| Abstract|| |
Background: For the management of connective tissue disorders (CTDs), antinuclear antibody (ANA) testing is essential, both from diagnostic and prognostic points of view. Usually, patterns obtained by ANA-IIF testing correlates to specific autoantibodies as obtained from the test for ENA (by LIA/ELISA, etc.). But to apply these data from western studies, we may need validation in the local population like our subjects in sub-Himalayan (Garhwal region) area where CTDs are common. Also, suppose ANA-IFA pattern's correlation is reliably known in our population, it can minimize the cost of managing CTDs by limiting ENA testing, which is 10 times costlier than ANA-IIF. Hence, this study was undertaken to know the specific autoantibody targets (ENA by LIA) against ANA-IIF patterns in our local population. Materials and Methods: In this retrospective cross-sectional work, serum samples of CTDs were tested for ANA by IIF (Euroimmune AG) and ENA by LIA (Euroline ANA-3G) continuously for 36 months. The manufacturer's kit insert was followed, and results were analyzed applying appropriate statistical methods. Results: Major ANA-IIF patterns were found to be associated with specific autoantibodies, for example, Nuclear homogenous with dsDNA, nucleosomes, histones; speckled pattern with nRNP/Sm, Sm, SSA/Ro-52, SSB; nucleolar pattern with Scl-70, Pm-Scl 100 and centromere pattern with CENP-B. Anticytoplasmic (ACA) are found to be linked with some ANA negative (by IIF) samples, emphasizing the need for careful observation for ACA especially where ANA is not found. Conclusions: In most subjects, specific ENA targets correlated well with ANA-IIF patterns, implying effective cost minimization in CTD management. Similar future prospective studies (with clinical data) can provide a database and reference for our population.
Keywords: ANA screening, ANA-IIF, ENA, extractable nuclear antigen, IFA pattern, immunoblot, nuclear homogenous, speckled
|How to cite this article:|
Kalita D, Rekha U S, Raj AK, Mahanta P, Gupta P, Deka S. Feasibility of Indirect immunofluorescence (IIF) alone as a screening method for antinuclear antibody in connective diseases in India's sub-Himalayan region. Indian J Pathol Microbiol 2022;65:873-8
|How to cite this URL:|
Kalita D, Rekha U S, Raj AK, Mahanta P, Gupta P, Deka S. Feasibility of Indirect immunofluorescence (IIF) alone as a screening method for antinuclear antibody in connective diseases in India's sub-Himalayan region. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Dec 7];65:873-8. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/873/359312
| Introduction|| |
Connective tissue diseases (CTDs) like Systemic Lupus Erythematosus (SLE), Rheumatoid arthritis (RA), Sjogren's syndrome (SS), Systemic sclerosis (SSc), Polymyositis (PM), Mixed connective tissue disease (MCTD), and so on. elicit a generalized autoimmune response with generation of Antinuclear Antibodies (ANAs). For ANA detection, the commonly used technique is Indirect Immunofluorescence (ANA-IIF) using substrate like HEp-2 (Human Epithelial tumor cell line) or its various modification at times in combination with other substrate like Primate Liver cell lines.,, For precise identification of these ANAs specialized techniques like ELISA, Western blot/Immuno-blot/Line immune assay (LIA), and so on. are needed.
Each CTD is linked to multiple specific antibodies, while immune response for antibody generation is influenced by genetic, racial, geographic location, environment, and so on.,, This leads to variation in autoantibody types from person to person, place to place, and probably from population to population. Most available data on antibody types and correlation with ANA-IIF patterns are derived from western studies. Such data from our region with CTDs being common will be very much useful for patient care. ANA-IIF tests are much cost-effective (Rs 1100.00 per test) compared to LIA (Rs 12000.00 per test). Thus, if ANA-IIF patterns alone can reliably predict important markers of CTDs (without adittional confirmatory tests like LIA), it will be very useful for a resource-limited set-up like ours in both diagnosis and prognosis domains.
