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A study of anti-neutrophil cytoplasm antibodies in systemic vasculitis and other related disorders. VD Pradhan, SS Badakere, YS Iyer, R Kumar, AF AlmeidaInstitute of Immunohematology, Indian Council of Medical Research, Parel, Mumbai - 400012, India., India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.936
BACKGROUND: Anti-neutrophil cytoplasm antibodies (ANCA) play an important role as specific and sensitive markers for small vessel vasculitis and in some other systemic disorders. Indirect immunofluorescence test, known as the "Gold Standard" for screening of ANCA, can be further substantiated by ELISA for confirmation and for identifying sub-specificities like anti-Myeloperoxidase (anti-MPO), anti-Proteinase 3 (anti-PR3) and anti-Lactoferrin (anti-LF). AIMS: The present study was undertaken to investigate the incidence, specificities and strength of ANCA in suspected vasculitis cases and to correlate their presence with that of these auto-antibodies and with the disease. SUBJECTS AND METHODS: Sera from 130 clinically suspected vasculitis patients were studied. Indirect immunofluorescence microscopy (IIF) was used to identify cytoplasmic (c-ANCA), perinuclear (p-ANCA) and atypical (X-ANCA) patterns using ethanol and formalin fixed polymorphonuclear cells (PMN) and HL-60 cells from a human promyelocytic leukaemic cell line as substrates. ELISA was performed for identifying ANCA sub-specificities to anti-MPO and anti-PR3 and HEp-2 cells were used for detection of anti-nuclear antibodies (ANA). RESULTS: ANCA positivity was noted in 42.3% of these patients, wherein p-ANCA positivity rate was 34.6% and c-ANCA positivity was noted in 5.4% subjects. Three patients showed the unusual X-ANCA positivity. ELISA determined the sub-specificities: Out of 45 p-ANCA positive patients, 38 patients (84.4%) had anti-MPO and out of 7 c-ANCA positive patients, 5 patients (71.4%) had anti-PR3 antibodies. One patient with Class IV Lupus Nephritis, showed both anti-MPO and anti-PR3 antibodies and 17.8% p-ANCA positive patients had anti-Lactoferrin antibodies. CONCLUSIONS: Use of the Immunofluorescence method coupled with identification of ANCA sub-specificities by ELISA, is recommended for detection of ANCA in clinically suspected cases of small vessel and other systemic vasculitis. Keywords: Antibodies, Antineutrophil Cytoplasmic, blood,Autoantibodies, immunology,Biological Markers, Enzyme-Linked Immunosorbent Assay, Human, Microscopy, Fluorescence, Neutrophils, immunology,Vasculitis, immunology,
Anti-neutrophil cytoplasm antibodies (ANCA) are directed against various components of neutrophil cytoplasm granules and monocyte lysozymes. Since the first description of ANCA[1] in patients with necrotising glomerulonephritis, its role as a sensitive marker for small vessel vasculitis associated disorders has been demonstrated. ANCA has been reported as a specific and sensitive serological marker for active Wegener’s granulomatosis (WG)[2] and ANCA was also found to be present in Churg Strauss Syndrome (CSS), Microscopic polyangiitis (MPA) and pauci-immune necrotising crescentic glomerulonephritis (NCGN).[3],[4] In most cases of systemic vasculitis, the primary target antigens for ANCA are Proteinase3 (PR3), a serine protease showing a cytoplasmic immunofluorescence pattern (c-ANCA) and Myeloperoxidase (MPO), an enzyme involved in the production of reactive oxygen intermediates and giving a perinuclear (p-ANCA) pattern on IIF.[5] A third mixed pattern, the ‘atypical’ or X-ANCA is rarely seen. The cytoplasmic enzymes are present in azurophilic granules of neutrophils and monocytes and antibodies against them, are known to have pathophysiological and diagnostic potential. The presence and changes in ANCA titers, as shown in human and experimental animal models, are known to correlate directly with changes in disease-activity indicating their usefulness as a diagnostic tool.[6],[7] ANCA associated vasculitis (AAV) has very minimal or no deposits of immune complex affected organs (‘pauci-immune’). Diagnosis is usually based on the clinical features, immunohistochemical studies, biopsies of lung or kidney tissues (necrotising vasculitis of small arteries, capillaries or veins) as well as the presence of some serological markers (either c-ANCA or p-ANCA by IIF microscopy and its subspecificities to either anti-MPO or anti-PR3 by ELISA).[7]
