Contribution of immunofluorescence to identification and characterization of antineutrophil cytoplasmic antibodies in inflammatory bowel disease

We evaluated the combined use of different fixatives for the identification of atypical perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA) in patients with inflammatory bowel diseases (IBD) by indirect immunofluorescence (IIF). Sera from 59 ulcerative colitis (UC) and 37 Crohn’s disease (CD) patients, and from 64 healthy controls were studied. The IIF on ethanol-, formalin-, and methanol-fixed neutrophils was used for the detection of ANCA. Enzyme linked immunosorbant assay (ELISA) was performed to identify the antigens recognized by ANCA. ANCAs were present in 35 of 59 (59.3%) UC patients and in 10 of 37 (27.02%) CD patients. Atypical p-ANCA positivity was strongly associated with UC disease (44.1% in UC vs. 8.1% in CD; p = 0.0002). The combined application of different fixatives contributed to make easy the differentiation between typical p-ANCA and atypical p-ANCA. Atypical p-ANCA determination appears to be a useful parameter for the distinction between UC and CD.     

Detection of anti-neutrophil cytoplasmic autoantibodies using the promyelocytic HL-60 cell-line.

IgG autoantibodies against antigen in the cytoplasm of cells of the neutrophil-monocyte cell lineage have been found in the sera of patients with Wegener's granulomatosis (WG). The indirect immunofluorescence test (IFT) is proving to be a valuable screening test for these antibodies, but obtaining neutrophils for substrate is time-consuming, and interpretation of the fluorescence patterns in ethanol-fixed cells requires considerable experience. We report an improved IFT using HL-60 cells as substrate. The myeloid reactivity of HL-60 cells was characterized and compared to that of neutrophils, with and without prior ethanol fixation. In contrast to neutrophils, myeloperoxidase (MPO) was more completely extracted from HL-60 cells by prior ethanol fixation, eliminating the confusion inherent in trying to distinguish anti-MPO antibodies from Wegener's granulomatosis associated anti-neutrophil cytoplasmic autoantibodies (WG-ANCA) in the IFT. The WG-ANCA reactivity remained intact with ethanol fixation, producing a distinct crescent and half-moon pattern of specific immunofluorescence. This WG-ANCA positive pattern was found in 25 sera from 11 WG patients and was absent in over 1200 control sera from patients referred for autoantibody testing.     

Clinical and pathophysiological significance of anti-neutrophil cytoplasmic autoantibodies in vasculitis syndromes

Necrotizing vasculitis of small blood vessels is a rare condition, but when it affects important organs it can lead to life-threatening organ damage and death. Thus, recognizing these conditions at an early stage before they spread to become systemic is a constant challenge to clinical medicine. The objectives of this review are: to give advice on clinical indications for ANCA diagnostics and laboratory procedures for highly specifically detecting the most important ANCA; to provide some data on the autoantigens involved in ANCA reactivity in small vessel vasculitides; and to discuss at the occurrence of ANCA in different vasculitic populations and at different stages of disease. One important task for the near future will be to standardize the assays used for ANCA detection/quantification and to harmonize the results given to clinicians by ensuring that international reference reagents are used by laboratories and the diagnostic industry. Finally, the author has attempted to summarize the role that ANCA are currently believed to play in the immuno-inflammatory events that take place in tissues and that affect small vessels in idiopathic vasculitis. The review concludes that the presence of ANCA is likely to become an important criterion for diagnosing idiopathic small vessel vasculitis.     

Clinical and pathophysiological significance of anti-neutrophil cytoplasmic autoantibodies in vasculitis syndromes

Abstract

Necrotizing vasculitis of small blood vessels is a rare condition, but when it affects important organs it can lead to life-threatening organ damage and death. Thus, recognizing these conditions at an early stage before they spread to become systemic is a constant challenge to clinical medicine. The objectives of this review are: to give advice on clinical indications for ANCA diagnostics and laboratory procedures for highly specifically detecting the most important ANCA; to provide some data on the autoantigens involved in ANCA reactivity in small vessel vasculitides; and to discuss at the occurrence of ANCA in different vasculitic populations and at different stages of disease. One important task for the near future will be to standardize the assays used for ANCA detection/quantification and to harmonize the results given to clinicians by ensuring that international reference reagents are used by laboratories and the diagnostic industry. Finally, the author has attempted to summarize the role that ANCA are currently believed to play in the immuno-inflammatory events that take place in tissues and that affect small vessels in idiopathic vasculitis. The review concludes that the presence of ANCA is likely to become an important criterion for diagnosing idiopathic small vessel vasculitis.     

