---
title: "ANCA testing in a large teaching hospital: decade-long single-centre experience and strategy impli"
id: "plos-one-22-anca-testing-strategies-a-single-centre-experience-over-the-past-decade-in-a"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-22-anca-testing-strategies-a-single-centre-experience-over-the-past-decade-in-a"
content_type: "clinical_feed_article"
specialty: "General"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357495"
published_at: "2026-09-09T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# ANCA testing in a large teaching hospital: decade-long single-centre experience and strategy impli
## Provenance & Clinical Metadata
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- **Specialty:** [General](https://medichelpline.com/clinical-feed/general.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357495)
- **Published At:** 2026-09-09T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Study objective: assess how the 2017 ANCA testing consensus—developed from tertiary referral centres—performs in a large teaching hospital with lower ANCA-associated vasculitis (**AAV**) prevalence, and adapt testing strategy to secondary care needs. - Population and timeframe: retrospective analysis of all patients screened for ANCA between 2012 and 2023 at a single large teaching hospital; statistical analyses performed in **R**. Exact patient selection criteria and some clinical detail are reported in the source. - Laboratory methods: indirect immunofluorescence (**IIF**) using EUROPLUSTM Granulocyte Mosaic 25 IIF; MPO/PR3 measured using ImmunoCAP®250 antigen-specific assay. - Testing volume and positivity: 5,518 patients were tested for ANCA; 286 (5.2%) tested ANCA-positive. Among ANCA-positive patients, 63% did not have AAV. - Diagnoses among non-AAV ANCA-positives: many had other autoimmune diseases, malignancies, infections, or drug exposure that can produce ANCA positivity; vasculitis can also occur in these settings, complicating diagnosis. - Test performance overview: when used as a first-line test, IIF and ELISA showed good negative predictive values (NPV). ELISA (antigen-specific assay) had higher positive predictive value (PPV) and higher specificity than IIF as a first test. Performing a second test increased specificity. - Antibody concentrations: MPO/PR3 concentrations were generally higher in patients with AAV than in non-AAV patients, but measured concentrations showed wide ranges and overlap between groups. - Prevalence impact: the authors highlight that lower AAV prevalence in secondary care reduces PPV even for high-specificity immunoassays; a figure demonstrates PPV increases markedly with higher disease prevalence while NPV is relatively stable. - Recommendations tailored to secondary care: in this lower-prevalence setting, antigen-specific **ELISA** performs best as a first-line test both to rule out and to rule in AAV. A second test is advised when clinical suspicion remains high despite a negative first test, when antibody concentrations are low, and when there is clinical doubt despite a positive first test—to increase specificity and reduce false positives. - Conclusion: the majority of ANCA-positive tests in this setting were not caused by AAV. Tailoring the 2017 consensus by emphasizing ELISA-first testing and selective second testing can improve specificity and PPV in secondary care. - Data availability and provenance: all relevant data are reported in the paper and supporting information; laboratory platforms and consensus years (1999, 2017) are specified in the source.
## Clinical Analysis & Structured Key Points
ANCA testing strategies: A single centre experience over the past decade in a large teaching hospital in the Netherlands | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract Objectives In 2017 a new consensus for antineutrophil cytoplasmic antibody (ANCA) testing was published, based on data from tertiary referral centres for ANCA-associated vasculitis (AAV). AAV-prevalence in their tested population was relatively high. We analysed how the consensus approach performs in a large teaching hospital and tailored it to secondary care settings with lower patient prevalences in their population. Methods Patients screened for ANCA between 2012–2023 were included; clinical data were retrospectively collected. Indirect immunofluorescence (IIF) was performed using EUROPLUS TM Granulocyte Mosaic 25 IIF. MPO/PR3 was detected by ImmunoCAP®250. Statistical analyses were performed in R. Results 286/5518 (5.2%) patients tested for ANCA were positive: 63% had no