Eliminating Interference from Autofluorescence in Flow Cytometry

项目总结/摘要 流式细胞术（FC）是一种强大的细胞分析工具，依赖于细胞的荧光标记。它可以提供 关于细胞计数、形状、大小、DNA含量、氧化还原状态、膜渗透性和表面受体的信息， 以及其他特征。FC是研究和临床领域的重要工具，用于肿瘤生物学研究、癌症 以及艾滋病诊断、干细胞治疗、免疫分型和癌症免疫治疗。领先的 FC的局限性在于外源添加的荧光团不是样品中唯一的荧光源。 在某些情况下，自体荧光（AF）可以是存在于每一种细胞中的信号的重要贡献者。 检测通道，通常与发射光谱重叠。AF限制检测灵敏度和原因 数据解释中的问题，这可能导致错误的科学结论。虽然有几种方法 虽然已经被用于消除AF，但是没有满意的解决方案可用。 Kinetic River一直在开发新的方法，利用荧光寿命（FLT）作为内在的 鉴别参数和分辨流式细胞术中的多指数寿命衰减。在我们完成的 第一阶段SBIR，我们成功地证明了自动去除细胞AF干扰的荧光团- 使用时间分辨发射寿命使用4色系统标记的人类细胞，建立了 法第二阶段的拨款将通过将系统扩展到以下领域来推动这些发现的商业化： 一个完全可操作的16色科罗拉多分析仪与实时数据处理。圆满完成 科罗拉多分析仪将导致第一个解决方案，自动消除干扰，从AF在流动 细胞计数器实验。它将成为一个改变游戏规则的平台，使细胞生物学家，免疫学家， 肿瘤学研究人员、诊断公司和其他FC用户，以获得更准确和定量的 结果去除细胞AF干扰将提高灵敏度，这对于检测罕见的 表面标记。科罗拉多平台的这一新增功能将为其提供显著的竞争优势， 与具有类似多路复用能力的当前商用FC分析仪相比，这将使 科罗拉多，以取代目前的中级多路复用仪器，目前占据了大片的 快速增长的48亿美元FC市场。

PROJECT SUMMARY/ABSTRACT Flow cytometry (FC) is a powerful cell analysis tool relying on fluorescently labeling of cells. It can provide information on cell count, shape, size, DNA content, redox state, membrane permeability, and surface receptors, among other features. FC is an essential tool in research and clinical fields for tumor biology research, cancer and AIDS diagnostics, stem cell therapy, immunophenotyping, and cancer immunotherapy. One of the leading limitations of FC is that exogenously added fluorophores are not the only source of fluorescence in a sample. Autofluorescence (AF) can be, in some circumstances, a significant contributor to the signal present in each of the detection channels, often overlapping with the emission spectra. AF limits assay sensitivity and causes problems in data interpretation, which can lead to erroneous scientific conclusions. While several approaches have been employed to eliminate AF, there are no satisfactory solutions available. Kinetic River has been developing novel methods utilizing fluorescence lifetime (FLT) as an intrinsic discriminating parameter and resolving multi-exponential lifetime decays in flow cytometry. In our completed Phase I SBIR, we successfully demonstrated the automated removal of cellular AF interference in fluorophore- labeled human cells using time-resolved emission lifetimes using a 4-color system, establishing feasibility of the method. This Phase II grant will advance these findings towards commercialization by expanding the system into a fully operational 16-color Colorado analyzer with real-time data processing. The successful completion of the Colorado analyzer will lead to the first solution that automatically eliminates interference from AF in flow cytometer experiments. It will become a game-changing platform enabling cell biologists, immunologists, oncology researchers, diagnostic companies, and other FC users to obtain more accurate and quantitative results. Removal of cellular AF interference will yield enhanced sensitivity, which is important for detecting rare surface markers. This added capability of the Colorado platform will provide it with a significant competitive advantage over current commercial FC analyzers of similar multiplexing capabilities. This will enable the Colorado to replace current mid-level multiplexing instruments, which currently occupy a large swath of the rapidly growing $4.8 billion FC market.