Threshold gating for flow cytometry methods

Abstract

Provided is a method of flow cytometry analysis that involves setting and using a static gating threshold that separates positive fluorescence from negative fluorescence for each fluorescence channel in the assay.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 126,509, filed December 16, 2020, entitled “THRESHOLD GATING FOR FLOW CYTOMETRY METHODS,” the contents of which are incorporated by reference in their entirety.

[0002] The present disclosure relates in some embodiments to flow cytometry analysis, including the setting and use of static gating thresholds that separate positive from negative fluorescence for each fluorescence channel in the assay. [Background technology]

[0003] Fluorescence Minus One (FMO) or isotype gating controls for each fluorochrome-conjugated antibody are the industry standard gating controls for flow cytometry methods used in quality control (QC) situations. The advantage of these gating approaches is that they allow objective gate placement, but they also have drawbacks, including reduced throughput, increased assay complexity (e.g., due to time constraints, failure rates, and limited sample volume), and increased cost of goods. There is a need for improved gating approaches. Provided are embodiments that meet such needs. Summary of the Invention

[0004] Provided herein is a method of determining a static fluorescence threshold gate, 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 at least two reference cell samples comprising: (a) a first plurality of reference cell samples each comprising a population of unstained cells that are not labeled with one or more fluorescent signals; and (b) a second plurality of reference cell samples, each of the second plurality of reference cell samples derived from the same cellular source type as the first plurality of reference cell samples, and each of the second plurality of reference cell samples comprising one or more fluorescent signals. and (2) setting a static fluorescence threshold gate for each of the one or more fluorescent signals such that the threshold gate for each fluorescent signal is (a) a fluorescence higher than the 90th percentile of the fluorescence of the plurality of first reference cell samples for each respective fluorescent signal of each of the one or more fluorescent signals; and (b) a fluorescence lower than the 10th percentile of the fluorescence of the plurality of second reference cell samples for the same respective fluorescent signal.

[0005] Also provided herein is a method of selecting a subset of cytometry events from flow cytometry data, the method including: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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, where 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) identifying a subset of cells from the test sample having a fluorescent signal above the static threshold gate for each of the one or more fluorescent signals.

[0006] Also provided herein is 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 comprises: (a) a first plurality of reference cell samples each comprising a population of unstained cells that are not labeled with the one or more fluorescent signals; and (ii) a second plurality of reference cell samples each comprising: and (b) applying a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal being determined from setting (i) a fluorescence higher than a 90th percentile of the fluorescence of the plurality of first reference cell samples for each respective fluorescent signal of each of the one or more fluorescent signals; and (ii) a fluorescence lower than a 10th percentile of the fluorescence of the plurality of second reference cell samples for the same respective fluorescent signal; and (3) identifying a subset of cells from the test cell sample having a fluorescent signal above the static threshold gate for each of the one or more fluorescent signals.

[0007] Also provided herein is a method of applying a static fluorescence threshold gate using flow cytometry, the method including: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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, where the static fluorescence threshold gate for each of the one or more fluorescent signals is determined according to any method of determining a static fluorescence threshold gate disclosed herein.

[0008] Also provided herein is a method of applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer; (2) individually applying a static fluorescence threshold gate for each of the one or more fluorescent signals, wherein the static fluorescence threshold gate for each of the one or more fluorescent signals comprises: (a) a first reference cell sample including a plurality of reference cell samples each sorted according to the one or more fluorescent signals, the first reference cell sample including a plurality of reference cell samples each including a population of unstained cells that are not labeled with the one or more fluorescent signals; a plurality of cell samples; and (ii) a plurality of second reference cell samples, each of the plurality of second reference cell samples derived from the same cellular source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals; and (b) applying a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal being determined from setting (i) a fluorescence higher than the 90th percentile of the fluorescence of the plurality of first reference cell samples for each respective fluorescent signal of each of the one or more fluorescent signals; and (ii) a fluorescence lower than the 10th percentile of the fluorescence of the plurality of second reference cell samples for the same respective fluorescent signal.

