Comparative Study of ELISA and Indirect Immunofluorescence for the Detection of Anti-Neutrophil Cytoplasmic Antibodies

Transcription

1 IMMUNOPATHOLOGY Comparative Study of ELISA and Indirect Immunofluorescence for the Detection of Anti-Neutrophil Cytoplasmic Antibodies Evaluation of the SCIMEDX/EURO Diagnostica ELISA Assay in a Clinical Setting MEDHA S. GODBOLE, MD, RAFAEL VALENZUELA, MD, PHD, SHARAD D. DEODHAR, MD, PHD, LEONARD CALABRESE, DO, 3 AND RAYMOND R. TUBBS, DO This study evaluates the performance of a commercial ELISA assay for detection of anti-neutrophil cytoplasmic antibodies (ANCA). Antiproteinase 3 (anti-pr3) and anti-myeloperoxidase (anti-) ELISA by (SCIMEDX/EURO Diagnostica, Denville, NJ) were compared with the indirect immunofluorescence (HF) method using ethanol-fixed neutrophils. Three hundred sixty serum samples were examined. There was complete correlation of results in 357 (99.%) cases between canca pattern and anti-pr3 results, and in 356 (98.9%) cases between panca pattern and anti- ELISA results. The specificity of ELISA for detection of vasculitis was 95.7% and sensitivity was 6%, whereas for HF, they were 95.7% and 65%, respectively. The ELISA showed good reproducibility with interassay variation (CV), ranging from 7.% to 3.%. Our results show that SCIMEDX/EURO Diagnostica ELISA for ANCA is a reliable and reproducible method that is as good as HF for detection of ANCA. (Key words: ANCA; canca; panca; Proteinase 3; Myeloperoxidase; ELISA; Indirect immunofluorescence) Am J Clin Pathol 995; 4: Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies against cytoplasmic antigens of neutrophils and monocytes. In 98, Davies and colleagues' demonstrated the presence of ANCA in patients with necrotizing glomerulonephritis. In 985, van der Woude and coworkers described ANCA (which they called ACPA), as a specific marker for Wegener's granulomatosis. Since 988, there have been six international workshops on ANCA and nomenclature, and methodology for detection of ANCA has been standardized. 3 " 9 The indirect immunofluorescence (IIF) test using alcohol-fixed human granulocytes as substrate is the standard method for determination of ANCAs. 78 Two types of immunofluorescence staining patterns are recognized: cyto- From the ' Departments ofclinical Pathology, and Rheumatic and Immunologic Disease, The Cleveland Clinic Foundation. Cleveland, Ohio; and 'Department of Pathology and Laboratory Medicine, University of Miami School of Medicine and VA Medical Center, Miami, Florida. Manuscript received February, 995; revision accepted July, 995. Address reprint requests to Dr. Godbole: Department of Pathology, Cuyahoga Falls General Hospital, 9, 3rd Street, Cuyahoga Falls, OH 443. plasmic (canca) and perinuclear (panca). canca is a bright, diffuse, granular cytoplasmic staining with no staining of the nucleus. panca yields an artifactual nuclear staining with accentuation at the periphery of the nucleus. Anti-neutrophil cytoplasmic antibodies has been shown to be associated with a group of clinicopathologic syndromes, including Wegener's granulomatosis, microscopic polyarteritis nodosa, Churg-Strauss syndrome, overlap vasculitis syndrome, and idiopathic pauci-immune necrotizing crescentic glomerulonephritis. " 5 Major target antigen for canca pattern of staining is a 9 kd serine protease or proteinase-3 (PR3) and common panca antigen is myeloperoxidase ().' 6 " Both of these antigens are present in the azurophilic granules of the neutrophils. Using these antigens, Rasmussen and colleagues, 3 Savage and associates, 4 Liidemann and coworkers, 5 and Niles and colleagues 6 have developed reliable solid phase immunologic assay systems, including RIA and ELISA. Although IIF is still considered the standard for detection of ANCA, there are many deficiencies with IIF. The IIF is a subjective test, and interpretation of IIF patterns requires a certain experience and expertise. Also, IIF ANCA can be posi- 667 Downloaded from on 5 February 8