To this end, the current study analyzed the serum samples from clinically diagnosed cases of CTDs sent to our lab to detect ANA by IIF (ANA-IIF). We further performed ENA (Extracted Nuclear Antigen or LIA)-profile using an immunoblot strip, and results were correlated.
| Method|| |
Serum samples of suspected connective tissue disorder patients (CTDs) sent for ANA-IIF testing and ENA profiling over a period of 36 months (from February 2017 to March 2020) were included in this study. Subjects were from sub-Himalayan region (Garhwal area) referred by various specialties (Rheumatology, Internal Medicine, Pediatrics, Dermatology, Nephrology, etc.) of our institute. Non-CTD/unknown diagnosis cases were excluded from the study. ANA-IIF was performed using Euroimmune Mosaic HEp-20-10 and Primate liver cell (PLC) substrate (Euroimmune AG, Germany, Lübeck) as per kit insert instruction. Serum was diluted in a ratio of 1:100. Slides were examined under a fluorescent microscope at 400× magnifications. The fluorescence intensity was scored semi-quantitatively from 1+ to 4+ relative to the positive control (4+). All samples were further tested by LIA using Euroline ANA profile 3 (Euroimmune AG, Germany, Lübeck) for ENA profile targets as per instruction in the kit insert [See [Supplementary Figure 1]]. Diluted sera (1:100) was used and the strip used had targets like nRNP/Sm (nuclear Ribonucleoprotein/Smith antigen), Sm (Smith antigen), SSA (Ro/Sjogren Syndrome A antigen), Ro-52 (Ro antigen subset with 52 KDa size), SSB (La/Sjogren Syndrome B antigen), Scl-70 (Scleroderma antigen of 70 KDa size), PM-Scl (Polymyositis Scleroderma antigen), PCNA (Poliferating cell nuclear antigen), Jo-1 (myositis specific antibody target), CENP-B (Centromere protein type B), dsDNA (double stranded DNA), nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (also called AMA-M2), and controls. The intensity of the reaction was analyzed using image analysis software EURO Line Scan (Ver. 3.4.30, Euroimmun AG, Germany, Lübeck). Distribution and correlation between ANA patterns and ENA profile findings were analyzed by various statistical methods like percentage descripton (for distribution), Wilcoxon signed-rank test/Kruskal test (Association study), Spearman's correlation, and so on using a SPSS package (Version 23, IBM, USA). Ethics Committee clearance for this retrospective analysis was obtained on 28 November 2020.
| Result|| |
During the study period, we received 2150 serum samples for ANA testing. Out of it, 312 cases [Figure 1] fulfilled the study criteria of patients with suspected CTDs, undergoing both ANA-IIF (Principal ANA-IIF patterns are shown in [Figure 2]) and LIA. As shown in [Figure 1], about 193 (61.9%) cases were both IIF and LIA positive, and in 40 (12.8%) subjects, both the results were negative with concordance in 233 (74.7%). IIF positive and LIA negative was found in 47 (15.1%), while IIF negative and LIA positive result was obtained in 32 (10.3%).
|Figure 2: Principal ANA-IIF patterns observed in the current study (Panel = a. Nuclear homogenous, b. Nuclear granular (Speckled), c. Centromere, d. Nucleus nuclear, e. Positive control, and f. Negative control)|
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[Table 1] is presenting the LIA results in 240 ANA positive samples.
The speckled and nuclear homogenous patterns followed by nucleolar and mitosis positive patterns were predominant in both LIA positive and LIA negative subgroups. A significant association was observed between ANA patterns and LIA positivity (p < 0.01) in ANA positive samples.
