Detection Of Antineutrophil Cytoplasmic Antibodies Research Articles

Anti-neutrophil cytoplasmic antibodies and their clinical significance

Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies that cause systemic vascular inflammation by binding to target antigens of neutrophils. These autoantibodies can be found in serum from patients with systemic small-vessel vasculitis and they are considered as a biomarker for ANCA-associated vasculitis (AAV). A conventional screening test to detect ANCA in the serum is indirect immunofluorescence study, and subsequently confirmed by enzyme-linked immunosorbent assay. A positive staining of ANCA can be classified into three main categories based on the staining patterns: cytoplasmic, perinuclear, and atypical. Patients with granulomatosis with polyangiitis (GPA) mostly have a positive cytoplasmic staining pattern (c-ANCA) whilst a perinuclear pattern (p-ANCA) is more common in microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA) patients. Atypical pattern (a-ANCA) is rarely seen in patients with systemic small-vessel vasculitis but it can be found in other conditions. Here, techniques for ANCA detection, ANCA staining patterns and their clinical significances are reviewed.

Positivity of antineutrophil cytoplasmic antibodies in children: prevalence and etiologies

In adults, anti-neutrophil cytoplasmic antibodies (ANCA) are considered as serological markers of several diseases, especially vasculitis and glomerulonephritis. Since ANCA are rarely positive in children, few data about the clinical relevance of these auto-antibodies in pediatric population have been reported. Therefore, our study aims to describe the spectrum of disorders associated with positive ANCA in Tunisian children. This study had been carried out over a period of 12 years and a half. All patients under the age of 15 for whom ANCA screening was performed in our laboratory were included. Clinical data were collected retrospectively. Indirect immunofluorescence (IFI) technique for ANCA detection was performed using PNN smears fixed with ethanol, formalin and, if necessary, methanol. Positive results were tested using immunodot to characterize the antigenic targets (myeloperoxydase (MPO) and proteinase 3 (PR3)). Our results showed that 410/5,990 (6.8%) laboratory requests for ANCA screening were for children. Forty (9.7%) requests were positive (24 children). Clinical data were available for 19 patients only. Sex-ratio (F/M) was 1.25. The mean age was 9 years and a half (3-15 years). The most frequent IIF patterns were x-ANCA (n=12) and p-ANCA (n=7). In our patients, the most frequent conditions associated to ANCA were treatment with benzylthiouracil for hypothyroidism (n=6), inflammatory bowel disease (n=4) and hemolytic anemia (n=4). In conclusion, the positivity of ANCA in children seems to be a rare event. Associated conditions include clinical disorders specific to the pediatric population. Treatment with benzylthiouracil is an etiology to be taken into consideration.

ANCA-ASSOCIATED VASCULITIS: HETEROGENEITY OF CLINICAL MANIFESTATIONS, PROGNOSIS, CURRENT OPPORTUNITIES OF PHARMACOTHERAPY

The article highlights the new views on classification and nomenclature of systemic vasculitis, the meaning of detection of antineutrophil cytoplasmic antibodies (ANCA) in the diagnosis and prognosis of the disease. The literature data and own research regarding clinical manifestations of ANCA-associated vasculitis are presented. Analyzing the first clinical manifestations in 41 patients with granulomatosis with polyangiitis, we identified four variants of the debut: with involvement of ENT organs (n ​=21), with lung lesions without involvement of the upper respiratory tract (n=8), with skin lesions, joint involvement and fever (n=7) and other variants (n=5). Fever (76%), involvement of ENT organs (51%), skin (41%), nervous system (39%) and arthritis/arthralgia (37%) were the most frequent first manifestations of granulomatosis with polyangiitis. In comparison with the presenting features there were more often lesions of the skin (66% vs. 41%), nervous system (51% vs. 39%), kidneys (41% vs. 10%), lungs (63% vs. 30%), eyes (32% vs. 10%) and myalgia (34% vs. 12%) throughout course of disease. Among the lesions of the ENT organs, sinusitis (n=19), rhinitis (n=8) and otitis (n=6) were predominate, laryngotracheitis, sublottic stenosis, saddle-shaped deformation of the nose, destruction of the walls of the paranasal sinuses and mastoiditis were diagnosed less frequently. The latest clinical recommendations for treating patients with ANCA-associated vasculitis, including the use of immunobiological therapy are presented, as well as describing of the results of our own experience in the using of immunobiological therapy in patients with granulomatosis with polyangiitis.