2 668 IMMUNOPATHOLOGY tive because of other unrelated antigens, including CAP57, elastase, lactoferrin unrelated and cathepsin G. These antigens have no diagnostic specificity for vasculitis. Many reports have described positive panca IIF pattern and negative anti- by ELISA in diseases distinct from vasculitis including rheumatoid arthritis, ulcerative colitis, and sclerosing cholangitis.7'8 ELISA is a more objective test and is designed specifically against PR3 and antigens. Currently, some laboratories have developed in-house immunoassays and perform both IIF and ELISA to detect anti-neutrophilic cytoplasmic antibodies. This study was conducted to evaluate a commercially available ELISA by SCIMEDX/EURO Diagnostica, Denville, NJ, which used purified fraction from neutrophil alpha granules as solid phase antigens. The purposes of this study were as follows: () to compare the ELISA results for anti-pr3 and anti- with IIF results; () to compare sensitivity and specificity of ELISA with IIF; and (3) to evaluate reproducibility of ELISA. To our knowledge, this is one of the first studies to evaluate a commercially available ANCA ELISA assay. MATERIALS AND METHODS All consecutive serum samples (n = 36) submitted for ANCA testing, to the Immunopathology Laboratory of the Cleveland Clinic Foundation from March 993 to July 993, were included in the study. Sera samples from 5 patients were received from other institutions, whereas were from in-house patients. Indirect Immunofluorescence Test All sera were examined by the standard indirect immunofluorescent technique introduced by the First International Workshop on ANCA in Copenhagen in Commercially available (INOVA Diagnostic, San Diego, CA) ethanol-fixed human neutrophil substrate slides were used. These multi-well slides by INOVA are prepared using short-term cell culture of purified human neutrophils. Each serum was first screened at : dilutions, and all positive cases were titrated by doubling dilutions up to :64. All positive results were reported as either canca or panca or combined canca and panca pattern along with their titers (Fig. ). To avoid confusion between panca IIF pattern with antinuclear antibody, a parallel IIF test on rat kidney for ANA was performed on every sample. ELISA Anti-neutrophil cytoplasmic antibodies (canca) quantitative test (ANC-4, SCIMEDX/Euro Diagnos- FIG.. Indirect immunofluorescence using alcohol-fixed normal human neutrophils as substrate to demonstrate the presence in serum of canca (A) and panca (B). tica) and anti-neutrophil perinuclear antibody (panca) quantitative test (-, SCIMEDX/Euro Diagnostica) were performed on all the samples according to manufacturer's instructions. The test uses microtitration strips containing wells coated with purified PR3 or antigen. Diluted serum was applied to the well and incubated at room temperature. If specific antibodies were present, they bind to the well. Unbound material was removed with a washing step. Any bound antibody was detected by adding alkaline phosphatase-labeled anti-human IgG, followed by a second washing step and then incubation with substrate, disodium p-nitrophenyl phosphate. A well containing canca/panca developed a color that was quantitated by reading the absorbance at 45 nm. The antibody titer of a patient's serum was determined by change in adsorption. The test samples were A.J.C.P.-December 995 Downloaded from on 5 February 8

3 GODBOLE ET AL. 669 ANCA, Comparison ofelisa, and IIF Methods TABLE. CORRELATION OF RESULTS FOR DETECTION OF ANCA BETWEEN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) AND INDIRECT IMMUNOFLUORESCENCE (IIF) UFcANCA (+) IIF canca H IIFpANCA (+) IIFpANCA M ELSAPR3(+) ELISAPR3H 5 * 34 ELISA (+) ELISA (-) * 344 ANCA = anti-neutrophil cytoplasmic antibodies: canca = cytoplasmic anti-neutrophil cytoplasmic antibodies; = myeloperoxidase antibodies: EIA = enzyme immunoassay: panca = perinuclear anti-neutrophil cytoplasmic antibodies. * One sample was canca negative but panca positive by IIF and both PR3 and positive by EIA. run in duplicates along with three standards ( EU/mL, EU/mL, and, EU/mL). Following manufacturer's instructions, all results were grouped into three groups. The antibody titers of < ELISA units were