Background Detection of auto-antibodies directed against nuclear antigens (antinuclear antibodies or ANA) have important diagnostic and prognostic implications in connective tissue diseases (CTD). The conventional indirect immunofluorescence assay on HEp-2 cell line (ANA-IIF) is the most commonly used method to detect ANA. The ANA-IIF can be labor intensive and suffers from lack of specificity.
Objectives To evaluate the utility of a new fluoroenzyme Immunoassay “EliA-CTD” as an alternative for screening patients suspected for autoimmune connective tissue diseases.
Methods Sixteen Hundread (1600) consecutive patients' sera submitted for anti-nuclear antibodies were tested using the ANA-IIF (Diasorin S.P.A, Saluggia, Italy) and the new EliA-CTD screen (Phadia GmbH, Frieiburg, Germany). ANA testing was ordered by both primary and secondary care physicians. The EliA-CTD screening assay is a fluoroenzyme immunoassay which is performed on the Phadia-250 automated platform. The EliA–CTD assay contains ANA-targeted recombinant antigens including dsDNA, Sm-D, Rib-P, PCNA, U1-RNP (70, A, C), SS-A/Ro, SS-B/La, Centromere B, Scl-70, Fibrillarin, RNA Polymerase III, Jo-1, Mi-2, and PM-scl. The test results are expressed as ratio, with >1.0 considered positive. For ANA-IIF, the cut off for positive results was 1:40 or greater. Additionally, further testing for dsDNA and other extractable nuclear antigens (ENA) was undertaken on a subset of sera that were ANA-IIF+ or whenever there was discrepancy between the two methods.
Results The overall agreement between the two methods was 84.2%. Three hundread and eight (308) out of 1600 (19.3%) samples tested positive by ANA-IIF positive as compared to 101/1600 (6.6%) for the EliA-CTD assay. Additional testing showed that 105 samples were positive for ENA including dsDNA. Of those, 101 were EliA-CTD positive and 81 were ANA-IIF positive. By incorporating the ENA results, the calculated sensitivity and specificity for the EliA-CTD were 97.1% and 99.7% respectively with positive and negative predictive values for the EliA-CTD assay of 96.1% and 99.8%, respectively. The corresponding sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the ANA-IIF assay at different dilutions is shown below:
Conclusions The new automated EliA-CTD assay shows superior sensitivity and specificity compared to the conventional labor intensive ANA-IIF. The EliA-CTD can be used as an upfront screening tool for connective tissue diseases. Depending on the clinical details, any EliA-CTD positive results could be reflexly followed by additional testing including ANA-IIF testing to elucidate the titer and pattern.
Hayashi N, Kawamoto T, Mukai M, Morinobu A, Koshiba M, Kondo S, Maekawa S, Kumagai S. Detection of Antinuclear Antibodies by Use of an Enzyme Immunoassay with Nuclear HEp-2 Cell Extract and Recombinant Antigens: Comparison with Immunofluorescence Assay in 307 Patients. Clinical Chemistry 2001; 47(9):1649–1959.
Disclosure of Interest None declared