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.

A novel strategy with combined assays for detection of anti-neutrophil cytoplasmic antibody (ANCA) in clinically ANCA-negative granulomatosis with polyangiitis patients

ObjectiveGranulomatosis with polyangiitis (GPA) that is localized to the upper airway presents a diagnostic challenge because of a tendency towards anti-neutrophil cytoplasmic antibody (ANCA)-negativity. The purpose of this study was to investigate whether positivity of ANCA detection might be elicited with combined use of enzyme-linked immunosorbent assay (ELISA) kits. MethodsTwenty-nine serum samples obtained from GPA patients were used in this study. In addition to routine biochemical investigation for ANCA, tests for detecting PR3-, MPO-ANCAs, and minor ANCAs were performed with commercially available ELISA kits. Cytoplasmic (C)-ANCA and perinuclear (P)-ANCA were evaluated using the indirect immunofluorescence (IIF) technique. ResultsTwelve patients were positive for PR3- or MPO-ANCA in the clinical laboratory test, and 17 patients were negative for both ANCAs. Of the 17 ANCA-negative patients, four were positive for PR3- or MPO-ANCA, and three were positive for minor ANCA according to results obtained from six different ELISA kits. These findings indicated that performing detection tests with six different ELISA kits might improve the positivity of ANCA and might contribute to establishing the diagnosis of ANCA-associated vasculitis. Together with results from IIF, the samples of eight patients with clinically ANCA-negative results (8/17, 47.1%) were converted to ANCA-positive results, and the ANCA detection rate was significantly improved from 12/29 (41.4%) to 20/29 (69.0%, p=0.03). ConclusionsAdditional detection techniques should be used to confirm the results of clinically ANCA-negative samples, particularly when vasculitis is suspected. Minor ANCAs should also be evaluated with detection tests when PR3- and MPO-ANCA are negative.

Use of a Granulocyte Immunofluorescence Assay Designed for Humans for Detection of Antineutrophil Cytoplasmic Antibodies in Dogs with Chronic Enteropathies.

BackgroundPerinuclear antineutrophil cytoplasmic antibodies (pANCA) previously have been shown to be serum markers in dogs with chronic enteropathies, with dogs that have food‐responsive disease (FRD) having higher frequencies of seropositivity than dogs with steroid‐responsive disease (SRD). The indirect immunofluorescence (IIF) assay used in previous publications is time‐consuming to perform, with low interobserver agreement.Hypothesis/ObjectivesWe hypothesized that a commercially available granulocyte IIF assay designed for humans could be used to detect perinuclear antineutrophil cytoplasmic antibodies in dogs.AnimalsForty‐four dogs with FRD, 20 dogs with SRD, 20 control dogs, and 38 soft‐coated wheaten terrier (SCWT) or SCWT‐cross dogs.MethodsA granulocyte assay designed for humans was used to detect pANCA, cANCA, and antinuclear antibodies (ANA), as well as antibodies against proteinase‐3 protein (PR‐3) and myeloperoxidase protein (MPO) in archived serum samples.ResultsSensitivity of the granulocyte assay to predict FRD in dogs was 0.61 (95% confidence interval (CI), 0.45, 0.75), and specificity was 1.00 (95% CI, 0.91, 1.00). A significant association was identified between positive pANCA or cANCA result and diagnosis of FRD (P < 0.0001). Agreement between the two assays to detect ANCA in the same serum samples from SCWT with protein‐losing enteropathy/protein‐losing nephropathy (PLE/PLN) was substantial (kappa, 0.77; 95% CI, 0.53, 1.00). Eight ANCA‐positive cases were positive for MPO or PR‐3 antibodies.Conclusions and Clinical ImportanceThe granulocyte immunofluorescence assay used in our pilot study was easy and quick to perform. Agreement with the previously published method was good.

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.

