Background/Purpose: Indirect immunofluorescence assay (IFA) on HEp-2 cells (HEp-2 IFA) remains a key tool in the diagnostic work-up of autoimmune connective tissue diseases (CTD). Traditionally performed manually, this method is labor-intensive, time-consuming, and subject to operator variability. While semi-automated and automated systems exist, there is still a need for fully automated platforms that integrate both slide processing and interpretation using computer-assisted image analysis. As a preliminary step in evaluating a novel fully automated IFA system (LumiQ®, AliveDx, Switzerland) in combination with the HEp-2 substrate Diagnostic Kit for ANA (investigational device), we assessed the analytical performance of the investigational device when used manually by a single operator, comparing results to historical IFA data from patients with suspected CTD.

Methods: Banked, de-identified human serum samples, previously manually characterized by the Mosaic Basic Profile (Euroimmun, Germany) (routine method) were included in the study. Samples were stored at -20°C for up to three months prior to being thawed for subsequent analysis with the investigational device. The study cohort consisted of 25 samples characterized as non-reactive and 60 samples characterized as reactive, with varying titers, covering a broad range of patterns, as per the International Consensus on ANA Patterns (ICAP). Samples were tested with the investigational device at a 1:80 screening dilution. The testing protocols for both investigational device and routine method were manually conducted following the manufacturer’s instructions for use (IFU), with all readings performed by a single operator using a manual fluorescent microscope to reduce inter-operator variability. Results were interpreted based on categorical classifications (reactive / non-reactive), pattern identification, and the grading of reactive samples from 1+ to 4+ at the screening dilution. Positive (PPA), negative (NPA) and overall percent agreement (OPA) were calculated for sample status (reactive or non-reactive). For reactive samples, percent agreement of each individual IFA pattern and overall pattern agreement were determined. From the 60 ANA reactive samples, 20 samples were selected to determine the end-point titer. Dilutions were prepared of these samples +/- one titer step around the pre-characterized end point titer (EPT).

Results: PPA was 100% (95% CI: 94.0, 100), with all 60 historically reactive samples yielding reactive results by the investigational device. NPA was 100% (86.3, 100), with all 25 historically non-reactive samples yielding non-reactive results by the investigational device. The overall agreement between methods was 100% (95% CI: 95.7, 100). The pattern agreement was 100% (95% CI: 86.3, 100). The end point titration of 20 positive samples showed EPT agreement of 100% (95% CI: 83.2, 100).

Conclusion: The manual preparation and readings using the investigational device showed high concordance with historical characterization. Future studies evaluating the fully automated capabilities of this solution will allow for an expanded assessment of the performance of the device and platform.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/analytical-performance-of-the-hep-2-substrate-diagnostic-kit-for-ana-as-an-initial-step-in-the-evaluation-of-a-novel-fully-automated-ifa-analyzer-in-a-laboratory-in-england/