Integration of multiple readouts into the z' factor for assay quality assessment

Methods that monitor the quality of a biological assay (i.e., its ability to discriminate between positive and negative controls) are essential for the development of robust assays. In screening, the most commonly used parameter for monitoring assay quality is the Z' factor, which is based on 1 selected readout. However, biological assays are able to monitor multiple readouts. For example, novel multiparametric screening technologies such as high-content screening provide information-rich data sets with multiple readouts on a compound's effect. Still, assay quality is commonly assessed by the Z' factor based on a single selected readout. This report suggests an extension of the Z' factor, which integrates multiple readouts for assay quality assessment. Using linear projections, multiple readouts are condensed to a single parameter, based on which the assay quality is monitored. The authors illustrate and evaluate this approach using simulated data and real-world data from a high-content screen. The suggested approach is applicable during assay development, to optimize the image analysis, as well as during screening to monitor assay robustness. Furthermore, data sets from high-content imaging assays and other state-of-the-art multiparametric screening technologies, such as flow cytometry or transcript analysis, could be analyzed.