Development of an indirect immunofluorescence based assay for diagnosis of ulcerative colitis in Indian population

The prevalence of Ulcerative Colitis (UC), once thought to be negligible, has increases exponentially in the Indian population. The development of novel, cost effective and time efficient Indirect Immunofluorescence (IIF) based assay for detection of anti-neutrophil cytoplasmic antibodies (ANCA) and diagnosis of UC in the Indian population is discussed. A novel IIF based assay was developed using intact nuclei from human neutrophils to detect atypical p-ANCA in patients suffering from UC. Sera from 45 patients diagnosed with UC, 45 healthy controls and one related disease control were tested using a novel UC-ANCA assay and validated by commercially available ANCA IIF assay. Prevalence of ANCA amongst UC patients in the Indian population was determined. Atypical p-ANCA was detected in 86.6% of the patients using the UC-ANCA assay as compared to 71.1% using the commercial ANCA assay. The validation of UC-ANCA assay with a commercially available ANCA IIF assay resulted in higher sensitivity. The UC-ANCA assay proved to be not only enhanced in terms of performance but also comparatively economical and rapid. The novel UC-ANCA assay may prove to be very useful in identification and differentiation of UC patients from typical ANCA positive subjects suffering from other autoimmune diseases at one tenth the cost of clinically available ANCA IIF tests which will immensely benefit the cost constrained diagnostic field of developing countries.

Simultaneous comprehensive multiplex autoantibody analysis for rapidly progressive glomerulonephritis.

Rapidly progressive glomerulonephritis (RPGN) is mainly caused by anti-glomerular basement membrane (GBM) antibody-mediated glomerulonephritis, immune-complex or anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides and leads to rapid loss of renal function. Detection of ANCA and autoantibodies (autoAbs) to GBM and dsDNA enables early diagnosis and appropriate treatment of RPGN aiding in preventing end-stage renal disease.Determination of ANCA on neutrophils (ANCA) as well as autoAbs to myeloperoxidase (MPO-ANCA), proteinase 3 (PR3-ANCA), GBM, and dsDNA was performed by the novel multiplex CytoBead technology combining cell- and microbead-based autoAb analyses by automated indirect immunofluorescence (IIF). Forty patients with granulomatosis with polyangiitis (GPA), 48 with microscopic polyangiitis (MPA), 2 with eosinophilic GPA, 42 with systemic lupus erythematosus (SLE), 43 with Goodpasture syndrome (GPS), 57 with infectious diseases (INF), and 55 healthy subjects (HS) were analyzed and findings compared with classical single testing.The CytoBead assay revealed for GPA, MPA, GPS, and SLE the following diagnostic sensitivities and for HS and INF the corresponding specificities: PR3-ANCA, 85.0% and 100.0%; MPO-ANCA, 77.1% and 99.1%; anti-GBM autoAb, 88.4% and 96.4%; anti-dsDNA autoAb, 83.3% and 97.3%; ANCA, 91.1% and 99.1%, respectively. Agreement with classical enzyme-linked immunosorbent assay and IIF was very good for anti-GBM autoAb, MPO-ANCA, PR3-ANCA, and ANCA, respectively. Anti-dsDNA autoAb comparative analysis demonstrated fair agreement only and a significant difference (P = 0.0001).The CytoBead technology provides a unique multiplex reaction environment for simultaneous RPGN-specific autoAb testing. CytoBead RPGN assay is a promising alternative to time-consuming single parameter analysis and, thus, is well suited for emergency situations.

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...

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.

A historical essay on detection of anti-neutrophil cytoplasmic antibodies

In this essay we describe a number of the known and not so known experiences of the early anti-neutrophil cytoplasmic antibodies (ANCAs) days, explaining why and how we reached consensus on the standard indirect immunofluorescence (IIF) techniques, the naming of the two principal C- and P-ANCA patterns, why we chose to use IIF as the standard technique, how the solid phase assays have developed and where we stand today, the use of ANCA for diagnosis and the importance of using several techniques for that purpose, how ANCA titres are related to disease activity and the clinical impact of this, and finally the implications of ANCA being a natural, polyclonal antibody response against various epitopes in relation to diagnostics and disease patterns.

