A historical essay on detection of anti-neutrophil cytoplasmic antibodies

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1 Nephrol Dial Transplant (2015) 30: i8 i13 doi: /ndt/gfv070 NDT Perspectives A historical essay on detection of anti-neutrophil cytoplasmic antibodies Niels Rasmussen 1, Allan Wiik 1 and David R. Jayne 2 1 Department of Autoimmune Serology, Statens Seruminstitut, Copenhagen, Denmark and 2 Medicine, Addenbrooke s Hospital, Cambridge, UK Correspondence and offprint requests to: Niels Rasmussen; nir@ssi.dk ABSTRACT 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. Keywords: ANCA, autoantibodies, biomarkers, crescentic glomerulonephritis, vasculitis THE FIRST OBSERVATIONS The indirect immunofluorescence (IIF) technique for detection of autoantibodies to neutrophil granulocytes was initially used to identify neutrophil-specific antibodies in patients with rheumatoid arthritis and Felty ssyndrome[1]. Some of these antibodies stained the nuclei and perinuclear region of neutrophils and monocytes and were therefore designated granulocyte-specific anti-nuclear antibodies (GS-ANA), but early attempts at demonstrating their antigenic targets were unsuccessful. By use of this technique, antibodies staining cytoplasmic granules of neutrophils (anti-pmn-cytoplasm autoantibodies) in a female patient with crescentic glomerulonephritis were incidentally found in 1973, and this technique was therefore included to screen for autoantibodies in the protocols for the Cattegat Study Group of Wegener s Granulomatosis (CSGWG) in In 1982, an Australian group published a short communication, describing similar anti-neutrophil cytoplasm autoantibodies in eight patients with pauci-immune segmental necrotizing glomerulonephritis [2]. With the subsequent paper from the CSGWG [3] and the ensuing paper by the Dutch- CSGWG collaboration [4] more specifically relating the neutrophil cytoplasmic staining autoantibodies, then called anticytoplasmic antibodies (ACPAs), to Wegener s granulomatosis/ granulomatosis with polyangiitis (Wegener s) (GPA) in 1985, testing for these autoantibodies, termed cytoplasmic staining anti-neutrophil cytoplasmic antibodies (C-ANCAs) in 1989, has become an important diagnostic tool for GPA. C-ANCAs in GPA are produced as a polyclonal response to an as yet unidentified agent or disease process and may belong to any of the immunoglobulin classes, IgG, IgM and IgA, but predominantly IgG in the active disease phases [2, 3]. C-ANCAs are typically present during the initial active disease phase, disappearing during remission (Figure 1) and reappearing at relapse [2 4]. ANCA can be detected not only using neutrophils and monocytes as substrate but also using the leukemic HL-60 cell line [5, 6]. It was originally speculated that Ross River virus could have a causal role for the disease [2],butnoparticularinfectiousagent has ever been shown to cause GPA. There is no cross-reactivity with neutrophil constituents from other species like rodents [2]or more specificallyrats,mice,rabbits,cows,horses,catsorhens[5]. THE IIF ASSAYS FOR ANCA Several IIF techniques have been used for the detection of ANCA, differing in source of neutrophils, purification, application on slides and fixation method. The most prominent The Author Published by Oxford University Press Downloaded from onhttps://academic.oup.com/ndt/article-abstract/30/suppl_1/i8/ behalf of ERA-EDTA. All rights reserved. i8

2 difference turned out to be the agent used for fixing the neutrophils. Using formalin would keep all substances fixed in their native position in the neutrophils, whereas ethanol would allow for some substances to be dissolved and redistributed in or outside the neutrophils. When using ethanol, myeloperoxidase (MPO) dissolves from the primary granules and attaches to the cell nucleus yielding a perinuclear staining pattern [7] like GS- ANA, whereas proteinase3 (PR3) does not and therefore yield a cytoplasmic staining pattern. At the first international workshop on ANCA in 1988, it was agreed to use this method with washed human leucocytes as substrate smeared onto glass slides and fixed with ethanol as a standard procedure for IIF detection of ANCA, as this method had already been fine-tuned for that purpose [8]. The nomenclature for these staining patterns, C-ANCA for the cytoplasmic pattern and P-ANCA for the perinuclear pattern (Figure 2), was agreed on at the second International ANCA Workshop in THE ANCA ANTIGENS MPO was the first ANCA antigen to be identified and reported in 1988 [7] as one of the ANCA antigens yielding a P-ANCA FIGURE 1: The first description of the disappearance of C-ANCA (anti-pmn-aab) titre in a GPA patient brought in complete remission on treatment, presented at the First International Academic Conference in Immunology and Immunopathology as applied to Otology and Rhinology, April 1984 in Utrecht, the Netherlands [3]. IIF pattern. Another P-ANCA antigen was identified as human neutrophil elastase (HNE) [9] and soon after, reports