Diagnostic performance of antineutrophil cytoplasmic antibody tests for idiopathic vasculitides: metaanalysis with a focus on antimyeloperoxidase antibodies

OBJECTIVE: The diagnostic value of tests for antimyeloperoxidase antibodies (anti-MPO) for systemic vasculitis is less established than that for cytoplasmic antineutrophil cytoplasmic antibody (cANCA)/antiproteinase 3 antibodies (anti-PR3). Controversy exists regarding the optimal utilization of indirect immunofluorescence (IIF) ANCA testing versus antigen-specific ANCA testing. To summarize the pertinent data, we conducted a metaanalysis examining the diagnostic value of ANCA testing systems that include assays for anti-MPO. METHODS: We performed a structured Medline search and reference list review. Target articles in the search strategy were those reporting the diagnostic value of immunoassays for anti-MPO for the spectrum of systemic necrotizing vasculitides that includes Wegener's granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, and isolated pauci-immune necrotizing or crescentic glomerulonephritis, regardless of other types of ANCA tests. Inclusion criteria required specification of a consecutive or random patient selection method and the use of acceptable criteria for the diagnosis of vasculitis exclusive of ANCA test results. Weighted pooled summary estimates of sensitivity and specificity were calculated for anti-MPO alone, anti-MPO + perinuclear ANCA (pANCA), and anti-MPO/pANCA + anti-PR3/cANCA. RESULTS: Of 457 articles reviewed, only 7 met the selection criteria. Summary estimates of sensitivity and specificity (against disease controls only) of assays for anti-MPO for the diagnosis of systemic necrotizing vasculitides were 37.1% (confidence interval 26.6% to 47.6%) and 96.3% (CI 94.1% to 98.5%), respectively. When the pANCA pattern by IIF was combined with anti-MPO testing, the specificity improved to 99.4%, with a lower sensitivity, 31.5%. The combined ANCA testing system (anti-PR3/cANCA + anti-MPO/pANCA) increased the sensitivity to 85.5% with a specificity of 98.6%. CONCLUSION: These results suggest that while anti-MPO is relatively specific for the diagnosis of systemic vasculitis, the combination system of immunoassays for anti-MPO and IIF for pANCA is highly specific and both tests should be used together given the high diagnostic precision required for these conditions. Because patients with ANCA associated vasculitis have either anti-MPO with pANCA or anti-PR3 with cANCA, and rarely both, a combined ANCA testing system including anti-PR3/cANCA and anti-MPO/pANCA is recommended to optimize the diagnostic performance of ANCA testing.     

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.     

Evaluation of antineutrophil cytoplasmic antibody seroconversion induced by minocycline, sulfasalazine, or penicillamine

OBJECTIVE: Case reports have suggested that minocycline, sulfasalazine, and penicillamine are associated with antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis. This study evaluated ANCA seroconversion due to these agents in serum samples prospectively collected in randomized, double-blind, controlled trials. METHODS: The sources of study sera were 3 clinical trials: 1) a 48-week trial of minocycline for early rheumatoid arthritis, with 64 patients receiving minocycline compared with 68 receiving placebo; 2) a 37-week trial of sulfasalazine for rheumatoid arthritis, with 51 receiving sulfasalazine compared with 38 receiving placebo; and 3) a 104-week trial of penicillamine for early systemic sclerosis, with 15 undergoing high-dose penicillamine treatment versus 12 receiving low-dose penicillamine. ANCA were measured in the baseline and study-end serum samples by indirect immunofluorescence (IIF) for perinuclear ANCA (pANCA) and cytoplasmic ANCA (cANCA) patterns, and by antigen-specific enzyme-linked immunosorbent assay (ELISA) for antibodies to myeloperoxidase (anti-MPO) and proteinase 3 (anti-PR3). Laboratory personnel were blinded to the group identity of the samples. ANCA results were interpreted using an ANCA scoring system that combines the results of IIF and ELISA testing. RESULTS: No patient in any of the active study drug groups demonstrated ANCA seroconversion according to the final interpretation of the combined IIF and ELISA results. Twelve of the 248 patients (5%) were positive for anti-MPO with pANCA at baseline. No subject was positive for anti-PR3 with cANCA. There were no findings suggestive of vasculitis in any of these patients. CONCLUSION: From our study results, there was no suggestion of ANCA seroconversion induced by minocycline, sulfasalazine, or penicillamine. However, these findings do not rule out the possibility of rare, sporadic cases of either ANCA seroconversion or true drug-induced vasculitis with these drugs.     

