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

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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 –20°C. 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.

View larger version (31K): [in this window] [in a new window]   Figure 1. Testing algorithm used for the final determination of the presence of antiproteinase 3 antibodies (anti-PR3). = positive final interpretation; \#8854; = negative final interpretation; ELISA = enzymelinked immunosorbent assay; P-ANCA = perinuclear pattern of staining for antineutrophil cytoplasmic antibodies.

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

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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 were commonly seen among study patients (range, 11% to 39%). An ANCA immunofluorescence pattern of any type was detected in 30% of the patients with connective tissue disease (range, 11% for patients with polymyositis to 70% for patients with systemic lupus erythematosus). An ANCA immunofluorescence pattern was also detected in 10 patients with the antiphospholipid syndrome (30%); 1 (3%) of these had P-ANCA, and 9 (27%) had an atypical pattern. Only 3 blood-donor controls (1.5%) had an ANCA immunofluorescence pattern; all of the immunofluorescence types were atypical.

The individual interpretations of immunofluorescence results that were used to determine the final immunofluorescence results for each serum sample were highly discordant. Twenty-three samples were determined to have C-ANCA by immunofluorescence. However, only 14 of the 23 samples (61%) were interpreted as showing C-ANCA on all three initial readings, even though the slides were read by highly experienced personnel. An additional 15 serum samples were found to have C-ANCA on at least one reading but were not found to have C-ANCA on the final interpretation. The interpretations of results of P-ANCA testing were even more discordant: Only 29 of 49 samples (59%) that had P-ANCA on the final interpretation were found to have P-ANCA on all three readings. An additional 41 serum samples showed P-ANCA on at least one reading but not on the final interpretation.

Many of the study patients with positive results by ANCA immunofluorescence also had positive results by tests for antinuclear antibodies. Of the 117 patients who had connective tissue disease and positive results by ANCA immunofluorescence, 91 (78%) were positive for antinuclear antibodies (0 patients with C-ANCA, 28 with P-ANCA, and 63 with atypical ANCA). In other words, 37% of the 249 patients who had connective tissue disease and were positive for antinuclear antibodies were also positive for ANCA by immunofluorescence. All 28 patients who were positive for P-ANCA and 63 of the 89 (71%) patients who had atypical ANCA immunofluorescence patterns were positive for antinuclear antibodies. The association between antinuclear antibodies and positivity by ANCA immunofluorescence was statistically significant (odds ratio, 2.459 [95% CI, 1.504 to 4.019]; P < 0.001).

Direct Enzyme-Linked Immunosorbent Assay for Antiproteinase 3 Antibodies

The results of direct testing for anti-PR3 are shown in the Table 1 and (Figure 2). Seven study patients (one with rheumatoid arthritis, one with systemic lupus erythematosus, one with polymyositis, and four with EUCTD) were found to have anti-PR3 by direct ELISA; none of the seven had the Sjogren syndrome. According to this assay, no patients with scleroderma or the antiphospholipid syndrome were positive for anti-PR3. Among the control groups, anti-PR3 was detected by direct ELISA in 25 of the 26 PR3-positive patients with vasculitis, 2 of the 26 MPO-positive patients with vasculitis, and none of the blood-donor controls.

View larger version (14K): [in this window] [in a new window]   Figure 2. Results of direct enzyme-linked immunosorbent assay (ELISA) for antiproteinase 3 antibodies (anti-PR3) in the serum of patients with connective tissue disease. The sample size (n) for each study group is given. A logarithmic scale is used for the ELISA results; the dotted line represents the positivity cutoff for the direct anti-PR3 ELISA (5.0 U). All measurements on or above the dotted line are considered to indicate positivity for anti-PR3. APS = the antiphospholipid syndrome; BD = blood-donor controls; EUCTD = early undifferentiated connective tissue disease; MPO+ = controls with vasculitis positive for antimyeloperoxidase antibodies; PM/DM = inflammatory myositis; PR3+ = controls with vasculitis positive for anti-PR3; PS = scleroderma; RA = rheumatoid arthritis; SJ = the Sjogren syndrome; SLE = systemic lupus erythematosus.

