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Background

ANCA stands for anti-neutrophilic cytoplasmic antibodies. These are a class of antibodies identified by immunofluorescence. In general, serologic testing for ANCA is recommended for patients with:

Glomerulonephritis
Pulmonary hemorrhage, especially Pulmonary-Renal syndrome
Cutaneous vasculitis with systemic features
Mononeuritis multiplex or other peripheral neuropathy
Long standing sinusitis or otitis
Subglottic tracheal stenosis
Retro-orbital mass

If a patient has 1 of these clinical features or other strong clinical evidence for small-vessel vasculitis, the positive ANCA test will provide over 90% confirmation of a vasculitis.

ANCA Microscopic Polyangiitis Wegener's Granulomatosis Churg-Strauss Syndrome
PR3-ANCA 40% 75% 10%
MPO-ANCA 50% 20% 60%
Negative 10% 5% 30%
ANCA TYPE IF PATTERN ANTIGEN CHARACTERIZATION
C-ANCA
Coarsely granular cytoplasmic pattern with central and interlobular accentuation PR3 Wegener's granulomatosis
Microscopic polyarteritis
Churg-Strauss syndrome
C-ANCA (atypical)
Flat, nongranular diffuse cytoplasmic pattern Mostly unknown, MPO and bactericidal/permeability increasing protein in some case ?
P-ANCA

Perinuclear cytoplasmic with nuclear extension

Caused by artifactual redistribution of the MPO antigen to the nucleus during substrate preparation

MPO

Microscopic polyarteritis
Necrotizing and crescentic glomerulonephritis
Churg-Strauss syndrome
Classic polyarteritis nodosa
Wegener's granulomatosis
SLE
Rheumatoid arthritis
Chronic IBD

  Without nuclear extension Elastase
Cathepsin G
Lactoferrin
Lysozyme
 
Atypical ANCA
(x-ANCA)
Snow-drift pattern, mixture of C- and P-ANCA pattern Nonspecific, or lactoferrin, lysozyme, beta-glucuronidase, cathepsin G, and so forth

Primary sclerosing cholangitis
Primary biliary cirrhosis
Autoimmune hepatitis
SLE
RA

OUTLINE

Reference Methods  
Clinical Utility Heart
Hemodialysis patients
Inflammatory bowel disease
Systemic lupus erythematosus
Vasculitis
Wegener's Granulomatosis
Interfering Diseases or Substances that Alter Levels  
Commonly Used Terms  
Internet Links  

 

LABORATORY CHARACTERIZATION
LABORATORY MARKERS  
Enzyme Immunoassays (EIA)
Utilizes purified MPO and PR3 as substrate
Indirect Immunofluorescence (IFA)
Should utilize a confirmatory EIA since there are a large number of false positive results with IFA alone

The value of indirect immunofluorescence and solid phase techniques for ANCA detection. A report on the first phase of an international cooperative study on the standardization of ANCA assays.

EEC/BCR Group for ANCA Assay Standardization. Hagen EC, Andrassy K, Chernok E, Daha MR, Gaskin G, Gross W, Lesavre P, Ludemann J, Pusey CD, Rasmussen N, et al.

Department of Nephrology, University Hospital Leiden, Netherlands.

J Immunol Methods 1993 Feb 26;159(1-2):1-16 Abstract quote

This study describes the results of phase I of an international effort to develop and standardize assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). 12 sera, four of which were selected for their potential to cause problems in the detection of various ANCA specificities, were analyzed in the standard indirect immunofluorescence (IIF) test and in ELISAs for ANCA routinely performed in the seven participating laboratories. The IIF methodology differed with respect to the dilution of the serum being screened and the concentration of the conjugate used. Results from sera with high ANCA titers were similar, although the quantitative values could not be compared. In sera containing rheumatoid factor and anti-nuclear antibodies (ANA), ANCA-unrelated staining patterns were observed. Six antigen preparations were used in ELISA for the detection of cANCA. In ELISA with purified proteinase-3 all three cANCA sera were positive, but not anti-myeloperoxidase (MPO) or anti-lactoferrin (LF) positive sera. The other assays were less sensitive or gave inconsistent results. Various preparations of purified MPO and LF used in ELISA were readily recognized by anti-MPO and anti-LF positive sera. From this study it can be concluded that the IIF test, although performed with different methods, shows comparable results using strongly positive sera. In general solid phase assays for cANCA detection are not well standardized and need improvement although the purified proteinase-3 ELISA is possibly an exception. MPO and LF can be used in ELISA procedures for the detection of pANCA-related antibodies.

Comparison of eight commercial kits for quantitation of antineutrophil cytoplasmic antibodies (ANCA).

Wang G, Csernok E, de Groot K, Gross WL.

Department of Rheumatology, University of Lubeck, Lubeck/Rheumaklinik Bad Bramstedt GmbH, Germany.

J Immunol Methods 1997 Oct 27;208(2):203-11 Abstract quote

Antineutrophil cytoplasmic antibodies (ANCA) are used as diagnostic markers for systemic vasculitis. However, the specificity and sensitivity of ANCA detection differs from centre to centre due in large part to variations in methodology.

We compared 8 commercial ELISA kits and an in-house method (HM) for their specificity and sensitivity in detecting ANCA against proteinase 3 (PR3-ANCA, 7 kits) and meyloperoxidse (MPO-ANCA, 8 kits).

Sera from 5 patients with systemic lupus erythematosus (SLE), 28 with Wegener's granulomatosis (WG), 22 with microscopic polyangiitis (MPA), 5 with idiopathic rapidly progressive glomerulonephritis (RPGN), and 5 healthy controls were examined by both the indirect immunofluorescence technique (IFT) and the ELISA kits. Sera from healthy controls and patients with SLE or cANCA-negative WG were shown to be PR3-ANCA negative by all 7 PR3-ANCA kits. In 25 cANCA-positive sera from WG patients, PR3-ANCA positivity ranged from 44% to 84%. An absolute concordance among the 7 kits was noted in 56% of the cANCA-positive samples. The PR3-ANCA levels in 5 of the 7 kits correlated with the cANCA titers in IFT. Sera from the healthy controls and 4 out of the 5 SLE and pANCA-negative patients were found to be MPO-ANCA negative in all 8 MPO-ANCA kits. In 20 pANCA-positive sera, MPO-ANCA positivity ranged from 25% to 75%. Thirty-five percent of MPO-ANCA-positive sera were confirmed by capture ELISA, immunoblot and inhibition assay.

The concordance rate was only 30% among pANCA-positive sera in the 8 MPO-ANCA kits. No significant correlation was observed between pANCA titers and MPO-ANCA levels. The HM showed that 65% of cANCA-positive sera were PR3-ANCA positive, and 45% of pANCA-positive sera were MPO-ANCA positive.