AAV. Non-AAV patients often had other autoimmune diseases, malignancies, infections or used specific drugs. IIF and ELISA as first test showed good negative predictive values. ELISA as first test showed higher positive predictive value and specificity; performing a second test increased specificity. ANCA concentrations were higher in AAV than non-AAV, but ranges were wide. Conclusion The majority of ANCA-positive patients did not have AAV. In our secondary care setting, ELISA performs best as first test to rule out and to rule in AAV. A second test should be considered with high clinical suspicion and a negative first test, or with low antibody concentrations, following the consensus. Additionally, we propose a second test when there is clinical doubt and a positive first test. This increases specificity and PPV, reducing false positives, which is especially relevant in secondary care settings. Citation: Wester Trejo MAC, Kuijper TM, Waverijn GJ, Bajema IM, van den Dorpel RMA, Kok MR, et al. (2026) ANCA testing strategies: A single centre experience over the past decade in a large teaching hospital in the Netherlands. PLoS One 21(9): e0357495. https://doi.org/10.1371/journal.pone.0357495 Editor: Alessandro Granito, University Hospital of Bologna Sant’Orsola-Malpighi Polyclinic Department of Digestive System: Azienda Ospedaliero-Universitaria di Bologna Policlinico Sant’Orsola-Malpighi Dipartimento dell’apparato digerente, ITALY Received: April 10, 2026; Accepted: August 18, 2026; Published: September 9, 2026 Copyright: © 2026 Wester Trejo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting information files. Funding: M.A.C. Wester Trejo (MACWT) is employed by Pathology Laboratory, Pathan B.V., Rotterdam, the Netherlands. I.M. Bajema (IMB) is employed by BiPath, Rotterdam, the Netherlands. These funders provided support in the form of salaries for MACWT and IMB but had no additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘Author Contributions’ section. Competing interests: The authors have read the journal’s policy and declare the following competing interests. M.A.C. Wester Trejo reports travel support from Otsuka for attendance at an educational meeting, outside the submitted work. T.M. Kuijper reports participation as a statistician on the Data Safety Monitoring Board of the TORPEDO-NL study and leadership or fiduciary roles as a statistician in ZonMw committees on Drug Rediscovery and Long COVID. I.M. Bajema reports research grants from Vifor and consulting fees from Vifor and Alentis. She serves as Vice-President of EUVAS. A.E. Berden reports honoraria for presentations at the EUVAS Symposium (Graz, Austria, October 3, 2024). She serves as President of the Dutch guideline working group for ANCA-associated vasculitis, established by the Dutch Society for Rheumatology, and as a member of the Presidential Council of EUVAS. The remaining authors declare that they have no competing interests. These declarations do not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. Introduction ANCA-associated vasculitides (AAV) are systemic small-vessel vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA). Most patients have proteinase 3 (PR3)-ANCA or myeloperoxidase (MPO)-ANCA [ 1 ]. AAV is subdivided into microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) and eosinophilic granulomatosis with polyangiitis (EGPA) [ 2 ]. ANCA are not specific for AAV and found in other conditions, including other autoimmune diseases, malignancies and infections, and they can be drug-induced. Vasculitis can also occur in many of these conditions, making it difficult to distinguish them from AAV [ 3 , 4 ]. ANCA were discovered in 1959 and associated with vasculitis in 1982 [ 5 – 7 ]. Different detection methods were developed, the most important being indirect immunofluorescence (IIF) and antigen-specific enzyme-linked immunosorbent assays (ELISAs). IIF can show a cytoplasmic (c-ANCA), perinuclear (p-ANCA) or atypical (x-ANCA) pattern. PR3 antibodies are mostly linked to a c-ANCA pattern and MPO to a p-ANCA pattern. IIF can also detect antibodies binding to autoantigens other than MPO and PR3 such as human neutrophil elastase, that can be found in drug-induced AAV [ 8 ]. The first international consensus on ANCA testing was published in 1999, providing ANCA screening recommendations when AAV is suspected [ 9 ]. The primary screening method was IIF, followed by an immunoassay testing for PR3- or MPO-ANCA in IIF-positive samples. It stated that ideally, both tests should be performed. In 2016, the European Vasculitis Society (EUVAS) performed