# US 10,371,640 B2 · Compositions and methods for leukocyte differential counting

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- Number: US 10,371,640 B2
- Document ID: US10371640B2
- Status: Granted
- Kind: B2
- Application number: 15/647,039
- Filed: 2017-07-11
- Published: 2017-11-02
- Granted: 2019-08-06
- Earliest claimed: 2011-11-28
- Assignees: California Institute of Technology
- Applicants: California Institute of Technology
- Inventors: Yu-Chong Tai; Wendian Shi; Harvey Kasdan
- Web page: https://patentgrep.com/patent/US10371640B2

## CPC

- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- 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 21/05: Flow-through cuvettes (G01N21/09 takes precedence; handling fluid samples G01N1/10)
- G01N 2021/0346: {Capillary cells; Microcells}
- G01N 2021/6441: {with two or more labels}
- G01N 2021/6482: {Sample cells, cuvettes}
- 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)

## Abstract

The present invention provide compositions and methods for classifying leukocytes in a leukocyte population using fluorescence detection. The methods include contacting a leukocyte population in a sample having one or more leukocyte types with a diagnostic composition, exciting the leukocyte population with a light source; and measuring emitted light from each of the one or more leukocyte types to classify the leukocyte population.

## Claims

### Claim 1

A method for classifying 5-leukocytes types in a leukocyte population with fluorescence detection, said method comprising:
contacting a leukocyte population in a sample having one or more leukocyte types with a diagnostic composition, said diagnostic composition comprising:
a first dye having a cationic charge, wherein said first dye is propidium iodide;
a second dye having less cationic charge compared to said first dye, wherein said second dye is Basic Orange 21; and
a third dye being reactive to a protein, wherein said third dye is fluorescein isothiocyanate having an affinity to a protein;
exciting the stained leukocyte population with a light source; and
measuring emitted light from each of the one or more leukocyte types to classify the leukocyte population, wherein the leukocytes in the population comprise a lymphocyte, a monocyte, a neutrophil, an eosinophil and a basophil.

### Claim 2 (depends on 1)

The method for classifying of claim 1, wherein the fluorescence emitted from each of the leukocyte types is distinguishable from each of the other types in the population.

### Claim 3 (depends on 1)

The method for classifying of claim 1, wherein the first dye primarily stains the nucleus of the one or more leukocyte types.

### Claim 4 (depends on 1)

The method for classifying of claim 1, wherein the second dye primarily stains the cytoplasm of the one or more leukocyte types.

### Claim 5 (depends on 1)

The method for classifying of claim 1, wherein the third dye primarily stains the protein content of the one or more leukocyte types.

### Claim 6 (depends on 1)

The method for classifying of claim 1, wherein the excitation light is emitted from a source selected from the group consisting of a diode laser, a light emitting diode (LED), an ion laser, a dye laser, and a lamp.

### Claim 7 (depends on 6)

The method for classifying of claim 6, wherein the emitted light is measured at a first wavelength and at a second wavelength to achieve two color fluorescence measurements.

### Claim 8 (depends on 7)

The method for classifying of claim 7, wherein the emitted light is measured at the first and the second wavelength.

### Claim 9 (depends on 8)

The method for classifying of claim 8, wherein the emitted light is measured simultaneously by two photomultiplier tubes.

### Claim 10 (depends on 1)

The method for classifying of claim 1, wherein the sample is selected from the group consisting of whole blood, plasma, and serum.

### Claim 11 (depends on 10)

The method for classifying of claim 10, wherein the sample is stained by the diagnostic composition and is flowed through a flow cytometer.

### Claim 12 (depends on 11)

The method for classifying of claim 11, wherein the one or more leukocyte types are exposed to the light source, and the intensities of the fluorescence is recorded.

### Claim 13 (depends on 10)

The method for classifying of claim 10, wherein the sample is stained by the diagnostic composition and is flowed through a microfluidic cytometer.

### Claim 14 (depends on 13)

The method for classifying of claim 13, wherein the sample is flowed through a detection channel.

### Claim 15 (depends on 13)

The method for classifying of claim 13, wherein the one or more leukocyte types are exposed to the light source one by one, and two color fluorescence emission from each individual leukocyte type is recorded.