Our results indicate that the sensitivities and specificities for ANCA detection differ significantly among the commercial kits tested and underline the necessity of establishing uniform international standards for ANCA ELISA procedures in order to permit more reliable interpretation and comparison of data.

Clinical evaluation of a capture ELISA for detection of proteinase-3 antineutrophil cytoplasmic antibody.

Westman KW, Selga D, Bygren P, Segelmark M, Baslund B, Wiik A, Wieslander J.

Department of Nephrology, Lund University, Sweden. Kerstin.

Kidney Int 1998 May;53(5):1230-6 Abstract quote

Detection of antineutrophil cytoplasmic antibodies (ANCA) has become a useful tool in the diagnosis of Wegener's granulomatosis and microscopic polyangiitis. However, the results obtained with indirect immunofluorescence (IIF) and by ELISA for ANCA demonstration do not always correlate. A possible explanation for this finding could be that proteins are denatured during the process of antigen purification or during coating onto the solid phase. To avoid this possibility, a monoclonal antibody to PR3 that is precoated on the plate can be used.

In the present study we have used the monoclonal antibody (MoAb) 4A3 for the capture of PR3 in an ELISA, and a clinical evaluation of the diagnostic properties of the new capture ELISA has been made. The sensitivity of the capture PR3-ANCA ELISA was 85% in a material of c-ANCA positive sera. A specificity of 90% was obtained in analyses from patients having various forms of glomerulonephritis. There was a significantly higher diagnostic sensitivity of the capture PR3-ANCA ELISA (85%) compared to c-ANCA by IIF (58%) in patients with Wegener's granulomatosis with renal involvement. Capture PR3-ANCA and direct ELISA for MPO-ANCA together gave a diagnostic sensitivity of 98%, versus 75% using IIF. In conclusion, the capture PR3-ANCA ELISA seems to be a valuable tool in the diagnosis of Wegener's granulomatosis with renal involvement.

Preliminary data suggest that the technique may have an advantage over direct ELISA for PR3-ANCA, as well as in the follow-up of c-/PR3-ANCA associated vasculitides. However, further prospective studies are needed to clarify this premise.

Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis.

EC/BCR Project for ANCA Assay Standardization. Hagen EC, Daha MR, Hermans J, Andrassy K, Csernok E, Gaskin G, Lesavre P, Ludemann J, Rasmussen N, Sinico RA, Wiik A, van der Woude FJ.

Department of Nephrology, University Hospital Leiden, The Netherlands.

Kidney Int 1998 Mar;53(3):743-53 Abstract quote

Anti-neutrophil cytoplasmic antibodies (ANCA) are widely used as diagnostic markers for Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), Churg-Strauss syndrome (CSS) and idiopathic rapidly progressive glomerulonephritis (iRPGN).

The objective of this study was to evaluate the diagnostic value of ANCA measurement by the indirect immunofluorescence (IIF) test, and by anti-PR3 and anti-MPO ELISA performed in different locations, in patients with idiopathic small vessel vasculitis.

Fourteen centers participated in a standardization study of ANCA assays, and entered a total number of 169 newly diagnosed and 189 historical patients with idiopathic systemic vasculitis or iRPGN. Patients were classified according to a pre-defined diagnostic classification system. Results were compared with those of 184 disease controls and 740 healthy controls. The IIF test was performed according to standard methodology; ELISAs had been standardized among the participants in a previous phase of the study. The sensitivities of assays in patients were as follows. The sensitivity in WG was: cANCA 64%, pANCA 21%, anti-PR3 66%, anti-MPO 24%. In MPA the sensitivity was: cANCA 23%, pANCA 58%, anti-PR3 26%, anti-MPO 58%. Sensitivity in iRPGN was: cANCA 36%, pANCA 45%, anti-PR3 50%, anti-MPO 64%. The specificity of assays (related to disease controls) was: cANCA 95%, pANCA 81%, anti-PR3 87%, anti-MPO 91%. When the results of the IIF test were combined with those of the ELISAs (cANCA/anti-PR3 positive, pANCA/anti-MPO positive), the diagnostic specificity increased to 99%. The sensitivity of the combination of cANCA + anti-PR3 or pANCA + anti-MPO for WG, MPA or iRPGN was 73%, 67% and 82%, respectively.

From this study we conclude that the value of the IIF test for ANCA detection can be greatly increased by the addition of a well standardized antigen-specific ELISA. In a significant number of patients with idiopathic small vessel vasculitis, however, the ANCA test results (either in IIF or ELISA) are negative.

PATTERN VARIATIONS  

A review of immunofluorescent patterns associated with antineutrophil cytoplasmic antibodies (ANCA) and their differentiation from other antibodies.

Savige JA, Paspaliaris B, Silvestrini R, Davies D, Nikoloutsopoulos T, Sturgess A, Neil J, Pollock W, Dunster K, Hendle M.

University Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg, Victoria, Australia.

J Clin Pathol 1998 Aug;51(8):568-75 Abstract quote

AIM: To describe the neutrophil fluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) with different antigen specificities, and by other auto- and alloantibodies.

BACKGROUND: Most sera from patients with active generalised Wegener's granulomatosis result in diffusely granular cytoplasmic neutrophil fluorescence with internuclear accentuation (cANCA) and proteinase 3 (PR3) specificity. About 80% of the sera from patients with microscopic polyangiitis result in perinuclear neutrophil fluorescence with nuclear extension (pANCA) and myeloperoxidase (MPO) specificity, or a cANCA pattern with PR3 specificity. However, many different neutrophil fluorescence patterns are noted on testing for ANCA in routine immunodiagnostic laboratories.

METHODS: Sera sent for ANCA testing, or containing a variety of auto- and alloantibodies, were studied. They were examined by indirect immunofluorescence according to the recommendations of the first international ANCA workshop, and for PR3 and MPO specificity in commercial and in-house enzyme linked immunosorbent assays (ELISA).

RESULTS: Sera with typical cANCA accounted for only half of all neutrophil cytoplasmic fluorescence. Other sera had "flatter" fluorescence without internuclear accentuation, and the corresponding antigens included MPO and bactericidal/permeability increasing protein (BPI), but were usually unknown. Peripheral nuclear fluorescence without nuclear extension occurred typically when the antigens were BPI, lactoferrin, lysozyme, elastase, or cathepsin G. Most types of ANA were evident on ethanol fixed neutrophil nuclei. AntidsDNA, antiRo, and antilamin antibodies resembled pANCA. Antimicrobial and antiribosomal antibodies produced cytoplasmic fluorescence, and antiGolgi antibodies, a pANCA. Sera from patients with anti-smooth muscle antibodies were associated with cytoplasmic fluorescence. There was no neutrophil fluorescence with anti-skeletal muscle and anti-heart muscle antibodies, anti-liver/kidney microsomal, antithyroid microsomal, or antiadrenal antibodies. Alloantibodies such as antiNB1 typically resulted in cytoplasmic fluorescence of only a subpopulation of the neutrophils.