a multicentre study, evaluating the value of IIF versus antigen-specific immunoassays, showing that the diagnostic use of the latter was equal to, or better than that of IIF [ 10 ]. A revised consensus was published in 2017, proposing preferred use of antigen-specific assays for ANCA-screening over IIF. With clinical suspicion of AAV and negative test results, a second antigen-specific test or IIF can be considered. Taking antibody level into account and considering a second test with low antibody levels is also recommended [ 11 ]. The 2017 consensus approach was devised using data from tertiary referral centres, where the proportion of AAV patients in the tested population (i.e., the prevalence) is expected to be higher compared to general hospitals. This impacts directly on pre-test probability and positive predictive value of the ANCA test: these are higher in a setting with a high AAV prevalence. Fig 1 shows the general relationship between different patient prevalences in a tested population and test characteristics. Download: PNG larger image TIFF original image Fig 1. General relationship between test characteristics for settings with different prevalences of AAV patients in the tested population (2% vs 20%). (A) Relationship between sensitivity and PPV (blue) and NPV (red); (B) relationship between specificity and PPV and NPV. Solid lines represent a 2% prevalence of AAV (approximating our setting), and dotted lines represent 20% prevalence (approximating the EUVAS multicentre setting). Higher prevalence results in higher PPV. At high specificity—such as typically observed for immunoassays measuring PR3 and MPO—the impact of prevalence on PPV is pronounced: with sensitivity fixed at 86.5% and specificity at 96.9%, PPV increases from ~35% at 2% prevalence to ~88% at 20% prevalence (≈50% absolute increase). The EUVAS multicentre study (Damoiseaux et al. 2017) tested different immunoassays including the ELISA used in our centre. From their data, we derived the sensitivity (86.5%) and specificity (96.9%) for our ELISA when used as a first-line test. These are indicated by vertical lines. To the right of these lines, increasing test performance shows that in a low-prevalence setting, increasing sensitivity results in only a minimal increase in PPV (A), whereas changes in specificity have a larger effect (B). NPV varies minimally across prevalence levels (<5%). These findings illustrate that while sensitivity and specificity are intrinsic test characteristics, PPV and NPV are strongly influenced by disease prevalence. Abbreviations : AAV (ANCA-associated vasculitis), ELISA (enzyme-linked immunosorbent assay), NPV (negative predictive value), PPV (positive predictive value), prev (prevalence). https://doi.org/10.1371/journal.pone.0357495.g001 As AAV prevalence in the tested population has direct influence on the diagnostic value of an ANCA test, it is important to evaluate test strategies in settings with different – i.e., lower – prevalences. We therefore assessed the different consensus approaches in a large teaching hospital in the Southwest of the Netherlands over the past decade. Three periods were discerned: a first period following the 1999 consensus, a second period validating automated ANCA read-out and a third period following the 2017 consensus. This study evaluates if the current consensus approach performs equally well in secondary care settings, where the prevalence of AAV is lower than in the tertiary care settings that the consensus was based on. We also take into account the clinician’s objective when requesting the test: is it used for ruling in AAV or for ruling it out . Finally, we assess the significance of MPO/PR3 concentrations. Combining our findings, we provide an external validation and propose a practical refinement of the 2017 international consensus in a low-prevalence secondary care setting. Materials and methods Patients and samples All patients (n = 5518) screened for ANCA in Maasstad Hospital between January 1, 2012 and August 1, 2023 were included in this study. Only diagnostic ANCA test requests, i.e., ‘first tests,’ were used in the analysis. Data were accessed for research purposes on 24-11-2023. Data were pseudonymised where identifying information was not part of the dataset, but a key to re-identify individuals was available. The study was conducted in accordance with the ethical principles stated in the Declaration of Helsinki. Data from the electronic medical record (ChipSoft version 6.1) were stored in a SQL-database and extracted using SQL Server Management Studio (version 