Evaluation Of A Semi-Automated Indirect Immunofluorescence Assay Using Validated In-House Hep-2 Cell Slides As A Reference Standard For Antinuclear Antibody Detection

Objective: To evaluate the analytical performance of a semi-automated assay for detection of antinuclear antibodies by indirect immunofluorescence. Study Design: Cross-sectional study Place and Duration of Study: Department of Immunology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from Jan to Jun 2023 Methodology: A total of thirty-six serum samples were selected from patients with antinuclear antibody-positive systemic autoimmune rheumatic diseases (n = 26) and from healthy controls (n = 10). Samples were applied on in-house HEp-2 cell slides and then subsequently stained and interpreted by two experienced observers independently. In parallel, ANA tests were also performed using a semi-automated indirect immunofluorescence assay (IFA) benchtop analyzer (Akiron NEO) and software using a standard kit as per the manufacturer's instructions. Positive/negative results, staining patterns, and intensities were compared between in-house HEp-2 cell slides as a reference standard and the semi-automated assay. Relative sensitivities, specificities, and degree of concordance were assessed by overall percentage agreement or by Cohen’s k. Results: Among a total of 36 samples, in-house HEp-2 cell slides yielded 26 (72.22%) positive and 10 (27.78%) negative. The semi-automated IFA system yielded 21(58.33%) positive samples and 15(41.66%) negative samples. Overall relative sensitivity, specificity, and concordance of the semi-automated IFA system were 65.38%, 60%, and 63.88% (k = 0.21), respectively (p = 0.26). Conclusions: The semi-automated IFA system (Akiron NEO) has fair agreement with in-house HEp-2 cell slides with respect to positivity/negativity and pattern recognition but poor agreement for intensity. This makes semi-automated evaluation of ANA relatively less suitable for routine testing.