considered negative. Results >5 EU were reported as positive, whereas samples with values between -5 EU were considered as borderline. According to SCIMEDX/ EURO Diagnostica, this negative limit and borderline limit have been established, in a clinical study of 54 patients with glomerulonephritis (GN) from the University of Lund, Sweden. Similar ELISA test for canca (anti- PR3) and panca (anti-) was also performed on normal human sera as negative controls. To evaluate reproducibility ofelisa, the intra-assay (n = 4) and interassay (n = 5) variation of low, medium, and high standards provided by manufacturer were noted. Clinical history of 9 in-house patients was available. Only biopsy or autopsy proven cases of Wegener's granulomatosis, or other systemic vasculitis known to be associated with ANCA were considered positive. RESULTS Correlation ofelisa Results With IIF Results Three hundred forty-two of the 36 samples (95%) were negative for ANCA by both IIF and ELISA. By IIF, 6 were positive for canca and 4 were positive for panca. By ELISA, 7 were positive for anti-pr3 and 4 were positive for anti- (Table ). The canca pattern of staining was compared with anti-pr3 ELISA results, whereas panca pattern of staining was compared with anti- ELISA. Fifteen cases were positive for both canca by IIF and anti-pr3 by ELISA, whereas 34 cases were negative for both. There a discrepancy in results in only 3 of 36 (.8%) cases. One of them was canca positive but anti-pr3 and anti- negative. One was anti-pr3 positive, but canca by IIF was negative. The third sample was canca negative but panca positive by IIF and anti- PR3 and anti- were both positive by EIA. Interpretation of panca pattern by IIF was more difficult. Totally 78 cases were positive for perinuclear/ nuclear staining by IIF. To avoid confusion between "panca" and ANA, parallel ANA screening was performed. Seventy of 78 cases were also positive for ANA. In those eight ANA-negative cases, the perinuclear staining was clearly due to panca staining pattern. In six additional cases, one of us (RV) noted difference in the perinuclear staining seen in neutrophils and nuclear staining of ANA in rat kidney and (RV) could identify these cases as panca positive. This information was taken into consideration to identify the 4 panca positive cases. Of these, six cases were positive for anti- by ELISA, but were also positive for ANA; six cases were positive for anti- by ELISA and negative for ANA; and two cases were negative for anti- and also negative for ANA (Table ). There was a discrepancy of results between panca by IIF and anti- ELISA in a total of 4 of 36 cases (.%). Specificity and Sensitivity ofelisa All normal human sera were negative (< EU/ inl) by ELISA for both anti-pr3 and anti-. Detailed clinical history was available for 9 in-house patients. There were cases with biopsy proven diagnosis of ANCA-related vasculitides including cases of Wegener's granulomatosis, 5 cases of pauci-immune crescentic glomerulonephritis, Churg-Strauss disease, and 3 polyarteritis nodosa (Table ). The remaining 7 cases had varied diagnosis including cases of respiratory tract diseases; cases of autoimmune disorders; cases of neurologic diseases; 9 cases of nephropathies; 6 with sinusitis and upper respiratory disorders; 5 cases of other vasculopathies; 4 cases of fevers of unknown origin; and 8 miscellaneous disorders. In Wegener's granulomatosis exclusively, anti-pr3 and canca positivity was noted, whereas cases of pauci-immune crescentic glomerulonephritis were positive for only anti- or panca. An interesting finding was dual positivity of anti-pr3 (canca) and anti- (panca) in both Vol. 4. No. 6 Downloaded from on 5 February 8

