ANCA Immunofluorescence

Special Precautions/Comments:

N.B.  ANCA Immunofluorescence is not routinely tested.  If this test is required please contact the Immunology Laboratory.

Interferences: ANA may be excluded utilising HEP 2 cells but if present may prevent the identification of a P-ANCA pattern.

Method: Indirect Immunofluorescence (IIF) on ethanol fixed human neutrophil slides. Calibration: N/A. EQA scheme: UK NEQAS scheme for Neutrophil cytoplasmic and GBM antibodies. IQC: Commercial preparations

Interpretation: Results are reported as POSITIVE or NEGATIVE along with a fluorescent intensity value (LIU). POSITIVE samples are assayed by ELISA for PR3 and MPO reactivity. Atypical P-ANCA patterns are reported as ATYPICAL PATTERN and these may be seen in primary sclerosing cholangitis and Crohn’s disease. Samples which may have an ANA present will have MPO and PR3 analysed have a comment stating that ANA is present on the report. This test should only be requested if there is a high suspicion of vasculitis due to the poor negative predictive value and potential for false positives.

Additional Information:

Indication: Can be used to aid in the diagnosis of small vessel vasculitis. Seen particularly in Wegener’s granulomatosis (WG), microscopic polyangitis (MPA) and its renal limited variant (pauci-immune crescentic glomerulonephritis) and Churg-Strauss syndrome.

Background Information: There are two major subclasses of ANCA, characterised by staining patterns found when using fixed human neutrophils as substrate under Indirect Immunofluorescence (IIF): 1. C-ANCA (Cytoplasmic or Classical Staining ANCA), denotes a granular cytoplasmic staining pattern on ethanol fixed neutrophils, with some interlobular accentuation. C-ANCA are principally directed against a 29,000 Dalton serine protease, proteinase 3 (PR3) present in the azurophil granules in the cytoplasm of human neutrophils. Other C-ANCA specificities have been detected for cationic protein 57 (CAP57) and cathepsin G. C-ANCA positive is suggestive but not diagnostic of WG, MPA (and its renal limited variant) and Churg-Strauss. C-ANCA positive results must be followed up by ELISA tests for anti-MPO and anti-PR3 [1]. 2. P-ANCA (Perinuclear Staining ANCA) denotes a staining pattern present in the perinuclear space of the cytoplasm of ethanol fixed neutrophils. P-ANCAs are principally directed against myeloperoxidase (MPO), which is also found in azurophil granules in the cytoplasm of human neutrophils. Other P-ANCA antigen specificities are for elastase and lactoferrin. P-ANCA is associated in MPA and in some cases of WG and Churg-Strauss syndrome although can also be seen in inflammatory bowel disease and other autoimmune diseases [1]. P –ANCA positive results must be followed up by ELISA tests for anti-MPO and anti-PR3 [1]. ANCA measurements are not closely associated with disease activity and should therefore not determine treatment increases or decreases [2]. However treatment withdrawal in patients with a persistently positive ANCA is associated with relapse [2]. Different ANCA patterns can be reported on the same sample in different laboratories due to variations in neutrophil substrate [3].

References: Sinclair D, Stevens JM. Role of antineutrophil cytoplasmic antibodies and glomerular basement membrane antibodies in the diagnosis and monitoring of systemic vasculitides. Ann Clin Biochem. 2007. 44(5):432-442. Lapraik C, et al. BSR and BHPR guidelines for the management of adults with ANCA associated vasculitis. Rheumatology. 2007. 46 (10):1615-1616. [Ref 2] Bosch X, Guilabert A and Font J. Antineutrophil cytoplasmic antibodies. Lancet. 2006. 368(9533):404-418. Seo P and Stone J. The Antineutrophil cytoplasmic antibody-associated vasculitides. Am J Med. 2004. 117:39-50. Reumaux D, Duthilleul P, Roos D. Pathogenesis of diseases associated with antineutrophil cytoplasm autoantibodies. Hum Immunol. 2004. 65(1):1-12. Savige J, et al. Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am. J. Clin. Pathol. 2003. 120(3):312-318. [Ref 1] Pollock W, et al. Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate. J. Clin. Pathol. 2002. 55:680-683. [Ref 3] Rao J K, et al. The role of antineutrophil cytoplasmic antibody (C-ANCA) testing in the diagnosis of Wegener granulomatosis: a literature review and meta-analysis. Annals of Internal Medicine. 1995. 123(12):925-932.

See Also: MPO; PR3

Gateshead Lab: 0191.4456499 Option 4, Option 1