# US 10,481,071 B2 · White blood cell analysis system and method

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

- Number: US 10,481,071 B2
- Document ID: US10481071B2
- Status: Granted
- Kind: B2
- Application number: 15/704,972
- Filed: 2017-09-14
- Published: 2018-05-10
- Granted: 2019-11-19
- Earliest claimed: 2011-05-04
- Assignees: Abbott Laboratories
- Applicants: Abbott Laboratories
- Inventors: Jiong Wu; Giacomo Vacca
- Web page: https://patentgrep.com/patent/US10481071B2

## CPC

- G01N 15/1434: Optical arrangements
- G01N 15/0205: by optical means
- G01N 15/1459: {the analysis being performed on a sample stream}
- 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 21/91: using penetration of dyes, e.g. fluorescent ink
- G01N 33/49: Blood {(chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80)}
- G01N 2015/016: {White blood cells}
- G01N 2015/1006: {for cytology}
- G01N 2015/1477: {Multiparameters}
- G01N 2015/1488: {Methods for deciding}
- G01N 2021/174: {either absorption-reflection or emission-fluorescence}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- 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

Systems and methods for analyzing blood samples, and more specifically for performing a white blood cell (WBC) differential analysis. The systems and methods screen WBCs by means of fluorescence staining and a fluorescence triggering strategy. As such, interference from unlyzed red blood cells (RBCs) and fragments of lysed RBCs is substantially eliminated. The systems and methods also enable development of relatively milder WBC reagent(s), suitable for assays of samples containing fragile WBCs. In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye, which corresponds in emission spectrum to an excitation source of a hematology instrument; (b) using a fluorescence trigger to screen the blood sample for WBCs; and (c) using measurements of (1) axial light loss, (2) intermediate angle scatter, (3) 90° polarized side scatter, (4) 90° depolarized side scatter, and (5) fluorescence emission to perform a differentiation analysis.

## Claims

### Claim 1

A hematology analyzer for conducting a white blood cell (WBC) differential analysis on a blood sample that contains a plurality of WBCs, the analyzer comprising:
an excitation source positioned to excite particles within the blood sample;
a detector unit comprising a fluorescence detector positioned to measure fluorescence emitted from the excited blood sample; and
a processor;
a non-transitory computer-readable memory medium comprising instructions that cause the processor to:
excite the blood sample with the excitation source in a flow cell;
collect a plurality of fluorescence emission signals from the excited sample; and
prior to performing a WBC differential analysis, exclude nuclei-free events and retain nuclei-containing events using only a fluorescence trigger that is limited to fluorescence emission signals and is set to a fluorescence magnitude that is greater than fluorescence emission signals from RBCs, including RBC fragments, and is less than fluorescence emission signals from WBCs; and
perform the WBC differential analysis on the nuclei-containing events.

### Claim 2 (depends on 1)

The hematology analyzer of claim 1, wherein the detector unit comprises an axial light loss detector that measures axial light loss at 0° scatter.

### Claim 3 (depends on 1)

The hematology analyzer of claim 1, wherein the detector unit comprises an intermediate angle scatter detector that measures light angle scatter at about 3° to about 15°.

### Claim 4 (depends on 1)

The hematology analyzer of claim 1, wherein the detector unit comprises one or more photomultiplier tubes.

### Claim 5 (depends on 1)

The hematology analyzer of claim 1, wherein the excitation source is a laser.

### Claim 6 (depends on 5)

The hematology analyzer of claim 5, wherein the blood sample comprises a fluorescent dye and the laser is configured to emit light at a wavelength corresponding to the fluorescent dye.

### Claim 7 (depends on 6)

The hematology analyzer of claim 6, wherein the fluorescent dye is selected to correspond with the excitation source, and wherein the fluorescent dye is cell membrane permeable, and nucleic acid binding.

### Claim 8 (depends on 1)

The hematology analyzer of claim 1, further comprising:
an incubation subsystem for diluting the blood sample with a WBC reagent.

### Claim 9 (depends on 8)

The hematology analyzer of claim 8, wherein the WBC reagent includes a fluorescent dye and a lysing agent.

### Claim 10 (depends on 8)

The hematology analyzer of claim 8, wherein the WBC reagent includes (a) at least one surfactant, (b) at least one buffer or at least one salt, (c) at least one antimicrobial agent, and (d) a fluorescent dye.

### Claim 11 (depends on 8)

The hematology analyzer of claim 8, wherein the incubation subsystem is configured to incubate the blood sample with the WBC reagent for a period of time of less than 25 seconds.

### Claim 12 (depends on 8)

The hematology analyzer of claim 8, wherein the incubation subsystem is configured to incubate the blood sample with the WBC reagent for a period of time of less than 17 seconds.

### Claim 13 (depends on 8)

The hematology analyzer of claim 8, wherein the incubation subsystem is configured to incubate the blood sample with the WBC reagent for a period of time of less than 9 seconds.

### Claim 14 (depends on 8)

The hematology analyzer of claim 8, wherein the incubation subsystem is configured to incubate the blood sample with the WBC reagent at a temperature ranging from 30° C. to 50° C.

### Claim 15 (depends on 8)

The hematology analyzer of claim 8, wherein the incubation subsystem is configured to incubate the blood sample with the WBC reagent at a temperature of 40° C.

### Claim 16 (depends on 1)

The hematology analyzer of claim 1, wherein the non-transitory computer-readable memory medium further comprises instructions that cause the processor to dilute the blood sample with a WBC reagent that includes a red blood cell (RBC) lysing agent and a cell membrane permeable, nucleic acid binding fluorescent dye.

### Claim 17 (depends on 16)

The hematology analyzer of claim 16, wherein the non-transitory computer-readable memory medium further comprises instructions that cause the processor to incubate the diluted blood sample for an incubation period of time.

### Claim 18 (depends on 17)

The hematology analyzer of claim 17, wherein the non-transitory computer-readable memory medium further comprises instructions that cause the processor to deliver the incubated sample to a flow cell in the hematology analyzer.