ANA were measured across four time periods as described previously (1 (link)) with indirect immunofluorescence at a 1:80 dilution using the NOVA Lite HEp-2 ANA slide with DAPI kit (INOVA Diagnostics, San Diego, CA) with a highly specific fluorescein isothiocyanate (FITC)-conjugated secondary antibody (goat anti-human IgG). Images were captured via the NOVA View automated fluorescence microscope system (INOVA Diagnostics) and stored digitally. Staining intensities were graded from 0 to 4 relative to a standard reference gallery, with non-zero values (e.g., values 1-4) considered indicative of ANA positivity (1 (link)). All samples from all years were assayed at the same time, using the same methods in a single laboratory. Readings were made independently by at least two experienced evaluators (blinded to sample characteristics and time period), who agreed on >95% of the intensities and patterns; differences were resolved by consensus or adjudicated by a third blinded rater. Repeat testing of random samples showed >98% concordance.
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