Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing

Abstract

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6–97.5% of patterns and 71.0–93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

Evaluation of a Fully Automated Antinuclear Antibody Indirect Immunofluorescence Assay in Routine Use

“…Considering results within ± one titer difference as acceptable, the overall agreement between automated and classical endpoint titer was 75.9% (Table 6), consistent with previous studies (23,24,29). For samples with the same single patterns by both Helios and visual readings (Table 7), the overall error rate was 26.0%, similar to previous reports (25,29,37,38). The high error rates may be due to the single-well titration method that most automated systems currently use.…”

“…The high error rates may be due to the single-well titration method that most automated systems currently use. Won suggested the multi-well (2 or 3 wells) based line slope titration method to improve the accuracy (37). Moreover, our analysis on pattern dependency of automated endpoint titer prediction demonstrated that Helios predicted higher titers for homogeneous patterns but lower titers for speckled patterns, consistent with a previous study (37).…”

“…Won suggested the multi-well (2 or 3 wells) based line slope titration method to improve the accuracy (37). Moreover, our analysis on pattern dependency of automated endpoint titer prediction demonstrated that Helios predicted higher titers for homogeneous patterns but lower titers for speckled patterns, consistent with a previous study (37). The centromere pattern (AC-3), which possesses a lower overall fluorescence than the other common patterns, had a lower concordance rate of 47.1%, and most of the error results had lower titers (41.2%), in line with Zeng et al (25).…”

A hierarchical bivariate meta-analysis of diagnostic test accuracy to provide direct comparisons of immunoassays vs. indirect immunofluorescence for initial screening of connective tissue diseases

“…cancer, infection) or in healthy individuals [1,[16][17][18], the test can have low specificity for CTD. Furthermore, IIF is a labour-intensive technique that requires highly skilled laboratory technicians to interpret the result and is therefore subject to high inter-observer variability [19][20][21]. Solid-phase immunoassays (IAs) offer an alternative to IIF.…”

Immunosensor‐based rapid quantitative detection of Newcastle disease virus antibodies using innovative gold immunochromatographic assay

“…2019) , immunofluorescence assay (IFA) (Ricchiuti et al . 2018) and the haemagglutination‐haemagglutination inhibition (HA‐HI) method (Peng et al . 2007; Chaka et al .…”