from several groups identified the C-ANCA pattern antigen in GPA as a 29 kda serine protease, PR3 [10 14] in 1989 and These antigens were all located to the primary/azurophilic granules in the cytoplasm of the neutrophils, along with cathepsin-g, bactericidal/permeability increasing protein (BPI), HNE and azurocidin. Lactoferrin is located in the secondary/ specific granules, and like alkaline phosphatase located in the tertiary/secretory granules and plasma membrane both have been implicated as P-ANCA antigens, too. Only PR3-ANCA and MPO-ANCA are important in relation to the small vessel vasculitides. Whereas PR3-ANCA/C-ANCA was identified from the very beginning as a marker for GPA, MPO-ANCA soon turned out to be a marker for microscopic polyangiitis (MPA). The importance of PR3-ANCA and MPO- ANCA as disease markers has recently been emphasized in a genomewide association study of ANCA associated vasculitis, as the most marked associations were differentially related to PR3-ANCA and MPO-ANCA [15]. MPO-ANCA, however, is also found in 30 38% of patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), primarily in patients with vasculitis symptoms [16], and as discussed below, PR3-ANCA and MPO-ANCA may also be detected in other diseases, although the sub-specificities of these ANCAs may differ from those closely related to GPA and MPA. THE EARLY SOLID PHASE ASSAYS FOR DETECTION OF ANCA The first radioimmunoassay for ANCA detection using neutrophil cytoplasm as antigen was described in 1987 [17], the first enzyme-linked immunosorbent assay (ELISA) using affinity purified antigen for C-ANCA detection was described in 1988 [18] and the first commercially available kit for C-ANCA detection using extract from the primary/azurophil/alpha granules of neutrophils was described in 1990 [19]. At the same time, several other solid phase assays were developed, including direct ELISAs and capture ELISAs (using a mouse-monoclonal catching antibody to present the antigen). Gradually, ELISAs became more specific using purified MPO or PR3 as antigens. NDT PERSPECTIVES FIGURE 2: The IIF staining patterns for C-ANCA (A) and P-ANCA (B) using ethanol fixed neutrophil granulocytes. Downloaded from History of ANCA i9

3 NDT PERSPECTIVES THE EC/BCR STANDARDIZATION OF SOLID PHASE ASSAYS At the second International ANCA Workshop in 1989, it became obvious that the emerging solid phase methodologies created for detecting and quantifying PR3-ANCA and MPO-ANCA gave rise to different results and thus necessitated efforts to standardize ANCA determination [20]. With a grant from the EU/EC in 1990 from the EC/BCR programme, 14 European laboratories participated, using three selected preparations of native human PR3 and one preparation of MPO [21 23] for their assays. These results were compared to results in the IIF. The IIF tests were comparable and after corrections of the solid phase methods, the coefficient of variation was reduced to 20%, concluding that the assays could be used for standardized PR3- and MPO-ANCA testing. ANCA DETECTION FOR DIAGNOSTIC PURPOSES However, it is not only necessary to standardize the ANCA assays but also necessary to secure that the clinical indications for ANCA testing are relevant in order to obtain a pre-test probability high enough to justify the test and in order to evaluate the sensitivity and specificity. The Lancet paper from 1985 showed a diagnostic sensitivity of 93% for active GPA [4]. Here, the sera were selected to make sure that the patients in each group were typical patients. In contrast, the sera for the EC/ BCR diagnostic study [23] were unselected sera obtained from consecutive series of patients retrospectively and prospectively in relation to a specific date. This included referred patients on treatment and patients with a suspected but not histopathology proven disease. The overall sensitivity for all PR3/C-ANCA assays was only 64% for GPA. This illustrates that a gating policy for requesting an ANCA test is necessary, as the diagnostic sensitivity and specificity depend entirely on the GPA population examined and the inflammatory disease control population used for selecting a suitably high specificity. This has complicated comparisons between reports on diagnostic sensitivity and specificity of the many different methodologies described since the end of the 1980s. THE DECISION TO USE IIF AS THE PRIMARY TEST FOR ANCA After the EC/BCR standardization, it became relevant to evaluate how, when and which assays should be used for clinical diagnostic use. At an international consensus meeting in Australia [24], it was decided to use the IIF test as the primary diagnostic screening test, based on the extensive experience with the diagnostic potential of the HEp2-cell assay for ANA screening and classification [25]. In accordance with the EC/ BCR study [23], a positive IIF test should always be followed by a specific MPO- or PR3-ANCA test, and ideally all three should be used in each case. This cluster of results would potentially