Sandwich Enzyme-Linked Immunosorbent Assay for Antiproteinase 3 Antibodies

The results of sandwich ELISA for anti-PR3 are shown in Table 1 and Figure 3. Four study patients (one with polymyositis and three with EUCTD) were found to have anti-PR3 by sandwich ELISA; only one of the four had the Sjogren syndrome (this patient also had EUCTD). No patients with rheumatoid arthritis, systemic lupus erythematosus, scleroderma, or the antiphospholipid syndrome had positive results for anti-PR3 by sandwich ELISA. Among the control groups, anti-PR3 was detected by sandwich ELISA in 23 of 26 PR3-positive patients with vasculitis, none of the 26 MPO-positive patients with vasculitis, and none of the blood-donor controls.

View larger version (13K): [in this window] [in a new window]   Figure 3. Results of sandwich enzyme-linked immunosorbent assay (ELISA) for antiproteinase 3 antibodies (anti-PR3) in the serum of patients with connective tissue disease. The sample size (n) for each study group is given. A logarithmic scale is used for the ELISA results; the dotted line represents the positivity cutoff for the sandwich anti-PR3 ELISA (20.0 U). All measurements on or above the dotted line are considered to indicate positivity for anti-PR3. APS = the antiphospholipid syndrome; BD = blood-donor controls; EUCTD = early undifferentiated connective tissue disease; MPO+ = controls with vasculitis positive for antimyeloperoxidase antibodies; PM/DM = inflammatory myositis; PR3+ = controls with vasculitis positive for anti-PR3; PS = scleroderma; RA = rheumatoid arthritis; SJ = the Sjogren syndrome; SLE = systemic lupus erythematosus.

Nine patients had positive results on at least one of the anti-PR3 ELISAs. However, in only two patients (one with polymyositis and one with EUCTD) was anti-PR3 found by both direct and sandwich ELISA, whereas seven patients (one with rheumatoid arthritis, one with systemic lupus erythematosus, and five with EUCTD) had discordant results on these two tests. The serum sample from the one PR3-positive control, which had negative results by immunofluorescence and by direct and sandwich anti-PR3 ELISAs, was retested and found to be repeatedly positive for anti-PR3 with C-ANCA and to have strongly positive results by direct anti-PR3 ELISA and sandwich anti-PR3 ELISA.

Direct Enzyme-Linked Immunosorbent Assay for Antimyeloperoxidase Antibodies

The results of direct anti-MPO testing are shown in Table 1 and Figure 4. Only two study patients (one with rheumatoid arthritis and one with systemic lupus erythematosus) were found to have anti-MPO by ELISA; neither patient had the Sjogren syndrome. No patients with scleroderma, polymyositis, EUCTD, or the antiphospholipid syndrome had positive results on tests for anti-MPO. Among the control groups, anti-MPO was detected in all of the MPO-positive patients with vasculitis but none of the PR3-positive patients with vasculitis or the blood-donor controls.

View larger version (13K): [in this window] [in a new window]   Figure 4. Results of direct enzyme-linked immunosorbent assay (ELISA) for antimyeloperoxidase antibodies (anti-MPO) in the serum of patients with connective tissue disease. The sample size (n) for each study group is given. A logarithmic scale is used for the ELISA results; the dotted line represents the positivity cutoff for the direct anti-MPO ELISA (2.8 U). All measurements on or above the dotted line are considered to indicate positivity for anti-MPO. APS = the antiphospholipid syndrome; BD = blood-donor controls; EUCTD = early undifferentiated connective tissue disease; MPO+ = controls with vasculitis positive for anti-MPO; PM/DM = inflammatory myositis; PR3+ = controls with vasculitis positive for antiproteinase 3 antibodies; PS = scleroderma; RA = rheumatoid arthritis; SJ = the Sjogren syndrome; SLE = systemic lupus erythematosus.