CONCLUSIONS: The ability to distinguish between different neutrophil fluorescence patterns, and the patterns seen with other auto- and alloantibodies is helpful diagnostically. However, the demonstration of MPO or PR3 specificity by ELISA will indicate that the neutrophil fluorescence is probably clinically significant, and that the diagnosis is likely to be Wegener's granulomatosis or microscopic polyangiitis.

Diagnostic value of classical and atypical antineutrophil cytoplasmic antibody (ANCA) immunofluorescence patterns.

Wong RC, Silvestrini RA, Savige JA, Fulcher DA, Benson EM.

Immunopathology Department, ICPMR, Westmead Hospital, NSW, Australia.

J Clin Pathol 1999 Feb;52(2):124-8 Abstract quote

BACKGROUND: The "classical" antineutrophil cytoplasmic antibody (C-ANCA) pattern seen on indirect immunofluorescence (IIF) is characterised by granular cytoplasmic staining showing central or interlobular accentuation, and is strongly associated with antiproteinase-3 antibodies (PR3-ANCA) and Wegener's granulomatosis. However, many laboratories report C-ANCA in the presence of any cytoplasmic IIF staining, regardless of pattern, which risks reducing the diagnostic value of this pattern.

AIMS: To classify different cytoplasmic ANCA patterns and thus determine whether stringent application of the classical criteria for C-ANCA would produce better correlation between C-ANCA and (1) PR3-ANCA enzyme linked immunosorbent assay (ELISA) results; (2) a diagnosis of systemic vasculitis (including Wegener's granulomatosis).

METHODS: 72 sera with cytoplasmic IIF collected over a two year period were analysed by IIF and a commercial PR3-ANCA ELISA kit.

RESULTS: Three IIF patterns were defined: "classical/true" C-ANCA as described above (n = 27 (37.5%)); "flat" ANCA with homogeneous cytoplasmic staining (n = 21 (29%)); and "atypical" ANCA which included all other cytoplasmic patterns (n = 24 (33.5%)). Twenty five of the 27 true C-ANCA sera (92.5%) contained PR3-ANCA (p < 0.0001), but none of the 21 with flat ANCA and only one of the 24 with atypical ANCA. From clinical data on 23 of the 27 true C-ANCA positive patients, 20 (87%) had evidence of Wegener's granulomatosis or systemic vasculitis (p < 0.0001 v the other two patterns). However, none of 19 sera with flat ANCA and clinical data had evidence of systemic vasculitis.

CONCLUSIONS: Restricting the term "c-ANCA" to the "classical" description of central/interlobular accentuation on IIF, will improve its correlation with PR3-ANCA positivity and a diagnosis of systemic vasculitis.

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

Radice A, Vecchi M, Bianchi MB, Sinico RA.

Department of Nephrology and Department of Allergy and Clinical Immunology, San Carlo Borromeo Hospital, Milan, Italy.

Clin Exp Rheumatol 2000 Nov-Dec;18(6):707-12 Abstract quote

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.

 

CLINICAL UTILITY CHARACTERIZATION
GENERAL  

Clinical significance of ANCA in 98 patients.

Geffriaud-Ricouard C, Noel LH, Chauveau D, Houhou S, Grunfeld JP, Lesavre P.

Departement de Nephrologie, Hopital Necker, Paris, France.

Adv Exp Med Biol 1993;336:273-9 Abstract quote

Clinical and histological data leading to precise diagnosis were retrospectively obtained in 98 patients with antineutrophil cytoplasmic antibodies (ANCA) detected by indirect immunofluorescence (IIF).

Specificity was determined by myeloperoxidase (MPO) and proteinase 3 (PR3) specific ELISA in all and a comparative study based on ANCA specificity was performed. Vasculitis was present in all cases. PR3-ANCA occurred predominantly in males (25/38) with WG (19/38). MPO-ANCA occurred predominantly in older women and were often associated with various autoimmune disorders. There was a high prevalence of lung hemorrhage (18/45) and mPA (26/45) in this group.

Patients with negative MPO and PR3 specific ELISA despite positive IIF (n = 15) were almost exclusively WG (13/15) and were characterized by a high prevalence of hepatic and digestive manifestations. Renal and patient survival at the 75th percentile was 15 months with MPO-ANCA and 16 months with PR3, and was similar for patients with WG and mPA.

With immunosuppressive treatment, ANCA disappeared in 66% of cases and this disappearance was always associated with absence of disease activity.

International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)

Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, Hagen EC, Jayne D, Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, van der Woude F, Wieslander J, Wiik A

. University Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg, Victoria, Australia.

Am J Clin Pathol 1999 Apr;111(4):507-13 Abstract quote

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO).

For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples. Inclusion of the most recent positive sample in the IIF or ELISA may help demonstrate a change in antibody level. Reports should use recommended terms.

Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of Wegener granulomatosis or microscopic polyangiitis and that decisions about treatment should not be based solely on the ANCA results.


Antineutrophil cytoplasmic autoantibody in the absence of Wegener's granulomatosis or microscopic polyangiitis: implications for the surgical pathologist.

Gal AA, Velasquez A.

Departments of Pathology and Laboratory Medicine (AAG) and Division of Critical Care and Respiratory Medicine (AV), Emory University School of Medicine, Atlanta, Georgia.

Mod Pathol 2002 Mar;15(3):197-204 Abstract quote

Antineutrophil cytoplasmic antibodies (ANCA) are useful serologic markers for the diagnosis and management of patients with Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). However, problems in diagnosis and classification may occur when patients with other disorders develop ANCA.

A 7-year review (1993-1999) disclosed 247 patients whose sera tested positively for ANCA by an indirect immunofluorescence method: 166 patients for cytoplasmic-ANCA (C-ANCA) and 81 patients for perinuclear-ANCA (P-ANCA) Twenty-seven patients had active pulmonary disease and underwent open-lung biopsy or transbronchial biopsy. Eight patients (30%) had a disease other than WG or MPA, and their clinical, pathological, and serological findings were reviewed. The patients, all women, ranged in age from 28 to 77 years (median, 37 y). Dyspnea (n = 6), cough (n = 6), chest pain (n = 2), and/or hemoptysis (n = 2) were present. The duration of symptoms lasted from 3 weeks to 6 years (median, 6 mo). ANCA titers were C-ANCA (n = 4; range, 1:40-1280) or P-ANCA (n = 4; range, 1:40-640). The lung biopsies disclosed nonspecific interstitial pneumonia (n = 4), bronchiolitis obliterans organizing pneumonia (n = 1), diffuse alveolar damage (n = 1), organizing diffuse alveolar hemorrhage without capillaritis (n = 1), and necrotic granuloma (n = 1). No cases showed characteristic histology for WG or MPA. The final diagnoses were various connective tissue disorders (n = 5), chronic hypersensitivity pneumonia (n = 1), postinfectious bronchitis/bronchiectasis (n = 1), and ulcerative colitis-related lung disease (n = 1).