[0009] In some of such embodiments, the test cell sample and each of the plurality of first and second reference cell samples are from the same cell source type. In some of such embodiments, the cell source type is a cell line. In some of such embodiments, the cell source type is a primary cell population from a subject. In some of such embodiments, the cell source type of each of the test cell sample and each of the plurality of first and second reference cell samples is from a different subject. In some of such embodiments, each different subject has the same or similar disease or condition. In some of such embodiments, the cell source type is a whole blood sample, an apheresis sample, or a leukapheresis sample. In some of such embodiments, the cell source type is an enriched population of cells, optionally an enriched population of T cells. In some of such embodiments, the cell source type is an engineered population of cells that includes a nucleic acid encoding a recombinant protein, optionally the recombinant protein has been introduced into the population of cells by gene transfer, optionally transduction. In some of any such embodiments, the source type of the cells is a cell therapy.

[0010] In some of such embodiments, the source type of cells is an enriched population of T cells. In some of such embodiments, the recombinant protein has been introduced into the population of cells by gene transfer. In some of such embodiments, the recombinant protein has been introduced into the population of cells by transduction.

[0011] In some of any such embodiments, the plurality of first reference cell samples comprises more than 2, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cell samples. In some of any such embodiments, the plurality of second reference cell samples comprises more than 2, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cell samples.

[0012] In some of such embodiments, each of the first and second reference cell samples is a cell source type from a subject with disease or condition in a clinical trial.In some of such embodiments, the cell source type is autologous cell therapy, and each of the first and second reference cell samples is a cell therapy sample from a subject in a clinical trial for testing the cell therapy.In some of such embodiments, the cell therapy is T cell therapy, optionally CAR-T cell therapy, TCR-T cell therapy, or TIL therapy.In some of such embodiments, the cell therapy is NK cell therapy.In some of such embodiments, the cell therapy is stem cell therapy.

[0013] In some of such embodiments, the cell therapy is CAR-T cell therapy. In some of such embodiments, the cell therapy is TCR-T cell therapy. In some of such embodiments, the cell therapy is TIL therapy.

[0014] In some of such embodiments, each of the test cell sample and the second reference cell sample is subjected to cell staining using one or more staining reagents for labeling cells with one or more fluorescent signals.In some of such embodiments, the one or more staining reagents include a binding agent specific to the marker and a fluorescent dye that can emit one of one or more fluorescent signals.In some of such embodiments, the marker is a cell surface marker or a viability marker.

[0015] In some of any such embodiments, the one or more staining reagents each include a binding agent specific for a marker and a fluorescent dye capable of emitting one of one or more fluorescent signals.

[0016] In some of any such embodiments, at least one marker is a prevalent attribute that is expressed or suspected to be expressed in at least 5% or more of the cells in the sample. In some of any such embodiments, at least one marker is a prevalence attribute that is expressed or suspected to be expressed in less than 5% of the cells in the sample. In some of any such embodiments, the samples are each of a test cell sample and a second reference cell sample.

[0017] In some of such embodiments, the one or more fluorescent signals include two or more different fluorescent signals. In some of such embodiments, the cell stain is a multicolor cell stain for labeling cells with two or more different fluorescent signals, each staining reagent labels a different marker with a different fluorescent signal. In some of such embodiments, the two or more different fluorescent signals include 2-10 fluorescent signals, optionally 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, or 6 or about 6 fluorescent signals. In some of such embodiments, each of the two or more different fluorescent signals has a different emission spectrum, and / or the peak emission spectra of each fluorescent signal do not overlap. In some of such embodiments, the two or more different fluorescent signals are signals emitted by a dye selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0018] In some of such embodiments, the two or more different fluorescent signals include 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, or 6 or about 6 fluorescent signals. In some of such embodiments, each of the two or more different fluorescent signals has a different emission spectrum. In some of such embodiments, the peak emission spectrum of each fluorescent signal does not overlap. In some of such embodiments, each of the two or more different fluorescent signals is a signal emitted by a dye selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0019] In some of any such embodiments, the cell staining of each of the test cell sample and the second reference cell sample is carried out with the same staining reagent and using the same protocol conditions.

