Background/Purpose:

Indirect immunofluorescence (IIF) on Hep-2 cells is still considered the gold standard for screening of antinuclear antibodies (ANA), the serological hallmark of connective tissue diseases (CTD). While this method is sensitive it lacks specificity. Moreover, low-titer ANA subspecificities may escape detection by IIF. It was therefore the aim of this study to investigate the diagnostic usefulness of an ANA screening assay containing 13 diagnostically relevant antigens.

Methods:

Sera from 265 consecutive patients presenting with symptoms characteristic of connective tissue diseases (but without a clear diagnosis yet) were analysed by both IIF and the EliA® CTD Screen (Thermo Fisher Scientific) containing the following antigens: dsDNA, U1-snRNP, Sm, Ro60/SSA, Ro52/TRIM21, La/SSB, ribosomal protein P (ribP), topoisomerase I (Scl-70), centromere B, RNA polymerase III, fibrillarin, Jo-1, Mi-2, Pm/Scl.

Results:

Among the 265 patients, 90 were positive in IIF and 78 in the CTD Screen; 61 sera were positive in both systems, 17 only in the CTD Screen and 29 only in the IIF assay. Thus, the CTD Screen increased diagnostic sensitivity of ANA testing by approximately 6%. In all double positive patients at least one diagnostically relevant antibody was detected, with anti-Ro/SSA (n=32), anti-Ro52/TRIM21 (n=21) and anti-dsDNA (n=15) antibodies being the most frequently detected ones. In addition to the antibodies determined by standard routine diagnostics (dsDNA, U1-snRNP, Sm, Ro60/SSA, Ro52/TRIM 21, La/SSB, topoisomerase I, centromere B, Jo-1) antibodies to ribP, RNA polymerase III and Pm/Scl were detected in one serum each. Importantly, antibodies were also detected in 15 of the 17 patients exclusively positive in the CTD Screen: anti-dsDNA (n=7), anti-Ro60/SSA (n=4), anti-U1snRNP (n=2), anti-La/SSB (n=1) and anti-Jo-1 (n=1). In contrast, among the 29 sera exclusively positive in IIF only two contained a diagnostically relevant antibody (low titwer anti-DNA and anti-Sm). Clinical evaluation suggested that the majority of CTD Screen pos/IIF negative patients were at high risk for developing a CTD, particularly primary Sjogren´s syndrome. Most common symptoms were arthralgia (n=13), sicca syndrome (n=12) and Raynaud´s phenomenon (n=5). These patients require careful monitoring during clinical follow-up and might have escaped early diagnostic detection due the negative IIF result.

Conclusion: CTD screening assays containing multiple antigens seem to be useful and highly specific diagnostic tools that increase sensitivity of ANA testing enabling the detection of disease-associated ANA subspecificities in IIF-negative sera. This may reduce the number of false negative diagnoses enabling the physician to diagnose and treat “ANA negative” connective tissue diseases at an earlier stage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/an-ana-screening-assay-containing-multiple-antigens-increases-the-sensitivity-and-specificity-of-ana-testing-by-indirect-immunofluorescence/