A novel way to evaluate autoantibody interference in samples with mixed antinuclear antibody patterns in the HEp-2 cell based indirect immunofluorescence assay and comparison of conventional microscopic and computer-aided pattern recognition

•About one third of positive routine HEp-2 IFA tests show mixed antinuclear patterns.•On these sera correct identification of ANA patterns is challenging.•We established a novel way to analyze the interference between two patterns.•Nuclear homogeneous disturb the recognition of speckled pattern more than vice versa.•Conventional/on-screen evaluation is superior to EUROPattern in pattern analysis. A major challenge of the HEp-2 cell-based indirect immunofluorescence (IIF) assays is the correct identification of the individual anti-nuclear antibodies (ANAs) if more than one is present in a sample. We created artificial mixes by pooling two different samples with a single autoantibody in different titers. Comparison of the expected and observed patterns and titers clarifies the interference between the two tested ANAs. Serum samples with a single homogeneous or speckled ANA pattern were serially diluted and mixed in 16 combinations, providing end-point titers of 1:5,120 to 1:80 for both patterns. These mixes were tested by a HEp-2 IIF assay and were evaluated by conventional evaluation, the EUROPattern (EPa) system and on-screen analysis. Homogeneous pattern can alter the identification of the speckled pattern much more than vice versa, but both has an interfering effect on the other. The effect of the interfering on the tested pattern was higher if the titer of the former one was higher. The pattern recognition efficacy of conventional and the on-screen evaluation was similar and superior compared to the EPa analysis. The application of artificial mixed samples can help the evaluation of the efficacy of manual and computer-aided ANA HEp-2 pattern recognition.