Evaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA)

doi:10.1016/j.autrev.2016.03.010

Autoimmunity Reviews

Volume 15, Issue 7, July 2016, Pages 736-741

https://doi.org/10.1016/j.autrev.2016.03.010 Get rights and content

Abstract

Objectives

The aim of this multicenter EUVAS study was to evaluate the diagnostic performance of multi-parametric indirect immunofluorescence (IIF) assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).

Patients and methods

The study included 912 samples from diseased controls and 249 diagnostic samples from GPA (n = 183) and MPA (n = 66) patients. The performance of two automated multi-parametric assays [Aklides (Medipan/Generic Assays) and EuroPattern (Euroimmun)] combining IIF on cellular and purified antigen substrates was compared with two manual IIF analyses and with commercially available ELISAs for MPO- and PR3-ANCA (Euroimmun).

Results

The area under the curve (AUC) of the receiver operating characteristics (ROC) curve to discriminate AAV from controls was 0.925, 0.848, 0.855 and 0.904 for, respectively, the two manual analyses, Aklides and EuroPattern, and 0.959, 0.921 and 0.886 for, respectively, antigen-specific ELISA, antigen-coated beads, and microdot, respectively. Variation in pattern assignment between IIF methods was observed.

Conclusion

The performance of IIF depends on the substrate used and the definition of IIF patterns. The performance of automated IIF is improved by multi-parameter testing (combined IIF and antigen-specific testing). Given the variability between IIF methods, the diagnostic importance of this technique is questioned.

Introduction

Anti-neutrophil cytoplasmic antibodies (ANCA) are recognized as important biomarkers for the diagnosis of ANCA-associated vasculitis (AAV), in particular, granulomatosis with polyangiitis (GPA) and microscopic polyangiitis [1], [2], [3]. According to the 1999 international consensus, indirect immunofluorescence (IIF) is the optimal assay for ANCA screening; if positive, reflex testing for both proteinase (PR)3- and myeloperoxidase (MPO)-ANCA is mandatory [4]. The methods for detection and clinical utility of ANCA are extensively reviewed [1], [2], [3]. The classical two-tier approach, i.e. IIF followed by antigen-specific immuno-assays, can be combined in a single multi-parametric IIF assay. The EuroPLUS™ ANCA biochip mosaic combines both ethanol- and formalin-fixed neutrophil substrates with PR3 and MPO antigen microdots in a single assay [5]. The Medipan Cytobead IIF assay combines ethanol-fixed neutrophil substrates with beads that have been differentially coated with PR3 and MPO [6]. Importantly, both multi-parametric IIF assays can be analyzed by automated pattern recognition software, the EuroPattern and Aklides, respectively [6], [7].

In the recently performed multicenter EUVAS study, we evaluated the performance of both the ANCA biochip mosaic and the Cytobead assay, as analyzed by the respective pattern recognition software.

Section snippets

Patients

The patients included are from a multicenter EUVAS (European Vasculitis Study Group) study. Patients (n = 249) with AAV (GPA and MPA) and controls (n = 912) with inflammatory disease were recruited at Klinikum Bad Bramstedt (Germany) (190 controls, 38 GPA, 26 MPA), Statens Serum Institute Copenhagen (Denmark) (237 controls, 38 GPA, 9 MPA), University Hospitals Leuven (Belgium) (235 controls, 39 GPA, 15 MPA) and Maastricht University Medical Center (The Netherlands) (250 controls, 68 GPA, 16 MPA).

Results

Table 1 gives an overview of (i) ANCA by manual [Bad Bramstedt and Copenhagen] and automated [Aklides (Medipan) and EuroPattern (Euroimmun)] IIF patterns and (ii) PR3-ANCA and MPO-ANCA by ELISA, antigen-coated beads (Aklides) and microdots (EuroPattern). The table shows all patterns in the way they were reported by their respective laboratories or by the respective software. For calculation of IIF performance characteristics, all reactivities were considered positive, except for EuroPattern for

Discussion

Nowadays, it is to be expected that automated ANCA IIF, which combines analysis of cellular and antigen-specific substrates, will increasingly be introduced in clinical laboratories. The benefits of these systems include a higher reproducibility and shorter hands-on time. In this EUVAS study we assessed the reliability of these multi-parametric assays by evaluation of two automated pattern recognition systems. Analysis of neutrophil substrates is combined with a bead-based assay in the Aklides

Take-home messages

•
Performance characteristics of indirect immunofluorescence for ANCA depend on the substrate used and the definition of patterns.
•
The performance of automated indirect immunofluorescence for ANCA is improved by multi-parameter testing.
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The highest likelihood ratio for ANCA-associated vasculitis was found for the combination C-ANCA/PR3-ANCA.
•
PR3- and MPO-ANCA ELISAs had the best performance characteristics for ANCA-associated vasculitis.
•
Given the variability between indirect immunofluorescence

Acknowledgments

We thank Medipan and Euroimmun for providing reagents and participating in the study.

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ReviewEvaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA)

Abstract

Objectives

Patients and methods

Results

Conclusion

Introduction

Section snippets

Patients

Results

Discussion

Take-home messages

Acknowledgments

Autoimmun Rev

J Immunol Methods

Antineutrophil cytoplasmic autoantibodies: how are they detected and what is their use for diagnosis, classification and followup?

Clin Rev Allergy Immunol

Current and emerging techniques for ANCA detection in vasculitis

Nat Rev Rheumatol

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

Am J Clin Pathol

Simultaneous automated screening and confirmatory testing for vasculitis-specific ANCA

PLoS One

EuroPattern suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics

Lupus

Review
Evaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA)