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US 12,590,882 B2Granted
Threshold gating for flow cytometry methods
AssigneeJuno Therapeutics, Inc.
InventorsRebecca Brunette, Ryan P. Larson, Kevin Hawkins
Life of this patent
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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.
Claims40
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 90th 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 10th percentile of fluorescence of the plurality of the second reference cell sample for the same respective fluorescent signal.
6 more elements
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 90th 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 10th 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.
10 more elements
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 90th 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 10th percentile of fluorescence of the plurality of the second reference cell sample for the same respective fluorescent signal.
8 more elements
Dependencies are inferred from the claim text.