系统性血管炎抗内皮细胞抗体与抗中性粒细胞胞质抗体的相关性初探

目的以来源于大小血管内皮细胞的两种永生细胞株为底物,检测系统性血管炎血清中抗内皮细胞抗体(AECA),分析其与抗中性粒细胞胞质抗体(ANCA)的相关性。方法细胞酶联免疫吸附试验(Cyto-ELISA)法检测血清中AECA;间接免疫荧光法(IIF)及抗抗体结合内皮细胞表面的蛋白酶3(PR3)、抗MPO-ELISA检测血清中ANCA;IIF及抗PR3、抗MPO-ELISA检测细胞株中PR3及MPO;反转录—聚合酶链反应(RT-PCR)法检测细胞株中PR3及MPOmRNA。结果AECAEA(EA.hy926为底物所测AECA)阳性率33.6%(41/122),AECAHMEC(HMEC-1为底物所测AECA)37.7%(46/122),ANCA35.3%(43/122),AECAEA或AECAHMEC与ANCA串联诊断系统性血管炎敏感性分别为59.8%(73/122)或60.7%(74/122)。AECAEA与ANCA,AECAHMEC与ANCA分别行配对字2检验,差异均无显著性(P>0.05),符合率仅分别为49.2%及51.6%。EA.hy926和HMEC-1中蛋白水平及mRNA水平均无PR3和MPO的表达。结论EA.hy926与HMEC-1中无PR3、MPO蛋白水平的表达,ANCA与AECA可能是两种相互独立的抗体,串联检测可提高诊断敏感性。     

Autoantik?rperdiagnostik rheumatologischer Systemerkrankungen mit pulmonaler Manifestation

The lung is frequently involved in disorders such as connective tissue diseases and small vessel vasculitides. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are often associated with alveolar haemorrhage and pulmonary granulomatous disease, whereas connective tissue disorders present with interstitial lung disease and pulmonary hypertension. Detection of ANCA directed against proteinase 3 or myeloperoxidase is helpful in the diagnosis of ANCA-associated vasculitis, and antinuclear antibodies (ANA) should be determined in cases of suspected connective tissue disease. Autoantibodies determined by ANA differentiation are often very specific for a certain type of connective tissue disease, e.g. antitopoisomerase-1 antibodies for systemic sclerosis. Autoantibody titres may correlate with disease activity or be associated with a special pattern of organ involvement and should be included in the diagnostics of connective tissue diseases and vasculitides.     

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.     

Anti-myeloperoxidase antibodies in patients with rheumatoid arthritis: prevalence, clinical correlates, and IgG subclass.

