Autoantibodies to proteinase 3 and myeloperoxidase in systemic sclerosis

OBJECTIVE: We evaluated the prevalence and clinical significance of proteinase 3 (PR3-) and myeloperoxidase (MPO-) antineutrophil cytoplasmic antibodies (ANCA) in 115 patients with systemic sclerosis (SSc, scleroderma). METHODS: Sera were assayed by 2 independent centers, which used indirect immunofluorescence (IIF) and direct ELISA as screening tests. Inhibition-ELISA for PR3- and MPO-ANCA and PR3 capture-ELISA experiments were also performed. The clinical features of the ANCA positive were compared with those of the ANCA negative scleroderma patients. RESULTS: The IIF test revealed 5 P-ANCA positive sera (4.34%). Surprisingly, by ELISA 2 of these were PR3-ANCA positive, one was MPO-ANCA positive, and 2 were both PR3- and MPO-ANCA positive. In addition, 3 IIF negative sera were ELISA positive, 2 for PR3- and one for MPO-ANCA. ELISA results were confirmed by fluid phase inhibition experiments. Only 2 out of the 6 PR3-direct ELISA positive sera were positive by PR3-capture ELISA at low titers. Neither PR3- nor MPO-ANCA were significantly associated to any clinical feature of patients with SSc. CONCLUSION: As well as the previously described MPO-ANCA, even PR3-ANCA may be detected in some sera from patients with SSc. The IIF pattern and the negative results obtained with PR3-capture ELISA suggest that different epitopes from those recognized by vasculitis sera might be involved with PR3-ANCA in SSc, and show the importance of combining IIF and ELISA tests for ANCA detection.     

Prediction of relapses in Wegener's granulomatosis by measurement of antineutrophil cytoplasmic antibody levels: a prospective study

OBJECTIVE: Prediction of relapses in Wegener's granulomatosis (WG) by measuring levels of antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase (MPO) remains a controversial issue. To assess the value of serial quantification of ANCA by indirect immunofluorescence (IIF) and antigen-specific enzyme-linked immunosorbent assay (ELISA) for monitoring disease activity in patients with WG, a prospective observational study was conducted in patients with WG attending an outpatient clinic in the Netherlands. METHODS: One hundred patients with WG (85 with PR3-ANCA, 15 with MPO-ANCA) were studied prospectively from 1996 to 1998. Serum samples were obtained and analyzed every 2 months for ANCA levels. Disease activity was prospectively assessed without knowledge of the ANCA levels. RESULTS: Relapses occurred in 37 of 100 patients (37%). Thirty-four (92%) of the 37 patients showed a rise in the level of ANCA preceding their relapse, as detected by ELISA or IIF. The predictive value of an increase in ANCA titers for relapse was 57% (17 of 30) for cytoplasmic/classic ANCA (cANCA; by IIF), 71% (27 of 38) for PR3-ANCA (by ELISA), and 100% (3 of 3) for MPO-ANCA (by ELISA). The predictive value of a rise in ANCA as measured by ELISA or IIF did not substantially improve following concomitant measurement of the IgG3 subclass of PR3-ANCA. Forty-three percent of patients who showed a rise in cANCA (by IIF) and 29% with a rise in PR3-ANCA (by ELISA) did not subsequently experience a relapse. CONCLUSION: Serial measurement of ANCA levels is valuable for the early prediction of relapses in patients with WG.     

Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus: prevalence, antigen specificity, and clinical associations

Fifty-five patients with systemic lupus erythematosus (SLE) were examined for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence (IIF). Enzyme-linked immunosorbent assay (ELISA) for ANCA against myeloperoxidase (MPO), lactoferrin (LF), proteinase 3 (PR3), elastase (HLE), and bactericidal/permeability-increasing protein (BPI) was performed. The prevalence of ANCA by IIF was 29.1% (16/55 patients). MPO-ANCA were found in 10.9% (6/55), LF-ANCA in 18.2% (10/55), PR3-ANCA in 12.7% (7/55), BPI-ANCA in 23.6% (13/55), and HLE-ANCA in 1.8% (1/55). The levels of BPI-, LF-, and PR3-ANCA correlated with disease activity. A significant association between serositis and the presence of BPI-, LF-, and PR3-ANCA was observed, and PR3-ANCA were found to be associated with arthritis as well. Our results demonstrate that ANCA of various specificities occur in SLE, and BPI appears to be an important target antigen.     

