Compensation-Free, Highly Multiplexed Flow Cytometer

无补偿、高度多重流式细胞仪

• 批准号: 9912775
• 负责人: Giacomo Vacca
• 金额: $ 71.3万
• 依托单位: KINETIC RIVER CORPORATION
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-10 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912775
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adoption; Algorithmic Analysis; Amplifiers; Antibodies; Benchmarking; Biological Assay; Cells; Clinical; Color; Complex; Coupled; Crowding; Cytometry; Data; Detection; Development; Dimensions; Discrimination; Disease; Electronics; Ensure; Experimental Designs; Financial compensation; Flow Cytometry; Fluorescence; Human; Immune; Immunology; Immunophenotyping; Kinetics; Label; Laboratory Diagnosis; Lasers; Malignant Neoplasms; Measurement; Measures; Molecular; Monitor; Monoclonal Antibodies; Noise; Oncology; Optics; Performance; Phase; Population; Protocols documentation; Research; Research Personnel; Resolution; Rivers; Scheme; Side; Signal Transduction; Specificity; Speed; Stem Cell Research; Surface Antigens; System; Techniques; Technology; Testing; Validation; base; clinical application; clinical diagnostics; commercialization; design; detector; drug development; experimental study; fluorophore; improved; innovation; instrument; instrumentation; novel; operation; prototype; research and development; success

PROJECT SUMMARY Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for diagnosis and monitoring of disease. It is particularly useful in distinguishing between populations of immune cells based on their expressed cell surface antigens. Standard flow cytometers use fluorescent tags, often conjugated to monoclonal antibodies, to give qualitative and quantitative information about specific molecules in the cell. This molecular specificity, coupled with the fact that information is obtained on a cell-by-cell basis with very high throughput (up to 30,000 cells per second), make this a powerful technique. The ability to multiplex (measure a variety of different molecular species in a single cell) further adds to its utility and to the complexity of the scientific questions that can be addressed using this technique. However, the level of multiplexing currently has limitations. Flow cytometry analysis typically relies solely on spectral information of the fluorescent tags and is thus limited by the spectral overlap of fluorophore emissions. Currently, employing even moderate levels of multiplexing requires complex instrumentation and careful experimental design, execution and analysis to compensate for spectral spillover of signal into multiple channels. This severely limits the range of scientific questions that can be addressed using current technologies, deters novices in the technique from attempting more complex yet scientifically relevant experiments, and is widely regarded as the major bottleneck in the field. To overcome this limitation, we propose to build on our results from Phase I where we demonstrated feasibility for an innovative approach that uses fluorescence lifetime as a separate, additional discriminating measurement parameter. Our scheme for using fluorescent lifetime for multiplexing is simple, scalable, and supported by preliminary data from our prototype instrument. Here we propose to upgrade our Phase I instrument to increase multiplexing capability, challenge that instrument with a battery of verification tests, and validate using a relevant biological assay, benchmarking results against a conventional flow cytometer. The result will be a system enabling compensation-free flow cytometry experiments of 12 colors, while requiring fewer lasers and detectors than similarly equipped commercial systems. Such a system would serve a large segment of the market, including clinical cytometry, and is expected to see broad adoption. This would pave the way for further development of an ultra-high (30+) parameter instrument suitable for immunophenotyping, yet requiring significantly less compensation than current systems, and pushing the boundaries of experimental complexity. Given flow cytometry’s wide-spread use and importance, this project will have a high impact in many biomedical and clinical applications. Several large instrumentation companies have already indicated an eagerness to engage in strategic partnerships aimed at commercialization.

项目摘要 流式细胞仪是研发以及临床实验室的一种主力技术 诊断和监测疾病。它在区分免疫种群方面特别有用 细胞基于它们表达的细胞表面抗原。标准流式细胞仪使用荧光标签，通常 与单克隆抗体共轭，以提供有关特定分子的定性和定量信息 在细胞中。这种分子特异性，再加上逐个细胞获得信息的事实 用非常高的吞吐量（每秒最多30,000个单元格），使其成为一种强大的技术。能力 多重（测量单个细胞中的各种不同分子物种）进一步增加了其效用和 可以使用此技术解决的科学问题的复杂性。 但是，当前的多路复用级别具有局限性。流式细胞仪分析通常仅依赖于 荧光标签的光谱信息，因此受荧光团排放的光谱重叠的限制。 目前，使用中等水平的多重速度需要复杂的仪器和谨慎 实验设计，执行和分析，以补偿信号的光谱间隔为多个 频道。这严重限制了可以使用当前解决的科学问题的范围 技术，确定技术中的小说，试图更复杂但科学上的相关性 实验，被广泛认为是该领域的主要瓶颈。 为了克服这一局限 对于使用荧光寿命作为单独的额外区分的创新方法 测量参数。我们使用荧光寿命进行多路复用的方案是简单，可扩展的，并且 由我们原型仪器的初步数据支持。在这里，我们建议升级第一阶段 提高多路复用能力的仪器，通过一系列验证测试来挑战该工具，然后 使用相关的生物测定验证，对常规流式细胞仪进行基准测试结果。 结果将是一个实现12种颜色的无补偿流式细胞仪实验的系统，而 比类似等效的商业系统所需的激光和探测器更少。这样的系统会 服务大部分市场，包括临床细胞仪，预计将看到广泛的采用。这 将为进一步开发超高（30+）参数仪器铺平道路 免疫表型，但需要比当前系统要少得多的补偿，并推动 实验复杂性的边界。鉴于流式细胞仪的广泛使用和重要性，该项目 将在许多生物医学和临床应用中产生很大的影响。几家大型仪器公司 已经表示渴望建立旨在商业化的战略合作伙伴关系。