Antinuclear antibodies (ANAs), also referred to as anti-cell antibodies, are hallmark biomarkers of systemic autoimmune rheumatic diseases (SARDs), a heterogeneous group of disorders characterised by immune-mediated inflammation and multisystem involvement, including systemic lupus erythematosus (SLE), Sjogren’s syndrome (SjS), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), and inflammatory myopathies [1]. Besides the HEp-2 cell indirect immunofluorescence assay (HEp-2 IFA), which remains a gold standard screening test for ANA, several high-throughput, solid-phase alternatives, such as enzyme-linked immunosorbent assay (ELISA) and line immunoassay (LIA), are also widely used. However, because solid-phase assays detect only the antigens incorporated into the assay, they may fail to identify autoantibodies directed against antigens not included in the antigen panel [2, 3]. In contrast, HEp-2 IFA can identify a broad range of ANA patterns, including nuclear, cytoplasmic, and mitotic reactivities. Nevertheless, conventional HEp-2 IFA requires specialized expertise and is subject to operator-dependent interpretation. To address these limitations, several automated platforms incorporating automated image acquisition and artificial intelligence-assisted pattern recognition have recently been developed, including the Euroimmun EUROPAttern Classifier 2.4, Werfen NOVA View, Medipan AKLIDES/akiron NEO, AESKU HELIOS, and Immunoconcept Image Navigator.

In high-resource settings, multiplex and array-based platforms have further improved diagnostic precision by enabling simultaneous detection of multiple autoantibodies. In contrast, laboratories in resource-limited regions often face financial, infrastructural, and technical constraints that restrict testing to a single assay, potentially leading to missed or delayed diagnoses. In such settings, the availability of an efficient, cost-effective complementary tool for ANA screening could improve diagnostic access and facilitate the timely evaluation of patients suspected of SARDs [4].

The EliA CTD Screen is a fluorescence enzyme immunoassay (FEIA) designed to detect autoantibodies associated with SARDs and has demonstrated good diagnostic accuracy for ANA screening in multiple clinical studies [5–8]. Several clinical studies have demonstrated that the CTD Screen assay provides good diagnostic sensitivity and specificity, while its fully automated format offers advantages in standardization and laboratory workflow [8–11]. However, its diagnostic performance remains incompletely characterized, particularly when directly compared with HEp-2 IFA and LIA. Additionally, its performance relative to the International Consensus on Antinuclear Antibody Patterns (ICAP)-standardized HEp-2 IFA interpretation, as defined by the ICAP, has not been systematically evaluated. To our knowledge, no previous study has simultaneously evaluated diagnostic accuracy, disease-specific performance, and optimal cutoff determination of the CTD Screen assay against both HEp-2 IFA and LIA in a large Indian cohort. This study was therefore designed to address this gap.

The present study extends existing literature in several important ways. First, it represents one of the largest comparative evaluations of CTD Screen performed in a clinical cohort. Second, to our knowledge, it is the first study to establish an optimized, population-specific cutoff validated against both HEp-2 IFA and LIA. Third, our disease-specific analysis demonstrates differential assay performance across SARD subtypes, a dimension rarely assessed in prior reports. We found CTD Screen is a reliable adjunct to conventional HEp-2 IFA for the serological evaluation of SARDs. Although it cannot replace HEp-2 IFA, its high specificity, automation, and rapid turnaround time make it an attractive complementary screening assay, particularly in laboratories where fluorescence microscopy is unavailable or cost-prohibitive [10].

