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

摘要 量化不同免疫细胞的存在、功能和命运在免疫学中是至关重要的。 免疫疗法的研究和开发。流式细胞术是一种单细胞技术， 广泛用于高通量检测免疫细胞表达的不同蛋白质标志物。 然而，荧光标记之间的光谱重叠使测量结果超过20 标记很困难，通常需要数月的优化，并限制了 标记物测量到40左右。这些限制妨碍了更全面的描述 对于免疫学和免疫肿瘤学研究至关重要的免疫细胞。质量 细胞测定法和基于rna-seq的方法测量的吞吐量有限且成本过高。 病人血液样本中含有数百万个细胞。此SBIR应用程序的目标是开发 循环流式细胞术--一种高通量、经济实惠且易于使用的高标记物解决方案 单细胞分析市场。循环流式细胞术利用专有激光粒子技术 对单个细胞进行条形码以进行重复的流量测量，从而实现复杂的流式细胞术 使用30个或更多标记进行实验，将其分解为多个更简单的循环 一次大约10个记分器。这一创新方法的可行性已在 第一阶段研究。此第二阶段研究的具体目标包括开发和测试 完整的工作流程和全功能的循环流式细胞仪原型。该系统将是 与外部调查人员就几种应用进行了验证。第二期工程完成后， 导致循环流式细胞仪的早期市场验证，并直接导致产品就绪的设计 样机，随后是商业发射。

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.