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Original Article

Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario
Wendy Sebastian, Atanu Roy1, Usha Kini1, Shalini Mullick1
School of Biotechnology, Chemical and Biomedical Engineering, Vellore Institute of Technology, Vellore 632 014, 1 Department of Pathology, St. Johns Medical College, Bangalore- 560 034, India
Address for correspondence: Dr. Usha Kini, Department of Pathology, St. Johns Medical College, Bangalore-560 034, India. E-mail: drushakini@gmail.com

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ABSTRACT Background: Immunity status, individual response to disease and types of antibodies produced are well known to vary from person to person, place to place and probably from population to population. A broad spectrum of specic auto antibodies that have so far been associated with specic rheumatic diseases, as noted in Western literature, has been well taken as a reference standard all over the world. There is neither research work nor any data correlating the auto antibodies and their antinuclear antibody (ANA) patterns with the immunoprole in the Indian population to date. Aims: To understand a denite association between ANA patterns and specic antibodies in the serum in the Indian study population and to document similarities / differences with the West. Settings and Design: This prospective and retrospective double blind study was undertaken on the South Indian population referred for ANA testing by Indirect Immunouorescence method and by immunoline methods. Materials and Methods: Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease were subjected for ANA testing by indirect immunouorescence (IIF) method and line immunoassay during the study period of 27 months. Serum samples were processed in dilution of 1:100 using HEp 2010 / liver biochip (Monkey) (EUROIMMUN AG). The serum samples which were further processed for line immunoassay were treated in 1:100 dilution on nylon strips coated with recombinant and puried antigens as discrete lines with plastic backing (EUROIMMUN AG) coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2) along with a control band. The analysis was done by comparing the intensity of the reaction with positive control line by image analysis. Results: The antinuclear antibody indirect immunouorescence (ANA IIF) patterns obtained were projectable to visualize a certain spectrum of specic antibodies such as homogenous (45.5%) with dsDNA, nucleosomes, histones, SSA / Ro-52, RIB and RNP / Sm, speckled pattern (35.6%) with Sm, RNP, SSA/Ro-52, SSB, Sm and RIB; nucleolar pattern with Scl-70, Sm, RNP and centromere pattern with CENP-B. The methodology indicated that, cytoplasmic pattern noted in ANA also needs to be correlated with primate liver in a biochip, which should prompt further decision for a request for line immunoassay and it is preferable for two pathologists to report independently and sign out a consensus ANA report for better predictive value. Conclusions: As a denite correlation between the ANA patterns and the group of antibodies was detected by line immunoassay, one could predict presence of certain specic auto antibodies for a particular ANA pattern identied. This may restrict one from requesting for line immunoassay, which is expensive and economizes on the cost of laboratory investigations in a developing country like India. Thus, screening of sera by ANA-IIF method alone may sufce and probably reduce the expense of detailed immunological work-up with minimal loss in diagnostic accuracy. This study, the rst of its kind in India, provides database and reference for the Indian subpopulation. INDIAN JOURNAL
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KEY WORDS: Antinuclear antibody test, immune prole, line immunoassay

