A Dual-Fixed Neutrophil Substrate Improves Interpretation of Antineutrophil Cytoplasmic Antibodies by Indirect Immunofluorescence

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1 AJCP / Original Article A Dual-Fixed Neutrophil Substrate Improves Interpretation of Antineutrophil Cytoplasmic Antibodies by Indirect Immunofluorescence Ming-Wei Lin, MBBS, 1,2 Roger A. Silvestrini, 1 Suzanne Culican, 1 David Campbell, 1 and David A. Fulcher, MBBS, PhD 1,2 From the 1 Department of Immunopathology, Pathology West, ICPMR, Westmead Hospital, Westmead, Australia, and 2 Discipline of Medicine, Sydney Medical School, University of Sydney, Sydney, Australia. Key Words: Antineutrophil cytoplasmic antibody (ANCA); Vasculitis; Indirect immunofluorescence (IIF); Formalin fixation Am J Clin Pathol September 2014;142: DOI: /AJCPG02FGQVAUSIU ABSTRACT Objectives: To determine whether the addition of a formalinfixed neutrophil substrate could improve interpretation and prediction of autoantigenic specificity in antineutrophil cytoplasmic antibody (ANCA) testing. Methods: Routine diagnostic samples sent for ANCA testing were analyzed prospectively on a dual substrate of both ethanol- and formalin-fixed neutrophils. Positive samples on ethanol-fixed neutrophils were deemed typical if formalinfixed neutrophils also stained, and atypical if not. Indirect immunofluorescence (IIF) results were correlated with antimyeloperoxidase (MPO) and anti proteinase 3 (PR3) results with an enzyme-linked immunosorbent assay (ELISA). Results: Of 1,426 samples, 201 from unique patients were ANCA-positive (200 on IIF, 1 on ELISA alone). Thirty-two (45%) of 71 typical ANCA staining patterns were positive for either an anti-mpo or anti-pr3 antibodies, whereas only one (0.8%) of 129 atypical patterns was ELISA-positive, in a patient without systemic vasculitis. Only one (3%) of 34 ELISA-positive samples had a negative IIF-ANCA (1/1,426 patients, 0.07%), and this patient did not have vasculitis. Conclusions: Concomitant staining on formalin fixation of IIF-positive ethanol-fixed ANCA samples improves the interpretation of ANCA testing and is predictive of vasculitis autoantigens MPO and PR3. Detection of antineutrophil cytoplasmic antibodies (ANCA) is critical for the diagnosis and monitoring of the ANCA-associated vasculitides (AAVs), which include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), pauci-immune crescentic glomerulonephritis, and Churg-Strauss syndrome (CSS, also called eosinophilic granulomatosis with polyangiitis or allergic granulomatosis). The test is usually performed with indirect immunofluorescence (IIF) using alcohol-fixed neutrophils as substrate, where the pattern of cytoplasmic staining has distinct diagnostic implications. Traditionally defined, cytoplasmic ANCA (c-anca), characterized by diffuse granular cytoplasmic staining with internuclear accentuation Image 1, is associated with anti proteinase 3 (PR3) antibodies, a specificity typical of GPA but also found in other AAVs. Similarly, a perinuclear ANCA (p-anca) gives rise to homogeneous perinuclear staining with nuclear extension (Image 1); this pattern occurs due to the relocation of negatively charged cytoplasmic autoantigens to the nuclear membrane upon alcohol fixation. It often reflects the presence of antimyeloperoxidase (MPO) antibodies, and is associated with various AAVs, particularly MPA, pauci-immune crescentic glomerulonephritis, and CSS. Subsequent to the description of these typical patterns, so-called atypical patterns were recognized. Although interpretations vary, a dull, flat, diffuse cytoplasmic staining without central accentuation is often reported as atypical c-anca (Image 1), whereas a broad pencillike perinuclear staining with no nuclear extension is often termed atypical p-anca (Image 1). The recognition of atypical patterns is important, because the target autoantigens that give rise to them are different and not associated with Am J Clin Pathol 2014;142: DOI: /AJCPG02FGQVAUSIU 325

Lin et al / Dual-Fixed Neutrophil Substrate for ANCA Testing Image 1 The four antinuclear cytoplasmic antibody (ANCA) fluorescence patterns based on staining of ethanol- and formalinfixed