One hundred and thirty clinically suspected cases of vasculitis were referred by clinicians from the Nephrology, Rheumatology and Medicine units of some major public hospitals in Mumbai for the detection of ANCA. This prospective study was carried out after obtaining the requisite Ethics Committee permission. Five ml of blood was aseptically collected and the separated serum was stored at -200C. Relevant clinical, histopathological and laboratory data was recorded in a proforma maintained by the investigators. Criteria published by the American College of Rheumatologists (ACR)8 was used for the classification of patients. The presence of renal vasculitis was confirmed by light microscopic examination of stained specimen (hematoxylin and periodic acid Schiff) of renal biopsy and by immunofluorescent microscopy using anti-IgG, anti-IgM, anti-IgA, anti-C3, anti-C4 and anti-fibrinogen FITC conjugates. For the latter examination renal tissue obtained at biopsy was collected in 10% buffered formalin and 3mm thick sections were prepared using paraffin embedded blocks. The identification of ANCA was done by IIF test which is considered as the ‘Gold standard’ for ANCA screening; using human neutrophils (PMN) as well as using a human promyelocytic leukemic cell line (HL-60) obtained from NCCS (Pune) and maintained in Minimal Essential Medium (MEM) as a continuous culture and harvested at log phase of growth. The cells were used to prepare a cytospun substrate using Hettich Universal 16A cytocentrifuge and fixing the slides with 96% ethanol and also formalin individually before coating with patient’s serum. Slides were probed using FITC tagged polyvalent anti-human globulin serum using a fluorescent microscope (Nikon, Optiphot II) and microphotography was done using an automated photography system (Nikon AFX II A). Ten controls of serum having anti-MPO and anti-PR3 antibodies were a gift from scientists from Germany, Denmark and Hongkong and were used as required. ANA was qualitatively and quantitatively tested by IIF using HEp-2 cells obtained from Enterovirus Research Center, Indian Council of Medical Research (ICMR), Mumbai. The cells were maintained in a continuous culture and harvested at log phase of growth. The results were interpreted in terms of titers i.e. test sera giving positivity for immunofluorescence at its highest dilution. A cut off for positivity was 1:10 dilution of test serum for ANCA and ANA testing. Rapid ELISA test, using ultrasonicated neutrophil cytoplasm extract (called as ? granules) was employed for detection of ANCA (method described by Rasmussen et al).9 In this method the ultrasonicated PMN extract after purification and estimation of protein concentration was adjusted to 50 mg/ml . 100 ml of this concentrate was coated onto the round bottom microtiter plates and after incubation and blocking, the test sera were reacted and the reaction was read using alkaline phosphatase conjugated anti-human globulin at 405nm. A cut off of normal human serum (NHS)+3SD was considered as positive. The specificity of the antibodies was identified by antigen binding ELISAs for anti-Myeloperoxidase (MPO) and anti-Proteinase3 (PR3) using kits from Genesis (UK) as per the directions in the kit. A value <3.0 u/ml was negative, 3-5 u/ml were equivocal and >5 u/ml were considered as positive. Anti-Lactoferrin (anti-LF) ELISA was developed in the laboratory using purified Lactoferrin from Sigma, USA and the assay was standardised as per the method described by Chikazawa et al.10