Plasma levels of soluble interleukin 2 receptor, soluble CD30, interleukin 10 and B cell activator of the tumour necrosis factor family during follow-up in vasculitis associated with proteinase 3-antineutrophil cytoplasmic antibodies: associations with disease activity and relapse

OBJECTIVES: To evaluate whether T cell activation, as reflected by levels of soluble interleukin 2 receptor (sIL2R), soluble CD30 (sCD30), IL-10 and B cell activator of the tumour necrosis factor family (BAFF) at diagnosis and during initial follow-up, is predictive for persistent or renewed antineutrophil cytoplasmic antibody (ANCA) positivity and clinical relapse in patients with vasculitis associated with proteinase 3-antineutrophil cytoplasmic antibodies (PR3-ANCA). METHODS: 87 Patients with PR3-ANCA-associated vasculitis and at least 2 years of follow-up were included in the study. At diagnosis, and at 3, 6, 12, 18 and 24 months after diagnosis, cytoplasmic ANCA titres were detected by indirect immunofluorescence (IIF), and PR3-ANCA, sIL2R, sCD30, IL-10 and BAFF levels were assessed by ELISA. 31 healthy volunteers provided plasma samples for comparison. Levels of immune markers were related to ANCA positivity and relapse during follow-up. RESULTS: Plasma levels of sIL2R, sCD30 and BAFF were higher in patients than in controls at all time points. Plasma levels of sIL2R, sCD30 and IL-10 were higher at diagnosis and relapse than during remission. At 18 months, sCD30 (p<0.001) and sIL2R levels (p = 0.01) were significantly higher in PR3-ANCA-positive patients (detected by ELISA) than in PR3-ANCA-negative patients. ANCA-positive patients detected by ELISA or IIF at 24 months had significantly higher plasma sCD30 levels (p = 0.02 and p = 0.03, respectively) than ANCA-negative patients. CONCLUSION: Increased T cell activation in patients with ANCA-associated vasculitis in remission during and after immunosuppressive treatment is associated with persistent or renewed ANCA positivity.     

Contribution of immunofluorescence to the identification and characterization of anti-neutrophil cytoplasmic autoantibodies. The role of different fixatives

OBJECTIVE: To study the sera from selected groups of antineutrophil cytoplasmic antibody (ANCA) positive patients by means of the indirect immunofluorescence test (ANCA-IIF) with different fixatives, in order to better discriminate among the various ANCAs (Ag-specificity and disease associations), especially those for which the antigen targets have not yet been identified. METHODS: Eighty pathological serum samples and 15 normal sera were evaluated. Pathological samples included sera from 30 ulcerative colitis (UC) ANCA positive patients, 30 P-ANCA/myeloperoxidase (MPO-ANCA) positive microscopic polyangiitis (MPA) patients, 10 C-ANCA/proteinase 3 (PR3-ANCA) positive Wegener's granulomatosis (WG) patients, and 10 antinuclear antibody (ANA) positive (ANCA negative) systemic lupus erythematosus (SLE) patients. ANCA were detected by IIF on ethanol, methanol and formalin-fixed granulocytes and by ELISAs specific for MPO, PR3, lactoferrin (LF) and bactericidal/permeability-increasing protein (BPI). Additionally, sera were tested for the presence of antinuclear antibodies on IIF. RESULTS: 96% of serum samples from UC patients, positive by IIF on ethanol-fixed granulocytes, became negative when tested on formalin-fixed neutrophil slides. On the contrary, 95% of sera from vasculitic patients showed a clear diffuse granular cytoplasmic pattern on the same substrate; sera from all 10 SLE patients did not show any reactivity when formalin was used as fixative. On methanol-fixed neutrophils, 100% of UC P-ANCA positive sera were positive with the same pattern versus only 20% of vasculitic P-ANCA positive (MPO positive). Methanol fixation had no effect on PR3-ANCA and ANA positive sera. CONCLUSION: The comparison of IIF patterns of sera tested on different fixed cells may be useful to distinguish vasculitis-related P-ANCA versus ANA and vasculitis-related P-ANCA versus UC-related P-ANCA.     

