Clinical optimization and multicenter validation of antigen-specific cut-off values on the INNO-LIA ANA update for the detection of autoantibodies in connective tissue disorders.

Objectives The INNO-LIA ANA Update is a qualitative multiparameter line immunoassay for detection of autoantibodies to several different antigens associated with connective tissue disorders. We sought to optimize and validate the cut-off values for its antigen-specific components: SmB, SmD, RNP-70k, RNP-A, RNP-C, SSA/Ro52, SSA/Ro60, SSB/La, Cenp-B, Topo-1, Jo-1, ribosomal P and histones. Our aim was to achieve 98% specificity for each of the markers, with respect to differential disease controls, while maintaining sensitivity. Methods For optimization, the cut-off value of the different antigen lines was fixed to achieve this specificity using an in-house set of 955 patient samples. Specificity was validated at multiple sites using a different set of 330 samples obtained from 158 apparently healthy blood donors, 100 patients with a variety of infections, 20 each with Wegener's granulomatosis, inflammatory bowel disease, and primary antiphospholipid syndrome, and 12 with psoricitic arthritis. Sensitivity was evaluated, using this optimized cut-off control, in 147 patients with scleroderma. 93 with Sjogren's disease, 40 with systemic lupus erythematosus, 40 with rheumatoid arthritis, 39 with mixed connective tissue disease, and 19 with polymyositis. Sensitivity and specificity of the INNO-LIA ANA Update were determined using the clinical diagnosis as reference. Results The optimized cut-off values resulted in a specificity 98% or more for all LIA markers except one (histones 97.8%) in the validation set of 330 samples. The sensitivity for each marker tested in 378 samples from the target patient groups was comparable to that reported in the literature. Conclusion The INNO-LIA ANA Update shows uniformly high specificities combined with sensitivities very similar to those of reference assays, in a single test format.