2 Lin et al / Dual-Fixed Neutrophil Substrate for ANCA Testing Image 1 The four antinuclear cytoplasmic antibody (ANCA) fluorescence patterns based on staining of ethanol- and formalinfixed neutrophils, as indicated, including cytoplasmic ANCA (c-anca), perinuclear ANCA (p-anca), atypical c-anca, and atypical p-anca Am J Clin Pathol 2014;142: DOI: /AJCPG02FGQVAUSIU

3 AJCP / Original Article vasculitis. They include lactoferrin, cathepsin G, elastase, lysozyme, bacterial permeability increasing protein, catalase, α-enolase, and lamin B1. 1 Such specificities are much less useful diagnostically, being found in various conditions including inflammatory bowel disease (IBD), 1 rheumatoid arthritis, Felty syndrome, primary sclerosing cholangitis, chronic bacterial infections, 2,3 and chronic liver disease. 4 However, distinguishing typical from atypical ANCA patterns is not straightforward, a difficulty that gives rise to significant discrepancies among laboratories in regard to interpretive criteria and reporting schemata. Interpretation may also be affected by interference from concomitant homogeneous ANA, which may mimic and therefore mask the presence of an underlying p-anca. To improve interpretation of IIF-ANCA patterns, several groups have proposed that concomitant consideration of the staining pattern on formalin-fixed neutrophils, in addition to the standard ethanol fixation, may be helpful Despite initial enthusiasm for this approach, other studies have identified inconsistencies, including false positivity owing to enhanced autofluorescence on the formalin-fixed substrate Given these controversies, the selective nature of the cohorts examined in these studies and the paucity of reports correlating IIF-ANCA patterns on a dual substrate with autoantigenic specificity, we examined the latter approach to ANCA testing in the routine diagnostic laboratory. 2. p-anca: Homogeneous perinuclear staining with nuclear extension on ethanol-fixed neutrophils, but a classic c-anca pattern (above) on formalin-fixed neutrophils (Image 1); 3. Atypical c-anca: Dull homogeneous cytoplasmic fluorescence without central accentuation on ethanolfixed neutrophils, but no fluorescence on formalin-fixed neutrophils (Image 1); 4. Atypical p-anca: Broad rim-like fluorescence of the nuclear periphery without nuclear extension on ethanolfixed neutrophils, but no fluorescence on formalin-fixed neutrophils (Image 1). The IIF-ANCA patterns were interpreted by two independent readers (R.A.S. and either S.C. or D.C.), who were blinded to the enzyme-linked immunosorbent assay (ELISA) results. ELISA Anti-MPO and -PR3 antibodies were detected using a commercial ELISA (INOVA Diagnostics, Werfen Group, Barcelona, Spain), performed according to the manufacturer s instructions. An anti-pr3 antibody result greater than 5 U/mL and anti-mpo antibody greater than 10 U/mL was considered positive. Results Materials and Methods Specimens Specimens were collected between February and July 2012 from diagnostic samples referred for ANCA testing to the immunopathology laboratory of the Institute of Clinical Pathology and Medical Research, Pathology West, Westmead Hospital, Sydney, Australia, a diagnostic laboratory servicing about one-quarter of public pathology in New South Wales, Australia. For patients with multiple ANCA requests over this time, only the first sample was included in the analysis. ANCA Interpretation IIF was performed using a combination of two substrates in a single well (Euroimmun Mosaic ANCA Biochip, ESL Biosciences, Lübeck, Germany), including ethanol- and formalin-fixed neutrophils. Samples were screened at 1/10 dilution, and four patterns were distinguished based on individual staining patterns, as follows: 1. c-anca: Finely granular diffuse fluorescence present throughout the cytoplasm with central accentuation, with similar appearance on ethanol- and formalin-fixed neutrophils (Image 1); The study included 1,426 unique patient samples, of which 200 were IIF-ANCA positive. Of these, 35 (18%) were c-anca, 36 (18%) were p-anca, 33 (17%) atypical c-anca, and 96 (48%) atypical p-anca Table 1. Positivity for anti-pr3 or anti-mpo by ELISA was almost completely restricted to the samples showing typical ANCA staining patterns on IIF. Thus, of the 35 c-anca samples, 17 (49%) were positive for anti-pr3, anti-mpo, or both (Table 1), and of the 36 p-anca positive samples, 15 (42%) were positive for one of these two specificities. Importantly, only one (0.8%) of 129 samples with an atypical IIF-ANCA pattern was positive on ELISA. This patient had an atypical p-anca on IIF, a low positive anti-pr3 level of 16 U/mL, but no clinical or radiologic evidence of vasculitis. The close correlation between typical ANCA staining patterns and autoantigenic specificity on ELISA prompted us to question whether IIF screening alone was able to detect all vasculitis-associated specificities, a finding that would allow testing for autoantigenic specificity to be reserved for those showing typical ANCA-IIF patterns. Consistent with this, only one (3%) of 34 ELISA-positive samples (1/1,426 [0.07%]) was negative on fluorescence. This sample was positive for both anti-mpo and anti-pr3 antibodies (both >100 U/mL); the patient had a clinical history of chronic Am J Clin Pathol 2014;142: DOI: /AJCPG02FGQVAUSIU 327

4 Lin et al / Dual-Fixed Neutrophil Substrate for ANCA Testing Table 1 Summary of the IIF-ANCA Pattern and ELISA Results Anti-PR3+/Anti-MPO+ IIF-ANCA Pattern No. of Patients Anti-PR3+ Anti-MPO+ Dual Specificity Negative PR3 and MPO c-anca p-anca Atypical c-anca Atypical p-anca Negative 1, ,225 ANCA, antinuclear cytoplasmic antibody; c-anca, cytoplasmic ANCA; ELISA, enzyme-linked immunosorbent assay; IIF, indirect immunofluorescence; MPO, myeloperoxidase; p-anca, perinuclear ANCA; PR3, proteinase 3. osteomyelitis with no evidence of systemic vasculitis, suggesting a false-positive result. Discussion ANCA testing is central to the diagnosis and classification of vasculitic diseases, yet the interpretation of IIF patterns based on a single substrate of ethanol-fixed neutrophils alone can be problematic in terms of distinguishing typical from atypical ANCA patterns, and in addressing interference from antinuclear antibodies. However, given the major differences in diagnostic implications, this distinction is important. Here we demonstrate that such differentiation can readily be made by considering IIF-ANCA staining patterns on formalinfixed, alongside ethanol-fixed, neutrophils. By defining a typical pattern as being positive on both substrates, we found a strong association with antibodies to PR3 and MPO, and by inference, with likelihood of AAV. 14,15 At the same time, atypical IIF-ANCA samples, defined as being positive on ethanol- but negative on formalin-fixed neutrophils, had a very low (<1%) positivity rate for anti-pr3 or anti- MPO antibodies in our study (Table 1), making a diagnosis of AAV much less likely. Widespread adoption of this approach would not only improve diagnostic accuracy, but also minimize intra- and interlaboratory variation, given its relatively straightforward interpretation by comparison with the often subtly different staining patterns on a single ethanolfixed neutrophil slide. Furthermore, the use of formalin-fixed neutrophils overcomes the problem of interference from antinuclear antibodies, which give a p-anca pattern on the ethanol-fixed neutrophil substrate, but are negative on formalin-fixed neutrophils. 6,16,20 Finally, given the confusion surrounding the various definitions of what constitutes a typical vs atypical IIF-ANCA pattern, 5,17 an argument could be made for abandoning the term atypical completely, and replacing it with a term such as nonspecific ANCA, with the potential to cause less confusion in diagnostic interpretation. Other researchers have previously examined the use of formalin fixation in differentiating the IIF-ANCA patterns. 