DOI: 10.4103/0377-4929.68262 PMID: 20699497

INTRODUCTION A systemic autoimmune response is a common manifestation of the rheumatic connective tissue disease (CTD) and its hallmark is the presence of antinuclear antibodies (ANA). Indirect immunofluorescence (IIF) on HEp-2 (human epithelial cell tumor line) is the classical technique used for the detection of ANA, which is also used as a screening test. Though positive fluorescence staining indicates the prescience of ANA, it does not, however, allow precise identification of these antibodies. For this purpose specialized techniques such as enzyme linked immunosorbant assay (ELISA), Western blotting or line immunoassay are employed. A broad spectrum of specific antibodies has been associated with each specific rheumatic disease entity as listed out in the literature.[1-3] Most of these have been prepared with data obtained from various studies on Western population, but one needs to recall that immunity status, individual response to disease, type of antibodies, all, varies from person to person, place to place and population to population. In this study, we have analyzed a cohort of serum samples from the south Indian population, referred to our laboratory, providing services to a tertiary health
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centre and teaching hospital for ANA testing by Indirect Immunofluorescence method and samples further processed for identification of the specific antibodies by line immunoassay in that population and the two were correlated with one another to establish any definite link between the two. The results of this study would, therefore, provide a reference database for the Indian population. If a definite correlation is found between the ANA patterns and the line immunoassay details, one could restrict performing line immunoassays which are expensive and use ANA-IIF fluorescent patterns to predict presence of auto antibodies to precisely diagnose a CTD. This would economize on the cost of laboratory investigations in a developing country like India. MATERIALS AND METHODS Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease as suspected by rheumatologists / in-house specialists/ internal medicine specialists/dermatologists/ nephrologists or from any hospital department for a diagnosis of CTD were subjected for ANA testing by indirect immunofluorescence (IIF) method and line immunoassay during the study period of 27 months. Fresh whole blood samples were collected with patient consent at the phlebotomy section. Serum separated from the clotted blood samples by centrifugation was stored at 4C if testing was planned within 72 hours or at -20C for testing after three days (without freezing and thawing). A fasting blood sample was most often used to avoid lipemic serum which could result in increased background fluorescence or unclear staining pattern. All serum samples, thus received with a request for ANA by IIF method and line immunoassay with a suspected diagnosis of rheumatic disease by treating clinicians were taken for the study. The samples with a request for ANA by any method other than IIF, or accompanied by a request with a non-rheumatic diagnosis were excluded from the study. Serum samples were processed in dilution of 1:100 using HEp 2010 / liver biochip (Monkey) (EUROIMMUN AG) and conjugated with specific antihuman IgG (EUROIMMUN AG).[4] The fluorescence intensity was scored at x 400, semiquantitatively from 1+ to 4+ relative to the intensity of the positive (4+) and negative control.[5] The test result was discarded if the positive control sample failed to show the precise results. The serum samples which were positive for ANA by IIF method or those which were negative by IIF method but requested by the rheumatologists on clinical grounds were further processed for line immunoassay. Nylon strips coated with recombinant and purified antigens as discrete lines with plastic backing (EUROIMMUN AG) coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2) were used, along with a control band. The
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nylon strip was incubated with serum at a 1:100 dilution. The test strips, thus, processed at a 1: 10 dilutions were analyzed by comparing the intensity of the reaction with positive control line by image analysis [Figure 1]. RESULTS In this prospective and retrospective study in a 27-month period, 5066 samples were received. Of these 5066, only 319 satisfied the specific criteria and were analyzed for both ANA by IIF method and line immunoassay in this study. Among these 319 samples, 122 (38.2%) were ANA-IIF positive in a 1:100 serum dilution. Of these positive ANA-IIF, 101 (82.8%) were also line immunoassay positive. Total number of samples with ANA-IIF and line immunoassay done: 319 Number of ANA-IIF positive with line immunoassay positive : 101 Number of ANA-IIF positive with line immunoassay negative : 21 Number of ANA-IIF negative with line immunoassay positive : 31 Number of ANA-IIF negative with line immunoassay negative : 166 The various ANA patterns seen in the positive samples with line immunoassay positivity are shown in Table 1. The homogenous pattern was the most common ANA pattern, seen in 46 (45.5%) of the positive 101 samples [Figure 2]. In comparison with line immunoassay results of these samples [Figure 3], various combinations of specific auto-antigens were observed and the most prevalent among these are shown in Figure 4. The second most commonly occurring ANA pattern in this series was the speckled (n = 36; 35.6%) pattern. Correlation with line immunoassay results exhibited combinations as shown in Table 2. Specific combination of antigens was not observed in centromere and nucleolar patterns [Table 3]. ANA-IIF results exhibiting centromeric pattern were 5 (4.9%). 3 / 5 (60%) samples showed positivity for CENP-B (centromeric protein B). Ro-

Figure 1: Incubated EUROLINE ANA prole 3 showing the panel of auto antibodies tested using EuroLineScan

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Figure 2: (a) HEp2010 / liver biochip showing ANA positivity of homogenous pattern (b) along with primate liver Table 1: Spectrum of ANA-IIF patterns observed in the study population
Ana patterns Homogenous Speckled Centromere Nucleolar Nucleolar + Homogenous Nucleolar + Speckled Rim
Values in paranthesis are in percentage

