# US 10,481,073 B2 · Basophil analysis system and method

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- Number: US 10,481,073 B2
- Document ID: US10481073B2
- Status: Granted
- Kind: B2
- Application number: 15/801,055
- Filed: 2017-11-01
- Published: 2018-05-17
- Granted: 2019-11-19
- Earliest claimed: 2011-05-04
- Assignees: Abbott Laboratories
- Applicants: Abbott Laboratories
- Inventors: Jiong Wu; Michael R. Buhl; Giacomo Vacca
- Web page: https://patentgrep.com/patent/US10481073B2

## 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 33/4915: {using flow cells (flow cytometry G01N15/14)}
- G01N 2015/011: {with lysing, e.g. of erythrocytes}
- G01N 2015/016: {White blood cells}
- G01N 2015/1006: {for cytology}
- G01N 2015/1402: {Data analysis by thresholding or gating operations performed on the acquired signals or stored data}
- G01N 2015/1477: {Multiparameters}
- G01N 2015/1486: {Counting the particles}
- G01N 2015/1488: {Methods for deciding}
- 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

Provided herein are systems and methods for analyzing blood samples, and more specifically for performing a basophil analysis. In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye; and then (b) using measurements of light scatter and fluorescence emission to distinguish basophils from other WBC sub-populations. In one embodiment, the systems and methods include performing a basophil cluster analysis of the blood sample, based on the combination of light scatter and fluorescence measurements.

## Claims

### Claim 1

A hematology analyzer for conducting a basophil analysis on a blood sample that contains a plurality of basophils and lymphocytes, 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 an excitation source in a flow cell;
collect a plurality of fluorescence emission signals from the excited sample;
prior to performing a basophil 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 white blood cells (WBCs); and
perform a basophil cluster analysis on the nuclei-containing events.

### Claim 2 (depends on 1)

The hematology analyzer of claim 1, wherein the basophil cluster analysis includes a coarse clustering of the received measurements.

### Claim 3 (depends on 2)

The hematology analyzer of claim 2, wherein the basophil cluster analysis further includes a fine clustering of the nuclei-containing events, utilizing a multi-dimensional, probability distance metric as a determinant to combine similar clusters.

### Claim 4 (depends on 1)

The hematology analyzer of claim 1, wherein the detector unit comprises a side scatter detector that is a polarized side scatter detector positioned to measure 90° polarized side scatter from the excited blood sample.

### Claim 5 (depends on 4)

The hematology analyzer of claim 4, wherein the detector unit further comprises an axial light loss detector and an intermediate angle scatter detector and wherein the processor is further configured to distinguish basophils within the blood sample from lymphocytes within the blood sample, based on the measurements of (1) axial light loss by the axial light loss detector, (2) intermediate angle scatter by the intermediate angle scatter detector, (3) 90° polarized side scatter by the polarized side scatter detector, and (4) fluorescence by the fluorescence detector.

### Claim 6 (depends on 4)

The hematology analyzer of claim 4, further comprising:
a depolarized side scatter detector positioned to measure 90° depolarized side scatter from the excited blood sample.

### Claim 7 (depends on 1)

The hematology analyzer of claim 1, wherein the detector unit further comprises an axial light loss detector, an intermediate angle scatter detector, a side scatter detector and wherein the processor is further configured to distinguish basophils within the blood sample from neutrophils, monocytes, and eosinophils within the blood sample, based on the measurements of (1) axial light loss by the intermediate angle scatter detector, (2) intermediate angle scatter by the intermediate angle scatter detector, and (3) 90° side scatter by the side scatter detector.

### Claim 8 (depends on 1)

The hematology analyzer of claim 1, wherein the detector unit further comprises an axial light loss detector, an intermediate angle scatter detector, a 90° polarized side scatter detector and wherein the processor is further configured to distinguish basophils within the blood sample from neutrophils, monocytes, and eosinophils within the blood sample, based on the measurements of (1) axial light loss, (2) intermediate angle scatter, (3) 90° polarized side scatter, and (4) fluorescence.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 13)

The hematology analyzer of claim 13, wherein the 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 15 (depends on 13)

The hematology analyzer of claim 13, wherein the incubation subsystem is configured to incubate the blood sample with the reagent for a period of time of less than 30 seconds.

### Claim 16 (depends on 13)

The hematology analyzer of claim 13, wherein the incubation subsystem is configured to incubate the blood sample with the reagent at a temperature ranging from 30° C. to 50° C.

### Claim 17 (depends on 13)

The hematology analyzer of claim 13, wherein the incubation subsystem is configured to incubate the blood sample with the reagent at a temperature of 40° C.

### Claim 18 (depends on 1)

The hematology analyzer of claim 1, wherein the processor is configured to perform a multi-dimensional basophil cluster analysis on the nuclei-containing events.

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 19)

The hematology analyzer of claim 19, wherein the non-transitory computer-readable memory medium further comprises instructions to incubate the diluted blood sample for an incubation period of time.

### Claim 21 (depends on 20)

The hematology analyzer of claim 20, wherein the non-transitory computer-readable memory medium further comprises instructions to deliver the incubated sample to a flow cell in the hematology analyzer.