OBJECTIVES--To determine the prevalence and clinical associations of autoantibodies to myeloperoxidase (MPO) in an unselected series of well-characterised outpatients with rheumatoid arthritis (RA) and to compare the distribution of IgG subclasses of anti-MPO antibodies in these patients with that in patients with systemic vasculitis. PATIENTS AND METHODS--A study was made of 97 patients with RA, who have been seen regularly in this department for up to 20 years, and 29 patients with anti-neutrophil cytoplasmic antibody (ANCA) positive systemic vasculitis. Anti-MPO antibodies were detected using a direct-binding enzyme-linked immunosorbent assay (ELISA) with MPO from human granulocytes as antigen. The IgG subclass of anti-MPO antibodies was determined by ELISA using isotype specific monoclonal antibodies. RESULTS--Anti-MPO antibodies were detected in 12% of patients with RA. Six sera contained IgG anti-MPO antibodies only, 1 IgM only and 5 antibodies of both classes. In the patients with RA the predominant subclasses were IgG1 and IgG3: only 2 sera contained detectable IgG4 antibodies. This was in contrast to patients with vasculitis, in whom most sera contained IgG1, IgG3 and IgG4 anti-MPO antibodies. Anti-MPO antibodies in sera from both patient groups bound only to the native protein. None of the patients studied with RA had evidence of vasculitis affecting the nerves or kidney: three patients (1 positive for anti-MPO antibodies and 2 negative) had cutaneous vasculitis. In the patients with RA, positivity for anti-MPO antibodies was associated with nodules and number of active joints. Three patients with anti-MPO antibodies, and none without, had pulmonary fibrosis. CONCLUSIONS--Twelve per cent of a group of unselected outpatients with RA, but without evidence of major systemic vasculitis, had anti-MPO antibodies in their serum. Positivity for anti-MPO antibodies was more common in patients with nodular disease and lung involvement but not in patients with cutaneous vasculitis. IgG4 sub-class anti-MPO antibodies were present in 90% of sera from patients with ANCA-positive vasculitis and only 2/11 (18%) of anti-MPO antibody containing sera from patients with RA.     

Lung vasculitis and alveolar hemorrhage: pathology

Pulmonary vasculitides are a diverse group of limited and systemic disorders associated with inflammation of pulmonary vessels and parenchyma. These diseases often have distinctive clinical, serological, and histopathological features-extrapulmonary sites of involvement, circulating autoantibodies, predispositions for small or large vessels, and others. Some have characteristic inflammatory lesions; others are characterized by the absence of such lesions. Frequently pathological findings overlap, rendering classification, and diagnosis a challenge. The anti-neutrophil cytoplasmic antibody (ANCA)-associated small-vessel diseases constitute the major pulmonary vasculitides. These include Wegener granulomatosis (WG), Churg Strauss syndrome (CSS), and microscopic polyangiitis (MPA). Less frequently, diseases such as polyarteritis nodosa, Takayasu arteritis, Beh?et syndrome, and connective tissue diseases may involve pulmonary vessels, but these entities are better associated with extrapulmonary disease. Diffuse alveolar hemorrhage (DAH) is a severe manifestation of pulmonary vasculitis. DAH is most commonly seen in small-vessel vasculitides, specifically MPA and WG. Other syndromes associated with DAH include Goodpasture syndrome, Henoch-Sch?nlein purpura, and systemic lupus erythematosus. Less commonly, DAH may be secondary to infection or drugs/toxins. Furthermore, in the absence of discernable systemic disease, DAH may be idiopathic-referred to as isolated pulmonary capillaritis (IPC) or idiopathic pulmonary hemosiderosis (IPH), depending on the presence of capillaritis.     

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.     

The antineutrophil cytoplasmic antibody-associated vasculitides

Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome are small- to medium-vessel vasculitides linked by overlapping pathology and the presence of antineutrophil cytoplasmic antibodies (ANCA). Commonly referred to as the ANCA-associated vasculitides, these diseases are challenging to diagnose and to treat. Distinguishing the ANCA-associated vasculitides from other forms of vasculitis or nonvasculitic processes (such as infection) can be particularly difficult. This review describes the clinical and pathologic hallmarks of the ANCA-associated vasculitides, discusses the role of ANCA assays in diagnosis and treatment, and outlines an approach to the evaluation and management of these diseases.     

Antineutrophil cytoplasmic antibodies: a still-growing class of autoantibodies in inflammatory disorders.