This study provides three key novel findings: 1) validation of CTD Screen performance in a large cohort, 2) derivation of an optimized diagnostic cutoff improving sensitivity by 7.6%, and 3) demonstration of disease-specific variability in assay performance. By directly comparing the manufacturer’s cutoffs with an in-house-optimized cutoff, this study evaluated the diagnostic performance of the CTD Screen across a large, clinically diverse cohort. Using the manufacturer’s cutoff, the assay achieved a sensitivity of 73.9% and a specificity of 92.6%. To maximize combined sensitivity and specificity, the Youden Index was used to determine the ROC-derived optimum cutoff of 0.6. Improved sensitivity may be beneficial in ANA screening workflows, as early detection of SARDs can prevent diagnostic and treatment delays. Its potential use as a screening threshold in clinically suspected cases is supported by the amended cutoff, which raised sensitivity from 73.9% to 81.5% while maintaining an acceptable specificity of 85.6%. Nevertheless, this improved threshold has not been validated externally and was obtained from a single-centre cohort. As a result, there is still uncertainty about its generalisability across various populations, ethnic groups, disease prevalence levels, and laboratory settings. To confirm this threshold’s clinical usefulness and reproducibility, more multicentre prospective investigations are needed.

While comparing with previous reports, the diagnostic findings in our cohort closely align with them. Previous studies have reported sensitivities ranging from 72% to 100% for automated CTD screening assays [5, 6, 9, 10, 12–14]. The observed variability among studies may reflect differences in study populations, disease spectrum, assay antigen composition, inclusion criteria, and methodological approaches. The substantial concordance between CTD Screen and HEp-2 IFA in our data (κ = 0.683) and the high AUC (0.845) confirm robust diagnostic agreement consistent with international studies.

In our study, specificity was 92.6% using the manufacturer’s cutoff and 85.6% using the in-house cutoff. These values are comparable with the specificity reported for the FEIA-based EliA CTD Screen assay in previous studies [9, 12]. Variation across studies may reflect demographic and clinical differences, including differences in the prevalence of overlapping autoimmune syndromes or in ethnicity-related antibody repertoires. It’s also important to recognize that ANA may be positive in several non-rheumatologic conditions and is not limited to classical SARDs. Even in the absence of overt autoimmune disease, situations like chronic infections, cancer, aging populations, and chromosomal diseases like Klinefelter syndrome have been linked to increased ANA prevalence. These findings emphasize the importance of carefully considering clinical correlation when interpreting ANA screening results, particularly from highly sensitive automated platforms. Therefore, rather than being used as independent diagnostic markers, CTD Screen and HEp-2 IFA results should always be interpreted in conjunction with clinical observations, complementary serology, and pre-test disease probability [15].

Disease-specific analysis further supports the CTD Screen’s diagnostic utility. SSc exhibited the highest sensitivity (97.4%) and NPV (99.7%), suggesting that a negative CTD Screen result effectively excludes this diagnosis in suspected cases. MCTD and OSs showed balanced diagnostic indices, likely attributable to their broader autoantibody spectrum. Interestingly, we observed an age-related pattern distribution: nuclear positivity predominated in younger individuals, while cytoplasmic and mitotic reactivities were more frequent in older patients. This may reflect immunosenescence-related antigenic drift or age-related changes in autoantibody expression.

Among CTD Screen-positive sera, nuclear antigen reactivity predominated (64.8), while cytoplasmic and mitotic patterns were less frequent, accounting for 33.7% and 1.4%, respectively. This distribution reflects the assay’s emphasis on nuclear antigens and may explain its lower detection rate for cytoplasmic patterns, particularly reticular or fibrillary types. Median fluorescence values further supported this distinction, being higher for nuclear patterns than for cytoplasmic patterns (3.5 vs. 1.0).

