This retrospective study includes 1289 patients who were tested for ANCA serology at Molecular Genetics laboratory in a large tertiary care center in the National Capital Region (NCR), India during 2011–2016. The study protocol conformed to the provisions of the 1975 Declaration of Helsinki (as revised in Seoul, Korea, October 2008). Samples were mainly received from patients undergoing secondary or tertiary referral to specialists, i.e., nephrologists, pulmonologists, rheumatologists, gastroenterologists, gynecologists, and general physicians. Whole blood was collected from the patients by venipuncture into plain vacuumed tubes, and separated serum was stored at −80°C until testing. However, to prevent repeated freezing and thawing, serum aliquots were thawed only once and immediately processed after thawing to prevent discrepancies in results. Based on standardized assays used in our laboratory, screening of ANCA was performed by IIFT and specific antigens [MPO, PR3, and (glomerular basement membrane) GBM] were identified using LIA.

Indirect Immunofluorescence test (IIFT): Screening of ANCAs was performed using ethanol fixed neutrophils from Immunoconcepts (Cat no: 10070-11). Serum diluted (1:20) in phosphate-buffered saline was incubated with ethanol fixed neutrophils for 30min at room temperature. Slides were washed twice for five minutes with PBS, incubated for an additional 30min with fluorescent-labeled conjugated antihuman IgG. Subsequently, a cover-slip was placed over the slide and analyzed using a fluorescence microscope at 40× magnification. The fluorescence of each sample was compared with the negative control and the pattern of fluorescence was determined and recorded. While reporting, the results were interpreted as (a) C-ANCA positive: diffuse granular cytoplasmic staining with interlobular accentuation (b) P-ANCA positive: peri-nuclear fluorescence with the nuclear extension (c) Atypical ANCA: peri-nuclear staining without nuclear extension, or diffuse flat cytoplasmic staining, or the combination of both cytoplasmic and nuclear/peri-nuclear staining on neutrophils (d) Atypical ANCA with granulocytes specific ANA: showing atypical staining with ANA interference (e) ANCA negative: shows no fluorescence.3–5

Line Immunoassay (LIA): Imtech ANCA LIA MAXX (Cat No: ITC92005) was used for the qualitative detection of three antigen panels (PR3, MPO, and GBM) according to manufacturer's instructions. Three antigens, cut-off control, and positive control are applied as lines on a nitrocellulose membrane. The nitrocellulose membrane was blocked by adding a blocking buffer for 5min to prevent non-specific reactions. Incubation of a strip with diluted patient samples was done for 30min to allow binding of the antigens on the strip with autoantibodies present in the sample. For the detection of the bound antibodies, an alkaline phosphatase labeled anti-human IgG antibody was added to strips for 30min. After the addition of the substrate solution for 30min, the appearance of brown lines indicated the existence of (auto) antibodies against the respective antigens. The intensity of brown lines was measured using Huma-Scan software. The test result is negative, if no band is to be recognized or if the band exhibits a smaller intensity in comparison to the cut-off control. The test is equivocal if the intensity of the band and the intensity of the cut-off control do not significantly differ. The test result is positive if a band exhibits stronger staining in comparison to the cut-off control.

To summarize the data, counts and percentages were used for categorical variables. Percentages were calculated using Microsoft Office Standard 2013.

Patient consent was obtained for diagnosis and treatment at the hospital. This was an observational study; no personal identifiers like name, addresses were used and no additional sample was drawn for this study. All investigation, treatment, and monitoring were according to the current ‘standard-of-care’.

Upon IIF investigation, ANCA was detected in 13.0% (168 out of 1289) at 1:20 dilution, of which 23.8% (40/168), 25.0% (42/168, and 47.6% (80/168) were positive with P-ANCA, C-ANCA and atypical pattern respectively. Atypical ANCA with granulocytes specific ANA was observed in 3.6% (6/168) cases (Fig. 1). On evaluation with a line immunoassay, 6.7% (86/1289) were positive out of which 52.3% (45/86), 41.9% (36/86), 8.8% (6/86) were positive for anti-MPO, anti-PR3, and anti-GBM respectively.

Fig. 1.

Summary of immunoassays in total patients (n=1289).

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The association of a specific autoantibody with specific immunofluorescence (IIF) pattern was observed. Out of 40 P-ANCA positive cases, 92.5% (37/40) were positive for anti-MPO and 5.0% (2/40) were positive for anti-PR3. Among 42 C-ANCA cases, 76.2% (32/42) and 16.7% (7/42) were positive for anti-PR3 and anti-MPO respectively (Fig. 1). Images of various ANCA IIF patterns are shown in Fig. 2.

Fig. 2.

ANCA patterns observed by IIFT in patients included in the study: (a) C-ANCA, (b) P-ANCA, (c) Atypical ANCA, (d) Atypical ANCA with granulocytes specific ANA.

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Incidentally, while studying the association of IIF patterns and specific autoantibodies, we found the distribution of anti-MPO and anti-PR3 in AAV (shown in Table 1). Anti-MPO antibody was predominant in 87.5% (7/8) of EGPA and 91.3% (21/23) cases of MPA/RLV. The anti-PR3 antibody was predominant in 87.5% (28/32) cases of GPA.

Out of 168 IIF positive samples 8, 32, and 23 cases were observed as EGPA, GPA, and MPA/RLV respectively. Six cases of rapidly progressive glomerulonephritis (RPGN) were identified as positive for GBM. 105 cases remained unclassifiable. A comparison of demographics among AAV diseases were shown in Table 1. Kidney and chest involvement was observed in most of the diseases. Table 2 indicates the involvement of different organs in AAV diseases.