Surgical pathologists should be aware that significantly elevated ANCA titers may be associated with diverse forms of pulmonary disease. ANCA positivity alone, in the absence of appropriate clinical or pathologic findings, should not be used to substantiate a diagnosis of WG or MPA.

DISEASE ASSOCIATIONS  
HEART  


Atypical manifestation of a cytoplasmic antineutrophil cytoplasmic antibody (PR3-ANCA)-associated vasculitis with involvement of aortic intima and parietal endocardium.

Schildhaus HU, Von Netzer B, Dombrowski F, Pfeifer U.

Institute of Pathology, University of Bonn, Germany.

Hum Pathol 2002 Apr;33(4):441-5 Abstract quote

The traditional classification of vasculitis, based on the size of affected vessels, has meanwhile been extended by using antineutrophil cytoplasmic antibodies (ANCAs) as seromarkers in the differential diagnosis of different types of vasculitis.

We report an autopsy case of fulminant generalized vasculitis positive for C-ANCA (1:320) and anti-proteinase 3 (PR3) antibodies (>100 U/mL) in a 63-year-old man. The unusually broad histologic spectrum included periarteritis nodosa-like lesions in medium-sized vessels and leucocytoclastic vasculitis in small vessels, as well as capillaritis. In addition, the left atrial and ventricular endocardium and the intima of the aorta thoracalis were patchily involved in the inflammatory process. Glomerulonephritis and/or immune complexes were not detectable by electron microscopy or immunohistochemistry.

To the best of our knowledge, involvement of the aortic intima ("intimitis") and the parietal endocardium has not been described in PR3-ANCA-positive vasculitis to date.

HEMODIALYSIS PATIENTS  

ANCA in haemodialysis patients.

Weidemann S, Andrassy K, Ritz E.

Department Internal Medicine, Ruperto Carola University, Heidelberg, Germany.

Nephrol Dial Transplant 1993;8(9):839-45 Abstract quote

The prevalence of positive ANCA as well as the prevalence of PR-3 and MPO antibodies were examined in a cross-sectional sample of 1277 haemodialysis patients from 16 German haemodialysis centres.

We found 32 patients positive for c-ANCA (median titre 1:40; range 1:20-1:320) and 65 for p-ANCA (1:80; 1:20-1:1280). Twenty-two percent of the c-ANCA-positive and 31% of the p-ANCA-positive patients had PR-3 and MPO antibodies by ELISA respectively. Clinical evidence of vasculitis was found in 11 of 32 c-ANCA-positive and 19 of 65 p-ANCA-positive patients. Of the 11 c-ANCA-positive, four had a known diagnosis of Wegener's granulomatosis (WG); WG was recognized after the test in a further five patients and two had renal limited RPGN. Of the 19 p-ANCA-positive patients, three had a clinical diagnosis of microscopic polyarteritis (MP), MP was newly diagnosed in a further 12, WG in one and renal limited RPGN in three. The patients had not received cyclophosphamide (the diagnosis had been non-specified 'systemic disease'). Thus false-positive ANCA, as defined by absence of vasculitis, was found in 5% of dialysis patients versus 0% in patients with preterminal renal failure (n = 152) or blood donors (n = 150).

Patients with vasculitis tended to have higher c-ANCA and p-ANCA titres respectively, but there was a considerable overlap. Titres were not higher in patients symptomatic at the time of examination (6 of 11 c-ANCA and 10 of 19 p-ANCA), but PR-3 and MPO ELISA were positive in all but two.(

INFLAMMATORY BOWEL DISEASE  

Neutrophil-specific autoantibodies in chronic inflammatory bowel diseases.

Wiik A.

Department of Autoimmunology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
Autoimmun Rev. 2002 Feb;1(1-2):67-72 Abstract quote  

This review intends to highlight important differences between neutrophil-specific autoantibodies (NSA) typically found in chronic inflammatory bowel diseases (CIBD) and anti-neutrophil cytoplasm antibodies (ANCA) associated with primary systemic small vessel vasculitides (SSVV).

Indirect immunofluorescence (IF) techniques alone cannot distinguish NSA from ANCA and special measures must be taken to separate these two autoantibody populations. Many autoantigens originating in all cell compartments may be targeted by NSA in CIBD, several of these being constituents of neutrophil nuclei. Apart from the use of NSA in the differential diagnosis between Crohn's disease (CD) and ulcerative colitis (UC), very limited clinical significance is ascribed to these antibodies in CIBD.

Laboratory reports on NSA-positivity must be clearly distinguishable from reports on ANCA to help avoid clinical misinterpretation.

Anti-neutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis (UC): no relationship with disease activity.

Reumaux D, Colombel JF, Masy E, Duclos B, Heresbach D, Belaiche J, Cortot A, Duthilleul P;

GETAID. Groupe d'Etude des Affections Inflammatoires du Tube Digestif. Departement d'Hematologie-Immunologie-Cytogenetique, Centre Hospitalier de Valenciennes, France.

Inflamm Bowel Dis 2000 Nov;6(4):270-4 Abstract quote

The relationship between anti-neutrophil cytoplasmic auto-antibodies (ANCA) and disease activity in inflammatory bowel diseases remains controversial.

The aim of this study was to highlight the relationship between ANCA presence or titers and disease activity in ulcerative colitis (UC). Three groups of patients with UC were studied: 1) group A included 39 patients who had not undergone colectomy, 2) group B, 43 patients with subtotal colectomy and ileo-rectal anastomosis, 3) group C, 98 patients with proctocolectomy and ileo-anal anastomosis, including 88% with pouchitis and 12% without pouchitis at the time of the study. Determination of ANCA was performed using the standardized indirect immunofluorescence assay. ANCA were positive in 59%, 65%, and 54% of patients from groups A, B, and C, respectively (NS). No relationship between ANCA presence or titers and UC activity could be detected within groups A and B. In group C, 45 of 86 patients (52%) without pouchitis and 8 of 12 patients (67%) with pouchitis, were ANCA positive (NS).

These results do not support a relationship between ANCA and UC activity in this cohort of 180 patients.


Are anti-neutrophil cytoplasmic antibodies (ANCA) clinically useful in inflammatory bowel disease (IBD)?

