Compensation-Free, Highly Multiplexed Flow Cytometer

无补偿、高度多重流式细胞仪

基本信息

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
项目类别：
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

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

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项目摘要

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而且吞吐量很高（每秒可以分析30,000个单元），使其成为强大的技术。多路复用的能力（在单个细胞中测量各种不同的分子物种）增加了它的效用以及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。通常，流式细胞仪分析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。当前，使用中等水平的多路复用单元内的参数需要仪器和分析的高度复杂性，并仔细设计和执行实验。相关的“补偿问题”（补偿来自荧光团进入多个通道 - 大多数荧光团的广泛光谱）也会引起显着的仪器的复杂性，繁琐的工作流程和结果不正确。这些因素对可以使用当前技术解决的科学问题范围，确定技术中的小说通过尝试更复杂但科学相关的实验，并被广泛认为是流式细胞仪中的主要电流瓶颈。为了克服这一限制，我们开发了一种创新的方法，该方法将荧光寿命作为一个单独的，附加的区分测量参数。我们使用荧光寿命的计划多路复用是简单，可扩展的，并且由我们原型仪器的初步数据支持。拟议的项目将通过修改我们的实验来确定基于终生的多路复用的可行性具有关键硬件和算法改进的平台，通过全面的验证和验证测试，以提高复杂性，并以A 在标准的四色细胞测定中，我们的技术与现有技术的比较。成功的结果将为我们计划开发的商业工具奠定基础（均分析仪和分类器）为最终用户提供了两个主要好处：（a）简单，交钥匙，无薪酬具有多重多路复用水平的仪器的操作；（b）具有两种的高端仪器当前最大多路复用能力的三倍。