6,8,11-13,18-21 Results have been highly variable, possibly because of differences in methodology, such as variable incubation times, concomitant use of acetone, different types of formalin (vapor vs liquid), and different types of alcohol fixative (ethanol vs methanol). Only five studies, performed in very selected patient populations, have specifically addressed the issue of PR3 and MPO specificity in relation to the use of formalin-fixed neutrophils, 8,11,16,20,21 and generated results consistent with those reported here in our otherwise unselected cohort. Radice et al 8 studied serum samples selected on the basis of disease and antigenic specificity, and found that samples from patients with vasculitis who were positive for anti-pr3 or anti-mpo antibodies were the only ones to show ANCA staining on formalin-fixed neutrophils, whereas serum samples from patients with ulcerative colitis did not. Similarly, studies by Lee et al 20 and Bang la Cour et al 11 also demonstrated that anti-mpo positive samples could readily be detected on formalin-fixed neutrophils. Pollock et al 21 selected patients with MPA and compared them with those with IBD or lupus, and found perfect correlation between those with active MPA and both ANCA staining on formalin-fixed neutrophils and MPO positivity; however, in those with vasculitis who received treatment, the MPO appeared to be slightly more sensitive. Finally, Damoiseaux et al 16 examined the usefulness of the Europlus ANCA Biochip mosaic, incorporating a substrate of both formalin- and ethanol-fixed neutrophils alongside PR3 and MPO antigen microdots, in a cohort of patients with AAV. Consistent with our results, they found a high correlation (>96%) between typical IIF-ANCA patterns and positivity for PR3 or MPO; furthermore, all IIF-ANCA samples that were negative on a formalin-fixed substrate were also negative for anti-mpo and anti-pr3. Although our study lacked clinical information on the samples studied, we showed that in the diagnostic laboratory environment, processing otherwise unselected patient samples, positivity on formalin-fixed neutrophils was very predictive of positivity for the two major vasculitis-associated antigens. Our study has deliberately focused on the detection of the vasculitis-associated specificities PR3 and MPO, fully 328 Am J Clin Pathol 2014;142: DOI: /AJCPG02FGQVAUSIU

5 AJCP / Original Article recognizing that IIF-ANCA is also occasionally used for the diagnosis of nonvasculitic diseases. For example, in IBD, IIF-ANCA patterns can help differentiate ulcerative colitis from Crohn disease, usually in conjunction with anti- Saccharomyces cerevisiae antibodies; thus patients with ulcerative colitis are usually negative for anti-s cerevisiae antibodies but have atypical p-anca, whereas patients with Crohn disease often have the opposite serologic findings. Although we did not examine patients with IBD specifically, the use of a dual substrate to define an atypical IIF-ANCA pattern in the manner outlined in our study should not detract from the usefulness of IIF-ANCA for this purpose. Indeed, a recent study showed that the distinction between typical and atypical p-anca using formalin fixation improved the sensitivity and specificity of ANCA testing in a cohort of 184 patients with IBD 1 ; MPO specificity was not reported, but seems unlikely to be relevant given the nature of the cohort. Finally, our study had significant implications for the optimal cost-effective approach for detecting and characterizing serum samples referred for ANCA testing. Although current international guidelines recommend that all serum samples be tested using IIF along with immunoassays for PR3 and MPO, 5 our data support the contention that ELISA testing could be restricted to samples showing a typical ANCA on IIF. Thus, only one ELISA-positive sample of our 1,426 referred samples (or 1/34 ANCA-positive samples) would have been missed with such an approach, and this patient did not have vasculitis. Our findings therefore have significant cost implications for laboratories, particularly given the large number of patients now being screened for ANCA. We propose that the term typical ANCA be used to refer to dual staining on both ethanol- and formalin-fixed neutrophil substrates, divided into c-anca and p-anca based on the ethanol-fixed slide (Image 1). All other neutrophil staining patterns could be reported as nonspecific ANCA, because they are rarely associated with PR3 or MPO specificity and thus unlikely to convey a diagnosis of vasculitis. Our laboratory now adopts this reporting schema. Reprint requests to Dr Lin: Dept of Immunopathology, Pathology West, ICPMR, Westmead Hospital, Westmead, New South Wales 2145, Australia; ming-wei.lin@sydney.edu.au. References 1. Barahona-Garrido J, Hernandez-Calleros J, Cabiedes J, et al. Distinguishing between anti-neutrophil cytoplasmic antibody patterns in inflammatory bowel disease: is the atypical pattern adding more information? Am J Gastroenterol. 