Antineutrophil cytoplasmic antibodies (ANCA) have been described as sensitive and specific markers for active Wegener's granulomatosis (WG). ANCA in WG produce a characteristic cytoplasmic staining pattern of neutrophils (c-ANCA) and are directed against proteinase 3 (Pr3), a serine protease from the azurophilic granules. c-ANCA, more or less equivalent to anti-Pr3, occur in more than 90% of patients with extended WG, in 75% of patients with limited WG without renal involvement, and in some 40% to 50% of patients with vasculitic overlap syndromes suggestive of WG such as microscopic polyarteritis. The presence of c-ANCA is highly specific for those diseases (greater than 98%). Changes of levels of c-ANCA precede disease activity and may be used as guidelines for treatment. Antibodies producing a perinuclear staining of ethanol-fixed neutrophils (p-ANCA) occur in a wide range of diseases. They are directed against different cytoplasmic constituents of neutrophils. Among those, antibodies to myeloperoxidase are found in patients with idiopathic crescentic glomerulonephritis, the Churg-Strauss syndrome, polyarteritis nodosa with visceral involvement, and vasculitic overlap syndromes. Their specificity for this group of necrotizing vasculitides is high (94% to 99%), although they may occur in patients with hydralazine-induced glomerulonephritis, anti-glomerular basement membrane disease, and possibly in some patients with idiopathic systemic lupus erythematosus. Antibodies to leukocyte elastase are incidentally found in patients with vasculitic disorders, whereas lactoferrin antibodies are detected in patients with primary sclerosing cholangitis with or without ulcerative colitis and in rheumatoid arthritis. Their diagnostic significance awaits further studies. p-ANCA of undefined specificity may distinguish ulcerative colitis (sensitivity of 75%) from Crohn's disease (sensitivity of 20%). p-ANCA also occur in autoimmune liver diseases: in 75% of patients with chronic active hepatitis, in 60% to 85% of those with primary sclerosing cholangitis, and in about 30% of patients with primary biliary cirrhosis. Finally, p-ANCA are detected in chronic arthritides and in some 5% of healthy controls. Assessment of their diagnostic value has to await further characterization of the antigens involved, allowing the development of antigen-specific assays.     

Clinical value of antineutrophil cytoplasmic antibodies

This article presents a brief review of the clinical value of antineutrophil cytoplasmic antibodies (ANCA) in the diagnosis and management of patients with various forms of vasculitis. ANCA assay methodology and testing recommendations are reviewed. The patterns of reactivity of ANCA observed by indirect immunofluorescence, the antigens recognized by ANCA, and the sensitivity, specificity, and positive predictive value of ANCA for diagnosis of different vasculitides are described. In addition, the spectrum of drugs and nonvasculitic diseases that are associated with ANCA and need to be differentiated from true ANCA-positive vasculitides are reviewed. When properly utilized and cautiously interpreted, ANCA are a useful, noninvasive diagnostic tool in the differential diagnosis of vasculitis.     

Circulating myeloperoxidase and anti-myeloperoxidase antibody in patients with vasculitis

To evaluate a role of myeloperoxidase (MPO) and antibody to myeloperoxidase (anti-MPO) in vasculitis, MPO and anti-MPO were determined by enzyme-linked immunosorbent assays in sera from 43 patients with vasculitis, 40 with rheumatoid arthritis, 36 with systemic lupus erythematosus (SLE), 23 with mixed connective tissue disease, 13 with systemic sclerosis, 22 with polymyositis/dermatomyositis, 18 with Sj?gren's syndrome, and 30 normal controls. Kidney and lung sections from patients with vasculitis were stained for MPO. Anti-MPO titers were significantly higher (p<0.005) in the patients with vasculitis (mean+/- SD absorbance at 405 nm: 0.53 +/- 0.37) than in any other groups (0.15 +/- 0.04 to approximately 0.21 +/- 0.11). MPO levels in patients with vasculitis were comparable with those in patients with other diseases except SLE. In two patients with vasculitis, anti-MPO decreased sharply with simultaneous increases in MPO 1-2 weeks after they developed pulmonary hemorrhage. Numerous cells positive for MPO infiltrated the Bowman's spaces. These results indicate that MPO may contribute to the pathogenesis of vasculitis and a sudden fall in anti-MPO may predict a poor prognosis in some cases.     