The lower proportion of CTD Screen positivity in HEp-2 IFA cytoplasmic and mitotic patterns should be interpreted in light of the differing antigenic breadth of the two assays. HEp-2 IFA can detect autoantibodies against a broad spectrum of approximately 100 cellular antigens, whereas the CTD Screen used in this study includes only 17 defined antigens [3, 5]. Consequently, some HEp-2 IFA positive samples may contain antibodies against antigens not represented in the CTD Screen panel, limiting direct comparison of overall positivity rates between the two methods. A more appropriate evaluation involves correlating specific HEp-2 IFA patterns with their corresponding autoantibodies included in the CTD Screen panel, as partially addressed in Table 3. In addition, the diagnostic performance of solid-phase assays cannot be assumed to be uniform across all ANA specificities solely because they rely on similar technological platforms. While certain autoantibodies, such as anti-centromere antibodies (AC-3 pattern), demonstrate good concordance between solid-phase assays and HEp-2 IFA, others, including anti-fibrillarin antibodies (AC-9 pattern), may show substantially lower sensitivity in solid-phase assays due to differences in antigen composition, presentation, and conformational epitope recognition. Previous studies have similarly reported limited detection of antibodies such as fibrillarin and RNA polymerase II in assays lacking these target antigens [16–18]. These observations reinforce the continued complementary role of HEp-2 IFA in comprehensive ANA screening and pattern characterization, particularly for uncommon, cytoplasmic, or mitotic autoantibody reactivities. Operationally, the CTD Screen may offer advantages such as full automation, high throughput, reduced subjectivity, and faster reporting times [5, 19]. However, the HEp-2 IFA remains the gold standard for ANA screening. Hence, its use may be justified only in resource-limited settings where a fluorescence facility is unavailable or remains suspended due to logistical or technical issues [19].

However, using CTD Screen as an initial screening assay, followed by LIA confirmation of positive results, offers an efficient and cost-effective workflow, particularly for guiding the selection of subsequent individual ANA specificity testing. Nevertheless, HEp-2 IFA remains indispensable for pattern recognition and the detection of novel cytoplasmic or other reactivities not captured by solid-phase assays [19]. Therefore, a combined or tiered testing strategy may provide optimal diagnostic accuracy [20]. Our proposed algorithm (Figure 4) represents a pragmatic diagnostic workflow integrating automation with confirmatory testing, potentially improving laboratory efficiency without compromising diagnostic performance.

While the study provides important insights, a few limitations merit consideration. An important methodological consideration is the initial use of a 1:40 HEp-2 IFA screening dilution, which is lower than the ≥ 1:80 threshold recommended by ICAP guidelines. Lower screening dilutions may improve analytical sensitivity and facilitate detection of weak antibody reactivities in early or evolving disease; however, they may also increase nonspecific staining and false-positive results, thereby reducing specificity. This may partly explain the discordance observed between HEp-2 IFA and CTD Screen/LIA results, particularly among low-intensity HEp-2 IFA-positive samples. To minimize this effect, all positive samples identified at 1:40 dilution were repeated at 1:80, and the results remained unchanged because faint fluorescence intensities had already been excluded during interpretation at 1:40. Nevertheless, using a lower screening dilution may limit direct comparability with studies that employ the internationally recommended ≥ 1:80 threshold. The 1:40 dilution was selected to reflect prevailing screening practices in many Indian laboratories, including ours, where higher analytical sensitivity is often prioritized in clinically suspected SARD cases. Another limitation is that LIA was not performed on double-negative samples, potentially leading to an underestimation of weak or rare autoantibody positivity. The inclusion of previously treated patients could have influenced antibody titers. The case-control design with equal numbers of HEp-2 IFA-positive and negative samples is another significant drawback, as it does not accurately reflect the prevalence of SARDs in standard clinical practice. As a result, prevalence-dependent metrics such as overall accuracy, PPV, and NPV may not be immediately applicable across different clinical contexts. To overcome this, we also focused on LR and ROC-AUC values, which provide more reliable measures of diagnostic performance and are less affected by disease prevalence. Future multicentre, prospective studies should validate the optimized cutoff in clinically stratified cohorts, include cost-benefit analyses, and assess the integration of automated platforms into routine laboratory workflows.

In conclusion, this study represents one of the largest comparative cohort studies to validate a population-specific CTD Screen cutoff against both HEp-2 IFA and LIA. The CTD Screen demonstrated high diagnostic accuracy and substantial agreement with HEp-2 IFA, while offering advantages of automation, reproducibility, and efficiency. However, CTD Screen should not be used as a standalone diagnostic tool, particularly in low-prevalence settings, and must be interpreted in conjunction with clinical evaluation and confirmatory testing. A tiered diagnostic approach integrating CTD Screen with HEp-2 IFA and LIA may improve diagnostic efficiency, optimize resource utilization, and support early and accurate detection of SARDs.