Determination of anti-neutrophil cytoplasmic antibodies in small vessel vasculitis: Comparative analysis of different strategies

doi:10.1016/j.cca.2008.07.026

Clinica Chimica Acta

Volume 397, Issues 1–2, November 2008, Pages 77-81

https://doi.org/10.1016/j.cca.2008.07.026 Get rights and content

Abstract

Background

Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with primary small vessel vasculitis (SVV). Proteinase-3 (PR3)-ANCA are primarily associated with Wegener granulomatosis, whereas myeloperoxidase (MPO)-ANCA are primarily associated with microscopic polyangiitis (MPA) and vasculitic Churg–Strauss syndrome. We evaluated whether a strategy that is based on screening with ELISA or fluoroenzymeimmunoassay (FEIA) is an accurate alternative to screening with indirect immunofluorescence (IIF).

Methods

C-ANCA and P-ANCA were determined by IIF and PR3-ANCA and MPO-ANCA were determined by ELISA (Inova) or FEIA (Phadia) on 326 patients (38 with newly diagnosed SVV and 288 diseased controls).

Results

Specificity and positive likelihood ratios were higher for ELISA and FEIA than for IIF. Post-test probability for SVV of a positive test result was higher for ELISA and FEIA than for IIF. Decision tree analysis in which several testing strategies were compared revealed that a testing strategy that is based on screening with ELISA or FEIA had an expected clinical utility that was comparable to screening with IIF and confirming with ELISA or FEIA. The highest expected clinical utility was found when both IIF and ELISA or FEIA were performed on all samples.

Conclusions

A strategy based on screening for ANCA with ELISA or FEIA (without prior IIF) is a valuable alternative to screening with IIF and confirming with ELISA or FEIA.

Introduction

Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with primary small vessel vasculitides (SVV), such as Wegener granulomatosis (WG), microscopic polyangiitis (MPA), and Churg–Strauss syndrome (CSS). Specifically serine proteinase-3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA are related to the ANCA-associated vasculitides (for review, see [1]).

In an International Consensus Statement [2], [3] it was agreed that indirect immunofluorescence (IIF) using ethanol-fixed neutrophils should be the basis for detection of ANCA. IIF shows two major staining patterns: a cytoplasmic pattern (C-ANCA) and a perinuclear pattern (P-ANCA). The C-ANCA pattern shows granular cytoplasmic fluorescence with central interlobular accentuation. This pattern is usually caused by PR3-ANCA and is associated with WG. The P-ANCA pattern shows a perinuclear or a nuclear staining. This pattern is associated with MPO-ANCA, but is also seen with antibodies to other neutrophil enzymes. MPO-ANCA is predominantly seen in patients with MPA. P-ANCA staining without specificity for MPO is associated with diseases such as ulcerative colitis, sclerosing cholangitis, autoimmune hepatitis, and rheumatoid arthritis.

In a large international study including vasculitis patients and controls with inflammatory diseases it was shown that the combination of IIF with PR3 ELISA and MPO ELISA had a specificity of 99% for the diagnosis of ANCA-associated vasculitis [4]. The sensitivities for newly diagnosed WG and MPA were 73% and 67%, respectively. Consensus guidelines were subsequently published [2], [3]. It was stated that positive results obtained with the IIF method should always be substantiated by a positive antigen-specific ELISA result for PR3 or MPO to support the diagnosis of small vessel vasculitis.

Although the International Consensus Statement [2], [3] proposed to screen with IIF and to confirm positive results with ELISA, an alternative strategy that uses a sensitive ELISA at the initial stage and IIF for confirmation has also been reported to reach high diagnostic accuracy [5]. ELISA or fluoroenzymeimmunoassay (FEIA) is easily automated and, therefore, more suited as a screening tool than IIF in clinical laboratories. In the present study we used a decision tree model to compare several testing strategies (screening with ELISA versus screening with IIF) for diagnosis of SVV in a routine clinical laboratory setting.

