Detection of antinuclear antibodies – a comparison of indirect immunefluorescence on HEp-2 cells and fluoroenzymeimmunoassay (EliA CTD screen, Thermo Fisher)

Lone Nørgård Troelsen1, Søren Jacobsen2, Lina Pedersen1,*

1Department of Clinical Immunology, 2Department of Rheumatology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark

Introduction: Testing for antinuclear antibodies (ANA) is important in the diagnosis of connective tissue disease (CTD). Indirect immunofluorescence (IIF) is considered the reference method for ANA screening. However, fluoroenzymeimmunoassay (EliA CTD screen, Thermo Fisher) testing for antibodies against 17 nuclear antigens has shown a good performance.

Objectives: To compare IIF to EliA CTD screen for the detection of ANA.

Methods: 1882 consecutive routine serum samples with request for ANA were examined by IIF on HEp-2 cells (Immunoconcept, Sacramento, CA, USA) and EliA CTD screen (Thermo Fisher, Freiburg, Germany). EliA CTD screen wells were coated with the following antigens: dsDNA, SSA/Ro (52, 60), SSB/La, U1-RNP (RNP-70, A, C), Sm, centromere B, Jo-1, Scl-70, Rib-P, fibrillarin, RNA Pol III, PM-Scl, PCNA, Mi-2. Antibodies to individual antigens were determined in all samples that turned positive in EliA CTD screen assay.

Results: 448  (24%) samples were IIF positive and 232 (12%) were EliA CTD screen positive. 56 EliA CTD screen positive samples did not exceed cu-off levels when examined with EliA single antigen assays.

115 samples were double positive (IIF and EliA single antigen). Agreement between ANA IIF patterns and EliA antigen specificity was found in 58%, partial agreement in 30% and disagreement in 12% of the samples.

Among the IIF negative and EliA single antigen positive samples (N=61), 30 samples were positive for antibodies against SSA and/or SSB, and 25 were positive for dsDNA antibodies.

The most common IIF patterns among the EliA CTD screen negative samples were homogeneous, speckled and nucleolar.

Conclusion: A positive test result by IIF was twice as frequent as a positive result by EliA CTD screen. Disagreement between ANA IIF patterns and reactivity to specific antigens could only partly be explained by a low expression of SSA/SSB on Hep-2 cells. Sensitivity and specificity of the two methods for the diagnosis of CTD are currently being examined.

Keywords: Biomarker, Method, Multiple organs