Value of anti-neutrophil cytoplasmic autoantibodies and other laboratory parameters in follow-up of vasculitis

The presence of anti-neutrophil cytoplasmic autoantibodies (ANCA), detected by indirect immunofluorescence, is of high sensitivity and specificity in the diagnosis of Wegener's granulomatosis and related diseases, associated with vasculitis. Titres of immunofluorescence are thought to closely reflect disease activity. In a retrospective series of 266 sera of 23 patients with at least one positive test for ANCA, disease activity was correlated with ANCA, assayed by immunofluorescence and by enzyme-linked immunosorbent assay; with rheumatoid factor and with erythrocyte sedimentation rate. All tests were of limited value in predicting disease activity or relapse. A normal sedimentation rate and, to a lesser extent, a negative result of ANCA-immunofluorescence, were useful in excluding active disease.     

Clinical features of patients with anti-neutrophil cytoplasmic autoantibodies targeting native myeloperoxidase antigen

Objectives

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are useful diagnostic markers in systemic vasculitic disorders with small-vessel involvement, but depending on the particular test used, the myeloperoxidase (MPO)-ANCA results are variable. In the present study, we performed a comparative analysis between our originally developed nMPO-ANCA assay that targets the native MPO antigen and other commercially available assays using sera of patients with clinical features of ANCA-associated vasculitis (AAV).

Methods

Sera of 24 patients strongly suspected of having AAV were examined for the presence of MPO-ANCAs by our nMPO-ANCA assay and by other commercial-based MPO-ANCA assays. These results were correlated to indirect immunofluorescence microscopy staining patterns and patient clinical parameters.

Results

Eighteen out of 24 patients (75 %) were positive for nMPO-ANCA, compared with 13 out of 24 patients (54 %) by one of the most frequently used commercial-based MPO-ANCA enzyme-linked immunosorbent assays (ELISAs) in Japan. Interestingly, the patients who tested positive with our nMPO-ANCA assay alone showed clinical features of AAV marked by continuous fever, polyarthritis, and mild nephritis. The titers of nMPO-ANCA decreased in association with clinical improvement after treatment.

Conclusions

Our data suggest that a positive nMPO-ANCA result, which identifies antibodies to human native MPO antigen, correlates with AAV disease activity. Moreover, the nMPO-ANCA test has clinical utility in detecting AAV-affected patients who have tested negative using commercially available assays.     

In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA-antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti-myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.     

Disease associations and pathogenic role of antineutrophil cytoplasmic autoantibodies in vasculitis.

Antineutrophil cytoplasmic autoantibodies are a useful diagnostic serologic marker for a variety of well-known vasculitic syndromes, including Wegener's granulomatosis, polyarteritis nodosa (especially microscopic polyarteritis nodosa), Churg-Strauss syndrome, and pulmonary-renal syndrome with alveolar capillaritis. Although most patients with antineutrophil cytoplasmic autoantibody-associated disease have systemic disease, disease limited to one organ does occur, eg, isolated necrotizing glomerulonephritis, isolated respiratory tract disease, or isolated orbital disease. Antineutrophil cytoplasmic autoantibody titers may be useful in modulating treatment regimens. There is in vitro evidence that antineutrophil cytoplasmic autoantibodies are directly involved in the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitides.     

Vasculitis associated with anti-neutrophil cytoplasmic autoantibodies in rheumatoid arthritis. Report of a case of microscopic polyangiitis and another case of Wegener's granulomatosis

Vasculitis is an uncommon complication of rheumatoid arthritis that is associated with a clear increase in morbidity and mortality, although systemic manifestations such as glomerulonephritis, cerebral vasculitis or pulmonary vasculitis are very rare. Systemic vasculitis with renal involvement is associated with overt polyarthritis in less than 5% and association with rheumatoid arthritis is exceptional. Determination of anti-neutrophil cytoplasmic autoantibodies (ANCA), used in the appropriate clinical context, has become an important diagnostic tool in small-vessel systemic vasculitides. We present two patients with rheumatoid arthritis who subsequently developed systemic vasculitis. ANCA determination was decisive in the early diagnosis of these patients.     

抗中性粒细胞胞质抗体相关小血管炎诊断和治疗中的问题与对策

抗中性粒细胞胞质抗体(ANCA)相关小血管炎(AAV)包括韦格纳肉芽肿病(WG)、显微镜下多血管炎(MPA)和变应性肉芽肿性血管炎(CSS),是西方国家常见的自身免疫性疾病.我国发病率不详,但是随着ANCA检测的推广和应用,发现我国也不少见[1].然而我国临床医生对此类疾病认识不足,常导致误诊、漏诊,部分患者确诊后在治疗上也存在较大随意性.本文拟对诊断和治疗中经常遇到的一些问题进行探讨.     