酶联免疫吸附测定法检测抗髓过氧化物酶IgG亚型分布及其临床意义

目的：观察抗髓过氧化物酶（MPO）IgG各亚型（IgG1、IgG2、IgG3、IgG4)在小血管炎患者的分布及其与临床病情的关系,以探讨IgG亚型是否能更准确地反映病情变化。方法：选取30例抗MPO抗体阳性的原发性小血管炎患者,对其活动期和缓解期血清采用抗原特异性ELISA法分别测定活动期抗MPO IgG各亚型的阳性率及缓解期的转阴率,并与抗中性粒细胞胞质抗体（ANCA）总IgG的变化进行对比。结果：临床活动期抗MPO－ANCA IgG的4种亚型均为阳性,以IgG4最高;而缓解期以IgG1和IgG3下降为著,其中IgG3的转阴率最高,且显著高于总IgG。结论：IgG3的变化与病情活动密切相关,在一定程度上可以代替总IgG监测病情的发展。IgG4在疾病的活动期与缓解期都显著增高,可能与反复的抗原刺激和慢性的免疫反应有关。     

Autoantibodies in systemic vasculitis

Abstract We have studied 495 sera that were referred to us from patients suspected on clinical and/or histological grounds to have a small vessel vasculitis. These sera were tested for antibodies against neutrophil cytoplasm antigens (anti-neutrophil cytoplasm antibodies, ANCA) using assays based on neutrophil acid extract, myeloperoxidase and elastase. Such antibodies are commonly found in Wegener's granulomatosis (WG) and microscopic polyarteritis (MPA), and sometimes in other small vessel vasculitides. One hundred and twenty-six of these sera (25%) were positive in the acid extract ELISA, 68 (14%) in the assay for anti-myeloperoxidase antibodies and 35 (16%) in the assay for anti-elastase antibodies. A total of 166 sera (34%) were positive for antibodies against neutrophil cytoplasm constituents. No ANCA, anti-myeloperoxidase or anti-elastase antibodies were detected in 26 convalescent sera from patients either with WG or MPA, or who had previously been positive. The mean time between positive and negative sera was eight weeks (range three weeks to six months) and three out of three who relapsed again developed ANCA of the same specificity as the original sera. Of the 228 sera also tested for anti-GBM antibodies, 13 (5.7%) were positive. All these contained antibodies against neutrophil cytoplasm constituents (three against the acid extract, eight against myeloperoxidase and two against elastase). Forty-nine of the 74 sera (66%) tested for ANA were positive. Twenty-nine (39%) had a speckled and 20 (27%) had a homogeneous pattern. Twenty of these were positive for ANCA or anti-myeloperoxidase or anti-elastase antibodies and 29 were negative. ANCA, anti-myeloperoxidase and anti-elastase antibodies are common in patients in whom a vasculitis is suspected on clinical and/or histological grounds. The presence of these antibodies correlates with disease activity. Anti-GBM antibodies and ANA are not uncommon in this group. A single ELISA for ANCA is insufficient to detect all antibodies directed against neutrophil cytoplasm constituents. (Aust NZ J Med 1991; 21: 433–437.)     

Significance of antineutrophil cytoplasmic antibody in adult patients with Henoch-Sch(o)nlein purpura presenting mainly with gastrointestinal symptoms