Figure 3: ANA prole 3 test strip showing antibodies (3+) against dsDNA, nucleosomes and histones read against control band (2+) in a sample showing a positive homogenous pattern for ANA Figure 4: Results with ANA-IIF and line immunoassay tests in the study population

Samples (n=101, %) 46, (45.5) 36, (35.6) 5, (4.9) 5, (4.9) 6, (5.9) 2, (1.9) 1, (0.99)

Table 2: Immunoassay details of 46 samples with ANA-IIF homogenous pattern

Line immunoassay specicity dsDNA, Nucleosomes, Histones dsDNA, Nucleosomes, Histones, SSA/Ro-52 dsDNA, Nucleosomes, Histones, RIB dsDNA, Nucleosomes, Histones, RNP/Sm RIB, SSA
Values in paranthesis are in percentage

Samples (n = 46) 16, (34.8) 10, (21.7) 6, (13.04) 4, (8.7) 12, (26.08)

Table 3: Immunoassay details of 36 samples with ANA-IIF speckled pattern

Line immunoassay specicity SSA/Ro-52, SSB RNP/Sm, SSA SSA, RIB RNP/Sm, SSA/Ro-52, SSB
Values in paranthesis are in percentage

Strangely, 21 (17.2%) of the ANA-IIF positive samples showed negativity with line immunoassay. The ANA pattern observed in these cases were mostly homogenous 11 (52.3%); 3 had (4+) intensity and 8 (3+) intensity. Speckled pattern was seen in 6 (28.6%) cases where 2 had (4+) intensity and 4 (3+) intensity. Two (9.5%) cases exhibited nucleolar pattern, one each showing (4+) and (3+) intensity. Two (9.5%) cases exhibited cytoplasmic positivity and identified AMA-M2 as borderline positive. ANA-IIF negativity was observed in 197 of the total 319 samples under study. Of these, 31 (15.7%) exhibited positivity with line immunoassay. Line immunoassay showed 26 sera, positive for SSA/Ro-52 (84%) and five were positive for Scl-70 (16%). There was a single case in the entire study that exhibited strong positivity for SSA/Ro-52, dsDNA, nucleosomes and histones on line immunoassay but remained consistently negative for ANA-IIF. 166 samples were negative for both ANA and line immunoassay, though these samples were from patients who had rheumatic disease. DISCUSSION The presence of ANA is a hallmark of rheumatic or autoimmune disease. Apart from dsDNA antibodies, antibodies against a variety of other nuclear antigens can cause a positive ANA-IIF.
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Samples (n = 36) 10, (27.8) 9, (25) 5, (13.9) 2, (5.5)

52 and SSA, Ro-52, RIB positivity was seen in the other two cases each. Nucleolar pattern was observed in 5 (4.95%) samples [Table 4]. No specific combinations were found. Line immunoassay showed positivity for PCNA in 2 (4%), Scl-70, SSB and RNP, SSA in 1 each (2%). ANA-IIF reported as nucleolar- homogenous in 6 (5.9%) showed strong Scl-70 positivity in all cases along with SSA/ Ro-52 positivity in 2 (33.3%) cases. ANA-IIF reported as nucleolarspeckled in 2 (1.98%) showed strong positivity for Scl-70 in both. A single sample with rim pattern showed positivity for SSA/Ro-52, dsDNA, nucleosomes and histones on line immunoassay.
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Table 4: Immunoassay details of 19 samples with ANA-IIF centromere, nucleolar and rim patterns
Pattern Centromere Samples (N) 5 Percentage (%) 4.9 Line immunoassay specicity CENP-B (centromeric Protein B) (2/5) RNP, Sm and SSA(1/5) Ro-52, RIB (1/5) PCNA (2/5) , Scl-70(1/5), SSB (1/5) RNP/ SSA (1/5) Scl-70 (4/6), SSA/Ro-52 (2/5) Scl-70 SSA/Ro-52, dsDNA, Nucleosomes and Histones.

