Compensation-Free, Highly Multiplexed Flow Cytometer

无补偿、高度多重流式细胞仪

• 批准号: 9348516
• 负责人: Giacomo Vacca
• 金额: $ 22.5万
• 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: 2017-04-05 至 2018-01-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9348516
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Algorithms; Alpha Cell; Amplifiers; Antigens; Biological; Biological Assay; CD3 Antigens; CD8B1 gene; Cells; Characteristics; Clinical; Collection; Color; Complex; Coupled; Data; Development; Development Plans; Discrimination; Disease; Financial compensation; Flow Cytometry; Fluorescence; Foundations; Goals; Human; Immune; Label; Laboratories; Laboratory Diagnosis; Lasers; Leukocytes; Malignant Neoplasms; Measurement; Measures; Molecular; Monitor; Monoclonal Antibodies; Noise; Outcome; PTPRC gene; Performance; Physiologic pulse; Population; Protocols documentation; Reproducibility; Research; Research Personnel; Running; Sampling; Scheme; Signal Transduction; Source; Specificity; Speed; Staining method; Stains; Surface Antigens; System; Techniques; Technology; Testing; Time; Validation; Width; base; clinical diagnostics; commercialization; design; digital; drug development; experimental study; fluorophore; improved; innovation; instrument; instrumentation; lens; operation; prototype; research and development; verification and validation

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 and that very high throughput is possible (30,000 cells per second can be analyzed), 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. Typically, flow cytometry analysis 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 for the simultaneous interrogation of multiple parameters within a cell requires high levels of complexity in instrumentation and analysis, and careful design and execution of experiments. The related "compensation problem" (compensating for spillover of signal from a fluorophore into multiple channels—due to the broad spectrum of most fluorophores) also causes significant instrument complexity, cumbersome workflow, and inaccurate results. These factors put severe limits on the range of scientific questions that can be addressed using current technologies, deter novices in the technique from attempting more complex yet scientifically relevant experiments, and collectively are widely regarded as the major current bottleneck in flow cytometry. To overcome this limitation, we have developed 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. The proposed project will establish the feasibility of lifetime-based multiplexing by modifying our experimental platform with key hardware and algorithm improvements, challenging the resulting prototype with a comprehensive set of verification and validation tests of increasing complexity, and culminating with a comparison of our technology to existing technology in a standard four-color cell-based assay. A successful outcome will lay the foundation for our planned development of commercial instruments (both analyzers and sorters) that offer two major benefits to end users: (a) simple, turnkey, compensation-free operation for instruments with low-to-medium levels of multiplexing; and (b) high-end instruments with two to three times the current maximum multiplexing capability.

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 并且可以实现非常高的吞吐量（每秒可以分析 30,000 个细胞），使其成为强大的 技术。进一步多重分析（测量单个细胞中多种不同分子种类）的能力 增加了它的实用性以及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。通常，流式细胞术分析仅依赖于 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，甚至采用中等水平的多路复用来同时询问多个 细胞内的参数需要高度复杂的仪器和分析以及精心的设计 和实验的执行。相关的“补偿问题”（补偿来自 荧光团进入多个通道（由于大多数荧光团的广谱）也会导致显着的 仪器复杂、工作流程繁琐、结果不准确。这些因素都严重限制了 使用现有技术可以解决的一系列科学问题，阻止了该技术的新手 来自尝试更复杂但与科学相关的实验，并且集体被广泛认为 目前流式细胞术的主要瓶颈。 为了克服这一限制，我们开发了一种创新方法，利用荧光寿命作为 单独的、附加的区分测量参数。我们利用荧光寿命的方案 多路复用简单、可扩展，并得到我们原型仪器的初步数据的支持。这 拟议的项目将通过修改我们的实验来建立基于生命周期的多路复用的可行性 具有关键硬件和算法改进的平台，挑战最终的原型 一套全面的验证和确认测试，其复杂性不断增加，最终以 在标准四色细胞检测中将我们的技术与现有技术进行比较。 成功的结果将为我们计划的商业工具开发奠定基础（包括 分析仪和分选机），为最终用户提供两大优势：(a) 简单、交钥匙、无补偿 适用于具有低至中等多路复用水平的仪器； (b) 具有两个至 目前最大复用能力的三倍。