Method for detecting low concentrations of specific cell from high concentrations of cell populations, and method for collecting and analyzing detected cell

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.

主权项

1. A method for enumeration of specific cells present in highly dense cells, characterized by comprising:
the pretreatment step of concentrating the specific cells, and fluorescence staining said cells, and the step of counting all the specific cells in the sample liquid by automatically identifying said cells based on fluorescence signal intensity thereof; wherein, in the counting step, the specific cells are counted by measuring a whole sample liquid by a flow cytometer having a disposable micro flow-path chip as a flow cell, the micro flow-path chip containing a sample reservoir, a collection reservoir, and a micro flow-path connected from the bottom of the sample reservoir to the bottom of the collection reservoir on a substrate; and an end point of the whole sample liquid is recognized by a detection of air bubbles generated in the micro flow-path just when the sample liquid in the sample reservoir flows out.