Current and emerging techniques for ANCA detection in vasculitis.

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.

Clinically-Irrelevant Positivity for Serum Proteinase 3-ANCA in HIV Disease

Diagnosis of small-vessel vasculitides (granulomatosis with polyangiitis, microscopic polyangiitis, Churg-Strauss syndrome, and their localized forms) is aided by the detection of serum antineutrophil cytoplasmic antibodies (ANCA). However, serum ANCA positivity does not always mean vasculitis. Here, we report on a 61-year-old female patient with very high serum levels of proteinase 3 ANCA, marked hypergammaglobulinemia, low complement levels, and a 16-mm lung nodule on chest CT scan, who was referred to our Institution with a provisional diagnosis of possible granulomatosis with polyangiitis. On admission, history-taking disclosed two recent episodes of viral reactivation (namely, cytomegalovirus and Varicella-Zoster virus), while physical examination revealed lingual and nail involvement suggestive of Candida infection. An immunodeficiency disorder was eventually suspected. Search for antibodies against human immunodeficiency virus (HIV) 1 and 2 turned positive. Western blot analysis confirmed HIV infection. Thus, although ANCA detection may be helpful in diagnosing small-vessel vasculitis in the appropriate clinical scenario, screening for HIV infection should sometimes be considered to discriminate clinically irrelevant serum ANCA positivity.

THU0418 Automated interpretation of ANCA patterns – a new approach in the serology of ANCA associated vasculitis

BackgroundIndirect immunofluorescence (IIF), utilizing ethanol-fixed neutrophils as substrate, is still the method of choice for detecting antineutrophil cytoplasmic antibodies (ANCA) in vasculitis. The conventional immunofluorescence microscopy for detection of ANCA...

Study about correlation of anti-neutrophil cytoplasmic antibodies and anticardiolipin antibodies with thromboangiitis obliterans

Doctors often have difficulties in clinical diagnosis and clinical stage of thromboangiitis obliterans (TAO). Immunolesion was important in the initiation and progression of various kinds of vasculitis diseases, including TAO. Several kinds of immune complexes were developed by immunolesion, including anti-neutrophil cytoplasmic antibodies (ANCA) and anticardiolipin antibodies (ACA). Our aim was to determine if it is an effective way for clinical diagnosis and clinical stage of TAO by detection of the presence of ANCA and ACA in blood serum of patients with TAO and the relationship among the presence of ANCA, ACA and patients with different grades of TAO. Blood samples and clinical characteristics were collected from 38 patients with Rutherford grade I TAO, 30 patients with Rutherford grade II–III TAO, 75 patients with arteriosclerosis obliterans (ASO) and 65 healthy volunteers. Their serum samples were investigated for ANCA by indirect immunofluorescent (IIF), and for ACA and ANCA specificity antigens including reactivity to proteinase 3(PR3), myeloperoxidase (MPO), cathepsin G (CG), bactericidal/permesbility-increasing protein (BPI), elastase (HLE) and lactoferrin (LF) by enzyme linked immunosorbent assay (ELISA). (1) ANCA positive rate and titre were much higher in cases with Rutherford grade I TAO (52.6%, 20/38, 0.386 ± 0.458) and Rutherford grade II–III TAO (73.3%, 22/30, 0.847 ± 0.658) than those in cases with ASO (4%, 3/75, 0.011 ± 0.002) and healthy volunteers (0%,0/65, 0.010 ± 0.002) (P < 0.01). ANCA positive rate and titre were higher in cases with Rutherford grade II–III TAO (73.3%, 22/30, 0.847 ± 0.658) than those in cases with Rutherford grade I TAO (52.6%, 20/38, 0.386 ± 0.458) (P < 0.05). (2) ACA concentration was much higher in cases with Rutherford grade I TAO (270.13 ± 13.05 IU/mL) and Rutherford grade II–III TAO (279.33 ± 19.98 IU/mL) than that in cases with ASO (236.85 ± 17.32 IU/mL) and healthy volunteers (229.16 ± 15.55 IU/mL) (P < 0.05) respectively. (3) In 42 cases of ANCA-positive samples, there were 20 cases reacted with MPO, 14 cases reacted with LF, five cases reacted with HLE, five cases reacted with BPI and no one reacted with PR3 and CG. All cases were Rutherford grade II–III TAO. Our results indicate that ANCA, ANCA specificity antigens and ACA were detected susceptibly and availably in patients with TAO. Thus, detection of ANCA, ANCA specificity antigens and ACA was helpful for clinical diagnosis of TAO and detection of ANCA and ANCA specificity antigens was helpful for clinical staging of TAO. They are important assistance for clinical diagnosis and stage of TAO.