[0020] In some of any such embodiments, the plurality of second reference cell samples comprises duplicate samples of the same stained cells. In some of any such embodiments, the plurality of second reference cell samples comprises triplicate samples of the same stained cells.

[0021] In some of such embodiments, the static fluorescence threshold gate for at least one of the fluorescent signals is a fluorescence greater than the 95th percentile of the fluorescence among the plurality of first reference cell samples for the fluorescent signal. In some of such embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence greater than the 97th percentile of the fluorescence among the plurality of first reference cell samples for the fluorescent signal. In some of such embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence greater than the 99th percentile of the fluorescence among the plurality of first reference cell samples for the fluorescent signal.

[0022] In some of such embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than the 5th percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some of such embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than the 3rd percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some of such embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than the 1st percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal.

[0023] In some of such embodiments, if the fluorescence of the population of unstained cells and the population of stained cells for the fluorescent signal differs by more than 2-fold, the static fluorescence threshold gate is set to less than 1 percentile of the fluorescence or less than all percentiles of the fluorescence between the plurality of second reference cell samples for the fluorescent signal.In some of such embodiments, if the fluorescence of the population of unstained cells and the population of stained cells for the fluorescent signal differs by more than 5-fold, the static fluorescence threshold gate is set to less than 1 percentile of the fluorescence or less than all percentiles of the fluorescence between the plurality of second reference cell samples for the fluorescent signal.In some of such embodiments, if the fluorescence of the population of unstained cells and the population of stained cells for the fluorescent signal differs by more than 10-fold, the static fluorescence threshold gate is set to less than 1 percentile of the fluorescence or less than all percentiles of the fluorescence between the plurality of second reference cell samples for the fluorescent signal.

[0024] In some of any such embodiments, for low frequency attributes, setting the static fluorescence threshold gate further includes identifying a mean fluorescence intensity (MFI) for one or more of the one or more fluorescent signals.

[0025] In some such embodiments, for each of the one or more fluorescent signals, the static fluorescence threshold gate is an even number on the logarithmic flow axis. In some such embodiments, for each of the one or more fluorescent signals, the static fluorescence threshold gate is rounded to the nearest 10, rounded to the nearest 100, or rounded to the nearest 1,000 on the logarithmic flow axis.

[0026] In some of any such embodiments, the method further includes identifying a subset of cells from the test cell sample having fluorescent signals above the static fluorescent threshold gate for at least two of the one or more fluorescent signals, at least three of the one or more fluorescent signals, at least four of the one or more fluorescent signals, at least five of the one or more fluorescent signals, at least six of the one or more fluorescent signals, or at least seven or more of the one or more fluorescent signals.

[0027] In some of any such embodiments, the method further includes identifying a subset of cells from the test cell sample having a fluorescent signal above a static fluorescent threshold gate for all of the one or more fluorescent signals.

[0028] In some of any such embodiments, the static fluorescence threshold gate includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells of the test cell sample. In some of any such embodiments, the static fluorescence threshold gate for each of the one or more fluorescent signals is set to include at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells of the test cell sample.

[0029] In some of the embodiments provided, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, the method further comprises evaluating the fluorescent intensity of at least one of the identified subset of cytometry events.In some of the embodiments provided, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, the method further comprises evaluating the intensity of the fluorescent signal of the identified subset of cytometry events.In some embodiments, the fluorescent intensity is the average fluorescent intensity.

[0030] In some of any provided embodiments, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, the method further comprises determining a percentage of the identified subset of cytometry events compared to the total number of collected cytometry events. In some embodiments, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, the method further comprises determining a percentage of the identified subset of cytometry events for the respective fluorescent signal compared to the total number of collected cytometry events.

