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

项目总结/摘要 定量不同免疫细胞的存在、功能和命运在免疫学和免疫学中至关重要。 肿瘤学研究以及血液病的诊断。流式细胞术是目前的方法 用于以高通量测量单细胞表达的不同蛋白质。然而，光谱重叠 荧光团标记之间的流动使得超过15种蛋白质标记物的流动测量困难，通常需要 几个月的优化，而测量超过40个标记是不可能的与当前的技术。这 上限阻止了对细胞状态和功能的更复杂的分析，并限制了免疫功能的发现。 治疗监测和诊断所需的生物标志物。其他高标记物分析方法 包括质谱细胞计数和基于RNA-seq的方法在通量上受到严重限制并且成本过高， 并且对于测量含有数百万个细胞的患者血液样品并不理想。长期目标是 应用是开发循环流式细胞术，一种高通量，低成本的解决方案，用于高标记单细胞 分析.循环流式细胞术利用专有的激光粒子（LP）技术对单个细胞进行条形码化， 重复的流量测量，一旦完全开发，预计将能够测量前所未有的 100个标记，高通量。第一阶段的具体目标包括制定所需的关键流程， 循环细胞计数法我们将优化LP进入人外周血单个核细胞（PBMC）的过程 并将建立一个同时测量激光和荧光发射的流动原型装置， 在多个流动循环中验证单细胞跟踪。第一阶段研究的完成将建立 循环流式细胞术的可行性。全流程的全面整合和100个标记的演示 分析将是第二阶段提交的主题。

Project Summary/Abstract Quantifying the presence, function, and fate of different immune cells is crucial in immunology and immuno- oncology research, as well as in the diagnosis of hematologic disorders. Flow cytometry is the current method of choice to measure different proteins expressed by single cells at high throughput. However, spectral overlap between fluorophore labels makes flow measurement of more than 15 protein markers difficult, often requiring months of optimization, while measuring more than 40 markers is not possible with current technologies. This upper bound prevents more sophisticated analysis of cell state and function and limits discovery of immune biomarkers needed for treatment monitoring and prognostication. Other methods for high-marker analysis including mass cytometry and RNA-seq based methods are severely limited in throughput and cost-prohibitive, and not ideal for measuring patient blood samples containing millions of cells. The long-term goal of this application is to develop cyclic flow cytometry, a high-throughput, low cost solution for high-marker single-cell analysis. Cyclic flow cytometry leverages proprietary laser particle (LP) technology to barcode individual cells for repeated flow measurements and, once fully developed, is expected to be capable of measuring unprecedented 100 markers at high throughput. The specific aims of this Phase I involve developing key processes required for cyclic cytometry. We will optimize the process of LP delivery into human peripheral mononuclear cells (PBMCs) and will build a flow prototype device for measuring laser and fluorescence emission simultaneously and validating single-cell tracking over multiple flow cycles. Completion of Phase I research will establish the feasibility of cyclic flow cytometry. Full integration of the entire processes and demonstration of 100 marker analysis will be the subject of a Phase II submission.