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Year : 2011  |  Volume : 54  |  Issue : 2  |  Page : 258-263
A decade long experience of anti-neutrophil cytoplasmic antibody testing in a tertiary care referral center in North India: Perspective from a developing country

1 Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Paediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication27-May-2011


Background: In a developing, tropical country like India, discontinuous power supply, high temperatures during summer, and lack of consistent cold chain and funds provide a challenging atmosphere for anti-neutrophil cytoplasmic antibody (ANCA) testing and reporting. However, a simple in-house test and testing algorithm are described here, which have been developed and tested over time. Materials and Methods: An analysis of a decade of testing and reporting of ANCA in the Department of Immunopathology in a tertiary referral health care center was performed to highlight the importance of testing for ANCA in proposed 1999 guideline recommended indications. Results: A total of 4195 ANCA tests were conducted from 2000 to 2009. Overall, 2060 (49%) requests had indications which met the 1999 guidelines, while the remaining 2135 (51%) fell outside the guidelines. A total of 350 samples (8.3%) were positive for ANCA on indirect immunofluorescence (IIF), out of which 212 were guideline recommended and 138 (3.2%) were non-guideline recommended ANCA requests; thus, 3.2% of non-small vessel ANCA associated vasculitis (non-SVAAV) conditions showed false positive results when the population was otherwise unselected. Maximum requests (1432) were for rapidly progressive renal failure/acute renal failure. Conclusions: The audit shows that compliance with guidelines for ANCA testing would decrease the number of false positive results. In-house screening for ANCA by IIF is cost-effective and must be performed at least twice on two different samples from the same patient or on two different sets of ANCA preparations in all the cases who requested ANCA testing with a proposed 1999 guideline recommended indication.

Keywords: Anti-neutrophil cytoplasmic antibody, autoimmune, immunofluorescence, vasculitis

How to cite this article:
Minz RW, Chhabra S, Rani L, Singh S, Jindal SK, Sakhuja V. A decade long experience of anti-neutrophil cytoplasmic antibody testing in a tertiary care referral center in North India: Perspective from a developing country. Indian J Pathol Microbiol 2011;54:258-63

How to cite this URL:
Minz RW, Chhabra S, Rani L, Singh S, Jindal SK, Sakhuja V. A decade long experience of anti-neutrophil cytoplasmic antibody testing in a tertiary care referral center in North India: Perspective from a developing country. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Jul 7];54:258-63. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/258/81587

   Introduction Top

Anti-neutrophil cytoplasmic antibody (ANCA) has provided doctors with a useful serological test to assist in the diagnosis of small vessel ANCA associated vasculitides (SVAAV). The guidelines for ANCA testing and reporting were developed as a part of 1999 international consensus statement on ANCA testing and reporting to minimize technical difficulties, produce a greater uniformity in the results issued by different laboratories and minimize the false positive rate. [1],[2] Various studies published indicate that ANCA testing should only be ordered on patients with an indication meeting these guidelines and a failure to follow them leads to increased cost and the risk of false positive results. [3],[4],[5] In light of this knowledge, it is imperative that an audit of testing strategy is undertaken to enforce the most cost-effective way of ANCA testing, thereby avoiding further unnecessary testing and consultation.

This study was undertaken to determine whether indications for ordering ANCA test in our clinical setting met the 1999 guidelines proposed for ANCA testing and to observe ANCA positivity time trends in a 10-year study period. This study also provides information on the spectrum of non-vasculitic conditions showing ANCA positivity. An attempt has also been made to offer a protocol for ANCA testing in a tropical, developing country like ours.

   Materials and Methods Top

A review of laboratory records identified 4195 consecutive patients for whom an ANCA test was ordered in a 10-year period from 2000 to 2009. The ANCA requests mainly involved patients undergoing secondary or tertiary referral to specialists, i.e., nephrologists, pulmonologists, rheumatologists, gastroenterologists, gynecologists and general physicians. All consecutive ANCA requisition forms received from both inpatient and outpatient units were examined and reviewed for clinical data and ANCA results [indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) results]. Data collected were used to determine whether the patients in this study fulfilled 1999 guideline criteria or not. [2] If a patient had one of the recommended clinical indications for ANCA testing at the time ANCA test was ordered, the patient was considered to have fulfilled the criteria for ANCA testing.

