公开(公告)号：US09341550B2

公开(公告)日：2016-05-17

申请号：US13885570

申请日：2011-11-21

申请人： Kazuo Takeda ,  Fumie Jimma ,  Masashi Takao

发明人： Kazuo Takeda ,  Fumie Jimma ,  Masashi Takao

IPC分类号： G01N33/543 ,  G01N33/566 ,  G01N33/577 ,  G01N35/08 ,  G01N33/574 ,  G01N1/30 ,  G01N15/14 ,  G01N15/10

CPC分类号： G01N33/57492 ,  G01N1/30 ,  G01N15/1436 ,  G01N15/1459 ,  G01N21/6428 ,  G01N33/54333 ,  G01N33/574 ,  G01N2015/1006 ,  G01N2015/1477 ,  G01N2021/6439 ,  G01N2201/06113 ,  G01N2333/705 ,  G01N2333/70596

摘要： Conventional CTC detection methods have been problematic in that 1) there is no technique for automatically determining and counting live CTCs in a brief period of time, 2) no process has been developed for detecting, counting, and thereafter collecting and culturing live CTCs, and 3) there exists no flow cytometer that is contamination free and is capable of measuring an entire sample. Provided is a CTC detection method which comprises a pre-treatment step for concentrating and fluorescence staining CTCs, and a step for identifying and counting CTCs. The pre-treatment step includes attaching magnetic beads to EpCAM antibodies expressed by epithelial cell-derived CTCs and concentrating the CTCs through the use of a magnet, fluorescently labeling an epithelia cell surface marker of the CTCs through the use of EpCAM antibodies or 5E11 antibodies, and performing two types of nuclear staining, one being cell membrane-permeable and the other being cell membrane-impermeable. The identifying and counting step includes evaluating the respective absolute concentrations of live and dead CTCs in a volume of blood by automatically identifying CTCs by the ratio of a plurality of fluorescence signal intensities using a flow cytometer, and differentiating between and counting the live CTCs and the dead CTCs. In the cytometer, an entire liquid-feeding system that includes a flow cell can be replaced for each sample, and the total amount of a liquid sample can be measured.

公开(公告)号：US20130302828A1

公开(公告)日：2013-11-14

申请号：US13885570

申请日：2011-11-21

申请人： Kazuo Takeda ,  Fumie Jimma ,  Masashi Takao

发明人： Kazuo Takeda ,  Fumie Jimma ,  Masashi Takao

IPC分类号： G01N1/30

CPC分类号： G01N33/57492 ,  G01N1/30 ,  G01N15/1436 ,  G01N15/1459 ,  G01N21/6428 ,  G01N33/54333 ,  G01N33/574 ,  G01N2015/1006 ,  G01N2015/1477 ,  G01N2021/6439 ,  G01N2201/06113 ,  G01N2333/705 ,  G01N2333/70596

摘要： Conventional CTC detection methods have been problematic in that 1) there is no technique for automatically determining and counting live CTCs in a brief period of time, 2) no process has been developed for detecting, counting, and thereafter collecting and culturing live CTCs, and 3) there exists no flow cytometer that is contamination free and is capable of measuring an entire sample. Provided is a CTC detection method which comprises a pre-treatment step for concentrating and fluorescence staining CTCs, and a step for identifying and counting CTCs. The pre-treatment step includes attaching magnetic beads to EpCAM antibodies expressed by epithelial cell-derived CTCs and concentrating the CTCs through the use of a magnet, fluorescently labeling an epithelia cell surface marker of the CTCs through the use of EpCAM antibodies or 5E11 antibodies, and performing two types of nuclear staining, one being cell membrane-permeable and the other being cell membrane-impermeable. The identifying and counting step includes evaluating the respective absolute concentrations of live and dead CTCs in a volume of blood by automatically identifying CTCs by the ratio of a plurality of fluorescence signal intensities using a flow cytometer, and differentiating between and counting the live CTCs and the dead CTCs. In the cytometer, an entire liquid-feeding system that includes a flow cell can be replaced for each sample, and the total amount of a liquid sample can be measured.

摘要翻译： 传统的CTC检测方法存在问题，即1）在短时间内没有自动确定和计数活CTC的技术，2）没有开发检测，计数，之后收集和培养活的CTC的过程， 3）不存在无污染的流式细胞仪，能够测量整个样品。 提供了一种CTC检测方法，其包括用于浓缩和荧光染色CTC的预处理步骤，以及用于鉴定和计数CTC的步骤。 预处理步骤包括将磁珠附着到由上皮细胞衍生的CTC表达的EpCAM抗体中，并通过使用磁体浓缩CTC，通过使用EpCAM抗体或5E11抗体荧光标记CTC的上皮细胞表面标志物， 并进行两种类型的核染色，一种是细胞膜可渗透的，另一种是细胞膜不可渗透的。 识别和计数步骤包括通过使用流式细胞仪自动识别多个荧光信号强度的比例来自动识别CTC，并且区分和计数活CTC和 死了四氯化碳。 在血细胞计数器中，可以为每个样品更换包括流通池的整个液体供给系统，并且可以测量液体样品的总量。