Evaluation of capture ELISA for detection of antineutrophil cytoplasmic antibodies directed against proteinase 3 in Wegener's granulomatosis: first results from a multicentre study

OBJECTIVE: To evaluate the performance characteristics of direct and capture ELISA for the detection of PR3-ANCA in Wegener's granulomatosis (WG) in international ANCA reference laboratories. METHODS: Serum samples were derived from patients with histological and clinical diagnosis of WG (n = 60), rheumatoid arthritis (RA) (n = 30) and healthy controls (n = 30). Each of them was tested for the presence of ANCA by indirect immunofluorescence technique (IFT), direct and capture ELISA in six international reference laboratories (Massachusetts General Hospital, Boston; Wieslab AB, Lund; University of Maastricht; University Hospital Groningen; Mayo Clinic, Rochester; Rheumaklinik Bad Bramstedt/University of Schleswig-Holstein Campus Lübeck). Each centre tested the sera according to their house protocols of IFT and ELISA. The diagnostic performance of each test was estimated by receiver operating characteristic curve analysis and sensitivity and specificity in detection of ANCA/PR3-ANCA were calculated for the respective methods. RESULTS: In patients histologically and clinically known as WG, the detection of ANCA by IFT varied between 52 and 83% among the participating centres. PR3-ANCA positivity with the different ELISAs ranged from 53 to 80% in direct ELISA and from 72 to 76% in capture ELISA. While most capture ELISAs successfully detected PR3-ANCA, there were significant differences between IFT and direct ELISA results between laboratories. ROC curve analysis demonstrated that in five of six laboratories the overall diagnostic performance of capture ELISA was superior to IFT and direct ELISA, respectively. CONCLUSION: Capture ELISA is a highly sensitive assay for detection of PR3-ANCA in WG and should be used in conjunction with compatible clinical picture and histological evidence.     

Rapid-detection GBM-ANCA ELISA. An emergency tool for the early diagnosis of type I and II rapidly progressive glomerulonephritis

Rapidly progressive glomerulonephritides (RPGN) are forms of necrotizing glomerulonephritis associated with anti-glomerular basement membrane (anti-GBM) and anti-neutrophil cytoplasmic antibodies (ANCA) against the antigens proteinase-3 (anti-PR3) and myeloperoxidase (anti-MPO). RPGN have a course of rapid progression to renal failure. We compared the results from the semiquantitative ELISAs for anti-GMB antibodies, PR3-ANCA and MPO-ANCA and the indirect immunofluorescence technique (IIF) against a new rapid assay (30 minutes) for the same antibodies in patients with clinically suspected RPGN. The semiquantitative ELISAs for anti-GBM antibodies and PR3-ANCA and MPO-ANCA have a proven diagnostic significance in patients with RPGN I and III. There were no significant differences between the ANCA-GBM screening test and the results from the semiquantitative ELISAs (p > 0.05). We did not find significant differences between the results for PR3-ANCA and MPO-ANCA from the ANCA-GBM screening test with C-ANCA and P-ANCA IIF values (p > 0.05). We also corroborated that the ANCA-GBM screening test is a diagnostic tool for RPGN I and III as useful as the semiquantitative ELISAs and the IFF technique. The ANCA-GBM ELISA screening test is a tool as useful as the semiquantitative ELISA against anti-GBM antibodies for diagnosis of RPGN I. The comparison of the screening ELISA with the IIF technique and the semiquantitative ELISAs against PR3-ANCA and MPO-ANCA showed similar utility for diagnosis of RPGN III. The advantages of the new screening assay are that three antibodies are tested at the same time, yielding results in only 30 minutes.     

Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA

OBJECTIVES: To evaluate the prevalence and association of antineutrophil cytoplasmic antibodies (ANCA) and their subtypes [proteinase 3 (PR3)-ANCA, myeloperoxidase (MPO)-ANCA] with distinct clinical features in various clinicopathological syndromes. METHODS: All consecutive ANCA-positive patients seen at the combined unit for rheumatology for Bad Bramstedt and the University of Lübeck between 1989 and 1999 were analysed. ANCA were detected by an immunofluorescence technique and ANCA subspecificities were determined by ELISA. Clinical features at presentation and diagnoses were recorded according to standardized procedures. RESULTS: Among 4620 patients tested, 333 were cytoplasmic ANCA-positive and 291 were perinuclear ANCA-positive. cANCA/PR3-ANCA were strongly associated with Wegener's granulomatosis (WG), whereas pANCA/MPO-ANCA were associated with a diverse disease spectrum. Further investigation of PR3-ANCA-positive (n=80) and MPO-ANCA-positive patients (n=40) revealed a greater extent of disease [disease extent index (DEI); median 8 vs 5, P<0.01] and more frequent involvement of the upper/lower respiratory tract and the eyes in PR3-ANCA-positive than in MPO-ANCA-positive patients. Fewer than 5% of WG patients were MPO-ANCA-positive. Compared with matched PR3-ANCA-positive WG patients, the MPO-ANCA-positive WG patients had a lower DEI (median 5 vs 8) and had a lower frequency of peripheral neuropathy. CONCLUSIONS: ANCA testing is useful due to its high sensitivity and specificity, especially for cANCA/PR3-ANCA in WG. We found a divergence in the disease spectrum between PR3- and MPO-ANCA-positive patients, characterized by higher DEI and extrarenal manifestations in the PR3-ANCA group. MPO-ANCA was rarely found in WG and was associated with less organ involvement.     

A critical evaluation of commercial immunoassays for antineutrophil cytoplasmic antibodies directed against proteinase 3 and myeloperoxidase in Wegener's granulomatosis and microscopic polyangiitis

OBJECTIVE: To evaluate the performance of 11 commercial enzyme-linked immunosorbent assay (ELISA) kits for the detection of antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) in patients with Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). METHODS: Serum samples were taken from 92 patients with a histological and clinical diagnosis of WG (n=50) or MPA (n=42) and from 30 disease controls (systemic lupus erythematosus, n=15; rheumatoid arthritis, n=15) and 30 healthy controls. Each of the sera was tested for the presence of ANCA directed against PR3 and MPO using 11 commercially available direct ELISA kits, our in-house PR3- and MPO-ANCA capture ELISAs, and the indirect immunofluorescence technique (IFT). RESULTS: In tests for WG using PR3-ANCA, the commercial direct ELISA kits differed widely in their sensitivity (from 22 to 70%) and negative predictive value (NPV) (from 43 to 70%), but only moderately in their specificity (from 93 to 100%) and positive predictive value (PPV) (from 93 to 100%). The highest sensitivity (74%) and specificity (100%) for PR3-ANCA were obtained with the in-house capture ELISA. Similar differences and trends were noted for MPO-ANCA assays. Diagnostic sensitivity was more than 60% for four and at least 50% for six of the 11 ELISA kits. The PPV varied from 84 to 100% and the NPV from 58 to 70%. In tests for MPA, the MPO-ANCA ELISA kit designated F and the in-house capture ELISA were best (both had sensitivity 62% and specificity 100%). For both WG and MPA, maximum sensitivity for ANCA was obtained with IFT (80 and 70% respectively). CONCLUSION: Determination of PR3-ANCA and MPO-ANCA with the commercial direct ELISA kits achieved poor sensitivity for both WG and MPA. The in-house PR3 and MPO-ANCA capture ELISAs performed better than the commercial ELISAs, combining higher specificity with similar sensitivity. IFT remains the best method for ANCA detection in both diseases.     

Absence of ANCA in Behçet's syndrome with large vessel involvement

The aim of this study was to examine the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Behcet's syndrome (BS) with large vessel involvement. The sera of 48 consecutive patients with BS with large vessel involvement, seven patients with Wegener granulomatosis (WG), and 10 healthy staff were studied for the presence of the cytoplasmic (c) and perinuclear (p) pattern of ANCA by indirect immunofluoresance (IIF). The sera were also examined for antibodies against proteinase 3 (anti-PR3) and myeloperoxidase (anti-MPO) by enzymelinked immunosorbent assay (ELISA). None of the sera from the patients with BS and healthy controls had detectable ANCAs, while all positive control sera from patients with WG showed ANCA positivity in some form. This study confirms that there are no detectable ANCAs in patients with BS with large vessel involvement by IIF and ELISA tests.     