AIM: To test the clinical significance of antineutrophil cytoplasmic antibody (ANCA) in evaluation of adult Henoch-Schonlein purpura (HSP) patients presenting mainly with abdominal symptoms.METHODS: Twenty-eight consecutive HSP patients who presented predominantly with abdominal symptoms were enrolled in this study. Control subjects included 27 age-and sex-matched patients with peptic ulcer disease, colon cancer, acute gastroenteritis, irritable bowel syndrome and colonic polyps. ANCA was measured by indirect immunofluorescence (IIF) in all patients, and follow-up ELJSA was performed in patients with positive IIF tests.RESULTS: ANCA was detected in 9 HSP patients by IIF (2 were positive for c-ANCA and 7 were positive for p-ANCA). No ANCA was found in the control group. The sensitivity and specificity of a positive ANCA test (either c- or p-ANCA) were 32.1% and 100% respectively. Only one out of the 9 patients with positive ANCA by IIF had positive ANCA by ELISA and the antigen was myeloperoxidase (MPO). The patients positive for ANCA had higher HSP clinical scores, and were more likely to have renal function impairment. Patients with late purpura development were also associated with more severe clinical manifestations.CONCLUSION: A positive ANCA test is associated with more severe symptoms in HSP. After inflammatory bowel disease is excluded, a positive ANCA test provides a clue to the diagnosis of HSP presenting predominantly with abdominal symptoms.     

What do antineutrophil cytoplasmic antibodies (ANCA) tell us?

Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against antigens found in the cytoplasmic granules of neutrophils and monocytes. ANCA testing is usually performed to help diagnose or exclude Wegener's granulomatosis and microscopic polyangiitis. The three most commonly used assays are indirect immunofluorescence (IIF) and the direct and 'capture' enzyme-linked immunosorbent assays (ELISAs) for ANCA directed against proteinase 3 (PR3) and myeloperoxidase (MPO). Although the International Consensus Statement for Testing and Reporting ANCA recommends that all sera are screened for ANCA by IIF and that IIF-positivity is confirmed by direct ELISAs, some laboratories test by direct ELISA alone, others screen with direct ELISA and confirm positive sera by IIF, and a few use capture ELISAs. This chapter discusses the various forms of vasculitis associated with ANCA, the usefulness of each of the ANCA assays and how ANCA testing can be used in the management of patients with small-vessel vasculitis.     

Significance of antineutrophil cytoplasmic antibody in adult patients with Henoch-Schönlein purpura presenting mainly with gastrointestinal symptoms

AIM： To test the clinical significance of antineutrophil cytoplasmic antibody （ANCA） in evaluation of adult Henoch-Schonlein purpura （HSP） patients presenting mainly with abdominal symptoms. METHODS： Twenty-eight consecutive HSP patients who presented predominantly with abdominal symptoms were enrolled in this study. Control subjects included 27 ageand sex-matched patients with peptic ulcer disease, colon cancer, acute gastroenteritis, irritable bowel syndrome and colonic polyps. ANCA was measured by indirect immunofluorescence （IIF） in all patients, and follow-up ELISA was performed in patients with positive IIF tests. RESULTS： ANCA was detected in 9 HSP patients by IIF （2 were positive for c-ANCA and 7 were positive for p-ANCA）. No ANCA was found in the control group. The sensitivity and specificity of a positive ANCA test （either c- or p-ANCA） were 32.1% and 100% respectively. Only one out of the 9 patients with positive ANCA by IIF had positive ANCA by ELISA and the antigen was myeloperoxidase （MPO）. The patients positive for ANCA had higher HSP clinical scores, and were more likely to have renal function impairment. Patients with late purpura development were also associated with more severe clinical manifestations. CONCLUSION： A positive ANCA test is associated with more severe symptoms in HSP. After inflammatory bowel disease is excluded, a positive ANCA test provides a clue to the diagnosis of HSP presenting predominantly with abdominal symptoms.     