Roozendaal C, Kallenberg CG.

Department of Clinical Immunology, University Hospital Groningen, Groningen, The Netherlands.

Clin Exp Immunol. 1999 May;116(2):206-13. Abstract quote  

Since the first detection of ANCA in IBD, numerous studies have dealt with their prevalence, antigenic specificities, clinical significance, pathophysiological role, and their induction. This review summarizes the information obtained from those studies and shows that ANCA are not directly useful as diagnostic and prognostic factors in IBD.

ANCA were detected in 50-85% of patients with ulcerative colitis (UC) and 10-20% of patients with Crohn's disease (CD). Multiple target antigens are recognized by these autoantibodies, including both cytoplasmic and nuclear proteins. A pathophysiological role for ANCA in IBD is far from clear. On the one hand, it is suggested that ANCA are genetic markers of susceptibility for IBD, and on the other hand, the induction of ANCA in those diseases may just be an epiphenomenon of chronic inflammation.

We discuss recent evidence that ANCA may be induced by a break-through of tolerance towards bacterial antigens.

Anti-neutrophil cytoplasmic antibodies in inflammatory bowel disease with special attention for IgA-class antibodies.

Gigase P, De Clerck LS, Van Cotthem KA, Bridts CH, Stevens WJ, Van Outryve M, Pelckmans PA.

University of Antwerp, Belgium.
Dig Dis Sci. 1997 Oct;42(10):2171-4. Abstract quote

Perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) of the IgG class have been reported in inflammatory bowel disease, mainly in ulcerative colitis. Since this disease affects the gastrointestinal tract, we determined whether IgA class ANCA were present in inflammatory bowel disease.

We used an indirect immunofluorescence assay for IgG and IgA ANCA testing. Sera from 34 patients with Crohn's disease and 29 patients with ulcerative colitis were collected together with clinical and laboratory data. We found IgA class ANCA of a perinuclear type in 52% of patients with ulcerative colitis and in 9% of Crohn's disease patients. There was a significant association between the presence of IgA ANCA and the occurrence of blood in the feces in the ulcerative colitis group (P = 0.03). IgG ANCA was found in 56% of patients with ulcerative colitis and in 7% of patients with Crohn's disease. Because of partial overlap between IgG and IgA ANCA positivity, the sensitivity of ANCA testing in ulcerative colitis increased from 56% up to 78% by combining IgG and IgA assays.

In conclusion, IgA ANCA occurs with a high prevalence in ulcerative colitis. Moreover there is a possible relationship between IgA ANCA and disease activity in ulcerative colitis.

Anti-neutrophil cytoplasmic antibodies in chronic inflammatory bowel disease. Prevalence and diagnostic role.

Hertervig E, Wieslander J, Johansson C, Wiik A, Nilsson A.

Dept. of Medicine, University Hospital, Lund, Sweden.

Scand J Gastroenterol. 1995 Jul;30(7):693-8. Abstract quote

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA), originally found to be associated with vasculitis, have been reported to be present in chronic inflammatory bowel disease. Most often the ANCA staining pattern is of the perinuclear type (p-ANCA), although nuclear and cytoplasmic stainings are seen. Single studies have shown some of the antibodies to react with lactoferrin or cathepsin G; however, most studies have not been able to determine a main antigenic specificity. We studied the prevalence of ANCA in sera from 155 patients with ulcerative colitis, 128 patients with Crohn's disease, and 51 patients with coeliac disease. The presence of ANCA was correlated to disease activity, extent, and age of onset of the diseases. Furthermore, we tried to characterize the antigen specificity by enzyme-linked immunosorbent assay (ELISA), using elastase, lactoferrin, myeloperoxidase, proteinase 3, and cathepsin G as antigens.

METHODS: The sera were screened for ANCA by indirect immunofluorescence. Anti-nuclear antibodies (ANA) were analysed on HEp2 cells, and ELISA for specific ANCA was performed using the antigens mentioned.

RESULTS: Most of the sera with positive immunofluorescence had the p-ANCA type of pattern. Seventy-eight of 155 (50.3%) of the patients with ulcerative colitis were ANCA-positive, compared with 31 of 128 (24.2%) of patients with Crohn's disease (p < 0.001). However, in the subgroup with Crohn's colitis, 16 of 44 (36.4%) were ANCA-positive. Only 4 of 51 patients (7.7%) with coeliac disease showed positive immunofluorescence (p < 0.001 compared with ulcerative colitis). Less than 10% of the samples were positive in the specific ELISA assays; thus other than the most well known granule proteins can be the target for ANCA in ulcerative colitis.

CONCLUSION: ANCA occur significantly more often in ulcerative colitis than in Crohn's disease. However, the prevalence of ANCA is rather high in Crohn's colitis. ANCA are thus of limited value in differentiating Crohn's colitis from ulcerative colitis. ANCA found in inflammatory bowel disease are different from those associated with vasculitis. The antigen(s) responsible remain to be determined.

Autoantibodies to neutrophil cytoplasmic (ANCA) and endothelial cell surface antigens (AECA) in chronic inflammatory bowel disease.

Romas E, Paspaliaris B, d'Apice AJ, Elliott PR.

Department of Clinical Immunology, St Vincent's Hospital, Melbourne, Vic., Australia.
Aust N Z J Med. 1992 Dec;22(6):652-9 Abstract quote  

Sera from 103 patients with chronic inflammatory bowel disease (IBD) were tested prospectively for antibodies against neutrophil cytoplasmic antigens (anti-neutrophil cytoplasm antibodies, ANCA) and endothelial cell surface antigens (anti-endothelial cell antibodies, AECA) by indirect immunofluorescence (IIF) and assays based on whole fixed neutrophils, purified neutrophil enzyme substrates and human umbilical vein endothelial cells.

Using IIF, ANCA were found in 26 IBD sera (25%) and in none of 51 controls. Twenty-two positive sera (85%) were from patients with ulcerative colitis (UC). The pattern of distribution of immunofluorescence was always perinuclear (P-ANCA). A majority of UC patients positive for these autoantibodies (68%) had active colitis, but none had evidence of vasculitis. Using a whole neutrophil ELISA, binding was demonstrable in 73% of UC sera compared to 27% of Crohn's (CD) sera and only 4% of controls. Unlike vasculitis sera, UC sera with P-ANCA did not bind strongly to myeloperoxidase (MPO). Forty-five per cent of IBD sera tested positive for IgG AECA in an endothelial cell ELISA, compared to seven of 51 (14%) controls. Binding correlated with both active and extensive colitis.

