May ELISA tests replace IIF in the diagnostic work-up of patients with suspected ANCA-associated vasculitis? The answer is clearly yes according to recently published results of the European Vasculitis Study Group.1
The authors performed a large multicenter study to evaluate the diagnostic accuracy of a comprehensive selection of immunoassay technologies that are available for detection of MPO-ANCA and PR3-ANCA. They compared the results obtained with these methods to two established IIF strategies: one based only on ethanol-fixed neutrophils, and the other based on the combination of ethanol-fixed neutrophils, formalin-fixed neutrophils and HEp-2 cells.
Diagnostic samples from 251 patients with two types of ANCA-associated vasculitis (AAV) were included in the analysis: 186 patients with granulomatosis with polyangiitis (GPA) and 65 with microscopic polyangiitis (MPA). In addition, 924 samples from patients with a wide spectrum of systemic rheumatic diseases, organ-specific autoimmune diseases drug-induced vasculitis, inflammatory and allergic diseases, infections, malignancies, and chronic fatigue syndrome served as controls.
According to the study authors their research will impact the future diagnostic approch to AAV:
“The data set obtained should enable to draw a firm conclusion about the role of ANCA IIF in the diagnostic work-up of AAV and is intended to be the basis of a novel international consensus on ANCA testing.”
Patients and controls were recruited at four different sites across Europe: Clinical Center Bad Bramstedt in Germany, Statens Serum Institute Copenhagen, Denmark, University Hospitals Leuven, Belgium and Maastricht University Medical Center, The Netherlands. The patient collective included 186 patients with GPA and 65 patients with MPA. Their diagnosis was based on the American College of Rheumatology classification criteria and the Chapel Hill Consensus Definitions. 2,3
Patients with suspected inflammatory bowel disease (IBD) were excluded, because several ANCA specifities that contribute to the differential diagnosis of IBD may be detected by IIF in these patients.
The IIF analyses were performed at two sites, in Bad Bramstedt and in Copenhagen. In Bad Bramstedt they used the combination approach with tests on ethanol-fixed neutrophils, formalin-fixed neutrophils and HEp-2 cells. In Copenhagen the single step procedure on ethanol-fixed eutrophils was established.
Seven commercial manufacturers of antigen-specific ANCA immunoassays participated in the study. They performed blinded analyses of the samples in their own laboratories and used the methods depicted in the table below:
Study resultsThe study revealed marked differences between IIF results obtained in the two laboratories and showed that most of the immunoassays outperformed IIF for ANCA determination.94 % and 78 % of the control population tested negative with IIF in Bad Bramstedt and Copenhagen respectively. 94 %-98 % of the controls tested negative with the different immunoassays, showing their higher specificity compared to IIF. The calculated overall accuracy of the single ANCA specifities ranged from 0,954 to 0.922, compared to 0.794 and 0.927 for the IIF tests.A small fraction of AAV patients (11-17 %) was ANCA negative in all assays. These patients were likely to present with localized disease.There were no significant differences between the individual immunoassay formats: the concordance between the assays ranged from 0.89-0.96 for anti-PR3, and from 0.77-0.96 for anti-MPO. Only one MPO test performed less well than all the other MPO immunoassays.In contrast to the consistency of the various immunoassay technologies, the corresponding IIF results showed high variability and significant differences of performance. Even in comparison to top-class laboratories with experienced and skilled personnel, the diagnostic performance of specific immunoassays equaled or surpassed the diagnostic performance of IFT.This evaluation demonstrates, that screening for ANCA with IIF has no further benefit to using high quality antigen specific immunoassays when AAV is suspected.The authors’ conclusion:
“Today, mounting evidence suggests that AAV should be classified based on the ANCA serotype as PR3-ANCA and MPO-ANCA disease. Recent studies have shown that PR3-ANCA and MPO-ANCA diseases are strongly associated with distinguishable genetic risk alleles, phenotypic differences and differences in risk of relapse. Therefore, these data suggest that perhaps a new classification is required centred around the antigen specificity of ANCA and strongly argue for the ANCA screening only by antigen-specific assays.”
- Damoiseaux J, Csernok E, Rasmussen N, et al. 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. Annals of the Rheumatic Diseases 2016.
- Jennette JC, Falk RJ, Bacon PA, et al. 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum 2013; 65(1): 1-11.
- Leavitt RY, Fauci AS, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of Wegener’s granulomatosis. Arthritis Rheum 1990; 33(8): 1101-7.