sort out those P-ANCA-/GS-ANA-positive sera that were relevant for small vessel vasculitis from those that could appear in a number of other connective tissue diseases. ANCADETECTIONINDRUG-INDUCED VASCULITIS, INTESTINAL BOWEL DISEASE AND CYSTIC FIBROSIS P-ANCA patterns and atypical (A-ANCA) patterns have been observed in a variety of other diseases than the systemic small vessel vasculitides. It is well-known that certain drugs given over prolonged periods of time can give rise to development of ANCA-positive vasculitis or lupus-like disease. In 1991, it was demonstrated that MPO-ANCA and NHE-ANCA were associated with hydralazine-induced glomerulonephritis [26], and later ANCA against lactoferrin, ANA [27] and ANCA against cathepsin-g was demonstrated in propylthiouracil-induced vasculitis, producing a picture of a multiple autoantibody response in these diseases. A special form of drug-induced disease with necrotizing inflammation is cocaine-induced midline destructive lesions, which is a differential diagnosis to localized GPA in the midface. This condition is highly correlated to HNE-ANCA with a consistent P-ANCA pattern, even though PR3-ANCA is also observed in about half of the cases [28]. The inflammatory bowel diseases, ulcerative colitis (UC) and Crohn s disease have been reported to be associated with a P-ANCA or A-ANCA pattern since the mid-1990s, but determination of the antigen specificity of these ANCA patterns has been difficult and has caused a number of conflicting results. Just lately, using a chemiluminescence immunoassay, the A-ANCA pattern has been correlated to PR3-ANCA in 31% of UC patients [29] and to 38% of Primary Sclerosing Cholangitispatients[30]. The finding of PR3-ANCA yielding an A-ANCA pattern, however, is discussed but not explained. In cystic fibrosis, the presence of ANCA against BPI is correlated to the severity of pulmonary involvement with vasculitis and is also associated with the load of chronic infection with Psudomonas aeruginosa (PA). These BPI-ANCAs, reflecting inflammation of the airways, are not only of IgG class but frequently also of IgA class. Operations to remove infected and diseased tissue in the form of a lung transplant or extensive image guided sinus surgery significantly reduced the IgG as well as the IgA BPI-ANCA levels [31]. Whether the decrease of the infectious burden and/or the removal of inflamed tissue is the reason for the decrease in BPI levels remains to be shown. The correlation between the load of chronic infection with PA and the presence of BPI-ANCA, however, suggests that the infection somehow triggers ANCA production. The presence of ANCA has been reported in a number of infectious conditions including bacterial endocarditis and HIV. Recently, it has been shown that bacterial DNA motifs may trigger production of PR3-ANCA as well as MPO- ANCA in vitro [32]. As new disease activity in GPA is frequently preceded by a flu-like condition, a transient infection may trigger or contribute to disease activity as well as ANCA production. i10 Downloaded from N. Rasmussen et al.

4 PR3-ANCA AND RELAPSE The RELANCA study [33] planned in 1995 as a part of the first series of randomized controlled trials by the European Vasculitis Study Group (EUVAS) using nine PR3-ANCA commercial solid phase assays in monthly serum samples from the NORAM trial, confirmed the original observations of reappearance of C-ANCA at relapse [2 4], but as already concluded in 1989, a rising titre/value was sensitive but not specific for the development of a relapse [34]. The correlation between a rise in titre and relapse is of interest from a pathogenic point of view, but from a clinical point of view, the relevant window for observing a rapid increase due to clinical relapse may be a few weeks. Decrease and increase due to change in therapy develop more slowly. Detecting a relapse therefore requires a sampling frequency of a few weeks, which is almost never the case in the clinical situation. This probably explains why increases in PR3- ANCA levels were not found to correlate to relapses in the WGET study, using three monthly sampling [35]. Nevertheless, an increase in PR3-ANCA titres may reflect a relapse. It is therefore recommended that increasing titres are used as an alert signal for a possible relapse, but that therapeutic action is taken only if a clinical relapse can be confirmed. So far, MPO-ANCA titre increases have not been linked to a similar risk of relapse, but recent studies of MPO-epitopespecific ANCA indicate that certain epitope specificities are linked to relapse as discussed below. CAN RESULTS OF SOLID PHASE ASSAYS FOR PR3-ANCA BE COMPARED? Although the many solid phase assays for PR3-ANCA have good performances for diagnostic sensitivity and specificity, they have turned out to have different performance patterns, i.e. highly variable correlations of PR3-ANCA values between assays [36, 37]. This can be illustrated by applying the dendrogram method on the changes of PR3-ANCA values from