One hundred and thirty patients were screened by both IIF and further tested by ELISA [Table - 1]. The aetiologies of the disorders encountered in this study are shown in [Table - 1]. ANCA were present in 55 patients, when tested by IIF test. Among these, 45 patients showed the classical perinuclear pattern (p-ANCA), 7 patients showed cytoplasmic fluorescence pattern (c-ANCA) on ethanol fixed substrates and also formalin fixed preparations while 3 patients had X-ANCA. All the 55 ANCA positive sera by immunofluorescence were found to be positive by the ? granule ELISA, and 38 patients had antibodies to MPO while 5 had anti-PR3 antibodies by ELISA also. There were no cases that were positive by ELISA but negative by IIF. Three cases showed the atypical pattern by IIF (also called as X-ANCA or the ‘Snow Drift’ pattern) and were interpreted to be a mixture of both p-ANCA and c-ANCA fluorescence patterns. On using formalin fixed PMN there was no alteration of pattern. However, they did not react with HL-60 cells at all. Of the 130 cases, 52 had ANA, with titers ranging between 1:40 and 1:160. It was observed that patients with MPA showed a higher incidence of 88.9% p-ANCA positivity with one case showing ‘X’ ANCA [Table - 1]. In a group of patients having MPGN with crescents, 8/13 patients (61.5%) had p-ANCA and one patient had c-ANCA and 7/21 patients (33.3%) in the RLV group, had p-ANCA pattern on IIF and twenty out of 41 patients (48.8%) in the SLE Class IV lupus nephritis group had p-ANCA positivity. Comparatively, a lower incidence of ANCA was seen in SLE patients having no renal involvement wherein out of 18 patients, only 2 (11.1%) patients showed p-ANCA positivity. It was observed that out of 4 patients having leukocytoclastic vasculitis, one showed a p-ANCA pattern and that in patients with other disorders like polyarteritis nodosa (PAN), Henoch-Schonlein purpura (HSP), autoimmune hemolytic anemia (AIHA) and RA vasculitis, ANCA was absent. Of 6 patients with suspected WG, four patients (66.7%) demonstrated the c-ANCA IIF pattern and one patient showed p-ANCA. Polymorphonuclear cells (PMN) and HL60 cells are the commonly used substrates for ANCA detection where initially ethanol fixed cells are used for observation of ANCA patterns and later, formalin-fixed cells are used to confirm all p-ANCA positive findings. The c-ANCA positives showed cytoplasmic fluorescence on formalin-fixation while the three cases of X-ANCA were negative on formalin fixation. HL-60 cells could detect none of X-ANCA. As shown in [Table - 3], when the antibody binding capacity of PMN and HL-60 cell extracts was compared, it was observed that ? granules prepared from PMN detected all ANCA positive samples by ELISA but the HL-60 sonicated extract could pick up only 40/45 (88.9%) of these ANCA positive cases. Therefore, ? granules of PMN was found to be a better substrate to be used as a broad-spectrum screening assay for ANCA detection, as three cases of X-ANCA which were positive on ? granules of PMN, were negative on HL-60 extract and also by anti-MPO and anti-PR3 ELISA. The anti-LF ELISA detected 8 out of the 45 cases showing perinuclear fluorescence and two of the atypical or X-ANCA were positive for anti-LF antibodies. As shown in [Table - 4], of the 88.8% positives detected by IIF in the MPA group, 5 patients were detected by ELISA (55.5%) having anti-MPO. In MPGN with crescents group, 4 patients (30.8%) had anti-MPO. Of the 7/21(33.3%) p-ANCA positives by IIF in RLV, 6 patients (28.6%) had anti-MPO antibodies and in class IV lupus nephritis, 12 patients (29.3%) were anti-MPO positives. Of the 6 Wegener’s granulomatosis cases, 4 (66.7%) had c-ANCA, all of which had anti-PR3 antibodies and one patient showed presence of anti-MPO antibodies. Analysing the strength of ANCA titers tested by two fold double dilution technique by IIF, it was seen that more number of cases with higher titers were observed in MPA,WG, RLV and MPGN with crescents where the titer values ranged from 80-320 while higher number of cases with lower titer values (40-80) were noted in the class IV LN and SLE groups. Similar phenomenon as in IIF was observed with ELISA.