A type of P-ANCA, in most cases distinct from MPO-specific P-ANCA observed in vasculitis, is detected in a significant proportion of patients with UC, but rarely Crohn's colitis and therefore may be of differential diagnostic value. IgG AECA are also frequent in CIBD sera but are less disease specific than ANCA.
SYSTEMIC LUPUS ERYTHEMATOSUS  

Anti-neutrophil cytoplasmic antibodies in 566 European patients with systemic lupus erythematosus: prevalence, clinical associations and correlation with other autoantibodies.

European Concerted Action on the Immunogenetics of SLE.

Galeazzi M, Morozzi G, Sebastiani GD, Bellisai F, Marcolongo R, Cervera R, De Ramon Garrido E, Fernandez-Nebro A, Houssiau F, Jedryka-Goral A, Mathieu A, Papasteriades C, Piette JC, Scorza R, Smolen J.

Istituto di Reumatologia, Universita di Siena, Italy

Clin Exp Rheumatol 1998 Sep-Oct;16(5):541-6 Abstract quote

OBJECTIVES: To evaluate, in a cohort of 566 patients with systemic lupus erythematosus (SLE) drawn from 11 European centres: (i) the prevalence of ANCAs and their subspecificities in a large series of European SLE patients; (ii) the possible associations of ANCA with the most common clinical manifestations of the disease; and (iii) whether ANCAs correlate with some of the autoantibodies commonly found in SLE.

METHODS: ANCA detection was performed by indirect immunofluorescence (IIF), and by ELISA for lactoferrin (LF), myeloperoxydase (MPO), proteinase3 (PR3) and lysozyme (LZ) subspecificities.

RESULTS: The prevalence of ANCA was 16.4% (IIF). The prevalence of LF was 14.3%, LZ 4.6%, MPO 9.3%, and PR3 1.7%. Our results show that ANCA is associated with certain clinical manifestations of SLE. In particular, positive correlations were found between IIF ANCA and serositis (p = 0.026), livedo reticularis (p = 0.01), venous thrombosis (p = 0.03) and arthritis (p = 0.04), while anti-LF antibodies were associated with serositis (p = 0.05) and livedo reticularis (p < 10(-3). Nevertheless, multivariate analysis demonstrated that other autoantibodies, such as aCL and SSA/Ro, are more closely correlated than ANCA with some of the aforementioned clinical features.

CONCLUSION: Our results demonstrate that ANCA are detectable in SLE sera and that some of them are associated with particular clinical manifestations. Whether ANCA plays a direct pathogenetic role in the vascular damage of SLE or only represents an epiphenomenon or a marker of disease activity remains to be elucidated.

VASCULITIS  

Relationship between ANCA and disease activity in small vessel vasculitis patients with anti-MPO ANCA.

Ara J, Mirapeix E, Rodriguez R, Saurina A, Darnell A.

Servei de Nefrologia, Hospital Clinic, Barcelona, Spain.

Nephrol Dial Transplant 1999 Jul;14(7):1667-72 Abstract quote

BACKGROUND: We analysed the usefulness of antineutrophil cytoplasmic antibodies (ANCA) as a marker of clinical activity in patients with small vessel vasculitis associated with anti-myeloperoxidase (MPO) ANCA.

METHODS: We studied a group of 25 patients, 15 with microscopic polyangitis and 10 with renal limited vasculitis, so-called rapidly progressive glomerulonephritis type III. The clinical and serological follow-up was accomplished quarterly over an average of 2.79 +/- 2.08 years (range 0.25-6 years). ANCA was analysed by indirect immunofluorescence and enzyme-linked immunosorbent assays (ELISAs).

RESULTS: At the time of diagnosis, all patients were ANCA positive (P-ANCA and anti-MPO). Following a standardized treatment, all patients except one achieved complete remission of vasculitis in <3 months. One patient suddenly died during the active phase (1 month of follow-up) and with positive ANCA. Seroconversion from positive to negative occurred in 24/25 patients (96%). Eighteen of these 24 patients (75%) achieved the seroconversion within the first 6 months. During the follow-up, two patients had four major relapses, all of them associated with positive ANCA. ANCA seroconversion from negative to positive was observed in one patient with microscopic polyangitis without clinical relapse of vasculitis.

CONCLUSION: ANCA should be used in conjunction with other markers of disease activity in the management of microscopic polyangitis and renal limited vasculitis patients with anti-MPO ANCA.

Outcome analysis of patients with vasculitis associated with antineutrophil cytoplasmic antibodies.

Brijker F, Magee CC, Tervaert JW, O'Neill S, Walshe JJ.

Department of Clinical Immunology, University Hospital of Groningen, The Netherlands.

Clin Nephrol 1999 Dec;52(6):344-51 Abstract quote

BACKGROUND: Objective scoring systems of disease activity and disease-associated damage have proven useful in the management of patients with systemic vasculitis.

PATIENTS AND METHODS: We used the recently designed Birmingham vasculitis activity score (BVAS; maximum score 63) and vasculitis damage index (VDI; maximum score 59) to assess initial activity and long-term damage, respectively, in ANCA positive patients from one center over a 3-year period. Thirty-two patients with ANCA vasculitis were identified and analyzed as an historic cohort. The median BVAS for all vasculitis patients at first presentation was 19 (range 6 - 36). Patients with Wegener's granulomatosis had a significantly higher total score and respiratory BVAS score compared to the 15 with microscopic polyangiitis. The majority of patients received standard cyclophosphamide/steroid treatment.

RESULTS: At the end of follow-up (mean 24.9 months), 4 patients had died; all patients had evidence of permanent organ damage. The median total VDI score at last follow-up was 4.0 (range 0-11), with no differences between patients with Wegener's granulomatosis and microscopic polyangiitis. The VDI was not associated with the number of relapses. A high initial BVAS was found to correlate with a later high vasculitis damage index (r = 0.56). Initial renal or respiratory involvement was also associated with longterm damage in the same organ system.

CONCLUSION: Although mortality from ANCA-associated vasculitis has decreased, morbidity remains a common problem. High early-disease activity may identify patients at high risk of long-term organ damage, allowing more effective individualized therapy. This hypothesis requires validation in a prospective, controlled study.

Serial measurements of antineutrophil cytoplasmic autoantibodies in patients with systemic vasculitis.

Kyndt X, Reumaux D, Bridoux F, Tribout B, Bataille P, Hachulla E, Hatron PY, Duthilleul P, Vanhille P.

Department of Nephrology, Internal Medicine, Centre Hospitalier de Valenciennes, France.

Am J Med 1999 May;106(5):527-33 Abstract quote

PURPOSE: To assess the value of serial determinations of antineutrophil cytoplasmic autoantibodies (ANCA) for monitoring disease activity in patients with systemic vasculitis.

