# US 10,481,072 B2 · Nucleated red blood cell analysis system and method

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- Number: US 10,481,072 B2
- Document ID: US10481072B2
- Status: Granted
- Kind: B2
- Application number: 15/704,974
- 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; Marilou Coleman; Emily H. Lin; Michael R. Buhl; Giacomo Vacca
- Web page: https://patentgrep.com/patent/US10481072B2

## CPC

- G01N 15/1434: Optical arrangements
- G01N 15/0205: by optical means
- G01N 15/1459: {the analysis being performed on a sample stream}
- G01N 15/147: {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 33/80: involving blood groups or blood types {or red blood cells (white blood cells G01N33/56972)}
- G01N 2015/011: {with lysing, e.g. of erythrocytes}
- G01N 2015/012: {Red blood cells}
- 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 2015/1488: {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

Systems and methods for analyzing blood samples, and more specifically for performing a nucleated red blood cell (nRBC) analysis. The systems and methods screen a blood sample by means of fluorescence staining and a fluorescence triggering strategy, to identify nuclei-containing particles within the blood sample. As such, interference from unlysed red blood cells (RBCs) and fragments of lysed RBCs is substantially eliminated. The systems and methods also enable development of relatively milder reagent(s), suitable for assays of samples containing fragile white blood cells (WBCs). In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye; (b) using a fluorescence trigger to screen the blood sample for nuclei-containing particles; and (c) using measurements of light scatter and fluorescence emission to distinguish nRBCs from WBCs.

## Claims

### Claim 1

A hematology analyzer for conducting a nucleated red blood cell (nRBC) analysis on a blood sample that contains a plurality of nRBCs, 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 an nRBC 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 white blood cells (WBCs) and nRBCs; and
perform the nRBC differential analysis on the nuclei-containing events.

### Claim 2 (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 3 (depends on 2)

The hematology analyzer of claim 2, further comprising:
a depolarized side scatter detector positioned to measure 90° depolarized side scatter from the excited blood sample.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

The hematology analyzer of claim 7, 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 9 (depends on 1)

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

### Claim 10 (depends on 9)

The hematology analyzer of claim 9, wherein the reagent includes a fluorescent dye and a lysing agent.

### Claim 11 (depends on 9)

The hematology analyzer of claim 9, 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 12 (depends on 9)

The hematology analyzer of claim 9, wherein the incubation subsystem is configured to incubate the blood sample with the reagent for a period of time of less than 25 seconds.

### Claim 13 (depends on 9)

The hematology analyzer of claim 9, wherein the incubation subsystem is configured to incubate the blood sample with the reagent for a period of time of less than 17 seconds.

### Claim 14 (depends on 9)

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

### Claim 15 (depends on 9)

The hematology analyzer of claim 9, 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 16 (depends on 9)

The hematology analyzer of claim 9, wherein the incubation subsystem is configured to incubate the blood sample with the reagent at a temperature of 40° C.

### Claim 17 (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 18 (depends on 17)

The hematology analyzer of claim 17, wherein the non-transitory computer-readable memory medium further comprises instructions to incubate the diluted blood sample for an incubation period of time.

### Claim 19 (depends on 18)

The hematology analyzer of claim 18, wherein the non-transitory computer-readable memory medium further comprises instructions to deliver the incubated sample to a flow cell in the hematology analyzer.