Improved flow cytometry panel design with a novel automated panel design system

PROJECT SUMMARY Cytometry experiments are increasingly complex, often involving more than 20 unique parameters. For example, the recent adaption of spectral flow cytometry means 64 or more channels with 40 simultaneous fluorescent parameters currently validated in Optimized Multicolor Immunofluorescence Panels (OMIP)-069 have >31 million combinations of commercially available products for these markers. Successful design of complex multi-param- eter experiments requires extensive knowledge of panel design principles to organize antigens based on biolog- ical density and cellular expression patterns. Insufficient user experience and poor panel optimization can lead to errors in experimental design, resulting in unreliable, internally inconsistent data. These errors can cost thou- sands of dollars and often waste rare or low-volume samples that may, in some cases, be irreplaceable. Recent work has estimated that there is nearly $28.2 billion in costs associated with irreproducible preclinical work in the U.S. alone that is predominantly attributed to poor antibody performance and study design. With the growing complexity of flow cytometry experiments, there is a need for automated software that accelerates and optimizes the experimental design process, reduces errors, and lowers the barrier to entry. With NIH Phase I support, FluoroFinder completed initial development and demonstrated proof-of-concept for IntelliPanel, a highly custom- izable tool for efficient and optimal design of flow cytometry assays that uses spectral overlap and biological application information to automatically generate a flow cytometry assay for a given instrument configuration. FluoroFinder developed the first version of IntelliPanel, conducted a comparison of autogenerated panels com- pared to published OMIPS, and surveyed expert cytometrists to demonstrate 92% approval of IntelliPanel results for up to 26 markers. In this Phase II project, FluoroFinder will integrate additional parameters, expand data- bases, and generate user friendly tools that significantly improve the accuracy of IntelliPanel recommendations for automated panels with 26 to 40 fluorescent markers and will advance features to improve standardization and reproducibility in the field through the following Aims: Aim 1: Improve the automated ease-of-use and accu- racy of recommendations through integration of the antigen density database and the expansion of additional experimental parameters. Aim 2: Integrate a “Cell Type to Panel” feature to improve experimental reproducibility. Aim 3: Optimize UX for comprehensive experimental recommendations based on user configuration. IntelliPanel is expected to yield significant time and cost savings, reduce or eliminate design errors, and lower the barrier to entry for developing successful flow cytometry experiments. Ultimately, these tools will enable the standardiza- tion of assays, platforms and analysis that will lead to more consistent and reliable high parameter flow experi- ments and emerging technologies (e.g., >26 color spectral flow cytometry).