as ELISA using purified proteins as antigens. The strongest association with ANCA is found in the pauci-immune small vessel vasculitides granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). There is compelling evidence that ANCA contributes to the pathogenesis in these conditions. ANCA also occurs in 30%-40% of patients with eosinophilic granulomatosis with polyangiitis (EGPA) and anti-GBM disease, but is uncommon in other forms of vasculitis. ANCA with different specificities have been described with varying frequencies in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, inflammatory bowel disease, endocarditis, chronic infections and hematopoietic malignancies. ANCA can also develop as an adverse event during pharmacological treatment. These entities are treated quite differently, with therapies ranging from immunosuppressive agents over antibiotics to simply removing the causative drug. A positive ANCA test thus requires a careful diagnostic work-up.

Antineutrophil cytoplasmic antibody (ANCA) testing: Audit from a clinical immunology laboratory.

Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with small vessel vasculitis now termed 'ANCA associated vasculitis' (AAV). ANCAs are reported in diverse diseases where they have no clinical utility. We carried out an audit in a clinical immunology laboratory and assessed if use of ordering practices could have improved utility of ANCA. All samples received for ANCA testing during 2014 were tested by indirect immunofluorescence (IIF) and automated enzyme-linked immunosorbent assay (ELISA). Clinical records of all samples positive by one or more assays were retrieved. We assessed the effect of applying proposed test ordering guidelines on performance of the tests. Of 1590 samples, 108 (6.8%) had a positive result by at least one method. IIF showed perinuclear pattern in 72 (21 were antinuclear antibody positive), cytoplasmic in 22, six had atypical pattern and eight were negative. By ELISA anti-myeloperoxidase antibodies were present in 33 samples, anti-proteinase 3 in 24, while five sera had both antibodies. ELISA and IIF were concordant in 45 samples. Twenty-seven patients had AAV of which 23 were both ELISA and IIF positive. Among these 27 with AAV all had at least one ordering criteria, while in 81 patients without AAV but with positive test, 38 had no ordering criteria. Reduction in false positive can be achieved by considering only those samples as ANCA positive that test positive both on IIF and ELISA and by following ordering guidelines before requesting ANCA testing, and by use of ordering criteria by clinicians.

Variability in Testing for Antineutrophil Cytoplasmic Antibodies: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

-Variability in testing for antineutrophil cytoplasmic antibodies (ANCAs) contributes to confusion and controversy related to testing for vasculitis and other ANCA-associated diseases. -To survey laboratory testing practices regarding ANCA testing and to investigate differences in testing algorithms. -Supplemental questions were sent to the 333 laboratories participating in the College of American Pathologists proficiency testing program for ANCA as part of the Special Immunology S2 Survey. -A total of 315 laboratories submitted responses to the supplemental questions. Only 88 of 315 participants (28%) reported using a combination of indirect immunofluorescence (IFA) and enzyme immunoassay (EIA) techniques as recommended by current guidelines, with a few additional labs using IFA and multiplex bead assay as an acceptable alternative to EIA. Other labs reported using only IFA, EIA, or multiplex bead assays. -A wide variety of testing algorithms are in use for ANCA testing despite evidence to suggest that a combination of IFA and EIA testing provides the most comprehensive information. Laboratories should inform clinicians clearly about testing practices and utility of testing in specific disease states.

Diagnostic ANCA algorithms in daily clinical practice: evidence, experience, and effectiveness.

Detection of antineutrophil cytoplasmic antibodies (ANCA) for ANCA-associated vasculitides (AAV) is based on indirect immunofluorescence (IIF) on ethanol-fixed neutrophils and reactivity toward myeloperoxidase (MPO) and proteinase 3 (PR3). According to the international consensus for ANCA testing, presence of ANCA should at least be screened for by IIF and, if positive, followed by antigen-specific immunoassays. Optimally, all samples are analyzed by both IIF and quantitative antigen-specific immunoassays. Since the establishment of this consensus many new technologies have become available and this has challenged the positioning of IIF in the testing algorithm for AAV. In the current paper, we summarize the novelties in ANCA diagnostics and discuss the possible implications of these developments for the different ANCA algorithms that are currently applied in routine diagnostic laboratories. Possible consequences of replacing ANCA assays by novel methods are illustrated by our data obtained in daily clinical practice. Eventually, it is questioned if there is a need to change the consensus, and if so, whether IIF can be discarded completely, or be used as a confirmation assay instead of a screening assay. Both alternative options require that ANCA requests for AAV can be separated from ANCA requests for gastrointestinal autoimmune diseases.