Cell selection method, cell detection method, cell selection apparatus, and cell detection apparatus

Patent metadata

Field	Value
Publication number	US-11060134-B2
Application number	US-201615363503-A
Country	US
Kind code	B2
Filing date	Nov 29, 2016
Priority date	Nov 30, 2015
Publication date	Jul 13, 2021
Grant date	Jul 13, 2021

Abstract

Disclosed is a cell selection method including a sample preparation step of preparing a sample by performing staining of nucleic acid in each of cells by a first fluorescent dye; and hybridization with respect to an evaluation target region in DNA in each cell by an evaluation probe including a second fluorescent dye; a light receiving step of applying light to the sample and receiving fluorescence from the first fluorescent dye and fluorescence from the second fluorescent dye; and a selection step of selecting an analysis target cell on the basis of intensity of the fluorescence from the first fluorescent dye and intensity of the fluorescence from the second fluorescent dye, wherein the first fluorescent dye is a dye that emits fluorescence having a first wavelength, and the second fluorescent dye is a dye that emits fluorescence having a second wavelength different from the first wavelength.

First claim

What is claimed is:

1. A cell selection method comprising:
a sample preparation step of preparing a sample by performing: staining of substantially an entirety of nucleic acid in each of cells by a first fluorescent dye; and hybridization with respect to an evaluation target region in DNA in each cell by an evaluation probe which comprises a second fluorescent dye;
a light receiving step of applying light to the sample and receiving fluorescence from the first fluorescent dye and fluorescence from the second fluorescent dye; and
a selection step of selecting an analysis target cell on the basis of intensity of the fluorescence from the first fluorescent dye and intensity of the fluorescence from the second fluorescent dye, wherein
the first fluorescent dye is a dye that emits fluorescence having a first wavelength,
the second fluorescent dye is a dye that emits fluorescence having a second wavelength different from the first wavelength, and
selecting as the analysis target cell a cell for which a proportion between the intensity of the fluorescence from the first fluorescent dye and the intensity of the fluorescence from the second fluorescent dye is evaluated as being in a predetermined range,
wherein the cells are human-derived cells,
wherein the hybridization forms a hydrogen bond between complementary nucleoside bases or complementary nucleotide bases, and
wherein the evaluation probe includes:
(i) a polynucleotide sequence complementary to a base sequence of the evaluation target region in DNA in the cell; and
(ii) a fluorescent substance.

2. The cell selection method of claim 1 , wherein
in the light receiving step, light is applied to the sample flowing in a flow cell, and the fluorescence from the first fluorescent dye and the fluorescence from the second fluorescent dye are received.

3. The cell selection method of claim 1 , wherein
in the light receiving step, light is applied to the sample disposed on a support, and the fluorescence from the first fluorescent dye and the fluorescence from the second fluorescent dye are received by means of a microscope.

4. The cell selection method of claim 1 , wherein
the analysis target cell is selected for detecting genomic abnormality in the cell, and
the genomic abnormality is one of gene amplification, deletion, translocation, and inversion.

5. The cell selection method of claim 4 , wherein
in a case of the genomic abnormality is gene amplification, the evaluation target region is, among DNA sequence regions in a nucleus of the cell, a part of a DNA sequence region excluding any DNA sequence region where amplification occurred due to the genomic abnormality, and
in a case of the genomic abnormality is deletion, the evaluation target region is, among DNA sequence regions in the nucleus of the cell, of a part of a DNA sequence region excluding any DNA sequence region where deletion occurred due to the genomic abnormality.

6. A cell selection method comprising:
a sample preparation step of preparing a sample by performing: staining of substantially an entirety of nucleic acid in each of cells by a first fluorescent dye; and hybridization with respect to an evaluation target region in DNA in each cell by an evaluation probe which comprises a second fluorescent dye;
an image capturing step of applying light to the sample and capturing an image of the cell in the sample;
determining, on the basis of the image of the cell captured in the image capturing step, brightness of an image of fluorescence from the first fluorescent dye and brightness of an image of fluorescence from the second fluorescent dye; and
selecting an analysis target cell on the basis of the brightness of the image of the fluorescence from the first fluorescent dye and the brightness of the image of the fluorescence from the second fluorescent dye, wherein a cell for which a proportion between the brightness of the image of the fluorescence from the first fluorescent dye and the brightness of the image of the fluorescence from the second fluorescent dye is evaluated as being in a predetermined range is selected as the analysis target cell, wherein
the first fluorescent dye is a dye that emits fluorescence having a first wavelength,
the second fluorescent dye is a dye that emits fluorescence having a second wavelength different from the first wavelength,
the cells are human-derived cells,
the hybridization forms a hydrogen bond between complementary nucleoside bases or complementary nucleotide bases, and
the evaluation probe includes:
(i) a polynucleotide sequence complementary to a base sequence of the evaluation target region in DNA in the cell; and
(ii) a fluorescent substance.

7. A cell detection method comprising:
a step of preparing a sample by performing: staining of substantially an entirety of nucleic acid in each of cells by a first fluorescent dye; hybridization with respect to an evaluation target region in DNA in each cell by an evaluation probe which comprises a second fluorescent dye; and hybridization with respect to a second target region in DNA in the cell by a detection probe which comprises a third fluorescent dye;
a step of applying light to the sample and receiving fluorescence from the first fluorescent dye, fluorescence from the second fluorescent dye, and fluorescence from the third fluorescent dye;
a step of selecting an analysis target cell on the basis of intensity of the fluorescence from the first fluorescent dye and intensity of the fluorescence from the second fluorescent dye, wherein a cell for which a proportion between the intensity of the fluorescence from the first fluorescent dye and the intensity of the fluorescence from the second fluorescent dye is evaluated as being in a predetermined range is selected as the analysis target cell; and
a step of identifying an abnormal cell from the analysis target cell on the basis of the fluorescence from the third fluorescent dye, wherein
the first fluorescent dye is a dye that emits fluorescence having a first wavelength,
the second fluorescent dye is a dye that emits fluorescence having a second wavelength different from the first wavelength,
the third fluorescent dye is a dye that emits fluorescence having a third wavelength different from the first and second wavelengths,
the cells are human-derived cells,
the hybridization forms a hydrogen bond between complementary nucleoside bases or complementary nucleotide bases, and
the evaluation probe includes:
(i) a polynucleotide sequence complementary to a base sequence of the evaluation target region in DNA in the cell; and
(ii) a fluorescent substance.

8. The cell detection method of claim 7 , wherein
in the step of applying light to the sample, light is applied to the sample flowing in a flow cell, and the fluorescence from the first fluorescent dye, the fluorescence from the second fluorescent dye, and the fluorescence from the third fluorescent dye are received.

9. The cell detection method of claim 7 , wherein
the abnormal cell is identified from the analysis target cell by comparing a ratio threshold with a ratio between intensity of the fluorescence from the third fluorescent dye and the intensity of the fluorescence from the second fluorescent dye.

10. The cell detection method of claim 9 , wherein
a cell in which a value determined by dividing the intensity of the fluorescence from the third fluorescent dye by the intensity of the fluorescence from the second fluorescent dye exceeds Rail the ratio threshold is detected as the abnormal cell.

11. The cell detection method of claim 7 , wherein
the second target region is, among DNA sequence regions in a nucleus of the cell, a DNA sequence re

Assignees

• Sysmex Corp

Inventors

• Takahashi Yusuke
• Yanagida Masatoshi

Classifications

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• C12Q2565/102

  Multiple non-interacting labels · CPC title

• C12Q2565/626

  being a flow cytometer · CPC title