Clinical course of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and systemic vasculitis. The Glomerular Disease Collaborative Network

OBJECTIVES: To determine the spectrum of clinical manifestations in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis; to determine renal and patient survival in these patients; to compare survival among patients treated with corticosteroids alone, corticosteroids plus intravenous cyclophosphamide or corticosteroids plus oral cyclophosphamide; and to assess the correlation of disease manifestations and treatment response with ANCA subtypes and serial autoantibody titers. DESIGN: Inception cohort study; mean follow-up of 24 months. SETTING: Collaborative network of 120 university and private practice nephrologists (The Glomerular Disease Collaborative Network). PARTICIPANTS: Seventy patients with ANCA and pauci-immune necrotizing and crescentic glomerulonephritis, of whom 59 were treated with either corticosteroids alone (14 patients), corticosteroids plus oral cyclophosphamide (30 patients), or corticosteroids plus intravenous cyclophosphamide (15 patients). MAIN RESULTS: Of the 70 patients, 18 had renal-limited disease (idiopathic crescentic glomerulonephritis); 15, nonpulmonary extrarenal disease consistent with polyarteritis nodosa; and 37, pulmonary disease consistent with Wegener granulomatosis or alveolar capillaritis. There were overlapping manifestations of disease between patients with autoantibodies producing a cytoplasmic pattern and patients with autoantibodies producing a perinuclear pattern; however, the perinuclear pattern occurred more frequently in patients with renal-limited disease. Renal and patient survival was 75% at 24 months, and no difference in survival was seen between patients with renal-limited disease and those with systemic disease. No differences in survival were seen between patients treated with oral cyclophosphamide and those treated with intravenous cyclophosphamide; however, the comparative data from patients treated with corticosteroids alone were inconclusive. In general, autoantibody titers correlated with response to treatment and disease activity, but there were exceptions. CONCLUSIONS: Patients with ANCA have various forms of necrotizing vascular inflammation, ranging from renal-limited disease to widespread systemic vasculitis, including polyarteritis nodosa and Wegener granulomatosis. Oral corticosteroids with either oral or intravenous cyclophosphamide appear to be equally effective therapy for ANCA-associated glomerulonephritis.     

炎症性肠病患者血清中自身抗体检测的临床意义

目的研究抗酿酒酵母抗体(ASCA)和抗中性粒细胞胞浆抗体(pANCA)在炎症性肠病诊断与鉴别诊断中的意义。方法间接免疫荧光生物薄片法检测29例溃疡性结肠炎(UC)、34例克罗恩病(CD)及25例正常对照者血清中ASCA和pANCA的表达。结果pANCA在CD组、UC组和正常对照组中的阳性率分别为47.1%、69.0%和16.0%,UC组显著高于CD组(P<0.05)和正常对照组(P<0.05)。ASCA在上述3组中的阳性率分别为11.8%、58.6%和8.0%,UC组亦显著高于CD组(P<0.05)和正常对照组(P<0.05)。ASCA /pANCA-诊断CD的敏感性、特异性、阳性预测值分别是0、89.7%和0;pANCA /ASCA-诊断UC的敏感性、特异性和阳性预测值分别是20.7%、64.7%和33.3%。结论ASCA和pANCA阳性有利于炎症性肠病的诊断却不能敏感地筛选患者;ASCA和pANCA联合检测不能作为汉族UC和CD鉴别诊断的指标。     

The diagnostic role and clinical association of serum proteinase 3 anti-neutrophil cytoplasmic antibodies in Chinese patients with inflammatory bowel disease

Abstract

Background and aim: Accurate differentiation of patients with ulcerative colitis (UC) or Crohn’s disease (CD) is important for appropriate therapy and prognosis. This study was designed to explore the utility of proteinase 3 anti-neutrophil cytoplasmic antibodies (PR3-ANCA) in the diagnosis of Chinese patients with inflammatory bowel disease (IBD).

Methods: Blood samples were collected from 216 Chinese patients, including 175 IBD and 41 colorectal polyps (disease control). Clinical characteristics were extracted from electronic medical records.

Results: Serum PR3-ANCA were increased in UC patients compared to those with CD or colorectal polyps (p?<?.0001). PR3-ANCA was negative in colorectal polyps and there was no significant difference between CD and colorectal polyps (p?>?.05). Using the cut-off value of 20 chemiluminescent units (CU) provided by manufacturer, the positive rate of PR3-ANCA was higher in UC than CD (41.7% vs. 1.1%; p?<?.0001). Receiver operating characteristic (ROC) analysis demonstrated an area under the curve (AUC) of 0.89 (95% CI: 0.84–0.95; p?<?.0001) for differentiating UC from CD and suggested an optimized cutoff of 7.3 CU which improved sensitivity from 41.7% to 57.1%, while maintaining a specificity of 98.9%. PR3-ANCA in severe UC patients were higher than those with moderate UC (p?<?.05), no difference was found between those in remission or with mild or moderate activity (p?>?.05).

Conclusions: Serum PR3-ANCA is a potentially useful clinical biomarker in Chinese patients with IBD. A modified cut-off value of 7.3 CU improves the performance for distinguishing UC from CD.