Automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells

Abstract

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

Recent Approaches To Optimize Laboratory Assessment of Antinuclear Antibodies

“…Although the overall performance for positive or negative results was acceptable, the accuracy of pattern recognition, restricted to the most common patterns reported (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots, and cytoplasmic patterns), was limited, ranging from 52% to 79%, with significant challenges in interpreting samples with more than one pattern (mixed patterns). Those findings appear to be consistent with others that report misinterpretation difficulties for the common and mixed patterns, in addition to false-negative results for cytoplasmic, nuclear dots, and nuclear membrane patterns (50)(51)(52)(53)(54)(55)(56)(57)(58). For the available ANA IFA automated readers, the final result is provided by the operator.…”

“…Automation of both ends of ANA testing (front-end processing and digital reading) significantly minimizes the manual and analytical challenges of ANA IFA testing. There are a number of commercially available systems for automated reading of ANA IFA results (Table 2) based on the principles outlined above, with some variations (13,(49)(50)(51)(52)(53)(54)(55)(56)(57)(58). These systems differ from each other with respect to the use of DNA-binding counterstains (4=,6-diamidino-2-phenylindole [DAPI], propidium iodide, or none), the cell substrate used (HEp-2 versus HEp-2000, with restriction to the manufacturer's slides), throughput (samples per hour), the number of patterns that can be identified, titer prediction, front-end automation (slide processors), and other software features (49,50).…”

Automated antinuclear immunofluorescence antibody analysis is a reliable approach in routine clinical laboratories

“…Similar to the results in our study, many studies have examined agreement between automated and conventional ANA IIF analyses. Regardless of the platform on which they are based, such as EUROPattern, AKLIDES software or NOVA View, automated interpretation systems all exhibit reliable discrimination between positive and negative results [11,13,16,[21][22][23].…”

“…Recently, many studies focused on the comparisons between automatic and manual microscope reading or discrepancies between various computer-aided ANA pattern diagnostic systems [11][12][13][14][15][16]. However, few studies evaluated the reliability of the autotiter evaluation system [17].…”

Comparative Study of Human and Automated Screening for Antinuclear Antibodies by Immunofluorescence on HEp-2 Cells

“…It is worth to note that in the comparative study of these commercialized systems used only single pattern sera [3]. In another study, including sera with mixed pattern, the global performance of the EuroPattern® system was estimated at 48.9% [7]. Therefore, recognizing mixed patterns is critical.…”