All ANCA tests were performed in the Department of Immunopathology. ANCA was tested using an IIF assay with "in-house" ethanol fixed neutrophil preparations (ANCA spots). Serum was diluted 1/40 and then overlayed on ANCA spots. A 1/40 serum dilution for adults and 1/20 serum dilution for children was used according to international guidelines. [6] ANCA was detected using fluorescein isothiocyanate conjugated antihuman immunoglobulins. Antinuclear antibodies (ANA) were also detected in all the patients using IIF of rat liver tissue (serum dilution 1/10) in the initial years and then Hep2 cell lines (serum dilution 1/40) in the later years before putting up ANCA tests. ANA positive sera were not tested for ANCA on IIF as there is great difficulty in interpretation of IIF when ANCA and ANA coexist. While reporting, the results were interpreted according to the international guidelines. [2]

  • C-ANCA positive
  • P-ANCA positive
  • Atypical C-ANCA positive
  • Atypical ANCA positive
  • ANCA negative
The reports of ELISA results to determine myeloperoxidase (MPO) and proteinase-3 (PR-3) specificity of ANCA were available in only 180 patients. Unfortunately, at that time we did not have the resources for ELISA analysis so we are unable to offer any comment on the performance characteristics of ANCA test. Multiple test requests were received in 45 patients but these requests were counted only once for the final data analysis.

   Results Top

A total of 4310 ANCA tests were conducted between 2000 and 2009. Forty-five patients had multiple tests with a total 115 ANCA tests done on these 45 patients. So, a total of 4195 ANCA test results were finally available for the analysis.

[Table 1] shows the clinical ANCA testing indications mentioned on the requisition forms in 4195 patients. Overall, 2060 (49%) had indications which met the 1999 guidelines. Indications for the remaining 2135 (51%) fell outside the guidelines. Where indications met the 1999 guidelines, 1432 ANCA requests were for rapidly progressive renal failure (RPRF)/acute renal failure (ARF)/chronic renal failure (CRF)/glomerulonephritis (GN), and where guidelines were not met, the most frequent indication was vasculitis not otherwise specified (NOS) in 671 cases.
Table 1: Indications for Anti-neutrophil cytoplasmic antibody testing

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[Table 2] and [Table 3] show the outcome of test results on the patients tested for ANCA. Overall, a total of 350 [212 (recommended) + 138 (non-recommended)] samples were positive on IIF. P-ANCA [Figure 1] was the most common pattern observed on IIF, present in 187 [112 (recommended + 75 (non-recommended)] samples. C-ANCA [Figure 2] was positive in 127 samples while atypical C-ANCA [Figure 3] was seen in 35 samples. Atypical ANCA was noted only in one non-guideline recommended patient.
Figure 1: IIF photomicrograph of ethanol fixed neutrophil preparation showing P-ANCA pattern of perinuclear fluorescence (×400)

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Figure 2: IIF photomicrograph of ethanol fixed neutrophil preparation showing C-ANCA pattern of granular cytoplasmic fluorescence with central lobular accentuation (×400)

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Figure 3: IIF photomicrograph of ethanol fixed neutrophil preparation showing atypical C-ANCA pattern of diffuse flat cytoplasmic fluorescence without central lobular accentuation (×400)

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Table 2: Anti-neutrophil cytoplasmic antibody patterns on IIF and ELISA results (wherever available) in patients with 1999 guidelines recommended indications"

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Table 3: ANCA patterns on IIF and ELISA results (wherever available) in patients with non-guideline recommended indications

