A high volume parallel acoustic flow cytometer for the detection of rare cells or particles in a large sample volume with a low background concentration (i.e. very dilute samples).

一种大容量并行声学流式细胞仪，用于检测低背景浓度的大样本量（即非常稀释的样本）中的稀有细胞或颗粒。

• 批准号: 9281078
• 负责人: James P Freyer
• 金额: $ 52.83万
• 依托单位: ETA DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9281078
• 关键词: Acoustics; Address; Algorithms; Animals; Area; Back; Bacteria; Beverages; Blood; Blood specimen; Buffers; Cells; Collection; Color; Data; Data Analyses; Detection; Diagnosis; Disease; Engineering; Ethics; Family; Flow Cytometry; Fluorescence; Food; Funding; Future; Gold; Health; Human; Image; L Cells; Lasers; Light; Liquid substance; Malignant neoplasm of urinary bladder; Measurement; Medical; Methods; Milk; Monitor; Neoplasm Circulating Cells; Optics; Performance; Population; Price; Recovery; Research; Sampling; Series; Speed; Sterility; Stream; System; Systems Analysis; United States National Institutes of Health; Urine; Water; Width; Work; base; cancer cell; computerized data processing; cost; data acquisition; detector; fetus cell; file format; fluorophore; high throughput screening; instrument; interest; new technology; novel; particle; pathogen; pathogenic bacteria; prevent; public health relevance; sample collection; sensor; success; tissue/cell culture; wasting

 DESCRIPTION (provided by applicant): Many biomedical applications require the detection of rare cells or particles in large sample volumes. These applications can be divided into those that require the detection of highly dilute cells in a large sample volume and those that require the detection of rare cells in a high background concentration of similar cells within a large volume. Though flow cytometry is the gold standard in the detection of cellular populations, its volumetric sample delivery rate dramatically limits its use for large volume samples and its analytical rate prevents its use for these applications. However, recent work funded by the NIH has resulted in several advances that can be used to develop a high volume flow cytometer that will be of immediate use for high volume rare cell detection in low cell backgrounds. Using a synergistic combination of parallel acoustic flow cells, a cutting edge high speed sCMOS camera, and a novel optical configuration, our high volume parallel acoustic flow cytometer (HVPAfc) will collect multicolor flow cytometry data at analytical rates of 100,000 cells per second and sample delivery rates of 50 mL/minute. This instrument will have immediate value for many applications, such as the detection of bladder cancer cells in urine or pathogenic bacteria in milk or water, and thus will have a large impact on the field and the marketplace. To create the GenI HVPAfc, we will first construct an acoustic flow cell that creates many parallel sample streams via standing waves. This flow cell will be constructed such that reversible flow will be possible and i will allow undiluted collection of the sample for re-analysis. Second, the flow cell will be coupld to an optimized optical system that uses a high-speed sCMOS camera for collection and a laser line that perpendicularly crosses the flow streams. The camera can collect 2048 x 8 pixels frames at rates of 25,655 fps, which will allow high speed collection of all flow streams using a single sensor. Importantly, we have developed a filter based optical dispersion system that allows collection of multiple colors within the same frame of the sensor. This will allow us to create a single detector instrument that can collect 3 spectral regions, thus reducing instrument cost and complexity. The third task will create a high-speed data acquisition that strips the data from the 2048 pixel x 8 pixel 16-bit data depth frames and converts it into standard flow cytometry file format. This will allow our instrument to be immediately implemented within the data analysis pipeline that exists in most labs worldwide. Fourth, the three subsystems will be integrated into a single flow cytometer that can sample at 50 mL/min, support particle analysis rates as high as 100,000 per second, collect three spectral regions of emitted light for fluorescence or scatter measurement, and provide sensitivities as low as few thousand fluorophores per particle. This cytometer will be engineered to be commercially robust and support real world users. Importantly, it will be constructed to be available at an average selling price of $100K per instrument. Finally, we will demonstrate the detection of mammalian and bacterial cells spiked in liquid samples at concentrations as low as 1 cell/L. Initial efforts will simply spike cells into clear buffers, but subsequent work will spike cells into clarified or lysed natural samples. This will be done to effectively mimic anticipated applications. Success in developing the HVPAfc will create an instrument that is immediately valuable for high volume applications and provide the basis for future flow cytometers that can address a myriad of additional application areas.

 描述（由申请人提供）：许多生物医学应用需要检测大量样品中的稀有细胞或颗粒。这些应用可分为需要检测大样品体积中高度稀释的细胞的应用和需要检测大体积内类似细胞的高背景浓度中的稀有细胞的应用。虽然流式细胞术是检测细胞群体的金标准，但其体积分析法不适用于细胞群体。 样品输送速率极大地限制了其用于大体积样品，并且其分析速率阻止了其用于这些应用。然而，最近由NIH资助的工作取得了一些进展，可用于开发高容量流式细胞仪，该流式细胞仪将立即用于低细胞背景中的高容量稀有细胞检测。 使用并行声学流式细胞仪、尖端高速sCMOS相机和新型光学配置的协同组合，我们的大容量并行声学流式细胞仪（HVPAfc）将以每秒100，000个细胞的分析速率和50 mL/分钟的样品输送速率收集流式细胞术数据。该仪器将对许多应用具有直接价值，例如检测尿液中的膀胱癌细胞或牛奶或水中的致病菌，因此将对该领域和市场产生巨大影响。 为了创建GenI HVPAf c，我们将首先构建声学流动池，其经由驻波创建许多平行样品流。该流动池的构造应确保能够实现可逆流动，并允许采集未稀释的样品进行再分析。 其次，流动池将耦合到优化的光学系统，该光学系统使用高速sCMOS相机进行收集，并使用垂直穿过流动流的激光线。该相机可以以25，655 fps的速率收集2048 x 8像素的帧，这将允许使用单个传感器高速收集所有流。重要的是，我们已经开发出一种基于滤光片的光学色散系统，该系统允许在传感器的同一框架内收集多种颜色。这将使我们能够创建一个单一的检测器仪器，可以收集3个光谱区域，从而降低仪器成本和复杂性。 第三个任务将创建一个高速数据采集，剥离数据 从2048像素× 8像素的16位数据深度帧中，并将其转换为标准流式细胞术文件格式。这将使我们的仪器能够立即在全球大多数实验室存在的数据分析管道中实施。 第四，这三个子系统将被集成到一个流式细胞仪中，该流式细胞仪可以以50 mL/min的速度采样，支持高达每秒100，000个的颗粒分析速率，收集发射光的三个光谱区域用于荧光或散射测量，并提供低至每个颗粒几千个荧光团的灵敏度。该血细胞计数器将被设计为具有商业稳健性，并支持真实的世界用户。重要的是，它将被建造成可在平均销售 每台仪器10万美元。 最后，我们将证明在低至1个细胞/L的浓度下，对液体样品中掺入的哺乳动物和细菌细胞的检测。初步努力将 简单地将细胞加入到澄清的缓冲液中，但随后的工作将细胞加入到澄清或裂解的缓冲液中， 天然样品这样做是为了有效地模拟预期的应用程序。HVPAfc的成功开发将创造一种对高容量应用立即有价值的仪器，并为未来的流式细胞仪提供基础，可以解决无数其他应用领域。