The prevalence and target antigens of antithyroid drugs induced antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with hyperthyroidism

OBJECTIVE: Antithyroid drugs such as propylthiouracil (PTU) and methimazole (MMI) are common medications in Chinese patients with hyperthyroidism and PTU-induced antineutrophil cytoplasmic antibody (ANCA) positive vasculitis has been reported. The current cross-sectional study aimed to investigate the prevalence and the target antigens of ANCA in Chinese patients with hyperthyroidism pre- and post-antithyroid medication therapy. METHODS: Sera from 216 patients with hyperthyroidism in our hospital were collected from January to July in 2002. Patients were divided into four groups: untreated (n = 34); treated with PTU (n = 62); treated with MMI (n = 77); and treated with both PTU and MMI (n = 43). Indirect immunofluorescence (IIF) assay was used to detect ANCA and ANA. Antigen-specific ELISAs were used to detect antigen specificities. The known antigens included myeloperoxidase (MPO), proteinase 3 (PR3), human leukocyte elastase (HLE), lactoferrin, bactericidal/permeability-increasing protein (BPI), cathepsin G and azurocidin. RESULTS: 33/216 sera were IIF positive, 20 of the 33 samples were ANCA positive, 11 samples were ANA positive, and two samples were both P-ANCA and ANA positive. The prevalence of positive ANCA in patients receiving PTU (14/62, 22.6%) was significantly higher than that of untreated patients (1/34, 2.9%) and patients treated with MMI (0/77, 0), P < 0.017. Of the 22 IIF-ANCA positive samples, 12 (54.5%) sera recognized lactoferrin, seven (31.8%) sera recognized HLE, four sera recognized MPO and azurocidin respectively, three sera recognized PR3 and cathepsin G respectively, and one serum recognized BPI. Six of the 22 (27.3%) patients with ANCA positive had clinical evidence of vasculitis. All patients with MPO-ANCA and two of the three patients with PR3-ANCA had clinical vasculitis. CONCLUSION: PTU is associated with the production of ANCA in patients with hyperthyroidism. PTU-induced ANCA are due to polyclonal activation of B cells. Anti-MPO and anti-PR3 antibodies may associate the occurrence of clinical vasculitis.     

Low seroprevalence and poor specificity of antineutrophil cytoplasmic antibodies in tuberculosis

OBJECTIVES: Recently published findings suggested that antineutrophil cytoplasmic antibodies (ANCA), particularly those with a cytoplasmic (C-ANCA) labelling pattern and targeting proteinase 3 (anti-PR3), might be markers of tuberculosis (TB). This is a critical issue, because C-ANCA/anti-PR3 were considered to be a highly specific hallmark of Wegener's granulomatosis or microscopic polyangiitis and because TB may clinically mimic Wegener's granulomatosis. We therefore undertook a study with the aim of investigating further the prevalence and specificity of ANCA in TB. METHODS: We evaluated serum samples from 67 patients diagnosed with culture-proven TB and 10 previously untested control samples from patients known to be ANCA positive (four Wegener's granulomatosis and two microscopic polyangiitides) or negative. All 77 sera were screened for ANCA using commercially available indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for anti-PR3 and antimyeloperoxidase (MPO). IIF-positive and anti-PR3- and anti-MPO-negative sera were also tested for bactericidal/permeability-increasing protein, lactoferrin, elastase and cathepsin G specificities with commercially available ELISA. RESULTS: IIF detected ANCA in seven (10%) of the TB sera, including three C-ANCA and four atypical perinuclear-labelling ANCA. Only one IIF-negative specimen was anti-PR3 positive in ELISA. ANCA testing of the control sera yielded IIF and ELISA results concordant with previous findings, except for one borderline ELISA. CONCLUSION: Our results indicate that TB is associated with low ANCA seroprevalence and poor specificity, with no test serum showing combined C-ANCA/anti-PR3 activity. In a clinical setting of Wegener's granulomatosis/TB mimicry, such combined reactivity would seem to be more suggestive of Wegener's granulomatosis.     

