Role of solid phase immunoassay as a screening test for antinuclear antibody – A comparative analysis on Indian population

Introduction

Antinuclear Antibody (ANA) testing is widely used as a screening test in Connective Tissue Diseases (CTD) such as Systemic Lupus Erythematosus, scleroderma, CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal motility abnormalities, sclerodactyly, and telangiectasia), Sjogren’s syndrome, mixed connective tissue disease (MCTD), polymyositis, and dermatomyositis. However, positive ANA results are seen in a significant proportion of the elderly population 1, 2, 3, 4 and the sensitivity of ANA testing varies widely from one clinical disease to another. 5 Indirect Immunofluorescence Assay (IFA) has been the gold standard for screening of ANA since its discovery more than 50 years ago. 6, 7 However, the IFA method is influenced by cell type, fixation procedure, dilution of patient serum, inspection time, day-to-day performance, the experience level of the microscopist, and the microscope itself. 8, 9 To overcome these issues, enzyme immunoassays including Solid Phase Immunoassays (SPI) were developed. 10, 11, 12 Several ANA antigens have been identified, and quantitative SPIs have been developed using either purified extracts or recombinant antigens. Most of the early manual SPI techniques have been widely replaced by automated versions. These newer methods are amenable to modern laboratories with high-throughput platforms, and they provide quantitative, reproducible results with minimal hands-on time and require less operator skill. In contrast to classical IFA methods using HEp-2 cells, which contain several hundred different antigens, the reactivity of SPI methods is limited to the relatively few individual antigens included in the assays. Although this factor may reduce SPI reactivity to some relevant antigens, it could also diminish reactivity toward irrelevant antigens. 13 The various methodologies discovered both as a screening test and to test for specific antibodies have led to a gamut of choices but without any consensus.

Objective

The objective of this study was to compare IFA and SPI as screening assays along with tests for specific antibodies in the context of the clinical diagnosis and propose a laboratory diagnostic algorithm.

Materials and Methods

Results

The 60 subjects recruited by Rheumatologists had a 1:4 Male to Female ratio with age distribution from 24 to 76 years. Out of the 60 cases, 38 were disease positive with 10 cases of SjS, 13 cases of UCTD, 13 cases of SLE and 2 cases of MCTD. The sensitivity, specificity, PPV, and NPV of the screening tests were compared (Figure 1). Although the results showed IFA to be a more sensitive test, the CTD Screen assay had better specificity, PPV and NPV. When we looked at the disease negative group (22 cases), CTD Screen had a lower false-positive rate (23%) than IFA (45%). The specific antibodies detected by three different methodologies were clustered against the disease groups (Table 1 and Figure 2 ). For statistical analysis, we chose SjS and SLE as disease groups to compare the different methodologies for detecting dsDNA, Sm and SS-A/Ro as these antibodies were part of the classification criteria - ACR/EULAR 2016 criteria for Sjogren’s syndrome 6 and SLICC 2012 criteria for SLE. 7 The results (Table 2) showed that for dsDNA, monospecific assays like ELISA or FEIA have a better detection rate than LIA. For Sm and SS-A/Ro, all three methods were comparable. While reporting the clustering of antibodies into disease groups, an antibody was considered to be non-specifically positive if it was found to be positive across all disease groups. U1RNP showed non-specific positivity across all disease groups along with SS-A/Ro and SS-B/La (Figure 2). Sm and Ribosomal P Protein were found to be clustered only to the SLE group (Figure 2). Antibody against Centromere proteins was found to be associated with UCTD as well as SLE. Antibody against Jo-1 and Scl 70 were found to be negative across all disease groups. In the disease negative group, SS-A/Ro, SS-B/La, dsDNA, and U1RNP antibodies were found to be positive. Out of these, isolated U1RNP positivity on LIA was the most consistent pattern (6 out of 22 cases). SLE and UCTD showed positive results for the largest group of antibodies among the disease groups.

Figure 1

Comparative analysis of sensitivity, Specificity, PPV and NPV

Figure 2

Comparison of antibody distribution across disease groups

Figure 3

Recommended Diagnostic Algorithm

Discussion

Comparison of the Screening Tests: The American College of Rheumatology had released a position statement titled “Methodology of Testing for Antinuclear Antibodies.” in 2011 which was reapproved by the board of directors in 2015. Although the statement supported immunofluorescence antinuclear antibody (ANA) test using Human Epithelial type 2 (HEp-2) substrate, as the gold standard for ANA testing, it also included a review of the literature related to clinical testing for ANAs, along with several recommendations for laboratories. One of these recommendations is that laboratories using newer methods need to provide data to physicians regarding clinical sensitivity and specificity of the chosen method in comparison to the traditional IFA. In our study, we have tried to fulfill that recommendation. When we compared the traditional IFA with newer SPI (CTD Screen) as a screening test for ANA, the results showed that IFA to be more sensitive than SPI but with lower specificity, PPV and NPV. These results were consistent with the studies carried out by Gniewek et al. 5 and Deng et al. 14 (Table 3). The results of our study showed that the use of IFA could still be considered the gold standard (highest sensitivity), but concurrent testing with SPI screen yielded a better specificity, PPV and NPV. This was particularly important in the disease negative group where the false-positive rate of IFA (45%) was much higher than CTD (22%).

Comparison of the specific tests: The results showed that LIA, ELISA and FEIA were comparable for the detection of specific antibodies in all the antibodies tested, except for dsDNA and U1RNP. In the case of dsDNA, monospecific assays like ELISA and FEIA showed better detection rates in patients with SLE. For U1RNP, LIA showed a very high false-positive rate in the disease negative group.

With the results of the above study and review of the literature, we recommended a diagnostic algorithm for detecting autoantibodies (Figure 3).

Conclusion

The diagnosis of Systemic Rheumatic Diseases is a clinical diagnosis with serological testing for Autoantibodies playing an adjunct role in confirming the diagnosis or as prognostic markers. With the development of new solid-phase assays, there is a lot of debate surrounding Indirect Immunofluorescence being the gold standard screening test. The various methodologies available for specific antibody tests have also added to this confusion. In our study, we have tried to objectively look into these methodologies with respect to the clinical diagnosis. Our findings from the study have led us to an algorithm based approach which maximizes the potential of each technique with specific recommendations of specific antibodies which will ultimately bring down the economic burden. In the end, we conclude by acknowledging that this is a pilot study with 60 samples from an Indian population and the requirement is to further test this algorithm and come up with recommendations for using solid-phase assays in concordance with Immunofluorescence.

Conflict of Interest

The authors declare that there are no conflicts of interest in this paper.

Source of Funding

None.