PATIENTS AND METHODS: Forty-three patients with histologically proven vasculitis (21 with Wegener's granulomatosis, 17 with microscopic polyangiitis, and 5 with renal-limited vasculitis) were studied for a median follow-up of 22 months. Disease activity was prospectively assessed and quantified by the Birmingham Vasculitis Activity Score. A total of 347 sera were analyzed for ANCA determination.

RESULTS: Relapses occurred in 23 (54%) of 43 patients. Diagnostic category (Wegener's granulomatosis vs micropolyangiitis and renal-limited vasculitis), severity of initial symptoms (mean vasculitis activity score, mean number of organs involved), and ANCA pattern [cytoplasmic-ANCA (c-ANCA) vs perinuclear-ANCA (p-ANCA)] did not significantly differ between relapsers and nonrelapsers. Lung involvement was more frequent at onset among relapsers [16 of 23 (70%) vs 6 of 20 (30%); P = 0.02]. Relapses were slightly, but not significantly, more frequent in patients with Wegener's granulomatosis or a c-ANCA pattern. The percentage of relapsers was greater in patients with persistently positive ANCA than in patients with negative or decreasing ANCA titers (86% vs 20%, P = 0.0001). However, the predictive value of an increase in ANCA titers for the occurrence of a subsequent relapse was only 28% (4 of 14) for c-ANCA, 12% (2 of 17) for anti-proteinase 3-ANCA, and 43% (6 of 14) for anti-myeloperoxidase-ANCA. An increase in ANCA occurred before or during relapse in 33% (10 of 30) of cases for c-ANCA/anti-proteinase 3 antibodies, and 73% (11 of 15) of cases for anti-myeloperoxidase antibodies.

CONCLUSION: The persistence of ANCA positivity is strongly associated with relapses. However, an increase in ANCA titers has a poor value for the early prediction of a subsequent relapse and should not be used as a sole parameter for therapeutic intervention. In addition, our results suggest that serial anti-myeloperoxidase determination may be useful as a prognostic marker in patients who are p-ANCA positive.

Antineutrophil cytoplasmic antibodies (ANCA) and systemic vasculitis: update of assays, immunopathogenesis, controversies, and report of a novel de novo ANCA-associated vasculitis after kidney transplantation.

Schultz DR, Diego JM.

Department of Medicine, University of Miami, School of Medicine, FL 33101, USA.

Semin Arthritis Rheum 2000 Apr;29(5):267-85 Abstract quote

OBJECTIVES: To characterize antineutrophil cytoplasmic antibodies (ANCA), their major autoantigens, disease associations, and pathophysiology in systemic vasculitides. To describe a patient with a novel de novo ANCA-associated vasculitis after kidney transplantation.

METHODS: We reviewed and compiled the literature on ANCA-related topics and systemic vasculitis. Laboratory and clinical data from a cadaveric kidney transplant patient who developed necrotizing vasculitis involving glomerular capillaries, with crescent formation associated with P-ANCA and myeloperoxidase, were analyzed.

RESULTS: Large-scale multi-center testing of patient and normal sera by the European ANCA Assay Standardization Project using immunofluorescence assays and enzyme immunoassays indicate the assays have good sensitivity and specificity, and diagnostic utility for ANCA-associated vasculitis. A few investigations covering basic and clinical research with ANCA remain controversial: whether endothelial cells do or do not express a 29-kd neutral serine protease termed proteinase-3 (PR-3), the target of ANCA in most individuals with Wegener's granulomatosis, and whether anti-myeloperoxidase (MPO) ANCAs recognize a restricted number of epitopes on MPO. This issue has relevance for using monoclonal antibodies to treat patients with vasculitis who have adverse effects from immunosuppressive drugs. The two allelic forms of FcgammaRIIa (H131/R131) and the two of FcgammaRIIlb (NA1/NA2) are discussed as possible inheritable genetic elements for vasculitic disorders and for signaling responses. Stimulatory and costimulatory molecules, and cytokine profiles of T lymphocytes are characterized to show that these cells are actively involved in the ANCA-associated vasculitides. The patient described had a de novo ANCA associated small vessel vasculitis which developed after renal transplantation.

CONCLUSIONS: There have been significant advances in the development of sensitive and specific ANCA assays. The immunopathogenetic mechanism of ANCA involves the constitutive FcgammaRs, ligands, and signaling responses to activate cytokine-primed neutrophils. This may lead to the generation of reactive oxygen intermediates, degranulation, and secretion of intracellular granule contents, and ultimately inflammation and vasculitis.

ANCA titres, even of IgG subclasses, and soluble CD14 fail to predict relapses in patients with ANCA-associated vasculitis.

Nowack R, Grab I, Flores-Suarez LF, Schnulle P, Yard B, van der Woude FJ.

Fifth Medical Clinic (Nephrology, Endocrinology), University-Clinic Mannheim, Faculty of Clinical Medicine of the University of Heidelberg, Mannheim, Germany.

Nephrol Dial Transplant 2001 Aug;16(8):1631-7 Abstract quote

BACKGROUND: Antineutrophil cytoplasmic autoantibodies (ANCA) are presumed to reflect disease-activity and to be useful for guidance of immunosuppressive therapy of ANCA-associated systemic vasculitis (AASV), but with respect to conventional ANCA assays this is controversial. ANCA titres, measured in the IgG3 subclass and modern capture ELISAs, have been said to be superior predictors of relapses of AASV.

METHODS: In this retrospective study serial measurements of ANCA parameters and soluble CD14 (sCD14) were performed in 169 consecutive sera over a median of 21 months in 18 patients with AASV and related to disease activity, assessed by Birmingham Vasculitis Activity Score (BVAS) for new or deteriorated (BVAS1), and for chronic disease activity (BVAS2). Fourteen patients had Wegener's granulomatosis (WG) and were C-ANCA positive with Pr 3-antibodies and four patients had microscopic polyangiitis (MPA) with P-ANCA and MPO-antibodies. In WG patients ANCA by IIF, Pr 3-ELISA for IgG, IgG1, IgG3, IgG4 and sCD14 were measured, as well as capture ELISA for Pr 3, and in MPA patients ANCA by IIF, MPO-ELISA for IgG and IgG1, IgG3, IgG4, and sCD14 respectively. In eight patients, data collection started at diagnosis, in 10 patients at remission.

RESULTS: The parameters predicted neither the nine major relapses (increase of immunosuppression necessary), nor the 26 minor relapses (increase of BVAS1>2) with sufficient sensitivity (>80%) or specificity (> 90%90%), and they also failed to predict relapses within the following 2 months. ANCA-IgG3 and capture ELISA for Pr 3 were not advantageous for prediction of relapses (sensitivity 0.45 and 0.19 respectively), and sCD14 remained elevated in all samples irrespective of disease activity.