Anti-neutrophil cytoplasmic antibodies (ANCA) in malaria

Various autoantibodies like anti-nuclear antibodies (ANA), anti-double stranded DNA (anti-dsDNA), anti-histone antibodies (AHA), anti-neutrophil cytoplasmic antibodies (ANCA), anti-myeloperoxidase (anti-MPO), anti-proteinase3 (anti-PR3) and anti-lactoferrin (anti-LF) antibodies were studied in 173 acute hospitalised patients suffering from malaria of which 160 patients had P. falciparum and remaining 13 had P. vivax infection. Standard methods like indirect immunofluorescence (IIF) microscopy along with Confocal microscopy and ELISA were used for identifying and quantifying the autoantibodies and IIF patterns on PMN and HL-60 cells were studied for ANCA classification. Also HEp-2 cells were used for ANA detection, while estimation of anti-dsDNA, AHA, anti-MPO, anti-PR3 and anti-LF were tested using ELISA. Sera from malaria patients showed prominent immunofluorescence staining patterns where 23.8% cases had ANA in P. falciparum group as compared to 15.4% in P. vivax group and ANCA was found to be present in 20% in P. falciparum and 15.4% in P. vivax group. An interesting observation was that, of the total ANCA positives, 59% had p-ANCA, 5.9% had c-ANCA and 44.1% of the cases showed the 'atypical' or X-ANCA pattern. When p-ANCA positivity was compared with c-ANCA positivity among these patients, a good statistical correlation was noted with OR = 16, chi 2 = 16.43, EF = 0.46 and p-value = 5.037E 0.5. ELISA showed 31.2% anti-MPO and 6.2% anti-PR3 in P. falciparum cases while the two ANCA positive cases in P. vivax had anti-MPO. Anti-LF was found to be present in 40.6% cases. Neither the P. falciparum nor P. vivax contained autoantibodies with specificities similar to the characteristic lupus autoantibodies such as double stranded DNA (dsDNA). ANCA positivity develops in some types of malarial infection also with the presence of various autoantibodies which is important from a clinical point of view and should be carefully evaluated in those geographic areas where malaria is endemic. It also alerts us to the fact, whether in cases of repeated malarial infections in susceptible individuals, vasculitic disorders, which through ANCA pathways develop, could lead to renal and other complications.     

Prevalence and clinical significance of antineutrophil cytoplasmic antibodies in Churg-Strauss syndrome

OBJECTIVE: Churg-Strauss syndrome (CSS) is classified among the so-called antineutrophil cytoplasmic antibody-associated systemic vasculitides (AASVs) because of its clinicopathologic features that overlap with the other AASVs. However, while antineutrophil cytoplasmic antibodies (ANCAs) are consistently found in 75-95% of patients with Wegener's granulomatosis or microscopic polyangiitis, their prevalence in CSS varies widely and their clinical significance remains uncertain. We undertook this study to examine the prevalence and antigen specificity of ANCAs in a large cohort of patients with CSS. Moreover, we evaluated the relationship between ANCA positivity and clinicopathologic features. METHODS: Immunofluorescence and enzyme-linked immunosorbent assay were used to determine the presence or absence of ANCAs in 93 consecutive patients at the time of diagnosis. The main clinical and pathologic data, obtained by retrospective analysis, were correlated with ANCA status. RESULTS: ANCAs were present by immunofluorescence in 35 of 93 patients (37.6%). A perinuclear ANCA (pANCA) pattern was found in 26 of 35 patients (74.3%), with specificity for myeloperoxidase (MPO) in 24 patients, while a cytoplasmic ANCA pattern, with specificity for proteinase 3, was found in 3 of 35 patients (8.6%). Atypical patterns were found in 6 of 30 patients with anti-MPO antibodies (20.0%). ANCA positivity was associated with higher prevalences of renal disease (51.4% versus 12.1%; P < 0.001) and pulmonary hemorrhage (20.0% versus 0.0%; P = 0.001) and, to a lesser extent, with other organ system manifestations (purpura and mononeuritis multiplex), but with lower frequencies of lung disease (34.3% versus 60.3%; P = 0.019) and heart disease (5.7% versus 22.4%; P = 0.042). CONCLUSION: ANCAs are present in approximately 40% of patients with CSS. A pANCA pattern with specificity for MPO is found in most ANCA-positive patients. ANCA positivity is mainly associated with glomerular and alveolar capillaritis.