4 67 IMMUNOPATHOLOGY TABLE. FREQUENCY OF POSITIVE CASES FOR ANCA IN VARIOUS DIAGNOSTIC CATEGORIES Diagnosis No. of Cases UFcANCA Pattern IIFpANCA Pattern ELISA PR3 Positive ELISA Positive Wegener's granulomatosis Active Inactive/limited Pauci-immune glomerulonephritis Churg-Strauss disease Polyarteritis nodosa Other conditions 9 5 * It 3 * 6 5 ANCA = anti-neutrophil cytoplasmic antibodies; IIF = indirect immunofluorescence: canca = cytoplasmic anti-neutrophil cytoplasmic antibodies: ELISA = enzyme-linked immunosorbent assay: = myeloperoxidase antibodies: panca = perinuclear anti-neutrophil cytoplasmic antibodies; PR3 = proteinase 3 antibodies. * Both cases of Churg-Strauss disease were positive for canca, panca, and PR3 and. t This case was positive for canca and PR3. t This case was positive for panca and. cases of Churg-Strauss syndrome. None of the other vasculitides (leukocytoclastic vasculitis, giant cell arteritis, or systemic lupus erythematosus) were ANCA positive. When both methods were compared for detection of ANCA, there was just one case of pauci-immune crescentic glomerulonephritis that was positive for panca by IIF, but was negative for anti- or anti-pr3 by ELISA. This is reflected in the specificity and sensitivity of these two methods. The combined specificity and sensitivity of anti-pr3 and anti- ELISA for diagnosis of these vasculitides was 95.7% and 6%, respectively. The specificity and sensitivity of the IIF technique was 95.7% and 65%, respectively (Table 3). Reproducibility of ELISA Test The reproducibility of ELISA assay was examined by evaluating interassay and intra-assay variation. Three different standards provided by the manufacturer with high, medium, and low ELISA values were used. The interassay variation for three standards was low with a standard variation of 7.4%,.78%, and 7.% for anti- and 9.9%, 3.5%, and 8.67% for anti-pr3, respectively. The intra-assay variation for anti- assay was also low, 4.%, 9%, and 4.4%. The intra-assay variation for anti-pr3 was moderately high, ranging from.6% to 34% (Table 4). DISCUSSION The discovery of ANCA has provided a serologic test that supports the diagnosis of systemic necrotizing vasculitis. 5 Anti-neutrophil cytoplasmic antibodies testing currently is commonplace, and indirect immunofluorescence is the most widely used method for detection of ANCA. 3 It has been demonstrated that a 9-3 kd serine protease from azurophilic granules designated as proteinase- 3 is the target antigen for canca, and myeloperoxidase is major target antigen for panca. Savage and colleagues 4 and Niles and coworkers 5 described an RIA method for detection of ANCA antibodies. In 987, Rasmussen and colleagues 3 described an ELISA method using ANCA antigens obtained from the alpha fraction of neutrophils by nitrogen-bomb cavitation and Percollgradient centrifugation. The ELISA values by this method correlated well with the values obtained by ELISA using affinity purified antigen. 3 Using these different techniques described in the literature, many laboratories have developed an in-house ELISA tech- TABLE 3. COMPARISON OF SENSITIVITY AND SPECIFICITY OF ENZYME IMMUNOASSAY AND IMMUNOFLUORESCENCE METHODS TO DETECT ANCA INDIRECT Indirect Immunofluorescence Enzyme Immunoassay % Specificity % Sensitivity % Specificity % Sensitivity Combined canca panca Combined PR ANCA = anti-neutrophil cytoplasmic antibodies: canca = cytoplasmic anti-neutrophil cytoplasmic antibodies; panca = perinuclear anti-neutrophil cytoplasmic antibodies; = myeloperoxidase antibodies. A.J.C.P.- December 995 Downloaded from on 5 February 8

5 GODBOLE ET AL. 67 ANCA, Comparison ofelisa, and IIF Methods TABLE 4. REPRODUCIBILITY OF ENZYME-LINKED IMMUNOSORBENT ASSAY Low Medium High PR3 PR} PR3 Interassay variation (n = 6) Mean SD %cv Intraassay variation (n = 4) Mean SD %cv ANCA = anti-neutrophil cytoplasmic antibodies: - myelopcroxidase antibodies; SD = standard deviation: CV = coefficient of variation: PR3 = proteinase 3 antibodies. The low. medium, and high standards were run as samples. Results are expressed as ELISA units/ml (EU/mL). nique for detecting anti-pr3 and anti To our knowledge, this is thefirststudy in which a commercially available ELISA kit for ANCA is evaluated. SCIMEDX/ EURO Diagnostica has developed an ELISA kit for canca (anti-pr3) and panca (anti-). The antigens for the ANCA kit are recovered from alpha granules of neutrophils by the methods described by Rasmussen. 3 To evaluate the performance of this kit, we compared the results with indirect immunofluorescence. For canca (anti-pr3), there was a correlation of 99.