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ORIGINAL CONTRIBUTIONS |
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| Year : 2002 | Volume
: 56
| Issue : 7 | Page : 335-339 |
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ANCA : Anti-neutrophil cytoplasmic antibodies and their role in vasculitis associated kidney disorders
SS Badakere, VD Pradhan
Institute of Immunohaematology, ICMR, KEM Hospital, 13th Floor, Mumbai 400012,
Correspondence Address:
S S Badakere
Institute of Immunohaematology, ICMR, KEM Hospital, 13th Floor, Mumbai 400012
PMID: 12645170
How to cite this article:
Badakere S S, Pradhan V D. ANCA : Anti-neutrophil cytoplasmic antibodies and their role in vasculitis associated kidney disorders. Indian J Med Sci 2002;56:335-9 |
How to cite this URL:
Badakere S S, Pradhan V D. ANCA : Anti-neutrophil cytoplasmic antibodies and their role in vasculitis associated kidney disorders. Indian J Med Sci [serial online] 2002 [cited 2013 May 24];56:335-9. Available from: http://www.indianjmedsci.org/text.asp?2002/56/7/335/11984 |
ANCA were first described in a few patients with necrotizing glomerulonephritis, but a few years later- their role as sensitive markers for small vessel vasculitis associated disorders was recognized.
The vasculitis syndromes comprise a group of inflammatory disorders of presumed autoimmune origin characterized by inflammation and necrosis of blood vessels frequently in combination with granuloma formation. Within the spectrum of vasculitides, small vessel vasculitides such as Wegener's granulomatosis, microscopic polyangiitis and Churg Strauss syndrome are strongly associated with ANCA. Basically the vasculitides include a heterogeneous group of clinicopathologic entities which have common histopathologic findings. i.e the inflammation within blood vessels resulting in vascular obstruction with tissue ischaemia and probably infarction.
Immunodiagnosis: The identification of ANCA is usually done by the indirect immunofluorescence microscopy, using human neutrophils (PMN) or a promyelocytic cell line HL-60 as the substrate which is coated with patients' serum and probed by using a fluorescent isothiocyanate (FITC) tagged antihuman globulin under a fluorescent microscope. The ANCA binds to certain target molecules in the cytoplasm of these cells, commonly seen are Proteinase-3 (PR-3), which shows a cytoplasmic immunofluorescence and it also binds to Myeloperoxidase (MPO), which shows a perinuclear immunofluorescence. Other antigens like Elastase, Lactoferrin, Cathepsin G or the BPI protein are also sometimes involved. The PR-3 and MPO specific ANCA which are detected by using ethanol fixed neutrophils are further confirmed by fixing them with formalin and the true ANCA patterns will be detected. Rarely another pattern is also seen, known as the snowdrift pattern or the "X" ANCA or atypical ANCA in some autoimmune disorders.
The standard immunofluorescence reactions are usually clear to read but sometimes a reaction with contaminating lymphocytes is seen, and this call be solved by performing an ELISA using purified PR-3 or MPO antigens or by using a commercially available kit. However, only p-ANCA results are easy to read in an immunofluorescence test, strongly positive samples often show a condensed nuclear fluorescence, but at higher dilutions it becomes perinuclear. The p-ANCA pattern is considered to be an artefact caused by charge interaction between the cationic lysosomal antigen and the anionic nucleus. Also, a positive p-ANCA result is usually interpreted with caution until confirmed by an ELISA for MPO-ANCA and only strong positive ELISA results are indicative of a vasculitic condition whereas weak MPOANCA results are common in other inflammatory disorders.
ANCA associated vasculitis share the characteristics of having very minimal or no immune complex deposits in affected organs therefore these have been termed as "pauciimmune". The diagnostic hallmarks consist of a) Clinical features. b) Immuno-histochemical studies. c) Presence of some serological markers. d) Biopsy of lung or kidney showing a necrotizing vasculitis of small arteries, capillaries or veins. The final diagnosis is usually done after taking these factors into consideration.
Various other criteria like the Chapel Hill Consensus Conference definitions or the Vasculitis Disease Activity Scoring Index are used for assessing severity of the disease but the actual vasculitis damage is measured according to Vascular Damage Index.
The rapidly advancing field of immunodiagnostics, in the area of autoimmune disorders has helped to a great extent in the diagnosis and prognosis of some autoimmune diseases which are known to have marker antibodies. This is especially true in the kidney disorders associated with small vessel vasculitis as there is a direct correlation of disease status with the presence and strength of the marker antibodies. Some of these diseases and the presence of either c-ANCA or p-ANCA is shown in [Table - 1].