17.1). The diagnosis of AAV was manually checked from medical records. All patients fulfilled the ACR 2022 classification criteria for MPA, GPA or EGPA. Comorbidities were extracted using registration of ‘DBC care products’ ( Diagnose Behandel Combinatie meaning Diagnosis Treatment Combination). The following comorbidities were extracted: malignancy (carcinoma, melanoma, haematological malignancy, sarcoma, glioma), infectious disease (hepatitis B, hepatitis C, lues/syphilis, tuberculosis, HIV, infective endocarditis, other), autoimmune disease (IBD, SLE, RA, sarcoidosis, other) and drug usage (minocycline, thyroid medication, hydralazine, immune suppressants, cocaine). For malignancy, auto-immune and infectious disease, those registered within 5 years before ANCA testing were included. For drug usage, 6 months preceding testing were considered. Registration of malignancies was checked manually to extract specific malignancies for which no DBC care product was present. Because reliable registration of DBC care products is not available for SLE, the diagnosis of SLE was also checked manually. Medication prescriptions were automatically extracted to check for specific drugs. Medication was included if the prescription period overlapped with the moment of ANCA testing. ANCA detection methods and testing strategies during different periods ANCA IIF was performed using EUROPLUS TM Granulocyte Mosaic 25 immunofluorescence (Euroimmun AG, Lübeck, Germany). Evaluation was performed manually by skilled technicians using a Zeiss Axiostar plus fluorescence microscope. When ANCA IIF was positive, ELISA was performed. MPO- and PR3-antibodies were detected by ImmunoCAP250 (Thermofisher Phadia, Sweden). Results were considered positive when above the upper limit of normal (ULN) of the manufacturer (MPO-ANCA 5 U/mL, PR3-ANCA 3 U/mL). In the first period (January 1, 2012-December 5, 2020), ANCA testing was performed according to the 1999 consensus [ 9 ]: i.e., a positive IIF screening test was followed by ELISA. In the second period (December 5, 2020-November 15, 2021), manual IIF read-out was replaced by automated IIF read-out using Europattern Suite (Euroimmun AG, Lübeck, Germany). The EUROStar III Plus LED fluorescence microscope was used with EUROLabOffice 4.0 software. During this period automated IIF and ELISA were done simultaneously for all patients and positive IIF was not a requirement to perform ELISA. This corresponds with ‘the ideal approach’ of the 1999 consensus. In the third period (November 15, 2021-August 1, 2023), ANCA testing was performed according to the 2017 consensus [ 11 ]. ELISA was performed as screening test. A second test - IIF - could be ordered upon clinicians’ request. During the entire study period IIF and ELISA were performed under stable laboratory conditions using commercial tests from the same manufacturers. The only difference in the testing methods themselves is that from period 2 onwards the read-out of IIF was automated. PR3- or MPO-ANCA concentrations Using all ELISA results obtained over the periods, the distribution of PR3- or MPO-ANCA concentrations in patients with and without AAV was analysed and graphically demonstrated using violin plots. Throughout the text we have stated ‘ANCA-positivity’ when this was the final result of the test strategy that was used. When ANCA-positivity was in fact ‘MPO/PR3 positivity’ by ELISA this is stated explicitly. Statistics Baseline characteristics for AAV and non-AAV patients were described and compared using simple descriptive statistics. Continuous variables were tested using student’s t-test or Wilcoxon ranksum test if distribution was skewed. Categorical variables were tested using Pearson chi-squared test or Fisher’s exact test. Diagnostic performance measures sensitivity (Sen), specificity (Spe), positive predictive value (PPV) and negative predictive value (NPV) were calculated; 95% confidence intervals (CI) were obtained by the Wilson score method implemented in the ‘binconf’ function [ 12 ]. Positive (LR+) and negative (LR-) likelihood ratios were calculated, 95% confidence limits were calculated by the Wald/Katz-log method implemented in the ‘BinomRatioCI’ function [ 13 ]. All analyses were performed using R version 4.2.2. [ 14 ]. Results Patients 5518 patients were tested for ANCA and 286 (5.2%) were positive. Of these, 107 patients (37%) were diagnosed with AAV, meaning that 1.9% of the tested population had AAV. This is shown graphically in Fig 2 . In the different periods the prevalences were 1.9.