Determining the prevalence of antineutrophil cytoplasmic antibody and the cut-offs of anti-PR3 and anti-MPO antibody in general population

ObjectivesAntineutrophil cytoplasmic antibodies (ANCAs) are serological hallmark of ANCA associated vasculitides (AAV); Wegener's granulomatosis (WG), Microscopic polyangiitis (MPA) and Churg–Strauss Syndrome. Conventionally, ANCA test is carried out on human neutrophils by indirect immunofluorescence (IIF) and antigenic determinant is confirmed with specific ELISA. Since most of the ELISA kits are manufactured outside India and cut-offs provided by the manufacturers are based on different ethnic population, their diagnostic performance may vary. Therefore, we aimed to define the optimum cut-off values that distinguish between the patient and healthy individual in Indian population.MethodsSera of patients with AAV, and healthy individuals were tested for anti-Proteinase 3 (PR3) and -Myeloperoxidase (MPO) using commercially available ELISA kits. Receiver operator characteristics (ROCs) curve was constructed to define the optimum cut-off value between the patients and healthy individuals.ResultsANCA was performed on 280 sera from healthy individuals, and 22 patients with active WG and 11 with active MPA. In healthy individuals, ANCA at dilutions 1:40, 1:80, 1:160, and 1:320 was positive in 7.14, 3.9, 1, and 0.3% respectively. ROC curves analysis showed that for PR3-ANCA antibody the best cut-off was 19 RU/ml at which the sensitivity was 81.8% and specificity was 100%. Similarly, for MPO-ANCA antibody, the best cut-off was 14.95 RU/ml at which sensitivity was 90.9% while specificity was 100%. Both of these cut-off values were different than the manufacturer's cut-off values.ConclusionOptimization of imported kits as per the local healthy population should be carried out to improve its diagnostic performance.     

抗嗜中性粒细胞胞浆抗体与狼疮性肾炎的关系

目的探讨抗嗜中性粒细胞胞浆抗体（ANCA）与狼疮性肾炎（LN）临床相关表现和发病机制的关系。方法分别应用间接免疫荧光法和酶联免疫吸附分析的方法,检测81例LN患者血清中的ANCA,并分析ANCA与LN临床表现和其它实验室检查结果之间的关系。结果单用间接免疫荧光法检测时,ANCA在LN中的阳性率是30.9％（25／81）。对间接免疫荧光法检测为阳性的血清,用酶联免疫吸附分析法进行验证,结果仅有72.0％（18／25）仍为阳性,全部是核周型ANCA（p-ANCA）,未见中央型ANCA（c-ANCA）出现。ANCA阳性组LN患者合并浆膜炎、神经系统累及、贫血、抗ds-DNA抗体阳性和低补体的频率均显著高于ANCA阴性组LN患者。结论ANCA在LN中的阳性率为30.9％,并与LN特定的临床表现相关,提示ANCA可能参与了LN的发病过程。     

Antineutrophil cytoplasmic antibodies in childhood systemic lupus erythematosus

We aimed to evaluate the presence of peripheral antineutrophil cytoplasmic antibodies (p-ANCA) and cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA) in children with SLE and to correlate its association of laboratory findings.Twenty-one children with SLE were studied. Serum samples in patients were tested by indirect immuno-fluorescence (IIF) slide kit (INOVA) for c-ANCA and p-ANCA and by ELISA for myeloperoxidase (MPO-ANCA) and proteinase 3 (PR3-ANCA). All the patients but two were quiescent for lupus at the time of samplingSixteen of 21 patients showed positive IIF staining whereas only 5 had MPO-ANCA and 2 of nine PR3-ANCA.The data suggests that SLE may be associated p-ANCA directed against additional target antigens rather than MPO and may be implicated in the pathogenesis of SLE or may be only non-specific antibodies developed in lupus.     