%. For panca (anti-), the correlation was 98.9%. The specificity and sensitivity were evaluated by gathering clinical information on 9 in-house cases. The specificity and sensitivity of anti-pr3 by ELISA was identical to canca by IIF technique. There was no significant difference between specificity and sensitivity of anti- by ELISA and panca by IIF. It is been suggested that because the PR3 antigen is obtained from alpha granule extract, small amounts of antigen may also be present with this PR3, giving rise to false positivity for sera containing high titers of anti-. 3 We had only one such case with positive anti-pr3 and anti- and panca but negative canca. The ELISA showed good reproducibility and the interassay variation was low. The higher intra-assay CVs for anti-pr3 as compared with interassay CVs indicate the value of running samples in duplicate for each run. Our results support two important findings: () SCIMEDX/Ferring Diagnostica kit for ANCA is a reliable and reproducible method, and () the ELISA is as good as indirect immunofluorescence for detection of ANCA. This is important because ELISA has certain advantages over indirect immunofluorescence. First, ELISA is specifically designed against anti-pr3 and anti-. These are the only two antigens shown to be associated with vasculitis. By contrast, nonspecific indirect immunofluorescence staining can be seen because of other autoantibodies against other neutrophil antigens such as cathepsin G, elastin, and lactoferrin. Various panca patterns have been described in ulcerative colitis, autoimmune liver disorders, and rheumatoid arthritis. 78 canca positivity (anti-pr3 negative) has also been reported in HIV-related diseases, cystic fibrosis, lymphoma, and bronchial carcinoma. 33 " 35 Secondly, panca can be indistinguishable from antinuclear antibodies. Parallel screening for ANA or additional screening on formalin-fixed neutrophils is necessary to differentiate between the two. ELISA for anticanca or anti-panca does not cross-react with ANA and so additional testing is not required. Most importantly, ELISA is an objective test that is easier to standardize and monitor. Most laboratories are well versed with ELISA technique, where as indirect immunofluorescence needs special setup, experience, and expertise. With this commercially available ANCA kit, even smaller laboratories will be able to detect ANCA. For these reasons, the use of ELISA as a screening test for ANCA and IIF for confirmation of positive cases should be strongly considered. CONCLUSIONS. SCIMEDX/EURO Diagnostica ELISA for anti-pr3 and anti- is comparable to indirect immunofluorescence for detection for ANCA.. This ELISA is a reliable and reproducible method. 3. Because ELISA is an objective test and is easier to perform, we recommend considering the use of ELISA as a screening test and IIF as a confirmatory test for detection of ANCA. REFERENCES. Davies DJ, Moran JE, Nlall JF, Ryan GB. Segmental necrotising glomerulonephritis with antineutrophil antibody: Possible arbovirus aetiology? BrMedJ 98; :66. Vol. 4-No. 6 Downloaded from on 5 February 8

6 67 IMMUNOPATHOLOGY. van der Woude FJ, Rasmussen N, Lobatto S, et al. Autoantibodies against neutrophils and monocytes: Tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 985; : van der Woude FJ, Kallenberg CGM, eds. Proceedings of the nd International Workshop on antineutrophil cytoplasmic antibodies (ANCA). Neth J Med 99;36: Rasmussen N. Summary of discussions at the clinical symposium on Wegener's granulomatosis and related vasculitic syndromes. Ada Pathol Microbiol Immunol Scand 99;98(suppll9): Falk RJ, Jennette JC. The third International Workshop on Antineutrophil Cytoplasmic Autoantibodies. Am J Kidney Dis 99; 8: Kekow J, Gross WL. Conference report Wegener's granulomatosis and ANCA-associated disease in 99. Br J Rheumatol 99; 3: Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. Ada Pathol Microbiol Immunol Scand 989; 97(suppl 6): Wieslander J. How are antineutrophil autoantibodies detected? Am J Kidney Dis 994; 8: Wiik