Anti-neutrophil cytoplasm antibodies (ANCA) often seen in pauci-immune small vessel vasculitis and some closely related disorders, is now assuming importance as a sensitive and specific serological marker for a group of systemic vasculitis and also for characterising some kidney disorders. Quantitative ANCA testing, too has been observed to be useful as an index of disease activity,[11] as a clinical relapse or exacerbation of clinical symptoms is almost invariably preceded by a rise in ANCA titre levels.[12] The utility of ANCA testing in determining the disease activity has been amply demonstrated by Jayne et al.[13] They demonstrated that in 83% ANCA positive cases, ANCA titres become negative during remission. They also observed that in as many as 57% of ANCA positive cases relapse was preceded by a rise in ANCA titres. Other reports have also noted that ANCA levels were lower during remission than during active disease.[14],[15] In our study, an overall incidence of 42.3% ANCA positivity by IIF was observed with the perinuclear pattern (p-ANCA) that was more commonly seen in 34.6% of the patients while the classical c-ANCA pattern was seen in 5.4% of the patients. However when tested by ELISA among the IIF positives, 84.4% had anti–MPO and 71.4% showed anti-PR3. Other investigators[16],[17],[18] have observed that only about 65-80% of IIF positive cases can be detected by ELISA. Higher incidence of presence ANCA was observed in MPA, WG and RLV. It may be noted that in these conditions presence of these antibodies is used for the establishment of diagnosis in conjunction with clinical observations and histopathological findings. Three unusual cases of ‘X’ or atypical ANCA which were negative by ELISA for anti-MPO and anti-PR3 were encountered. Clinical and histopathological investigations showed that two of them had vasculitis with focal proliferative glomerulonephritis (FPGN) and one had vasculitis with acute tubular interstitial disease (ATID). Atypical or ‘X’ ANCA, detected mainly by IIF is often undetected by anti-MPO or anti-PR3 ELISA, but shows specificity for anti-Lactoferrin or anti-Cathepsin G, and are seen in a few clinical conditions[19] often showing a confusing immunofluoresence pattern. Initially, the ‘X’ ANCA was identified as a mixed pattern on IIF using ethanol fixed neutrophils and formalin fixation produced no change in the IIF pattern. Ludemann[19] also observed that IIF using various fixatives like ethanol, formalin, formaldehyde etc. cannot solve the identification problem of ‘X’-ANCA as the specificity of such reacting sera could be directed towards certain other than MPO or PR3 cytoplasmic antigens, like Cathepsin G or Lactoferrin or may be to other antigens in the neutrophil cytoplasm. The indirect immunofluorescence test known earlier as the ‘Gold standard’ is a good screening test for detection of all ANCA, but combining it with ELISA for anti-MPO and anti-PR3 would be more accurate for ANCA detection and identification.[20],[21] In this study we have observed the positives picked up by IIF could include cytoplasmic antibodies other than anti-MPO or anti-PR3 antibodies. Hence, using ELISA that detect antibodies against MPO and PR3, may give a lower incidence of positive tests. In fact, this should prompt the investigators to test for specificities to other cytoplasmic granules. Inclusion of a PMN ? granules ELISA, which detects all IIF, would act as a broad spectrum screening assay for the detection of antibodies to cytoplasmic components of primary, secondary and tertiary granules of the cytoplasm, as compared to HL-60 sonicated extract which is known to have only primary granules in cytoplasm.[22] In our patients, the incidence of p-ANCA was found to be higher than that of c-ANCA. The reason for this observation is not known. It is a matter of speculation if this could be related to the predisposition to common microbial infections.
[Table - 1], [Table - 2], [Table - 3], [Table - 4]
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