% (period 1), 1.2% (period 2) and 2.5% (period 3). Of the 107 ANCA-positive AAV patients, 52 (48.6%) had MPO-ANCA and 55 (51.4%) PR3-ANCA. Download: PNG larger image TIFF original image Fig 2. People graph visualizing the numbers of ANCA-positive patients with and without AAV in the tested population. A total of 5518 patients were tested for ANCA, of whom 286 (5.2%) tested positive. Of those, 107 (37.4%) were diagnosed with AAV (orange) and 179 (62.6%) had a positive ANCA test without having AAV (blue). Abbreviations : AAV (ANCA-associated vasculitis), ANCA (antineutrophil cytoplasmic antibodies). https://doi.org/10.1371/journal.pone.0357495.g002 In our cohort, the majority of ANCA-positive patients (63%) did not have AAV. Table 1 summarizes clinical characteristics of ANCA-positive patients without AAV. Of those patients, 51 (28.5%) had another autoimmune disease, 30 (16.8%) a malignancy, 18 (10.1%) an infection and 60 (33.5%) used drugs associated with presence of ANCA. Infections included Hepatitis B/C, HIV, syphilis and tuberculosis. Malignancies included mainly carcinomas, but also haematological malignancies, sarcomas and melanomas. Of patients with other autoimmune diseases 22 (12.3%) had IBD, 18 (10.1%) RA and 7 (3.9%) SLE. Strikingly, five patients had a clinical diagnosis of RA before getting diagnosed with AAV. These patients all presented with arthritis years before onset of vasculitis. Two of these patients had positive RF, but all were anti-CCP antibody negative. It cannot be ruled out that arthritis was the first presenting feature of AAV in these patients. Download: PNG larger image TIFF original image Table 1. Characteristics of patients with a positive ANCA test, with and without vasculitis. https://doi.org/10.1371/journal.pone.0357495.t001 Test results over the different periods During period 1, the 1999 consensus was followed and 3846 manual IIF tests were performed. Considering only the IIF results ( Table 2 ), sensitivity was 92.0% (95%CI 83.6–96.3), specificity 96.9% (95%CI 96.3–97.4), PPV 37.3% (95%CI 30.7–44.5), NPV 99.8% (95%CI 99.6–99.89) and LR + 29.9 (95%CI 24.7–36.). IIF followed by ELISA led to an increase in PPV to 76.2% (95%CI 66.1–84.0) and LR+ to 162.6 (95%CI 104.1–254.1). Download: PNG larger image TIFF original image Table 2. ANCA test strategies in the different testing periods. https://doi.org/10.1371/journal.pone.0357495.t002 In period 2, automated IIF was validated and 366 patients were tested with both automated IIF and ELISA. Specificity and NPV were high, as in period 1. However, due to the limited number of AAV cases, confidence intervals for sensitivity, PPV and LR+ were wide ( Table 2 ). In period 3, the 2017 consensus was followed and 829 patients were tested with ELISA. No additional IIF tests were requested. Sensitivity (100%, 95%CI 0.85–1) and specificity (98.5%, 95%CI 0.97–0.99) values were high, PPV was 63.6% (95%CI 46.7–77.8) and LR + 67.3 (95%CI 38.4–118.1). Table 3 shows that, when comparing the first step in screening (IIF in period 1 and ELISA in period 3), ELISA as a first test shows a significantly higher PPV (63.6 vs 37.3) and LR+ (67.3 vs 29.9) than IIF as a first test ( p = 0.008 and p = 0.039 respectively). However, when IIF is followed by ELISA as a second test, specificity increases significantly (99.5%; p = 0.007) compared to ELISA alone. Also, a significant increase in LR+ (162.6; p = 0.047) is observed when two tests are performed. Download: PNG larger image TIFF original image Table 3. Test periods compared. https://doi.org/10.1371/journal.pone.0357495.t003 MPO- and PR3-ANCA concentrations Overall, MPO- and PR3-ANCA concentrations were higher in AAV than in MPO/PR3-positive patients without AAV ( p < 0.001). Median concentrations in AAV were 63 U/mL (IQR 25–210) for MPO-ANCA and 49 U/mL (IQR 19–129) for PR3-ANCA, compared to 9 U/mL (IQR 5.5–21) for MPO-ANCA and 8.4 U/mL (IQR 4.8–15) for PR3-ANCA in non-AAV patients ( Fig 3 ). The lowest concentration in AAV patients for MPO-ANCA was 5.3 U/mL and for PR3-ANCA 3.3 U/mL; the highest concentration for MPO-ANCA was 1553 U/mL and for PR3-ANCA 2175 U/mL. Conversely, in ANCA-positive patients without AAV, the lowest concentration was 5.1 U/mL for MPO-ANCA and 3.1 for PR3-ANCA; the highest concentration in these patients was 81 U/mL for MPO-ANCA and 98 U/mL for PR3-ANCA. Download: PNG larger image TIFF original image Fig 3. Violin plot showing MPO/PR3 concentrations for MPO/PR3-positive patients with and without ANCA-associated vasculitis. Both MPO-ANCA and PR3-ANCA concentrations were higher for patients with vasculitis
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