Clinical significance of detection of anti-neutrophil cytoplasmic antibodies in patients with autoimmune hepatitis

Clinical significance of detection of anti-neutrophil cytoplasmic antibodies in patients with autoimmune hepatitis

Antineutrophil Cytoplasmic Autoantibodies: How Are They Detected and What Is Their Use for Diagnosis, Classification and Follow-up?

Antineutrophil cytoplasmic antibodies (ANCA) are traditionally detected by an indirect immunofluorescence technique. According to the international consensus on ANCA testing, ANCA should also be tested by antigen-specific tests for myeloperoxidase-ANCA and proteinase 3-ANCA. The direct noncompetitive enzyme-linked immunosorbent assay (ELISA) used to be the method of choice. Nowadays, these assays are called "first-generation" assays. Second-generation tests (capture ELISA) or third-generation tests (anchor ELISA) are more sensitive and specific for ANCA testing. We postulate that ANCA as detected by these newer ANCA tests may replace the need to perform indirect immunofluorescence-based assays. For classification of patients, ANCA serotype seems more important than classifying patients according to their clinical subtype, since genetics, clinical manifestations and response to therapy are more related to ANCA serotype than to clinical subtype. Detection of ANCA to monitor disease activity is still a controversial issue. Treatment based on ANCA levels is at present only experimentally performed in those patients who are treated with B-cell depletion therapy with rituximab. Future studies are needed to establish whether this way of monitoring patients is warranted.

Chromogranin A — A comparison of serum and plasma results from different ELISA kits

Antineutrophil cytoplasmic antibodies (ANCA) are used as diagnostic markers for systemic vasculitis. However,the specificity and sensitivity of ANCA detection differs from centre to centre due in large part to variations in methodology. We compared 8 commercial ELISA kits and an in-house method (HM) for their specificity and sensitivity in detecting ANCA against proteinase 3 (PR3-ANCA, 7 kits) and meyloperoxidse (MPO-ANCA, 8 kits). Sera from 5 patients with systemic lupus erythematosus (SLE), 28 with Wegener's granulomatosis (WG), 22 with microscopic polyangiitis (MPA), 5 with idiopathic rapidly progressive glomerulonephritis (RPGN), and 5 healthy controls were examined by both the indirect immunofluorescence technique (IFT) and the ELISA kits. Sera from healthy controls and patients with SLE or cANCA-negative WG were shown to be PR3-ANCA negative by all 7 PR3-ANCA kits. In 25 cANCA-positive sera from WG patients, PR3-ANCA positivity ranged from 44% to 84%. An absolute concordance among the 7 kits was noted in 56% of the cANCA-positive samples. The PR3-ANCA levels in 5 of the 7 kits correlated with the cANCA titers in IFT. Sera from the healthy controls and 4 out of the 5 SLE and pANCA-negative patients were found to be MPO-ANCA negative in all 8 MPO-ANCA kits. In 20 pANCA-positive sera, MPO-ANCA positivity ranged from 25% to 75%. Thirty-five percent of MPO-ANCA-positive sera were confirmed by capture ELISA, immunoblot and inhibition assay. The concordance rate was only 30% among pANCA-positive sera in the 8 MPO-ANCA kits. No significant correlation was observed between pANCA titers and MPO-ANCA levels. The HM showed that 65% of cANCA-positive sera were PR3-ANCA positive, and 45% of pANCA-positive sera were MPO-ANCA positive. Our results indicate that the sensitivities and specificities for ANCA detection differ significantly among the commercial kits tested and underline the necessity of establishing uniform international standards for ANCA ELISA procedures in order to permit more reliable interpretation and comparison of data.