A, Van der Woude FJ. The new ACPA/ANCA nomenclature. Neth J Med 99; 36:7-8.. Falk RJ, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis.iv >ig/./ate/988;38: Liidemann G, Gross WL. Autoantibodies against cytoplasmic structures of neutrophil granulocytes in Wegener's granulomatosis. Clin Exp Immunol 987;69: Nolle B, Specks U, Ludemann J, Rohrbach MS, DeRemee RA, Gross WL. Anticytoplasmic autoantibodies: Their immunodiagnostic value in Wegener granulomatosis. Ann Intern Med 989; : Specks U, Wheatley CL, McDonald TJ. Rohrbach MS, DeRemee RA. Anticytoplasmic autoantibodies in the diagnosis and follow-up of Wegener's granulomatosis. Mayo Clin Proc 989; 64: Savage COS, Tizard J, Jayne D, Lockwood CM, Dillon MJ. Antineutrophil cytoplasmic antibodies in Kawasaki disease. Arch Dis Child 989;4: Fienberg R, Mark EJ, Goodman M, McCluskey RT, Niles JL. Correlation of antineutrophil cytoplasmic antibodies with the extrarenal histopathology of Wegener's (pathergic) granulomatosis and related forms of vasculitis. Hum Pathol 993; 4: Niles JL, McCluskey RT, Ahmad MF, Aranout MA. Wegener's granulomatosis autoantigen is a novel serine proteinase. Blood 989;74: Jennette FC, Hoidal JH, Falk RJ. Specificity of anti-neutrophil cytoplasmic autoantibodies for proteinase 3. Blood 99;75: Bini P, Gabay JE, Teitel A, Melchior M, Zhou J-L, Elkon K. Antineutrophil cytoplasmic autoantibodies in Wegener's granulomatosis recognize conformational epitope(s) on proteinase 3. J Immunol 99; 49: Cohen Tervaert JW, Goldschmeding R, Elema JD, et al. Autoantibodies against myeloid lysosomal enzymes in crescentic glomerulonephritis. Kidney Int 99;37: Cohen Tervaert JW, Elema JD, Kallenberg CGM. Clinical and histopathological association of 9kD-ANCA and -ANCA. Ada Pathol Microbiol Immunol Scand 99;98(suppll9):35.. Lesavre P. Antineutrophil cytoplasmic autoantibodies antigen specificity. Am J Kidney Dis 99; 8: Rasmussen N, Sjolin C, Isaksson B, Bygren P, Wieslander J. An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA). J Immunol Methods 99; 7: Rasmussen N, Ludermann J, Utecht B. ELISA examination for IgG ANCA in sera submitted for the first international workshop on ANCA. Ada Pathol Microbiol Immunol Scand 989;97(suppl6):-. 4. Savage CO, Winearls CG, Jones S, Marshall PD, Lockwood CM. Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet 987; : Ludemann J, Utecht B, Gross WL. Detection and quantification of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by ELISA using affinity-purified antigen. J Immunol Methods 988; 4: Niles JL, Pan G, Collins AB, et al. Value of antigen-specific radioimmunoassays for measuring anti-neutrophil cytoplasmic antibodies (ANCA) in the differential diagnosis of rapidly progressive glomerulonephritis. J A m Soc Nephrol 99; : Saxon A, Shanahan F, Landers C, Ganz T, Targan S. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 99; 86:-. 8. Hardarson S, Labrecque DR, Mitros FA, Neil GA, Goeken JA. Antineutrophil cytoplasmic antibody in inflammatory bowel and hepatobiliary diseases. High prevalence in ulcerative colitis, primary sclerosing cholangitis, and autoimmune hepatitis. Am J Clin Pathol 993; 99: Goldschmeding R, Tervaert JW, Dolman KM, von dem Borne AE, Kallenberg CG. ANCA: A class of vasculitis-associated autoantibodies against myeloid granule proteins: Clinical and laboratory aspects and possible pathogenetic implications. Adv Exp Med Biol 99; 97: Ulmer M, Rautmann A, Gross WL. Immunodiagnostic aspects of autoantibodies against myeloperoxidase. Clin Nephrol 99; 37: Niles JL. Value of tests for antineutrophil cytoplasmic autoantibodies in the diagnosis and treatment of vasculitis. Current Opinion Rheumatol 993; 5: Rasmussen N, Daha MR. Concluding remarks on solid phase assays. Neth J Med 99,36: Koderisch J, Andrassy K, Rasmussen N, Hartmann M, Tilgen W. False-positive anti-neutrophil cytoplasmic antibodies in HIV infection. Lancet 99,335: Finnegan MJ, Hinchcliffe J, Russel-Jones D, et al. Vasculitis complicating cysticfibrosis.qj Med 989; 7: Schmitt WH, Csernok E, Gross WL. ANCA and infection. Lancet 99;337: A.J.C.P.* December 995 Downloaded from on 5 February 8