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Reports of ELISA for PR-3 and MPO were available in only 180 of 350 patients. ELISAs for either PR-3 or MPO or both were positive in 87 patients. Remaining 93 patients were negative by ELISA despite the fact that they showed positivity on IIF. A total of 54 patients showed PR3-ANCA, 39 of whom had a 1999 guideline recommended indication; remaining 15 patients presented with a clinical impression of PAN (1), vasculitis NOS (7), systemic lupus erythematosus (SLE, 2), connective tissue disorders (CTDs, 2), cirrhosis (1), pyrexia of unknown origin (PUO, 1), and Takayasu's arteritis (1). Out of 54 patients showing PR3-ANCA positive ELISA, 45 showed a C-ANCA, 6 showed P-ANCA, 2 showed an atypical C-ANCA and 1 showed an atypical ANCA pattern on IIF.

Only 34/180 patients had MPO-ANCA, 24 of whom had recommended indications; the remaining 10 patients presented with a clinical indication of PAN (1), vasculitis NOS (4), SLE (3) and CTD (1) and autoimmune pancreatitis (1). Out of 33 MPO-ANCA positive ELISA patients, 29 showed P-ANCA, 2 showed C-ANCA, 1 showed an atypical C-ANCA and 1 showed an atypical ANCA pattern on IIF.

In all cases where SVAAV was diagnosed, the indication for testing was either known SVAAV or a 1999 guideline approved indication. No case of SVAAV was diagnosed from a test requested outside the scope of the guidelines.

The query of atypical C-ANCA pattern could be resolved in only three patients where ELISA reports were available. It included two patients who requested ANCA with an indication of WG and one patient with CTD. Both the patients with an indication of WG showed PR-3-ANCA and the patient with CTD showed MPO-ANCA. One of the SLE patients who showed atypical ANCA pattern on IIF showed dual specificity for both MPO-ANCA and PR3-ANCA and ++++ diffuse ANA pattern on Hep2 cell lines. The remaining three cases of SLE that showed ANCA positivity on IIF were ANA negative.

The bar diagram [Figure 4] depicts the time trends of ANCA positivity in a tertiary referral health care center in North India where an average of 420 ANCA tests are performed annually.
Figure 4: Bar diagram showing total ANCA positive cases on IIF vs. total number of cases tested in year by year distribution. Series 1 shows total ANCA positive cases, while Series 2 shows total number of cases

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   Discussion Top

ANCA testing has been promoted as a potentially important diagnostic tool. After performing 4310 clinician ordered ANCA testing and reporting at our institute in North India, it was imperative to audit the endeavor to assess its contribution to diagnose SVAAV and to propose an algorithm for ANCA testing.

False positive ANCA test results (3.2% in our study) have been reported with a differing frequency in a number of conditions. [7],[8],[9],[10],[11],[12],[13],[14],[15] These frequencies differ between laboratories because of cohort variation and difference in detection methods. ANCA results in patients without good clinical evidence for vasculitis should be interpreted with care. If ANCA are to remain useful tools in the diagnosis and monitoring of patients with SVAAV, we must be aware of the relative frequencies of other diseases which can result in a positive ANCA. [16]

A negative ANCA test result cannot be used to rule out vasculitis in general. [2] Edgar et al. demonstrated in their audit that ANCA testing should not be used as the only screening investigation for vasculitis as it cannot confirm or exclude a diagnosis but should only be used as a part of a rational investigative scheme and the interpretation of a positive ANCA result must take into account the presence of other autoantibodies and the full range of non-vasculitic conditions when the clinical situation is not typical of vasculitis. [16]

There are several methods available for the detection and further identification of ANCA, but the method of choice for initial testing remains IIF. This is a labor-intensive test that depends on the expertise of the technicians and requires observer experience and consistency. To avoid false positive results and operator dependence in IIF, the consensus documents recommend that all ANCA positive immunofluorescence results should be investigated further using a second technique like ELISA, specifically for MPO and PR-3. A positive IIF test result is not specific for SVAAV as also observed in this audit. Some authors even suggest that IIF testing be avoided altogether, [17] but the current knowledge of multiple target antigens of ANCA demonstrates that several suitably standardized assays would be required to replace IIF.