As shown in [Table 2], ds-DNA, Nucleosome, and Histones were the primary ENA targets in the nuclear homogenous pattern, while SSA, SSB, nRNP/Sm, Sm, and PCNA were the predominant ENA detected in the speckled pattern [Table 3]. However, [Table 4] is showing ENA targets linked to other ANA-IFA patterns. Three CENP-B positive cases had centromere patterns, though four more centromere-patterned cases had no ENA target detected [see [Table 1]]. Scl-70 and Pm-Scl were the principal ENA targets detected in the nucleus nucleolar ANA-IIF pattern, though no targets were detected in 11/30 (36.7%) of such cases [see [Table 1]]. About 3/11 (27.1%) mitosis positive cases had detectable ENA targets [Table 1] and [Table 3], while in other patterns (cytoplasmic, dotted, etc.), predominantly cytoplasmic (AMA-M2, Jo-1, Ro-52, etc.) and mixed targets were detected [Table 4]. In ANA-IIF negative cases, ds-DNA was detected predominantly 12/32 (37.5%), followed by SSA in 7/32 (21.9%) and PCNA 4/32 (12.5%) [see [Table 5]].
|Table 4: ENA profiled linked to ANA-IIF patterns like Centromere, Nucleolar, mitosis positive, and others|
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A significant difference in detection of ENA targets among different ANA patterns was observed, with the highest detection of double ENA targets in speckled (nuclear granular) pattern followed by Nuclear homogenous and Nucleolar patterns [Table 6]. Single ENA targets were highly observed among Nuclear homogenous IIF patterns followed by Speckled and other patterns. Speckled patterns were also found to have three or more ENA targets. ENA target detection frequency in speckled patterns was significantly different from that of Nuclear homogenous, Nucleolar, and Other ANA-IIF patterns (p < 0.01) (Wilcoxon).
Correlation between ENAs among all ANA patterns
Spearman's correlation was performed for inter-relationship between ENAs [Supplementary Table 1] - nRNP/sm was significantly correlated with Sm, and SSA was found to be significantly correlated with Ro52 and SSB. A significant correlation between Ro52 and Ribosomal Protein P was observed, while dsDNA and Histones were significantly correlated with Nucleosome.
| Discussions|| |
ANA-IIF patterns are linked to autoantibodies to nuclear antigens., Additional tests targeting specific antibodies (to nuclear/cytoplasmic antigens) are often asked for – for a more specific diagnosis (specificity) or to delineate a subset with a different course of action (prognostic markers) and different management approaches. In certain situations, the ANA-IIF pattern may throw up a wide array of diagnostic possibilities; additional tests may be needed for pinpoint diagnosis. These additional tests can exclude some grave systematic diseases (negative predictive value) or may help to settle down to a particular condition (disease specificity). ANA-IIF is most commonly performed on HEp-2 cell lines from cultured human laryngeal epithelial carcinoma as these are more sensitive to ANAs than many other cell lines used earlier. Biochips incorporating HEp-2 cells and primate liver cells can be useful to obviate false positivity. Transfected cell line HEp-20-10 makes the system more sensitive to specific targets like SSA, SSB, and so on. In a developing country like India with testing being performed in both CTDs and those without features of CTDs, it is very pertinent to resort to a price-conscious reliable screening test protocol. ANA-IIF is much cheaper than LIA and can be an ideal screening test for our situation. Given the situation, this study evaluated the diagnostic value and cost-effectiveness of ANA-IIF.
Earlier, one study in India found a homogenous nuclear pattern (45.5%) in the presence of LIA finding of dsDNA, Nucleosome and histone; speckled pattern (35.6%) was observed with Sm, nRNP, SSA/Ro-52, SSB, and so on; nucleolar pattern with Scl-70, Sm and centromere pattern with CENP-B. A definitive correlation between ANA patterns and LIA finding was observed. In Bangladesh also, a strong association between Nuclear homogenous pattern with anti-dsDNA (p < 0.05), speckled pattern with antibodies like anti RNAP, anti-SSA & SSB were detected. Another study found speckled (42.5%) in maximum cases followed by homogenous (41.4%) and nucleolar (10.6%) pattern. Definite association of the speckled pattern with targets like Sm, RNP, SSA/Ro, or SSB/La was established. The other report in Iran also concluded a similar association between speckled pattern and ribonucleoprotein. They identified anti-dsDNA antibodies exclusively linked to samples with homogeneous ANA pattern. The nucleolar pattern showed an association with anti-Scl-70. However, one recent study from Pakistan found no specific ANA pattern was associated with any particular LIA/ENA targets. They found Speckled to be the most common ANA pattern and Anti SS-A the commonest ENA.