Nucleolar Nucleolar- homogenous Nucleolar- speckled Rim

5 6 2 1

4.9 5.9 1.98 0.99

Table 5: Comparison of immunoassay details of the present study population with Western literature
ANA pattern Homogenous Speckled Centromere Nucleolar Rim Western Catalogue[5] nDNA, Histone, deoxyribonuclear protein (DNP) Sm, RNP, Scl-70, SSA, SSB Centromere protein 4-6 S RNA nDNA, DNA-histone complex Western text book[2,3] DNA-histone complex Sm, RNP, Scl-70, SSA/Ro-52, SSB, RNA pol I and II and other antigens Centromere protein Nucleolar RNA DNA Present study dsDNA, nucleosomes, histones, SSA/ Ro-52, RNP/Sm, RIB Sm, RNP, SSA/Ro-52, SSB Centromere protein B Scl-70, SSA/Ro-52, SSB SSA/Ro-52, dsDNA, nucleosomes, histones

Although some IIF patterns strongly suggest distinct specificities, additional tests are requested to demonstrate antibody reactivities against specific nuclear and cytoplasmic antigens. These tests are used to either support the diagnosis (disease specificity) or to identify subsets of patterns that are prone to particular disease manifestation (prognostic marker). Further, the results of the test could be used in patterns with a wide differential diagnosis, so that the results of the tests may exclude systemic autoimmunity (negative predictive value) or may assist the diagnostic process by meeting the diagnostic criteria of a particular rheumatic disease (disease specificity).[6] Detection of ANA by traditional assays based on hemagglutination reactions and immunodiffussion are being replaced by newer technologies such as ELISA, ANA-HEp-2 which focus on short hands-on time and lower costs. HEp-2 cell lines from cultured human laryngeal epithelial carcinoma are more sensitive than animal (mouse line or rat kidney) tissue sections to the presence of antinuclear antibodies both in patients and in controls. Because of its high sensitivity rates, a high false positive rate for antinuclear antibodies is expected and that makes interpretation of a positive test result very difficult. To overcome this problem, a biochip incorporating HEp-2 cells and primate liver is used, thus incorporating the good of both the old and the new technologies. The high sensitivity response by Hep-2 cells needs to be confirmed on the primate liver. Thus, false positives are dramatically reduced by the use of biochips for ANA by IIF method in this study. Accepting that in daily life, many tests are requested for patients with or without rheumatic disease and with or without manifestation of autoimmune process and that laboratory management should be price conscious, especially in a
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country like India, the laboratory needs adequate and reliable screening tests that are relatively cheap in comparison to the third generation tests like line immunoassay. Hence, this study was undertaken to compare the diagnostic value and cost effectiveness of ANA pattern (costing approximately Rs 150 per test) with line immunoassay (Rs 1100 per test). Perhaps the study that most closely resembles ours is from Albania, reported in 1992, by Sulcebe and Morcka.[7] This study reports the results of sera tested on ANA using rat liver substrate and a screening dilution of 1:100 on rheumatic as well as nonrheumatic diseases. In another study by Slater and Shmerling,[8] ANA was performed on Hep-2 cell substrate at a titre of 1: 40. In this study, 319 of the 5066 serum samples satisfied the definite selection criteria and processed for both ANA and line immunoassay tests. 101 / 319 (31.7%) of serum samples were both ANA and line immunoassay positive wherein the florescent ANA pattern could be correlated with the presence of specific antibodies depicted by the immunostrip. Homogeneous, the most common ANA pattern observed in this study (n = 36, 45.5%) showed an association with dsDNA, nucleosomes and histones (n = 26) with variable intensities of SSA / Ro-52 (n = 10), RIB (n = 6) and RNP / Sm (n = 4). Thus, with a homogenous pattern, one could project further that the serum had antibodies against dsDNA, nucleosomes and histones in 75% cases. The next common speckled pattern (n = 36, 36.6%) showed an association with SSA/Ro-52, SSB in all cases in varying combinations with RIB and RNP / Sm. Thus, one could predict the presence of SSA/Ro-52 and SSB in 100% of cases with speckled pattern.