Section snippets

Methods

ANCA was determined by IIF using 6-well ethanol-fixed human neutrophil substrate slides and reagents (anti-human IgG conjugate [goat], fluorescein labeled) from INOVA Diagnostics Inc. (San Diego) according to the instructions of the manufacturer. All samples were screened at 1:40 dilution using a Nikon Eclipse E400 fluorescence microscope. Titration was performed on positive samples (using 2-fold dilutions). PR3-ANCA and MPO-ANCA were determined by FEIA [EliA™] (Phadia, Uppsala, Sweden) and by

Descriptive analysis of the results

The study population included 23 patients with newly diagnosed WG. Of these 23 WG patients, 18 patients had C-ANCA and PR3-ANCA (with FEIA and ELISA), 3 patients had P-ANCA (one with MPO-ANCA, one with PR3-ANCA with FEIA [not with ELISA], and one without PR3 or MPO-ANCA), and 2 patients were IIF negative (one of them had PR3-ANCA with FEIA [not with ELISA]).

In addition to patients with WG, C-ANCA and PR3-ANCA (with FEIA and ELISA) were found in a patient with polyarteritis nodosa and in a

Discussion

Many clinical laboratories screen for ANCA in patients with, or suspected for, vasculitis by IIF and confirm positive results with ELISA. This conforms to international consensus guidelines [2], [3]. Screening with a sensitive ELISA and confirming with IIF have been proposed as an accurate alternative [5]. ELISA can be easily automated and, therefore, is an attractive alternative to IIF as a screening method. In the present paper we used a decision tree model to compare several testing

Acknowledgments

We thank INOVA and Phadia for providing reagents to perform this study. We thank S. Fieuws for helping with StatXact analysis.