Our finding of 47 cases (14.9%) of positive results in ANA-IIF but negative in LIA [Table 1] is similar to some other works and it was ascribed to an excess of dsDNA., But in the latter studies (including the current one), LIA strips were incorporated with dsDNA indicating some other target (other than dsDNA). In the current study, most LIA negative ANA-IIF positive cases belong to nuclear homogenous (12/47, i.e., 36.2%) and nucleolar (11/47, i.e., 27.7%). There may be a sensitivity issue in the LIA strip for nuclear homogenous pattern targets (dsDNA etc.), as detected by a recent Bangkok (Thailand)-based study. Similarly, some important targets for the nucleolar pattern (e.g., fibrillarin, NOR-90, etc.) are not included in this LIA strip (ENA-blot assay) used in current work.,
We found 32 cases (10.2%) with LIA blot providing positive result, but ANA-IIF was negative. The majority of such cases (12/32, i.e., 37.5%) were dsDNA positive followed by SSA and Ro-52 detection, singly or in combination, (7/32, i.e., 21.9%) in the blot [Table 5]. Similar dsDNA finding was observed by Sharmin et al., which was explained by the rise of antibodies during disease flares. Earlier, Hoffman et al. found that the LIA assays could detect SSA/Ro-52 more efficiently than ANA-IIF, even after using modified HEp-2000. About four cases of PCNA was negative in ANA- IIF, which can be explained by the fact that speckled pattern due to antibody to PCNA is often difficult to elicit in IIF preparation. Out of 10 LIA (ENA) positive PCNA cases, we detected six (60%) speckled pattern and missed four (40%) in ANA-IIF [see [Table 2] and [Table 5]. We may miss crucial systemic sclerosis-related anti-Scl-70, anti-Jo-1, etc., in ANA-IIF unless cytoplasmic patterns are also given priority in routine work. Hence, cytoplasmic patterns are essential to look for, especially when ANA are reported negative.
Earlier, a study from north India in 2019 found clear associations of ANA-IIF patterns and specific autoantibodies to have high predictibilty for an accurate diagnosis of CTDs. With availability of clinical information our current study data can reveal similar information, extremely useful for patient care set-up. Another study from Northern India (few years back) found targets like SSA, SSB, SSA/SSB, Ro-52, Jo-1 and dsDNA, nRNP/Sm may be missed (i.e. no pattern) by ANA-IIF in a minority of cases (only 3.4%) – a finding very relevant to our results and also conforming other studies., In this regard to ward off errors, one recent study in United States found specific need to enhance competency in reporting two patterns—mitosis and cytoplasmic. Both the patterns (especially first one) are very relevant in Indian perspective. One study from Brazil earlier found some relevance of titer (esp. in ruling out healthy individuals) beside ANA pattern predicting CTDs.
| Conclusions|| |
In our population, ANA-IFA can be useful for the initial prediction of clinically relevant antibodies in suspected CTDs. This could be a cost-effective and accurate screening test for patients with an autoimmune disease in our set-up (Suggested flow is given at [Figure 3]). Anti-cytoplasmic antibody screening during ANA-IIF testing can provide important information, especially in ANA–negative cases. Immunoblot (ENA-profile) can be reserved for clinically suspected cases with uncommon presentation or overlap syndrome.
We are thankful to the authority of AIIMS Rishikesh for allowing us to do this study. Also, we express our thankfulness to CPC diagnostics for technical bits of help.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of Microbiology, AIIMS Rishikesh, Virbhadra Road, Rishikesh - 249203, Uttarakhand
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]