In spite of published guidelines for ANCA testing and reporting, the clinical practice in our institute till date has been to order ANCA testing in a large number of non-guideline conditions (2135/4195) like PUO, liver diseases, SLE, rheumatoid arthritis (RA), stroke, etc., as shown in [Table 1]. In the present audit, the decision to test for ANCA, therefore, reflects actual patterns of ANCA test ordering in clinical practice at our institute. Test ordering outside of the guidelines is not restricted to one specialist group as ANCA requests were received from almost all specialists.

Sinclair et al. in their study applied a "gating policy" which refused analysis on requests that were not supported by clinical data suggestive of vasculitis, which thereby resulted in a large cost saving. [4] Mandll et al.[3] and Robinson et al.[5] had earlier demonstrated that this exercise would have resulted in significant cost savings without any loss of diagnostic yield. Thus, it would be appropriate to follow this gating policy at our institute too. The rate of 1999 appropriate tests was 2060/4195 (49%) in the present study which is closer to that observed by Robinson and Steele [5] from New Zealand, i.e., 35.5%. This reflects propensity of clinicians to use ANCA testing to diagnose conditions other than SVAAV, where the exact role of ANCA positivity has not been clearly worked out as yet.

The biggest drawback of the study is its retrospective nature that has a potential for error due to lack of documentation of signs, symptoms and follow-up data. Also, ELISA tests were not done in all the patients due to non-availability of resources during the initial years of setting-up of autoimmune laboratory, so the actual performance characteristics (positive predictive value) of the test could not be evaluated.

Although initially thought to be an excellent tool for diagnosing SVAAV, the ANCA testing proved to be an imprecise indicator of disease when applied widely to heterogeneous populations. Limiting ANCA testing to patients whose clinical signs and symptoms are most suggestive of SVAAV could effectively increase the prevalence of SVAAV in the group being tested, thus improving ANCA's positive predictive value. This is one rationale behind the development of guidelines. [3] It was suggested by the guideline authors that if ANCA testing was restricted to patients meeting the guideline criteria, ANCA testing would be "probably 95% sensitive and 90% specific for SVAAV, with a much higher positive predictive value than when ANCA testing is used in unselected patients". [2] Thus, limiting ANCA test ordering according to the guidelines could result in cost savings without compromising patient care and will have a major impact on the efficiency of ANCA testing.

Through this paper we would also like to share our experience on technical aspects as well as interpretation of ANCA test results in a tropical environment like ours. [Figure 5] shows an algorithm for ANCA testing in our setting. In-house testing using ethanol fixed neutrophil preparations (ANCA spots) is a labor-intensive procedure for ANCA screening. Because of frequent power cuts, thawing of these ANCA spots can give false negative results. So, we recommend that ANCA screening by IIF should be done at least twice on two different samples from the same patient and/or on two different sets of ANCA spots in all cases where ANCA testing is requested for a proposed 1999 guideline recommended indication. Also, performance of ELISA is directly affected by strict maintenance of a cold chain during transit and storage which is often difficult in a tropical country. Laboratories should do strict internal quality control using known positives and negatives in each test run. Well-characterized disease cohorts will help define cut-off values and sensitivity and specificity of ANCA for the population under study.
Figure 5: An algorithm for ANCA testing in a developing and tropical country

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The experience of testing and reporting of 4152 ANCA tests has been audited here to develop an algorithm for ANCA testing in a tropical and developing country like India and for also highlighting the importance of testing ANCA in proposed 1999 guideline recommended indications.

   Acknowledgment Top

We acknowledge the technical expertise of Mrs. Shashi Anand and Mrs. Ranjit Bhardwaj for helping us with ANCA tests and ELISAs.