2009;104: Bonaci-Nikolic B, Andrejevic S, Pavlovic M, et al. Prolonged infections associated with antineutrophil cytoplasmic antibodies specific to proteinase 3 and myeloperoxidase: diagnostic and therapeutic challenge. Clin Rheumatol. 2010;29: Csernok E, Lamprecht P, Gross WL. Clinical and immunological features of drug-induced and infectioninduced proteinase 3-antineutrophil cytoplasmic antibodies and myeloperoxidase-antineutrophil cytoplasmic antibodies and vasculitis. Curr Opin Rheumatol. 2010;22: Wong RC, Silvestrini RA, Savige JA, et al. Diagnostic value of classical and atypical antineutrophil cytoplasmic antibody (ANCA) immunofluorescence patterns. J Clin Pathol. 1999;52: Savige J, Gillis D, Benson E, et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol. 1999;111: Lock RJ. ACP Broadsheet No 143: January 1994 detection of autoantibodies to neutrophil cytoplasmic antigens. J Clin Pathol. 1994;47: Papp M, Altorjay I, Lakos G, et al. Evaluation of the combined application of ethanol-fixed and formaldehydefixed neutrophil substrates for identifying atypical perinuclear antineutrophil cytoplasmic antibodies in inflammatory bowel disease. Clin Vaccine Immunol. 2009;16: Radice A, Vecchi M, Bianchi MB, et al. Contribution of immunofluorescence to the identification and characterization of anti-neutrophil cytoplasmic autoantibodies: the role of different fixatives. Clin Exp Rheumatol. 2000;18: Bird AG. Is there life in the formalin fixed neutrophil for ANCA testing? no! J Clin Pathol. 1999;52: Terjung B, Worman HJ, Herzog V, et al. Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-anca) using immunofluorescence microscopy. Clin Exp Immunol. 2001;126: Bang la Cour B, Wiik A, Hoier-Madsen M, et al. Clinical correlates and substrate specificities of antibodies exhibiting neutrophil nuclear reactivity: a methodological study. J Immunol Methods. 1995;187: Chowdhury SM, Broomhead V, Spickett GP, et al. Pitfalls of formalin fixation for determination of antineutrophil cytoplasmic antibodies. J Clin Pathol. 1999;52: Spickett GP, Broomhead V. Formalin fixation and patterns of antineutrophil cytoplasmic antibodies. J Clin Pathol. 1995;48: van der Woude FJ. Anticytoplasmic antibodies in Wegener s granulomatosis. Lancet. 1985;2: Falk RJ, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med. 1988;318: Damoiseaux J, Steller U, Buschtez M, et al. EUROPLUS ANCA BIOCHIP mosaic: PR3 and MPO antigen microdots improve the laboratory diagnostics of ANCA-associated vasculitis. 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6 Lin et al / Dual-Fixed Neutrophil Substrate for ANCA Testing 17. Savige J, Dimech W, Fritzler M, et al. Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies: quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am J Clin Pathol. 2003;120: Yang P. Comparative evaluation of unfixed and fixed human neutrophils for determination of antineutrophil cytoplasmic antibodies by indirect immunofluorescence. J Clin Pathol. 1997;50: Billing P, Tahir S, Calfin B, et al. Nuclear localization of the antigen detected by ulcerative colitis-associated perinuclear antineutrophil cytoplasmic antibodies. Am J Pathol. 1995;147: Lee SS, Lawton JW, Chak W. Distinction between antinuclear antibody and P-ANCA. J Clin Pathol. 1991;44: Pollock W, Trevisin M, Savige J. Testing on formalin-fixed neutrophils is less sensitive and specific for small vessel vasculitis, and less sensitive for MPO-ANCA, than most ELISAs. J Immunol Methods. 2008; 339: Am J Clin Pathol 2014;142: DOI: /AJCPG02FGQVAUSIU

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