The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases

Aim This study aimed to investigate whether it is useful both for diagnostic purposes and for cost savings to associate a solid-phase monotest called CTD screen to the immunofluorescence (IIF) test, to detect anti-nuclear and anti-cytoplasmic antibodies (ANA). Methods Fifteen Italian laboratories participated in the study enrolling at least 250-each consecutive unselected samples from patients routinely referred for ANA testing. They all used the conventional IIF method on HEp-2 cells associated to a solid phase CTD screen method. Seven centers used the EliA FEIA CTD screen method on the Phadia 250 instrument (ThermoFisher), five centers used the QUANTAFlash CTD Screen method (Inova Diagnostics) on the BioFlash CLIA instrument, while three centers used both methods, albeit in two different series of patients. Results A total of 5043 samples were tested. We focused attention to the 1674 discrepant results: 106 (2%) were ANA-IIF negative/CTD screen positive and 1568 (31%) were ANA-IIF positive/CTD screen negative. Specific antibodies were detected in 87/106 (82%) of the ANA-IIF negative/CTD screen positive samples, mainly Ro52, Ro60, dsDNA, PM/Scl and Jo1. Thirty four of these patients were diagnosed with an ANA-associated autoimmune rheumatic disease (AARD) (seven SLE, 15 Sjӧgren's syndrome, nine autoimmune inflammatory myositis, three systemic sclerosis). Among 1568 ANA-IIF positive/CTD screen negative discrepant patients, a specific antibody was found in 94 (6%) samples (28 patients with AARD, 13 with undifferentiated connective tissue disease, two with undifferentiated arthritis, and 51 without a rheumatic disease). Conclusions This study is the first that compares the IIF method vs. solid-phase assays in the detection of ANA, on a very large sample size from the routine work-up (real-life approach). Results show that the association of solid-phase CTD screen tests to the ANA-IIF test increases the sensitivity (from 89.2% to 97.4%) and the specificity (from 64.6% to 98.4%) of serological tests for ANA screening. Analysis of costs demonstrated that combination of the two tests is also cost-effective, reducing global costs for the immunoserological diagnosis of AARD by 22%.