Prevalence of Antineutrophil Cytoplasmic Antibodies in a Large Inception Cohort of Patients with Connective Tissue Disease

Antineutrophil cytoplasmic antibodies (ANCA) are strongly associated with the spectrum of vasculitis that includes Wegener granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, idiopathic necrotizing and crescentic glomerulonephritis, and related or overlapping forms of vasculitis [1-3]. Other forms of vasculitis, including Takayasu arteritis, Henoch-Schonlein purpura, and cryoglobulinemia, are not associated with the presence of ANCA. Different assays have been used to test for ANCA, including indirect immunofluorescence and immunoassays that use either crude or highly purified preparations of specific antigens. Although several ANCA antigens have been described [2, 4], only antiproteinase 3 antibodies (anti-PR3) and antimyeloperoxidase antibodies (anti-MPO) have been shown to be of value in the diagnosis of vasculitis [1-3]. When used to stain ethanol-fixed, cytocentrifuged, normal human neutrophils by indirect immunofluorescence, anti-PR3 produce a cytoplasmic pattern of staining (C-ANCA) and anti-MPO produce a perinuclear or nuclear pattern (P-ANCA). At presentation, the clinical features of patients with Wegener granulomatosis, microscopic polyangiitis, and the Churg-Strauss syndrome may include glomerulonephritis, alveolar hemorrhage, tracheobronchitis, sinusitis, palpable purpura, arthritis, ocular inflammation, and neuropathy. Patients with connective tissue diseases may also display many of these features. Therefore, testing for ANCA, if highly specific, could be of great importance in the initial diagnostic evaluation of patients with a differential diagnosis that includes both connective tissue disease and vasculitis. Determination of the specificity of tests for ANCA in the diagnosis of vasculitis is crucial because the decision of whether to pursue biopsies or initiate potentially toxic immunosuppressive therapy may be made on the basis of results of such testing. We report the results of a blinded, controlled study to determine the prevalence of ANCA in a unique group of patients with various connective tissue diseases who were followed for as long as 5 years. A standard testing system, including indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA) for anti-PR3 and anti-MPO, was used to determine the prevalence of ANCA. Methods Patients The Early Undifferentiated Connective Tissue Disease project, a multicenter study funded by the National Institutes of Health through the Cooperative Systematic Studies of the Rheumatic Diseases Program, was designed to develop and prospectively follow a large cohort of patients with rheumatologic disease early in their clinical course. All patients were enrolled within 1 year of the onset of signs, symptoms, or serologic abnormalities that suggested connective tissue disease. Patients were evaluated at study entry and at years 1, 3, and 5. More than 800 clinical and laboratory variables were recorded for each patient according to a standardized protocol. Details of the original project and other study results have been published elsewhere [5-8]. Enrollment began in 1982 and was completed in June 1987. Patients with systemic lupus erythematosus, rheumatoid arthritis, inflammatory myositis, polymyositis or dermatomyositis, or scleroderma had to meet standardized criteria for the diagnosis of these diseases [9-12]. Early undifferentiated connective tissue disease (EUCTD) was diagnosed if patients did not meet criteria for the other connective tissue diseases and met specific criteria that have been described elsewhere [5]. We used serum samples that had been collected from the study patients at baseline. The original study enrolled 410 patients; for 386 (94%) of these, enough serum was available so that the patients could be included in our study. Final diagnoses were determined at the last visit and were therefore based on the cumulative data that had been collected. All analyses and results were based on the final diagnosis; as a result, patients were separated into the following diagnostic groups: systemic lupus erythematosus (n = 70), rheumatoid arthritis (n = 70), scleroderma (n = 45), polymyositis (n = 36), and EUCTD (n = 165). Within the original group, a subgroup of patients who had the Sjogren syndrome was identified. The Sjogren syndrome was defined by the presence, at any time during the study, of xerophthalmia (as determined by positive results on a Schirmer test); xerostomia; and positive results for any one of the following tests: antinuclear antibodies, rheumatoid factor, anti-Ro (anti-SS-A) antibody, or anti-La (anti-SS-B) antibody. All patients in the subgroup with the Sjogren syndrome also had a diagnosis of a primary connective tissue disease as outlined above. Forty-four patients met our definition for the Sjogren syndrome; these patients were drawn from all five diagnostic groups: systemic lupus erythematosus (n = 6), rheumatoid arthritis (n = 9), scleroderma (n = 5), polymyositis (n = 1), and EUCTD (n = 23). Serum specimens from 33 patients who were known to have the antiphospholipid syndrome [13, 14] with medium-to-high titers of IgG or IgM anticardiolipin antibodies (provided by EN Harris) were also studied. Serum samples from 200 random blood donors were collected through the Massachusetts General Hospital Blood Transfusion Service; these donors served as a control group. Serum samples were also collected from 52 patients with Wegener granulomatosis, microscopic polyangiitis, or related forms of vasculitis who had positive results on tests for ANCA; these patients were selected as positive controls for the ANCA assays. This control group of patients with vasculitis included 26 patients with anti-PR3 and 26 patients with anti-MPO; patients with high, low, and intermediate antibody titers were included. Serum Serum samples were both stored and shipped at 20C. All 671 samples, each of which had a unique identifier based on its original source, were assigned new, randomized, study identification numbers and were redivided and relabeled. The laboratory investigators who did the ANCA assays were thus blinded to the diagnosis for each patient's sample. The code for the serum samples was not revealed until all data were collected and the analysis was ready to begin. Indirect Immunofluorescence for Antineutrophil Cytoplasmic Antibodies Indirect immunofluorescence was done as described elsewhere [15]. The results of staining were classified as having one of four patterns: C-ANCA (cytoplasmic), P-ANCA (perinuclear), atypical (neither cytoplasmic nor perinuclear), or negative. Because of the subjective nature of scoring the results of immunofluorescence for ANCA, each sample was stained twice and interpreted independently. Results of the first round of staining were interpreted by one observer, and results of the second round were interpreted by this observer and a second observer; both observers had considerable experience in interpreting the results of immunofluorescence staining of ANCA. If all three readings were the same, the interpretation was considered final. If the interpretations differed, a third slide was prepared and reexamined by the two observers. If at least three of the five interpretations matched, the results were considered final; if not, the staining results were considered to be atypical ANCA. Both observers were blinded to the previous results of immunofluorescence and ELISAs. Testing for Antineutrophil Cytoplasmic Antibodies by Enzyme-Linked Immunosorbent Assay We used direct antigen-specific ELISAs to detect anti-PR3 and anti-MPO, as described elsewhere [15-17]. A sandwich ELISA was also done on each sample. In the sandwich ELISA, monoclonal antibody 1E8 [18] was adhered to the wells of microtiter plates and used to bind proteinase 3. Subsequent steps were the same as those of the direct ELISA. An additional control in the sandwich ELISA for anti-PR3 was performed with selected serum specimens. To control for antibodies to the monoclonal catching antibody, additional wells were coated with monoclonal anti-PR3 catching antibody 1E8 but were not subsequently incubated with cytoplasmic extract of granulocytes. The reactivity of the serum to the monoclonal 1E8 alone was then subtracted from the reactivity to the 1E8-PR3 complex. The result is the titer for a revised sandwich ELISA for anti-PR3. Final Interpretation of Results of Testing for Antineutrophil Cytoplasmic Antibodies A final interpretation of ANCA test results was determined for each patient by using the results of immunofluorescence and ELISA. The set of decision rules used for the final interpretation of testing for anti-PR3 is outlined in Figure 1. A final interpretation for the presence of anti-MPO was considered positive only if samples were positive on immunofluorescence for P-ANCA or atypical ANCA patterns and on direct ELISA for anti-MPO. This is the same system that we use to provide a final interpretation for clinical samples submitted to our laboratory. Figure 1. Testing algorithm used for the final determination of the presence of antiproteinase 3 antibodies (anti-PR3). Statistical Analysis Comparisons between groups were analyzed by the Fisher exact test for categorical variables using a two-tailed significance level of 0.05. All data were stored on a SUN SPARC-5 workstation (Sun Microsystems, Mountain View, California) and analyzed using SAS software (SAS Institute, Cary, North Carolina) for UNIX. The 95% CIs for test specificity were determined using the methods described by Collett [19]. Results Indirect Immunofluorescence for Antineutrophil Cytoplasmic Antibodies The final results of immunofluorescence are shown in the (Table 1). None of the study patients or controls had C-ANCA by immunofluorescence staining. The rate of P-ANCA positivity by immunofluorescence was low for all study groups except patients with systemic lupus erythematosus, who had a rate of 31%. However, atypical patterns of ANCA immunofluorescence we