How and why should we detect ANCA?

Antineutrophil cytoplasmic antibodies (ANCA) have become an established tool for the diagnosis of systemic vasculitis. The major role for ANCA testing is in diagnosing renal insufficiency of unknown origin, where a positive test indicates whether the patient will benefit from immunosuppressive treatment or not. A negative test result almost completely rules out the presence of systemic vasculitis. In this clinical setting the major antigens for ANCA are proteinase 3 and myeloperoxidase, and antibodies to these antigens can best be tested by ELISA. In other clinical settings like inflammatory bowel disease, arthritis and so on, several other ANCA specificities have been described and the IIF test is preferred. However, the clinical value of these somewhat more esoteric specificities is doubtful. New developments in assay techniques and better knowledge of specific epitopes will lead to tools for the improved diagnosis as well as follow up of patients during treatment, as has already been seen with the capture assay for PR3-ANCA.     

Relationship between anti-neutrophil cytoplasmic antibody determined with conventional binding and the capture assay, and long-term clinical course in vasculitis

OBJECTIVES: To evaluate the relationship between anti-neutrophil cytoplasmic antibody (ANCA) measured with two different methods and long-term clinical course in vasculitis. DESIGN: Retrospective determination of ANCA with two different assays for detection of PR3-ANCA, conventional direct binding ELISA and capture ELISA using monoclonal antibodies against PR3. The 245 ANCA determinations were performed from frozen blood samples collected three to four times a year in each patient. SETTING: Department of Nephrology at a Swedish University Hospital. SUBJECTS: A total of 10 ANCA-positive patients with vasculitis caused by Wegener's granulomatosis (WG) or microscopic polyarteritis (MPA) and a very long follow-up time (mean 9 years, range 5-15.5 years). RESULTS: The total number of episodes with active vasculitis was 29 and all of them (100%) were detected by the capture technique whilst the conventional technique detected 23 (79%). The mean number of episodes with active disease requiring treatment with steroids and cytotoxic drugs was three per patient (range 1-6). At the time of clinical relapse of the vasculitis disease, the ANCA titre using the capture technique was either increasing or showed a very high value in all cases. The pattern of capture ANCA response could be subdivided into three categories: a close (four patients), an intermediate (three patients), and no (three patients) relationship between capture ANCA level and long-term clinical course. CONCLUSION: Detection of PR3-ANCA by the capture ELISA showed a higher sensitivity than that obtained by the direct ELISA in diagnosing relapse during follow-up of patients with vasculitis. The specificity of the capture ANCA was, however, low, as high levels occurred in patients without clinical disease activity.     

ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS

Antineutrophil cytoplasmic antibodies (ANCA) with specificityfor proteinase-3 (PR3) are associated with Wegener's granulomatosis,and ANCA directed to myeloperoxidase (MPO) with other idiopathicvasculitides. Inflammation of small-sized blood vessels is ahallmark of systemic lupus erythematosus (SLE). We evaluatedthe prevalence of ANCA in SLE, their antigenic specificities,and their possible relation to clinical disease patterns andactivity. Plasma samples from 84 patients with SLE were testedfor ANCA during remission. Plasma samples from the 25 patientswho relapsed during a follow-up of 32 months were serially analysedfor ANCA in a 6 month period preceding and including the relapse.The presence of ANCA was assessed by indirect immunofluorescence(IIF) and ELISA for antibodies to PR3, MPO, lactoferrin (LF),elastase (HLE) and cathepsin-G (CG). We related the presenceof ANCA to disease patterns, activity and duration. ANCA byIIF were difficult to interpret due to the presence of antinuclearantibodies (ANA). By ELISA, we found no anti-PR3 or anti-HLE.Anti-MPO (n = 7), anti-LF (n = 13) and anti-CG (n = 10) weredetected, generally in low titres. The presence of ANCA of definedspecificity was not associated with specific clinical subsets.The prevalence of ANCA was higher in patients who developedrelapses than in those who did not (P < 0.01). However, levelsof ANCA did not fluctuate in the period preceding the relapse.ANCA of various specificities occur in SLE. Their presence isnot associated with specific clinical disease entities. Thehigher frequency of ANCA in relapsing patients compared to thosewho do not relapse may suggest that ANCA are involved in diseaseexpression. Their diagnostic significance is limited. KEY WORDS: ANCA, SLE, Disease activity, ELISA     

Variations in performance characteristics of commercial enzyme immunoassay kits for detection of antineutrophil cytoplasmic antibodies: what is the optimal cut off?

