Threshold gating for flow cytometry methods

Patent metadata

Field	Value
Publication number	US-12590882-B2
Application number	US-202118267744-A
Country	US
Kind code	B2
Filing date	Dec 15, 2021
Priority date	Dec 16, 2020
Publication date	Mar 31, 2026
Grant date	Mar 31, 2026

Abstract

Provided are methods for flow cytometry analysis, including the setting and use of static gating thresholds separating positive fluorescence from negative fluorescence for each fluorescence channel in the assay.

First claim

The invention claimed is:

1 . A method of determining a static fluorescence threshold gate, the method comprising:
(1) measuring cytometry events by flow cytometry for one or more fluorescent signals for a plurality of at least two reference cell samples, the plurality of the at least two reference cell samples comprising:
(a) a plurality of a first reference cell sample each comprising a population of unstained cells that have not been labeled with the one or more fluorescent signals; and
(b) a plurality of a second reference cell sample, wherein each of the plurality of the second reference cell sample is derived from the same source type of cells as one of the plurality of the first reference cell sample, and wherein each of the plurality of the second reference cell sample comprises at least one population of stained cells that have been labeled with at least one of the one or more fluorescent signals; and
(2) setting a static fluorescence threshold gate for each of the one or more fluorescent signals, such that for each fluorescent signal the threshold gate is a fluorescence that:
(a) is greater than the 90 th percentile of fluorescence of the plurality of the first reference cell sample for the respective fluorescent signal of each of the one or more fluorescent signals; and
(b) is less than the 10 th percentile of fluorescence of the plurality of the second reference cell sample for the same respective fluorescent signal.

2 . A method of selecting a subset of cytometry events from flow cytometry data, the method comprising:
(1) receiving flow cytometry data comprising a plurality of cytometry events from a test cell sample comprising labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer; (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescent signals, wherein the static fluorescence threshold gate for each of the one or more fluorescent signals is determined according to the method of claim 1 ; and (3) for each of the one or more fluorescent signals, identifying a subset of cytometry events from the test sample that have a fluorescence signal above the static threshold gate.

3 . A method of selecting a subset of cytometry events from flow cytometry data, comprising:
(1) receiving flow cytometry data comprising a plurality of cytometry events from a test cell sample comprising labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer; (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescent signals, wherein the static fluorescence threshold gate for each of the one or more fluorescent signals is determined from:
(a) cytometry events measured by flow cytometry for the one or more fluorescent signals for a plurality of at least two reference cell samples each sorted according to the one or more fluorescent signals, the plurality of the at least two reference cell samples comprising:
(i) a plurality of a first reference cell sample each comprising a population of unstained cells that have not been labeled with the one or more fluorescent signals; and
(ii) a plurality of a second reference cell sample, wherein each of the plurality of the second reference cell sample is derived from the same source type of cells as one of the plurality of the first reference cell sample, and wherein each of the plurality of the second reference cell sample comprises at least one population of stained cells that have been labeled with at least one of the one or more fluorescent signals; and

(b) setting a static fluorescence threshold gate for each of the one or more fluorescent signals such that for each fluorescent signal the threshold gate is a fluorescence that:
(i) is greater than the 90 th percentile of fluorescence of the plurality of the first reference cell sample for the respective fluorescent signal of each of the one or more fluorescent signals; and
(ii) is less than the 10 th percentile of fluorescence of the plurality of the second reference cell sample for the same respective fluorescent signal; and

(3) for each of the one or more fluorescent signals, identifying a subset of cytometry events from the test cell sample that have a fluorescence signal above the static threshold gate.

4 . A method of applying a static fluorescence threshold gate using flow cytometry, comprising:
(1) receiving flow cytometry data comprising a plurality of cytometry events from a test cell sample comprising labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer; and (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescent signals, wherein the static fluorescence threshold gate for each of the one or more fluorescent signals is determined according to the method of claim 1 .

5 . A method of applying a static fluorescence threshold gate using flow cytometry, comprising:
(1) receiving flow cytometry data comprising a plurality of cytometry events from a test cell sample comprising labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer; (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescent signals, wherein the static fluorescence threshold gate for each of the one or more fluorescent signals is determined from:
(a) cytometry events measured by flow cytometry for the one or more fluorescent signals for a plurality of at least two reference cell samples each sorted according to the one or more fluorescent signals, the plurality of the at least two reference cell samples comprising:
(i) a plurality of a first reference cell sample each comprising a population of unstained cells that have not been labeled with the one or more fluorescent signals; and
(ii) a plurality of a second reference cell sample, wherein each of the plurality of the second reference cell sample is derived from the same source type of cells as one of the plurality of the first reference cell sample, and wherein each of the plurality of the second reference cell sample comprises at least one population of stained cells that have been labeled with at least one of the one or more fluorescent signals; and

(b) setting a static fluorescence threshold gate for each of the one or more fluorescent signals, such that for each fluorescent signal the threshold gate is a fluorescence that:
(i) is greater than the 90 th percentile of fluorescence of the plurality of the first reference cell sample for the respective fluorescent signal of each of the one or more fluorescent signals; and
(ii) is less than the 10 th percentile of fluorescence of the plurality of the second reference cell sample for the same respective fluorescent signal.

6 . The method of claim 2 , wherein the test cell sample, and each of the plurality of the first and second reference cell sample, are from the same source type of cells.

7 . The method of claim 1 , wherein the source type of cells is a cell line.

8 . The method of claim 1 , wherein the source type of cells is a primary cell population from a subject.

9 . The method of claim 2 , wherein the source type of cells of each of the test cell sample, and each of the plurality of the first and second reference cell sample, are from a different subject.

10 . The method of claim 9 , wherein each different subject has the same or similar disease or condition.

11 . The method of claim 1 , wherein the source type of cells is a whole blood sample, an apheresis sample, or a leukapheresis sample.

12 . The method of claim 1 , wherein the s

Assignees

• Juno Therapeutics Inc

Inventors

• Brunette Rebecca
• Larson Ryan P
• Hawkins Kevin

Classifications

• G01N2015/1014

  Constitution of reference particles · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• G01N2015/1488

  Methods for deciding · CPC title

• G01N2015/1402

  Data analysis by thresholding or gating operations performed on the acquired signals or stored data · CPC title

• G01N15/1012 Primary

  Calibrating particle analysers; References therefor · CPC title