Patients with Connective Tissue Disease and Positive Results on Testing for Antineutrophil Cytoplasmic Antibodies

The clinical characteristics of the study patients with EUCTD and positive results on anti-PR3 or anti-MPO ELISA were studied to determine whether any evidence suggested vasculitis, especially renal disease, at any time during follow-up that lasted as long as 5 years. Data that were available for patients from each of their study visits included serum creatinine level measurements, results of dipstick and microscopic urinalysis, and a list of secondary diagnoses (diseases other than systemic lupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis, or EUCTD). No study patients who had positive results by ELISA for ANCA were found to have evidence of vasculitis or renal disease.

Final Interpretations

The final interpretations of results of testing for anti-PR3 and anti-MPO using immunofluorescence and ELISA are shown in Table 1. No study patients were found to have anti-PR3 by final interpretation. Only two study patients (one with rheumatoid arthritis and one with systemic lupus erythematosus) were found to have anti-MPO by final interpretation. Both of these patients had moderately high titers of anti-MPO but no evidence of vasculitis at baseline or at follow-up visits.

Discussion

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This blinded, controlled study measured the prevalence of ANCA in the serum of patients with various connective tissue diseases. Indirect immunofluorescence, antigen-specific ELISA, and a combined ANCA scoring system were used to determine the presence of anti-PR3 and anti-MPO. Serum samples from 419 patients with connective tissue disease and from control groups of 26 patients with known anti-PR3, 26 patients with known anti-MPO, and 200 normal blood donors were redivided, randomly ordered, and renumbered before testing. Among patients with connective tissue disease, we found a high prevalence of antibodies to neutrophils detected by immunofluorescence but a low prevalence of anti-PR3 or anti-MPO detected by antigen-specific ELISA. Although many immunofluorescence patterns were found, no patient's serum consistently produced C-ANCA. Many serum samples produced P-ANCA, but this could always be accounted for by antinuclear antibodies. Eleven serum samples (2.6%) were positive for anti-PR3 or anti-MPO by direct ELISA, sandwich ELISA, or both. Most of these samples had low titers. However, in a final interpretation in which samples were considered positive only if the results of immunofluorescence were consistent with those of ELISA, no sample was positive for anti-PR3 and only two samples were positive for anti-MPO. This scoring system provided a combined specificity for anti-PR3 and anti-MPO of 99.5% (CI, 98.1% to 99.9%).

Patients with connective tissue diseases have a high prevalence of autoantibodies and clinical characteristics that may mimic the spectrum of vasculitis that includes Wegener granulomatosis, microscopic polyangiitis, and the Churg-Strauss syndrome. Our finding that testing for anti-PR3 and anti-MPO was highly specific for diagnosing such types of vasculitis in this population allows for increased confidence in the diagnostic accuracy of a positive test result. This accuracy may be particularly useful early in the course of an autoimmune disease.

Our results show that testing for ANCA by immunofluorescence, even if it is done by highly trained and experienced personnel, is fairly subjective and is less reproducible and less specific for vasculitis than antigen-specific ELISA testing for anti-PR3 and anti-MPO. A strong association was seen between serum samples that were positive for antinuclear antibodies and those that were positive for P-ANCA or atypical ANCA immunofluorescence patterns. Immunofluorescence testing for P-ANCA is problematic because antinuclear antibodies can produce staining that cannot be distinguished from the P-ANCA that is produced by anti-MPO. Only serum with anti-MPO, and not other serum that produces P-ANCA, has been associated with vasculitis. In contrast, C-ANCA has generally been found to be a reliable marker of Wegener granulomatosis, microscopic polyangiitis, and related types of vasculitis. Indeed, all of our serum samples that were given a final interpretation of C-ANCA by immunofluorescence were found to have anti-PR3 by ELISA. However, several samples were interpreted as having C-ANCA on at least one reading but were not shown to have anti-PR3 or to be associated with vasculitis. Compared with immunofluorescence testing, antigen-specific ELISA for anti-PR3 and anti-MPO among the patients with connective tissue disease produced few false-positive results.

To ensure that the high diagnostic specificity and clinical utility of the presence of ANCA as a screening or confirmatory test for certain types of vasculitis are retained, clinicians should insist that samples be processed in diagnostic laboratories that have substantial experience in testing for ANCA. They should also ensure that immunofluorescence and antigen-specific ELISA tests for anti-PR3 and anti-MPO be done. It seems inappropriate at this time for clinicians to diagnose vasculitis and treat affected patients on the basis of the results of ANCA immunofluorescence alone; proper testing for anti-PR3 or anti-MPO should become standard for ANCA testing.

