# US 10,309,900 B1 · Biological substance quantitation method, pathological diagnosis support system and recording medium storing computer readable program

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,309,900 B1
- Document ID: US10309900B1
- Status: Granted
- Kind: B1
- Application number: 16/256,224
- Filed: 2019-01-24
- Published: 2019-05-23
- Granted: 2019-06-04
- Earliest claimed: 2019-01-24
- Assignees: Konica Minolta, Inc.
- Applicants: Konica Minolta, Inc.
- Inventors: Hideki Gouda; Kensaku Takanashi; Kohsuke Gonda; Noriaki Ohuchi; Mika Watanabe
- Web page: https://patentgrep.com/patent/US10309900B1

## CPC

- G01N 21/6428: {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)}
- G01N 21/64: Fluorescence; Phosphorescence
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 21/93: Detection standards; Calibrating {baseline adjustment, drift correction}
- G01N 33/48: Biological material, e.g. blood, urine (G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence); Haemocytometers (counting blood corpuscules distributed over a surface by scanning the surface G06M11/02)
- G01N 33/483: Physical analysis of biological material
- G01N 33/5023: {on expression patterns}
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/582: {with fluorescent label}
- G06T 7/0012: {Biomedical image inspection}
- G06T 7/0016: {involving temporal comparison}
- G06T 7/90: Determination of colour characteristics
- G06T 2207/10024: Color image
- G06T 2207/10056: Microscopic image
- G06T 2207/10064: Fluorescence image
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- G06T 2207/30242: Counting objects in image
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL

## Abstract

A biological substance quantitation method includes the following. A fluorescent image is input, which represents an expression of a specific biological substance in a sample stained with a fluorescent substance by a florescent bright spot. A quantitative evaluation value of the florescent bright spot is calculated. A standard fluorescent image of a standard sample stained under a same condition as the sample and representing an expression of the biological substance in a standard sample, is input under a same condition as the fluorescent image. A quantitative evaluation value in the standard fluorescent image is calculated under a same condition as the fluorescent image. Based on a correlation between an expression amount of the biological substance in a standard sample measured in advance and the evaluation value in the standard fluorescent image, the evaluation value in the fluorescent image is converted to an expression amount of the biological substance in the sample.

## Claims

### Claim 1

A method for quantitating an amount of a target substance in a sample stained with a staining reagent which stains the target substance with a fluorescent substance, the method comprising:
inputting a fluorescent image which represents a presence of the target substance in the sample by a fluorescent bright spot;
performing fluorescence quantitation which includes calculation of an evaluation value by quantitative evaluation of the fluorescent bright spot in the fluorescent image;
inputting a standard fluorescent image under a same condition as the inputting of the fluorescent image, wherein
the standard fluorescent image represents a presence of the target substance in a standard sample by a fluorescent bright spot based on staining under a same condition as the sample, and
an amount of the target substance in the standard sample is measured in advance;
performing standard fluorescence quantitation which includes calculation of an evaluation value by quantitative evaluation of the fluorescent bright spot in the standard fluorescent image under a same condition as the quantitation of fluorescence;
calculating a correlation between the amount of the target substance in the standard sample and the evaluation value of the fluorescent bright spot in the standard fluorescent image; and
converting the evaluation value of the fluorescent bright spot in the fluorescent image to an amount of the target substance in the sample based on the calculating of the correlation.

### Claim 2 (depends on 1)

The method according to claim 1, wherein
a calibration curve is prepared in calculating the correlation, wherein the calibration curve represents the evaluation value of the fluorescent bright spot of the standard fluorescence image corresponding to the amount of the target substance in the standard sample, and
in the converting, the evaluation value of the fluorescent bright spot of the fluorescent image is converted to the amount of the target substance in the sample based on the calibration curve.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the standard sample is a cell cultured on a substrate.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the staining reagent comprises a fluorescent particle in which a plurality of molecules of the fluorescent substance are accumulated.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the florescent substance emits a light having a wavelength in the range of 400 to 1100 nm when excited by a light having a wavelength in the range of 200 to 700 nm.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the staining reagent comprises a plurality of the florescent particle, and the plurality of the florescent particle have an average particle diameter in a range of 40 to 280 nm.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the amount of the target substance in the standard sample is measured in advance by one of ELISA, flow cytometry, and Western blotting.

### Claim 8

A pathological diagnosis support system which quantitates an amount of a target substance in a sample stained with a staining reagent which stains the target substance with a fluorescent substance, the system comprising:
a storage which stores a program for quantitating the amount of the target substance; and
a processor, the program causing the processor to function as:
a fluorescent image inputting unit which inputs a fluorescent image which represents a presence of the target substance in the sample by a fluorescent bright spot;
a fluorescence quantitation unit which calculates an evaluation value by quantitative evaluation of the fluorescent bright spot in the fluorescent image;
a standard fluorescent image inputting unit which inputs a standard fluorescent image under a same condition as the fluorescent image inputting unit, wherein
the standard fluorescent image represents a presence of the target substance in a standard sample by a fluorescent bright spot based on staining under a same condition as the sample, and
an amount of the target substance in the standard sample is measured in advance;
a standard fluorescent quantitation unit which calculates an evaluation value by quantitative evaluation of the fluorescent bright spot in the standard fluorescent image under a same condition as the fluorescence quantitation unit;
a correlation calculator which calculates a correlation between the amount of the target substance in the standard sample and the evaluation value of the fluorescent bright spot in the standard fluorescent image; and
a converter which converts the evaluation value of the fluorescent bright spot in the fluorescent image to an amount of the target substance in the sample based on the correlation.

### Claim 9

A non-transitory recording medium storing a computer readable program causing a computer which quantitates an amount of a target substance in a sample stained with a staining reagent which stains the target substance with fluorescent substances, to function as:
a fluorescent image inputting processor which inputs a fluorescent image which represents a presence of the target substance in the sample by a fluorescent bright spot;
a fluorescence quantitation unit which calculates an evaluation value by quantitative evaluation of the fluorescent bright spot in the fluorescent image;
a standard fluorescent image inputting unit which inputs a standard fluorescent image under a same condition as in the fluorescent image inputting unit, wherein
the standard fluorescent image represents a presence of the target substance in a standard sample by a fluorescent bright spot based on staining under a same condition as the sample, and
an amount of the target substance in the standard sample is measured in advance;
a standard fluorescent quantitation unit which calculates an evaluation value by quantitative evaluation of the fluorescent bright spot in the standard fluorescent image under a same condition as the fluorescence quantitation unit;
a correlation calculator which calculates a correlation between the amount of the target substance in the standard sample and the evaluation value of the fluorescent bright spot in the standard fluorescent image; and
a converter which converts the evaluation value of the fluorescent bright spot in the fluorescent image to an amount of the target substance in the sample based on the correlation.