Advancing High Throughput Flow Cytometry

描述（由申请人提供）：流式细胞术（FCM）是一种用于多参数分析的多功能流式细胞术技术，其在生物测定多路复用（在单个样品体积中进行多种测定）的设施和能力方面是无与伦比的。通过将多样品通量提高20倍，最近开发的HyperCyt技术克服了FCM在高通量筛选（HTS）药物和探针发现企业中应用此功能和其他强大功能的历史障碍。这里的具体目标是实现一个强大的FCM平台的长期目标，该平台能够在HTS环境中进行扩展的全自动操作。在目标1中，将修改HyperCyt平台，以平行处理1536孔板的4个384孔象限，使用4个采样探针通过正压样品输送将样品发送到4个单独的流式细胞仪。这种方法将利用功能强大、低成本的流式细胞仪，将通量提高四倍，达到约8,400个样本/小时。目标2将评价一种替代方法，其中样本将被拉而不是被推过细胞仪观察室。利用使用负压进样的细胞仪，自动XYZ载物台将气泡分离的生物测定样品从1536孔板直接引入细胞仪，而不需要HyperCyt蠕动泵。这将消除细胞暴露于压缩泵送力，缩短样品传输距离以减少样品之间的流体携带，并允许样品流体以更高的速度移动而不损失光学分辨率，以实现单个细胞仪的预期吞吐量加倍。Aim 3将在新墨西哥州大学分子发现中心（UNMCMD）对每个平台进行beta测试。已建立的多重检测将用于优化和验证平台性能与成熟的384孔FCM HTS技术。含有20个或更多个单重元素的样品（即，e20-plex）将用于定义多重组成和样品通量之间的限制和平衡。到项目结束时，这些新平台将被整合到UNMCMD的自动化流程中，使HTS FCM能够在分子库探针中心网络和国际科学界的强大HTS功能中占有一席之地。公共卫生相关性：流式细胞术已被证明是健康和疾病（艾滋病，基因组计划，干细胞，自身免疫性疾病和癌症，仅举几例）的生物学研究中的关键技术。提出的两个创新平台承诺互补的解决方案，通过这些解决方案，可以显着提高流式细胞术在小分子高通量筛选中的速度和效率，推进新药和生物探针的发现。

DESCRIPTION (provided by applicant): Flow cytometry (FCM) is a versatile multicolor technology for multi-parameter analysis which is unparalleled in its facility and capacity for bioassay multiplexing, the performance of multiple assays in a single sample volume. By increasing multi-sample throughput 20-fold, recently developed HyperCyt technology has overcome historic barriers to application of this and other powerful features of FCM in the high throughput screening (HTS) drug and probe discovery enterprise. Specific aims here address the long term goal of a robust FCM platform capable of extended, fully automated operation in the HTS environment. In Aim 1 the HyperCyt platform will be modified to process four 384-well quadrants of a 1536-well plate in parallel, using four sampling probes to send samples to four separate flow cytometers by positive pressure sample delivery. This approach will take advantage of powerful, low-cost flow cytometers to quadruple throughput to about 8,400 samples/hour. Aim 2 will evaluate an alternative approach in which samples will be pulled rather than pushed through the cytometer observation chamber. Exploiting a cytometer that uses negative pressure for sample intake, an automated XYZ stage will introduce air-bubble separated bioassay samples directly into the cytometer from 1536-well plates without need of the HyperCyt peristaltic pump. This will eliminate cell exposure to compressive pump forces, shorten sample transit distance to reduce fluid carryover between samples, and allow sample fluid to be moved at higher velocity without loss of optical resolution to achieve an expected doubling of throughput for a single cytometer. Aim 3 will beta-test each platform in the University of New Mexico Center for Molecular Discovery (UNMCMD). Established multiplexed assays will be used to optimize and validate platform performance vs. mature 384 well FCM HTS technology. Samples containing 20 or more single-plex elements (i.e., e 20-plex) will be used to define the limits of and balance between multiplex composition and sample throughput. By the end of the project period, these novel platforms will be positioned for integration into the automated process stream of the UNMCMD, allowing HTS FCM to take its place in the armamentarium of robust HTS capabilities for the Molecular Libraries Probe Centers Network and for the international scientific community. PUBLIC HEALTH RELEVANCE: Flow cytometry has proven to be a pivotal technology in biological investigations of health and disease (AIDS, Genome Project, stem cells, autoimmune disease, and cancer, to name a few). Two proposed innovative platforms promise complementary solutions by which to significantly increase the speed and efficiency of flow cytometry in small molecule high throughput screening, advancing discovery of novel drugs and biological probes.