The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test

Abstract

According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight (n = 84), Helios (n = 85) and NOVA View (n = 92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A (k = 0.82) and B (k = 0.72), and almost perfect for C (k = 0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C (k = 0.86; k = 0.82) and substantial agreement for A (k = 0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k = 0.62; B: k = 0.65; C: k = 0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform.

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

“…To our knowledge, most literatures focused on reporting positive or negative rate on single devices [11,12,[26][27][28], even less data attempted to compare patterns between different assay kits [9,29]. Therefore, we further evaluated the discrepancy caused by the HEp-2 assay kits in our clinical laboratory.…”

Recent Approaches To Optimize Laboratory Assessment of Antinuclear Antibodies

“…Correct determination of ANA patterns is important for confirming a number of autoantibody specificities and for timely diagnosis, to minimize severe outcomes. While ANA IFA automated readers have been demonstrated to improve intra-and interlaboratory correlations (59,60), it is likely that factors such as the HEp-2 substrate and secondary antibodies may also significantly affect the appearance of certain ANA specificities. In addition, the different systems for acquiring and analyzing ANA HEp-2 images are likely based on different programming parameters (61).…”

Biomarkers and Pathogenic Mechanisms in Autoimmunity

“…In a ''real-life'' study, the comparison of computer-aided diagnosis (CAD) systems from three different vendors (Zenit G-sight, Helios, and NOVA view) was compared to indirect immunofluorescence (IFA) readings of ANA slides by two to three expert physicians. When 261 consecutive samples with suspected autoimmune diseases were tested for ANA, CAD systems were proven to represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra-and inter-laboratory variability in a new vision of the diagnostic autoimmune platform [8]. In another study, automated IFA methods were compared with established manual IFA screening.…”