Cyclic Flow Cytometry for Ultra-High Marker Single-Cell Analysis

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Abstract Quantifying the presence, function, and fate of different immune cells is crucial in immunology research and development of immunotherapies. Flow cytometry is a single-cell technique that is widely used to measure different protein markers expressed by immune cells at high throughput. However, spectral overlap between fluorophore labels makes measurement of more than 20 markers difficult, often requiring months of optimization, and limits the maximum number of markers measured to around 40. These limitations prevent more comprehensive characterization of immune cells that is critically needed for immunology and immune-oncology research. Mass cytometry and RNA-seq based methods are throughput-limited and cost-prohibitive for measuring patient blood samples containing millions of cells. The goal of this SBIR application is to develop cyclic flow cytometry, a high-throughput, affordable and easy-to-use solution for the high-marker single-cell analysis market. Cyclic flow cytometry leverages proprietary laser particle technology to barcode individual cells for repeated flow measurements, enabling complex flow cytometry experiments with 30 or more markers to be broken down into multiple, much easier cycles with around 10 markers at a time. The feasibility of this innovative approach has been verified in the Phase I research. The specific aims of this Phase II research involve developing and testing a complete workflow and fully functional prototype for cyclic flow cytometry. The system will be validated with external investigators for several applications. Completion of the Phase II work will lead to early market validation of cyclic flow cytometry and directly lead to design of product-ready prototypes, followed by commercial launch.