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The centromeric and nucleolar ANA patterns were seen only in five cases each (4.95%). Sixty per cent of cases (n =3) showed positivity for centromeric protein-B. Thus one could predict the presence of CENP-B in 60% o centromere cases. PCNA (n = 2, 4%), Scl-70 (n = 1, 2%), SSB (n = 1, 2%), RNP, SSA (n = 1, 2%) was seen with nucleolar pattern. As the numbers are very small, no definite correlation could be drawn between the nuclear pattern and the antibodies present in case of rim pattern. Positive ANA-IIF with negative line immunoassay was interestingly noted in 17.2% of cases (n = 21). A positive result with ANA-IIF, together with negative results in line immunoassay, was also noted earlier and attributed to the presence of anti-dsDNA antibodies.[9] Vos et al.[6] have found 3/32 ANA positive samples negative with line immunoassay but found positive for anti-dsDNA antibodies by FARR assay. This problem has been overcome in this study by incorporating anti-dsDNA as one of the 14 antigenic lines for testing on line immunoassay. Therefore, in the 21 cases studied here, the serum has probably, some antibodies other than the most commonly encountered 14 antigens. Review of the ANA patterns of these cases had shown that homogenous pattern was in 13, speckled in six and nucleolar in two. As clinically important reactivities on line immunoassay are tested out, it is unlikely that the ANA is of importance in those 21 cases. However, they need to be followed up to understand the significance before one attributes to ANA as a too sensitive test in comparison to line immunoassay which is considered as a gold standard in this study. On the contrary, 31 sera positive for line immunoassay were negative by ANA-IIF. 26 of these sera showed SSA/Ro-52 positivity while five showed positivity for Scl-70. A similar observation was noted by Vos et al.[6] and Hoffman et al.[10] This is explained by the fact that line immunoassay is more sensitive for the detection of SSA/Ro-52 than ANA-IIF even when Hep-2000 cells are used. Screening with ANA-IIF also missed Scl-70 antibodies in a patient, which is of relevance in polymyositis. Scl-70 reactivity goes undetected or unreported with ANA-IIF as these antibodies give a cytoplasmic positivity rather than nuclear staining pattern on IIF and that ANA could have been reported as negative. This drives home a message that cytoplasmic staining pattern identification is equally important even though ANA is reported negative to pick up Scl-70 antibodies. Two sera with CENP-B positivity on line immunoassay were read negative on ANA-IIF and the review of the wells showed a centromeric pattern. These incidences conclude that a certain degree of expertise is required by the reporting pathologists while reporting ANA and to have consistency in reporting, a protocol could be drawn where in at least two pathologists report independently and a consensus taken before the report is signed out. This also highlights that a careful reporting of ANA is mandatory to identify rare but specific ANA patterns so as to predict auto antibodies. It may also be important to run the sera for line immunoassay if ANA is negative, but rheumatic
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disease is strongly suspected as is seen in 26 cases, which were ANA-negative but line immunoassay-positive. The ANA patterns with their corresponding antibodies in sera are available for ones reference from the Western literature.[2,3] Table 5 compares these results with those obtained in this study. The findings are comparable with those in western literature but in case of homogenous anti-dsDNA and histone complex, nucleosomes, RNP / Sm and RIB are also significantly positive in sera of our south Indian population. There is no difference in the correlation between speckled, nucleolar and centromere patterns and their corresponding antibodies in sera. A single case of Rim pattern was insufficient to draw a comparison with line immunoassay in our study. In conclusion, the most cost - effective test that does not harm patients interests in investigating rheumatic disease is ANA by IIF method using biochip wells. It may be used for screening purposes for patients with or without any clinical criterion for autoimmune disease in daily clinical practice. Its fluorescent pattern could also predict the presence of certain specific antibodies in the sera. These correlations are of relevance for the diagnosis of a specific rheumatic disease and probably keep line immunoassay as a special investigation particularly for those patients who need them for prognostic significance. Cytoplasmic pattern in ANA-IIF are as important as the nuclear pattern though they are read as ANA-negative and those sera must be subjected for line immunoassay as they help in the diagnosis of a specific rheumatic disease or autoimmune disease. ACKNOWLEDGMENT
The authors acknowledge CPC Pharmaceuticals for the material support of ANA and immunoblot kits used in this study.

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Source of Support: Nil, Conict of Interest: None declared.

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