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Anti-neutrophil cytoplasmic antibody testing by indirect immunofluorescence: Computer-aided versus conventional microscopic evaluation of routine diagnostic samples from patients with vasculitis or other inflammatory diseases
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Detection of anti-neutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence assays (IFA) is of diagnostic importance in vasculitides and some other inflammatory diseases. Automation of IFA may be beneficial in high-throughput clinical laboratories. An analytical appraisal of the EUROPattern (EPa) automated microscope and image analysis system has not been reported in a routine clinical laboratory setting testing samples from both vasculitis and non-vasculitis patients.
Results of EPa and on-screen ANCA pattern recognition of 568 consecutive routine serum samples were compared to those of conventional visual evaluation.
Agreement of discrimination between negative and non-negative samples was 86.1% comparing EPa and conventional reading, and it increased to 96.7% after on-screen user validation. Importantly, from the 334 samples classified as negative by EPa 328 (98.2%) were also negative by conventional evaluation. Pattern recognition showed ‘moderate’ agreement between classical microscopic and EPa analysis (κ = 0.446) and ‘very good’ agreement after user validation (κ = 0.900). Misclassification by EPa was dominantly due to the presence of anti-nuclear/cytoplasmic antibodies (incorrect pattern, 80/568) and the lower fluorescence cut-off of the automated microscope (false positives, 73/568).
Automated ANCA testing by EPa is a reliable alternative of classical microscopic evaluation, though classification of sera needs correction by trained personnel during on-screen validation.
Discrepancies between two immunoassays for the determination of MPO and PR3 autoantibodies
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Citation Excerpt :
Therefore, a sensitive and highly specific ANCA test is important for clinicians to classify ANCA associated diseases in order to provide optimal patient care. Although dual testing with IIF and solid-phase ELISA are currently recommended for ANCA detection, ELISA and other immunoassays offer many advantages over IIF including easy automation and lower cost [7]. Among the FDA-approved immunoassays, Bio-Plex® multiplex method and INOVA QUANTA Lite® ELISA tests are utilized in many clinical laboratories, but a head-to-head comparison of these two methods is lacking.
Testing 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.
117 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).
Comparison 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%).
This 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.
Performance of two strategies for urgent ANCA and anti-GBM analysis in vasculitis
2014, European Journal of Internal Medicine
Citation Excerpt :
In patients with AAV, these patterns are usually associated with antibodies against proteinase 3 (PR3) and myeloperoxidase (MPO) which are strongly associated with the presence of granulomatosis with poly-angiitis (GPA) or microscopic poly-angiitis (MPA) [3–5]. However, C-ANCA and P-ANCA as detected by IIF are not equivalent to the presence of anti-PR3 and anti-MPO antibodies respectively, as ANCA with different or unknown antigenetic specificities do occur in other diseases as well [2,4–8]. The diagnosis of ANCA-associated vasculitis is of course made by clinical symptoms and biopsy, so the presence of C-ANCA or P-ANCA only gives a clue to the diagnosis [5].
In anti-neutrophil cytoplasmic antibodies (ANCA) associated small vessel vasculitis (AAV), rapid testing for ANCA and anti-glomerular basement membrane (GBM) antibodies may be beneficial for therapeutic purpose.
We analysed the diagnostic performance of two rapid ANCA and anti-GBM test methods in 260 patients with suspected AAV.
Between January 2004 and November 2010, we analysed 260 samples by qualitative Dotblot (Biomedical Diagnostics); retrospective analysis followed with directly coated highly sensitive automated Phadia ELiA and ELiA anti-GBM.
Results were related to the final clinical diagnosis and compared with routine capture ELISA.
Seventy-four patients had a final diagnosis of AAV (n = 62) or anti-GBM disease (n = 12).
Both Dotblot and ELiA detected all 12 cases of anti-GBM disease; 2 false positive results were found.
Dotblot detected ANCA in 56 of 62 AAV patients (sensitivity 90%, NPV 97%), and showed 5 false positives (specificity 97%, PPV 90%). The Phadia ELiA anti-PR3^s or anti-MPO^s was positive in 57 of 62 AAV patients (sensitivity 92%, NPV 97%), and had 5 false positives (specificity 97%, PPV 88%). Routine capture ELISA was equally accurate (sensitivity 94%, specificity 97%, PPV 88%, NPV 98%).
The Dotblot and Phadia ELiA on anti-GBM, anti-PR3^s and anti-MPO^s performed excellently; results were almost identical to routine ELISA. When suspicion of AAV or anti-GBM disease is high and diagnosis is urgently needed, both tests are very powerful for rapid serological diagnosis. Further studies have to confirm the test performances in samples routinely presented for ANCA testing and in follow-up of positive patients.
Development and performance evaluation of novel chemiluminescence assays for detection of anti-PR3 and anti-MPO antibodies
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However, contradictory data has been published by different groups [12,23]. As a consequence of the high sensitivity of the novel methods, it has been proposed that screening for MPO- and PR3-ANCA could be done with sensitive immunoassays [24] such as ELISA and the results confirmed by ANCA IFA [25]. Based on our findings we recommended to test for PR3- and MPO-ANCA by IIF on human neutrophils and using a sensitive PR3- and MPO assays such as the QUANTA Flash CIA.
The detection of anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies represents a serological hallmark in the diagnosis of small vessel vasculitis such as granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). We evaluated novel chemiluminescence assays (CIAs) for PR3- and MPO-ANCA detection and investigated their utility for disease activity monitoring.
Sera collected from GPA (n = 41) and MPA (n = 30) patients were tested by QUANTA Lite® PR-3 and MPO ELISAs (INOVA Diagnostics) and by the QUANTA Flash™ PR3 and MPO CIAs (INOVA). Precision and linearity were analyzed following reference guidelines. The recently launched reference sera for PR3-and MPO-ANCA (Centers of Disease Control and prevention, CDC) were used to establish international units for the new assays. Disease activity was determined using the Birmingham Vasculitis Activity Score.
The international standards for PR3-and MPO-ANCA yielded results of 403 CU and 332 CU in the novel CIAs, respectively. The linearity analysis showed linear regression values > 0.97 with slopes between 0.96 and 1.04. Total variation obtained from the precision study showed CV% of ≤ 7.4 for PR3-ANCA and ≤ 12.8 for MPO-ANCA. Good agreement (Spearman rho ≥ 0.89) was observed between CIA and ELISA. PR3-ANCA determined by CIA, but not by ELISA, was correlated with disease activity. No correlation was found for MPO-ANCA.
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¹: These authors equally contributed.

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Determination of anti-neutrophil cytoplasmic antibodies in small vessel vasculitis: Comparative analysis of different strategies

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Methods

Descriptive analysis of the results

Discussion

Acknowledgments

Kidney Int

Kidney Int

Clin Immunol

International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)

Am J Clin Pathol

Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies

Am J Clin Pathol