Serum samples were shipped with dry ice and stored at −80°C until evaluation by 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. Immunofluorescence staining intensities were graded 0-4 compared to standard references (8 (link)). Values of 1-4 indicated ANA positivity; those graded 3 or 4 were further assessed by sequential ANA titers up to 1:1280 dilution. ANA patterns (including nuclear, cytoplasmic, or mitotic) were defined according to international consensus (15 (link)). All samples were assayed 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.