CONCLUSIONS: There is no rationale for serial measurements of ANCA in AASV. For changes of therapy, the ANCA parameters should only be used in conjunction with clinical information.

WEGENER'S GRANULOMATOSIS  

Clinical spectrum associated with positive ANCA titres in 94 consecutive patients: is there a relation with PR-3 negative c-ANCA and hypergammaglobulinaemia?

Blockmans D, Stevens E, Marien G, Bobbaers H.

Department of Internal Medicine, University Hospital, Leuven, Belgium.

Ann Rheum Dis 1998 Mar;57(3):141-5 Abstract quote

OBJECTIVE: To calculate the positive predictive value (ppv) of cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCAs) and anti-proteinase 3 (PR 3) antibodies for Wegener's granulomatosis (WG) and to evaluate their association with other diseases.

METHODS: The clinical files of all 94 patients who had a positive c- or perinuclear (p)-ANCA test, or both, in the laboratory of the University Hospital, Leuven between April 1995 and March 1996 and who attended the Internal Medicine Department of the hospital were retrospectively studied.

RESULTS: Of the 94 patients with ANCAs (fluorescence titre > or = 1/40), 57 were c-ANCA positive and 45 p-ANCA positive (eight were simultaneously c- and p-ANCA positive). Of the 57 c-ANCA positive patients, 23 had WG. The ppv for WG thus was 40%. This value did not increase by defining a higher threshold for a positive ANCA. There was not a good relation between ANCA titres and disease activity in the WG patients, nor was there a relation between anti-PR 3 antibody levels and WG disease activity. The ppv of anti-PR 3 antibodies for WG however was very high (85%). There was a positive correlation between the level of (hyper) gammaglobulinaemia and c-ANCA titres in those patients with final diagnoses not known to be associated with c-ANCA. Forty five patients had positive p-ANCAs. The largest group were those with inflammatory bowel disease (n = 20, of whom the majority had colitis ulcerosa or primary sclerosing cholangitis, or both); the great majority of these patients had no anti-myeloperoxidase antibodies. Vasculitis was present in eight patients, of whom two had WG (both were also c-ANCA positive).

CONCLUSION: There is a low ppv of c-ANCAs for WG, caused by a high percentage of PR 3 negative, positive c-ANCA determinations, possibly related to hypergammaglobulinaemia. Anti-PR 3 antibodies have a high ppv for WG. However, neither c-ANCA titre, nor the level of anti-PR 3 antibodies correlated with the activity of the disease.

 

INTERFERING DISEASES OR SUBSTANCES THAT ALTER LEVELS CHARACTERIZATION
GENERAL  

False-positive myeloperoxidase binding activity due to DNA/anti-DNA antibody complexes: a source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic autoantibodies.

Jethwa HS, Nachman PH, Falk RJ, Jennette JC.

Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7525, USA.

Clin Exp Immunol 2000 Sep;121(3):544-50 Abstract quote

Anti-myeloperoxidase antibodies (anti-MPO) are a major type of anti-neutrophil cytoplasmic antibody (ANCA). While evaluating anti-MPO monoclonal antibodies from SCG/Kj mice, we observed several hybridomas that appeared to react with both MPO and DNA. Sera from some patients with systemic lupus erythematosus (SLE) also react with MPO and DNA.

We hypothesized that the MPO binding activity is a false-positive result due to the binding of DNA, contained within the antigen binding site of anti-DNA antibodies, to the cationic MPO. Antibodies from tissue culture supernatants from 'dual reactive' hybridomas were purified under high-salt conditions (3 M NaCl) to remove any antigen bound to antibody. The MPO and DNA binding activity were measured by ELISA. The MPO binding activity was completely abrogated while the DNA binding activity remained. The MPO binding activity was restored, in a dose-dependent manner, by the addition of increasing amount of calf-thymus DNA (CT-DNA) to the purified antibody. Sera from six patients with SLE that reacted with both MPO and DNA were treated with DNase and showed a decrease in MPO binding activity compared with untreated samples. MPO binding activity was observed when CT-DNA was added to sera from SLE patients that initially reacted with DNA but not with MPO.

These results suggest that the DNA contained within the antigen binding site of anti-DNA antibodies could bind to the highly cationic MPO used as substrate antigen in immunoassays, resulting in a false-positive test

HIV INFECTION  

Anti-nuclear, anti-neutrophil cytoplasmic and anti-glomerular basement membrane antibodies in HIV-infected individuals.

Savige JA, Chang L, Horn S, Crowe SM.

University Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia.

Autoimmunity 1994;18(3):205-11 Abstract quote

Many autoantibodies have been described in HIV-infected individuals.

We have examined the incidence, associations and prognostic significance of anti-nuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (GBM) antibodies in individuals with HIV infections.

One hundred and five patients, with asymptomatic infections (n = 37), AIDS-related complex (n = 32) or AIDS (n = 36) were studied.

Plasma from 24 of these (23%) were positive for ANA: most demonstrated speckled fluorescence (n = 21) and were of low titre (1+ in 18). ANCA were demonstrated by IIF in 18 individuals (17%) and all fluorescent patterns were seen; 6 of these plasma were also positive in the ELISAs for antibodies to proteinase 3, myeloperoxidase or elastase. Thirteen plasma were positive for ANCA in the neutrophil cytoplasm ELISA; 10 of these were also positive in the specific ELISAs. A total of 30 plasma bound to proteinase 3, myeloperoxidase or elastase in specific ELISAs, in 6 cases with 2 specificities. Finally, 18 plasma (17%) contained anti-GBM antibodies by ELISA, but none of 4 plasma tested in inhibition assays was specific.

ANA, ANCA and anti-GBM antibodies were not uncommon in HIV-infected individuals but the presence of these antibodies was not associated with the clinical manifestations of the corresponding autoimmune diseases. In addition, there was no correlation between the demonstration of these antibodies and the immunological status of the individual (apart from a correlation between CD4 counts less than 400/microliters with anti-GBM antibodies), the presence of an opportunistic infection, the development of malignancy or reduced survival. Some of these antibodies may arise from polyclonal activation, or be due to "sticky" serum since we have shown that the presence of anti-GBM antibodies correlated with the demonstration of ANCA by ELISA. These antibodies are not more common in hypergammaglobulinemic plasma but some may be due to heat-treatment of the plasma.

The clinician caring for HIV-infected individuals needs to be aware of these "false-positive" antibody results.

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Rosai J. Ackerman's Surgical Pathology. Eight Edition. Mosby 1996.
Sternberg S. Diagnostic Surgical Pathology. Third Edition. Lipincott Williams and Wilkins 1999.
Robbins Pathologic Basis of Disease. Sixth Edition. WB Saunders 1999.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.


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