Our system for determining a final interpretation of ANCA test results attempts to make the best use of the specificity of the ELISAs for anti-PR3 and anti-MPO while still retaining the clinical utility of immunofluorescence for ANCA. Although this system is labor-intensive, even small improvements in specificity result in appreciable improvement in the test's positive predictive value, especially given the low prevalence of ANCA-associated vasculitis among patients who present with features of connective tissue disease. Unfortunately, assays are not standardized in the United States. Different assays have different specificities and sensitivities [20]. Therefore, clinicians must look at the operating characteristics of the particular assay that is used by their diagnostic laboratory.

Our study has several important strengths, including the use of data that involve a large and carefully studied group of patients with connective tissue disease. Our sample size is especially large for a research project involving such patients. The 165 patients with EUCTD is a unique group with clinical features of connective tissue disease that cannot be classified by traditional case definitions; this group may represent the largest collection of such patients ever studied systematically and prospectively. The tests for ANCA were done on serum samples that had been collected during the first year of the patients' disease. This enabled us to evaluate the utility of ANCA testing at a time in the patients' clinical courses when physicians would be most likely to order such diagnostic tests. Moreover, patients were given diagnoses on the basis of rigorously standardized criteria, and data from follow-up lasting as long as 5 years were available to help confirm the diagnoses.

Another strength of our study is the extensive experience of the ANCA testing laboratory at our institution. The assays that were used in this project have been used in our laboratory since 1989 on more than 20 000 samples. The cutoff values for the ELISAs were determined in previous studies of disease and control patients [15]. The combined immunofluorescence and ELISA scoring system that we used had been used in our laboratory before this study began.

Our study also has some limitations. Although the clinical data were obtained prospectively, we added the ANCA data retrospectively. The EUCTD project was not designed to follow patients for the development of vasculitis, per se. However, the original project was designed to accommodate additional research, and this was the purpose of establishing the serum bank and collecting more than 800 clinical variables for each patient at each visit. As a result, we do not think that important clinical data were missed during the study.

Our study expands on previous studies of the prevalence of ANCA in patients with connective tissue disease [21-27]. Several of these studies found a relatively high rate of positivity on immunofluorescence, especially for P-ANCA. Not all studies measured anti-PR3 and anti-MPO, and laboratory personnel were not blinded to group in any of the studies. The latter limitation is important, given the subjective nature of the interpretation of immunofluorescence results. Moreover, not all studies described standardized diagnostic criteria and most did not include normal controls. In previous reports, the stage and duration of illness varied considerably among the study patients. Finally, relatively few patients with scleroderma or polymyositis were previously studied; to our knowledge, the prevalence of ANCA in patients with EUCTD has not been reported. Thus, ours is the first comprehensive, blinded, controlled study to determine the prevalence of ANCA among patients in whom connective tissue disease was diagnosed according to strict criteria and to use a full set of laboratory tests for ANCA.

Although our results show that testing for anti-PR3 and anti-MPO can be highly specific in patients with connective tissue disease, it is possible that ANCA assays may be less specific in other clinical settings. For example, ANCA immunofluorescence has been reported to yield positive results in patients with various infections [28-32]. However, other studies of patients with infections that used antigen-specific immunoassays for anti-PR3 and anti-MPO found ANCA testing to be very specific for vasculitis [33, 34]. Although additional studies of the specificity of testing for ANCA in various additional disease categories are warranted, the specificity of appropriate antigen-specific immunoassays for anti-PR3 and anti-MPO seems to be high overall.

In conclusion, our data show that if a rigorous system for detecting and confirming the presence of anti-PR3 and anti-MPO is used, testing for ANCA has a high specificity for diagnosing such types of vasculitis as Wegener granulomatosis and microscopic polyangiitis among patients who present with signs and symptoms of connective tissue disease.

Drs. Chang and Skates: Medical Practices Evaluation Center, 50 Staniford Street, Boston, MA 02114.

Dr. Niles: Immunopathology, Cox 5, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114.