   References Top

1.Savige J, Dimech W, Fritzler M, Goeken J, Hagen C, Jennette C, 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;120:312-8.  Back to cited text no. 1
2.Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, et al. International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). Am J Clin Pathol 1999;111:507-13.  Back to cited text no. 2
3.Mandl LA, Solomon DH, Smith EL, Lew RA, Katz JN, Shmerling RH. Using antineutrophil cytoplasmic antibody testing to diagnose vasculitis: Can test-ordering guidelines improve diagnostic accuracy. Arch Inter Med 2002;162:1509-14.  Back to cited text no. 3
4.Sinclair D, Saas M, Stevens JM. The effect of symptom related "gating policy" on ANCA requests in routine clinical practice. J Clin Pathol 2004;57:131-4.  Back to cited text no. 4
5.Robinson PC, Steele RH. Appropriateness of antineutrophil cytoplasmic antibody testing in a tertiary hospital. J Clin Pathol 2009;62:743-5.  Back to cited text no. 5
6.Wilk A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS Suppl 1989;6:12-3.  Back to cited text no. 6
7.Koderisch J, Andrassy K, Rasmussen N, Hartmann M, Tilgen W. False positive antineutrophil cytoplasmic antibodies in HIV infection (letter). Lancet 1990;335:1227-8.  Back to cited text no. 7
8.Peeters M, Joossens S, Vermeire S, Vlietinck R, Bossuyt X, Rutgeerts P, et al. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies in inflammatory bowel disease. Am J Gastroenterol 2001;96:730-4.  Back to cited text no. 8
9.Xu J, Yang CH, Chen XY, Li XH, Dai M, Xiao SD. A subset of ulcerative colitis with a positive proteinase-3 antineutrophil cytoplasmic antibody. World J Gastroenterol 2008;14:7012-5.  Back to cited text no. 9
10.Choi HK, Lamprecht P, Miles JL, Gross WL, Merkel PA. Subacute bacterial endocarditis with positive cytoplasmic antineutrophil antibodies and antiproteinase 3 antibodies. Arthritis Rheum 2000;43:226-31.  Back to cited text no. 10
11.Afeltra A, Sebastiani GD, Galezzi M, Caccavo D, Ferri GM, Marcolongo R, et al . Antineutrophil cytoplasmic antibodies in synovial fluid and in serum of patients with rheumatoid arthritis and other types of synovitis. J Rheumatol 1996;23:10-5.  Back to cited text no. 11
12.Ensault VL, Jayne DR, Keogan MT, Brownlee AA, Testa A, Lecarrer D, et al. Antineutrophil cytoplasmic antibodies in patients with monoclonal gammopathies. J Clin Lab Immunol 1990;335:1227-8.  Back to cited text no. 12
13.Pradhan VD, Badakere SS, Ghosh K, Pawar AR. Spectrum of antineutrophil cytoplasmic antibodies in patients with pulmonary tuberculosis overlaps with that of Wegener's granulomatosis. Indian J Med Sciences 2004;58:283-8.  Back to cited text no. 13
14.Pradhan V, Badakere SS, Kumar US. Increased incidence of cytoplasmic ANCA (C-ANCA) and other autoantibodies in Leprosy patients from Western India. Lepr Rev 2004;75:50-6.  Back to cited text no. 14
15.Pradhan VD, Badakere SS, Bichile LS, Almeida AA. Antineutrophil cytoplasmic antibodies (ANCA) in systemic lupus erythematosus: Prevalence, clinical associations and correlation with other autoantibodies. J Assoc Physicians India 2004;52:533-7.  Back to cited text no. 15
16.Edgar JD, McMillan SA, Bruce IN, Conlan SK. An audit of ANCA in routine clinical practice. Postgrad Med J 1995;71:605-12.   Back to cited text no. 16
17.Harris A, Gilles D, Vadas M, Chang G. ELISA is the superior method for detecting antineytrophil cytoplasmic antibodies in the diagnosis of systemic necrotizing vasculitis [letter]. J Clin Pathol 2000;53:644-5.  Back to cited text no. 17

Correspondence Address:
Seema Chhabra
Department of Immunopathology, PGIMER, Chandigarh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.81587

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

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