Standardized Indirect Immunofluorescence Assay

Serum samples were evaluated by indirect immunofluorescence at a 1:80 dilution using the NOVA Lite HEp-2 ANA slide with DAPI kit (INOVA Diagnostics, San Diego, CA, USA) and a highly specific fluorescein isothiocyanate conjugated secondary antibody (goat anti-human IgG). Images captured via the NOVA View automated fluorescence microscope system (INOVA Diagnostics) were stored digitally. Immunofluorescence staining intensities were assigned integer grades from 0 to 4, relative to standard references, with nonzero grades indicating ANA positivity (Dinse et al. 2020 (link)). All samples were assayed in a single laboratory, using identical methods. At least two experienced evaluators made independent readings, blinded to participant characteristics and time period, and they agreed on over 95% of the grades; differences were resolved by consensus or adjudicated by a third blinded rater. Repeat testing of 200 random samples showed over 98% concordance.

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Dinse G.E., Co C.A., Parks C.G., Weinberg C.R., Xie G., Chan E.K., Birnbaum L.S, & Miller F.W. (2022). Expanded assessment of xenobiotic associations with antinuclear antibodies in the United States, 1988–2012. Environment international, 166, 107376.

Publication 2022

NOVA Lite HEp-2 ANA slide with DAPI kit NOVA View automated fluorescence microscope system

Anti igg Antibody Dapi Diagnostics Dilution Fluorescein Fluorescence microscope Goat Human Immunofluorescence Indirect immunofluorescence Isothiocyanate Serum

Corresponding Organization : National Institutes of Health

Other organizations : Social and Scientific Systems (United States), University of Florida

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Variable analysis

independent variables

Serum sample dilution (1:80)

dependent variables

Immunofluorescence staining intensity (integer grades from 0 to 4)

control variables

Assay method (indirect immunofluorescence using NOVA Lite HEp-2 ANA slide with DAPI kit and fluorescein isothiocyanate conjugated secondary antibody)
Microscope system (NOVA View automated fluorescence microscope system)
Laboratory (single laboratory, using identical methods)
Evaluators (at least two experienced evaluators, blinded to participant characteristics and time period)
Consensus/adjudication (differences in evaluations were resolved by consensus or adjudicated by a third blinded rater)
Repeat testing (200 random samples showed over 98% concordance)

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