entry to relapse for each patient for each of the nine commercial solid phase assays used in the RELANCA study [33]. The dendrogram method applied on the changes from active disease at entry to active disease at relapse revealed that some assays had similar performance, whereas other assays were completely discordant (Figure 3). As PR3-ANCA are normal human, polyclonal autoantibodies [38], ANCA from different persons must be expected to behave differently in different assays depending on the binding profiles of these assays in relation to distribution of IgG subclasses [39], specificity for the epitopes exposed over time [40] and in relation to disease activity [41], binding to free PR3 complexed to alpha-1-antitrypsin in serum [42] and binding to reverse PR3-ANCA [43]. This means that PR3-ANCA values from one assay cannot necessarily be compared with values from another assay and that values from discordant assays cannot be standardized. The marketing of a PR3-ANCA standard from CDC can therefore only be used to standardize each single assay and secure FIGURE 3: Dendrogram illustrating the concordance/discordance between the performance of nine solid phase assays with the level of the horizontal bars illustrating the concordance of the connected assays, where the level of 1 (height) is good concordance and levels >2 are discordance. constant performance of that assay when new batches of antigen are introduced. The dendrogram results support previous findings that combining results from several PR3-ANCA assays may increase the diagnostic potential of PR3-ANCA detection [44]. A screening of PR3-ANCA assays using the dendrogram method can identify each assay as belonging to a certain cluster of assays, which could be helpful to guide the local clinical laboratory to pick out a relevant panel of assays from the assays available in that region, in order to optimize sensitivity and specificity. EPITOPE SPECIFICITY OF ANCA In 1995, it was demonstrated that at least four different epitopes on PR3 were relevant for PR3-ANCA binding [45]. Since then, several groups have been working on the implications of PR3-ANCA epitope specificity. The major problem is that the relevant PR3 epitopes are conformational, complicating the possibilities to use linear epitopes for meaningful diagnostic purposes and pathogenic studies. Production of chimeric human/mouse PR3 recombinant proteins made way for epitope-mapping of anti-pr3 antibodies in 2004 [46] and in 2010, using a refined construct of their original recombinant proteins, the presence of four major surface epitopes with functional importance in relation to enzymatic activity and disease activity was described [41]. A design of specific PR3- epitope-anca assays was therefore suggested to be of potential clinical interest. In contrast to PR3, some meaningful epitopes on MPO are linear. In 1998, it was reported that five recombinant fragments of human MPO identified with rabbit antibodies could sub-classify MPO-ANCA disease in Japan [47]. These findings have been further confirmed in 2007 [48]. In 2006, the technique of producing chimeric human/ mouse constructs was translated to MPO, but the results suggested that further refinements using shorter fragments of the NDT PERSPECTIVES Downloaded from History of ANCA i11

5 NDT PERSPECTIVES epitopes had to be performed [49]. This was described in 2013, using a highly sensitive epitope excision/mass spectrometry approach, reporting on 5 linear as well as 20 conformational epitopes relevant for MPO-ANCA, some being related to disease activity [50]. These findings make it relevant to consider not only the implications for setting up specific functional PR3- and MPO- ANCA assays, but also the implications for further research in the possible pathogenic role of ANCA. Depending on the functional aspects of the individual epitope-specific autoantibodies, these might differ in capacity to exert the pathogenic functions suggested to be effective according to in vitro and in vivo experiments. ACKNOWLEDGEMENTS The EUVAS organization that grew out from the group of colleagues assembled at the first International ANCA Workshop in Copenhagen in Without the numerous contributions from this mass of talent, none of the selected results mentioned in this review could have been achieved. MSc. Severin Olesen Larsen, Statens Serum Institute, has contributed with the statistical handling of data from the EUVAS Serum Bank using the dendrogram method. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1. Wiik A. Granulocyte-specific antinuclear antibodies. Possible significance for the pathogenesis, clinical features and diagnosis of rheumatoid arthritis. Allergy 1980; 35: Davies DJ, Moran JE, Niall JF et al. Segmental necrotizing glomerulonephritis with antineutrophil antibody: possible arbovirus aetiology? Br Med J 1982; 285: Rasmussen N, Wiik A. Autoimmunity in Wegener s granulomatosis. In: Veldman JE, McCabe BF, Huizing EH et al. (eds). 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