BACKGROUND: Previous studies have shown considerable variation in diagnostic performance of enzyme linked immunosorbent assays (ELISAs) for measuring antineutrophil cytoplasmic antibodies (ANCA) specific for proteinase 3 (PR3) and myeloperoxidase (MPO). OBJECTIVE: To analyse the performance characteristics of different commercially available direct ANCA ELISA kits. METHODS: ELISA kits for detecting PR3-ANCA and MPO-ANCA from 11 manufacturers were evaluated. Serum samples were taken from patients with Wegener's granulomatosis (15), microscopic polyangiitis (15), other vasculitides (10), and controls (40). RESULTS: were compared with data obtained by indirect immunofluorescence (IFT). The diagnostic performance of the tests was analysed and compared by receiver operating characteristic (ROC) curve analysis.Results: Applying the manufacturers' cut off resulted in great variation in sensitivity of the commercial PR3-ANCA kits for diagnosing Wegener's granulomatosis (ranging from 13.3% to 66.7%), and of the MPO-ANCA kits for diagnosing microscopic polyangiitis (ranging from 26.7% to 66.7%). Specificities were relatively constant (from 96.0% to 100%). IFT was superior to all ELISAs (C-ANCA for Wegener's granulomatosis: sensitivity 73.3%, specificity 98%; P-ANCA for microscopic polyangiitis: sensitivity 86.7%, specificity 98%). The sensitivities of PR3-ANCA and MPO-ANCA ELISA kits were increased by lowering the cut off values. This reduced specificity but increased overall diagnostic performance. CONCLUSIONS: The low sensitivity of some commercial kits reflects the high cut off levels recommended rather than methodological problems with the assays. Comparative analyses using sera from well characterised patients may help identify optimum cut off levels of commercial ANCA ELISA tests, resulting in better comparability of results among assays from different manufacturers.     

Alpha1-antitrypsin phenotypes and anti-neutrophil cytoplasmic auto-antibodies in inflammatory bowel disease

OBJECTIVES: Alpha1-antitrypsin (alpha1-AT) is encoded by a highly polymorphic gene with over 75 codominantly expressed alleles at the protease inhibitor (Pi) locus classified as normal, deficient, dysfunctional or null. The aim of this study was to determine in patients with inflammatory bowel disease: (i) the prevalence of anti-neutrophil cytoplasmic auto-antibodies (ANCA) and their antigen specificities; (ii) alpha1-AT Pi phenotypes; and (iii) possible associations between ANCA, disease activity and deficient alpha1-AT alleles. DESIGN: Study of 95 consecutive patients with ulcerative colitis (UC) and 63 patients with Crohn's disease (CD). METHODS: Diagnosis and disease activity were determined by clinical, endoscopic and histological criteria. ANCA by indirect immunofluorescence (IIF) and Pi phenotyping by isoelectric focusing were performed for all patients. Positive IIF sera were tested in antigen-specific enzyme-linked immunosorbent assay: proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin (LF), lysozyme, human leucocyte elastase (HLE), cathepsin G and bactericidal/permeability increasing protein (BPI). RESULTS: Sixty-one patients with UC (64.2%) and only 11 with CD (17.5%) had ANCA (P < 0.001). Antigen specificities were PR3 (7/61), MPO (3/61), LF (6/61), HLE (1/63) and BPI (10/61) in UC, and PR3 (2/11) and BPI (2/11) in CD. Three PiZ alleles were found, matching the prevalence in the normal French control population. No relationship was found between the presence of ANCA, antibody specificity, disease activity and deficient alpha1-AT alleles. CONCLUSION: ANCA are more frequent in UC than CD and do not correlate with disease activity. ANCA and protease/antiprotease imbalance do not appear to modulate the clinical course of inflammatory bowel disease.