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.

项目概要 流式细胞术是研究和开发以及临床实验室的主力技术 疾病的诊断和监测。它对于区分免疫群体特别有用 细胞基于其表达的细胞表面抗原。标准流式细胞仪通常使用荧光标签 与单克隆抗体偶联，提供特定分子的定性和定量信息 在细胞中。这种分子特异性，加上信息是在逐个细胞的基础上获得的事实 具有非常高的吞吐量（每秒高达 30,000 个细胞），使其成为一项强大的技术。有能力 多重检测（测量单个细胞中多种不同的分子种类）进一步增加了其实用性和 使用该技术可以解决的科学问题的复杂性。 然而，目前的复用水平存在局限性。流式细胞术分析通常仅依赖于 荧光标签的光谱信息，因此受到荧光团发射的光谱重叠的限制。 目前，即使采用中等水平的多路复用也需要复杂的仪器和仔细的操作。 实验设计、执行和分析，以补偿信号的频谱溢出到多个 渠道。这严重限制了使用当前技术可以解决的科学问题的范围 技术，阻止该技术的新手尝试更复杂但科学相关的 实验，并被广泛认为是该领域的主要瓶颈。 为了克服这一限制，我们建议以第一阶段的结果为基础，我们已经证明了可行性 寻求一种创新方法，该方法使用荧光寿命作为单独的、额外的区分 测量参数。我们使用荧光寿命进行多重分析的方案简单、可扩展且 我们的原型仪器的初步数据支持了这一点。在这里我们建议升级我们的一期 仪器以提高复用能力，通过一系列验证测试来挑战该仪器，以及 使用相关的生物测定进行验证，将结果与传统流式细胞仪进行基准测试。 结果将是一个能够进行 12 种颜色的无补偿流式细胞术实验的系统，同时 与装备类似的商业系统相比，需要更少的激光器和探测器。这样的系统将 服务于很大一部分市场，包括临床细胞术，预计将得到广泛采用。这 将为进一步开发适用于的超高（30+）参数仪器铺平道路 免疫表型，但需要比现有系统少得多的补偿，并推动 实验复杂性的界限。鉴于流式细胞术的广泛使用和重要性，该项目 将对许多生物医学和临床应用产生重大影响。几家大型仪器仪表公司 已经表示渴望参与旨在商业化的战略伙伴关系。