Therefore, a technique having a good sensitivity, specificity and a positive predictive value is useful for diagnosis and prognosis. However, even in international laboratories they are yet to identify methodologies that are good. It is said that indirect immunofluorescence technique using PMN is the "Gold Standard" followed by ELISA for confirmation, but variations among laboratories in the techniques used and also inter kit variations for ELISA have indeed created problems. The first international workshop on ANCA for the standardization of methodologies was convened by Dr. Allan Wiik in 1989 and soon after the second workshop was held by Dr. van der Woude and Dr. Allan Wiik in 1990. In 1992, a project for standardizing ANCA was held with 9 international laboratories participating. In 1996, another international workshop was conducted in Europe and laboratories from Germany, Netherlands, UK, Sweden, France, Italy and Greece participated. It was called "The workshop for evaluation of techniques for ANCA detection". Again in 1999, an international meeting was convened and the International Consensus Statement on testing and reporting of anti-neutrophil cytoplasmic antibodies was drafted. An important point that emerged was that for a proper diagnosis a .sensitive and specific immunofluorescence test using PMN in combination with a standard antigen specific ELISA was more accurate than either technique alone. It was also deemed necessary to establish uniform International Standards at the global level for ANCA testing which is lacking at present.
Pathophysiology of ANCA: The pathophysiology of small vessel vasculitis is guite difficult to understand. In-vitro experiments by research workers have shown that ANCA have the potential to further activate proactivated neutrophils to produce reactive oxygen species and release lytic enzymes. Also in-vitro ANCA is known to induce increased expression of CD 11 b and CD 18 on neutrophils.
During an inflammatory response in vivo, various mechanisms of vascular injury may operate. One is that the adhesion molecules mediate the interaction of neutrophils with endothelial cells. Pre-activated neutrophils pass over the endothelial layer, and get further activated by local mediators, which makes them adhere to and `transmigrate through the endothelium. "Shwartzman Phenomenon" is the neutrophil activation and adhering to endothelium causing vascular injury, In biopsies of ANCA associated vasculitis cases, there is upregulation of endothelial intercellular adhesion molecule-1 (ICAM-I) and vascular cell adhesion molecule. (VCAM- I). The TNF - a is also involved. One of the possible mechanisms of vasculitis is the activation of neutrophils intravascularly which results in upregulation of CDIIb/CD 18 expression followed by degranulation and production of oxygen radicals after cells adhere to activated endothelium.
Role of activation markers: In patients with vasculitis, the expression of markers CD 66b, CD 63, CD 64 on neutrophils is increased as compared to normal healthy controls. Degranulation of azurophilic granules may explain the expression of PR-3 on the surface of neutrophils. Also, vasculitis cases have increased expression of CD 63, a marker for azurophil granule degranulation which correlates with disease activity but not with ANCA titers. Therefore, the hypothesis that primed neutrophils which bind to the endothelium are activated by ANCA and release lyric enzymes and "O" radicals at the surface of endothelial cells resulting in endothelial cell lysis and vasculitis may be true.
In the presence of vasculitis, the terms c-ANCA or anti-PR-3 associated vasculitis and p-ANCA or anti - MPO associated vasculitis are useful concepts. The former one is a sensitive marker for Wegener's granulomatosis, MPA, or the Churg Strauss syndrome, while the latter is a marker for necrotizing crescentic glomerulonephritis (NCGN) and microscopic polyangiitis. Some diseases like Henoch Schonlein purpura, essential cryoglobulinemic vasculitis are not associated with ANCA. Antibodies to MPO are seen in patients with pauci immune NCGN where more renal limited disease cases are seen while in anti-PR3 cases there is also upper and lower respiratory tract involvement in combination with renal disease.
Clinical features : The clinical differences between anti-PR-3 and anti-MPO associated vasculitis are that anti-MPO associated NCGN may follow a more severe course of loss of renal function to complete renal failure. There is also some association between proteinuria and renal outcome in NCGN associated with anti-MPO. The renal function loss could be prevented: by strict BP control, salt restriction and use of angiotensin converting enzyme inhibitors to reduce proteinuria. As the relapse rate is lower in anti-MPO positive patients, the duration of immunosuppressive treatment is shorter than that of anti-PR-3 positive patients.
Extra renal organ involvement and respiratory granulomas and deterioration of renal function are more frequent in patients with anti-PR-3 than anti-MPO. Therefore, early recognition of these patients by clinical, histopathological and serological diagnosis and their subsequent treatment and follow up by repeat serological testing is absolutely essential. It has been proved that the antibodies to PR-3 and MPO and also other cytoplasmic antigens, are in raised amounts during exacerbation and in lower amounts when the patient is in remission and identification of such parameters would possibly affect renal outcome and its subsequent treatment in a favourable way.
Tables
[Table - 1]
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