[0031] In some of any provided embodiments, the method includes normalizing one or more fluorescent signals by a flow cytometer. In some embodiments, normalizing is performed at least once a day. [Brief description of the drawings]

[0032] [Figure 1A]Figure 1A and Figure 1B depict graphical representations of analysis of flow cytometry events. Figure 1A depicts multiple cell populations from different samples, identifies the upper boundary of the negative, unstained cell population and the lower boundary of the positive, stained cell population, and also depicts the fluorescence threshold set to separate the negative unstained cell population from the positive stained cell population. Figure 1B depicts representative histogram analysis of the negative unstained cell population (left) and the positive stained cell population (right), where the mean fluorescence intensity (MFI) and the 95th and 99th percentiles of fluorescence were determined for the negative unstained cell population (left), and the MFI and the 1st and 5th percentiles of fluorescence were determined for the positive stained cell population (right). [Figure 1B] Figure 1A and Figure 1B depict graphical representations of analysis of flow cytometry events. Figure 1A depicts multiple cell populations from different samples, identifies the upper boundary of the negative, unstained cell population and the lower boundary of the positive, stained cell population, and also depicts the fluorescence threshold set to separate the negative unstained cell population from the positive stained cell population. Figure 1B depicts representative histogram analysis of the negative unstained cell population (left) and the positive stained cell population (right), where the mean fluorescence intensity (MFI) and the 95th and 99th percentiles of fluorescence were determined for the negative unstained cell population (left), and the MFI and the 1st and 5th percentiles of fluorescence were determined for the positive stained cell population (right). [Figure 2A] 2A and 2B depict analysis of the negative unstained cell population (FIG. 2A) and the positive stained cell population (FIG. 2B) for frequency attributes using the AF700 fluorophore. [Figure 2B] 2A and 2B depict analysis of the negative unstained cell population (FIG. 2A) and the positive stained cell population (FIG. 2B) for frequency attributes using the AF700 fluorophore. [Diagram 3]FIG. 3 depicts the application of static fluorescence gating thresholds to the fluorescence minus one (FMO) gating controls for each of the six tested attributes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Provided herein is a method for determining a static fluorescence threshold gate in the context of gating cells in flow cytometry methods. In some embodiments, the provided method provides an automated gating method that can be performed on cells in a fast and efficient and, in some aspects, high-throughput manner. The provided method is particularly useful for quality control evaluation of cells, for example, for the purpose of evaluating certain attributes or features of cells. In some embodiments, the provided method for setting a static gate threshold utilizes a reference cell population that is known or suspected to have similar characteristics or features as the test cell sample of interest. Once set, the static threshold gate can be applied to one or more test cell samples. In some embodiments, the provided method is particularly useful in the evaluation of similar cell samples (e.g., cells of the same source type) in clinical trials or studies or between subjects that are treated according to the same or similar treatment regimen. In certain embodiments, the methods provided may be used to evaluate or characterize cell therapies (e.g., T cell therapies, e.g., CAR-T cells), particularly cell therapies (e.g., T cell therapies, e.g., CAR-T cells) manufactured or produced according to a similar protocol or procedure or from a similar group of subjects (e.g., subjects having the same underlying disease or condition).

[0034] In some embodiments, the method of determining the static gate threshold comprises: (1) measuring cytometry events by flow cytometry for one or more fluorescent signals for at least two reference cell sample pluralities, the at least two reference cell sample pluralities comprising: (a) a first reference cell sample pluralities, each of which comprises a population of unstained cells that are not labeled with one or more fluorescent signals; and (b) a second reference cell sample pluralities, each of which is derived from the same cell source type as the first reference cell sample pluralities, and each of which comprises at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals; and (2) setting a static fluorescent threshold gate for each of the one or more fluorescent signals. In some embodiments of the provided method, the static fluorescent threshold gate is set to maximize the inclusion of positive (stained) cells and minimize the inclusion of negative (unstained) cells. In some embodiments, for each fluorescent signal of a labeled attribute (e.g., stained with a marker-specific fluorophore dye), a static gate threshold is set based on an upper boundary of cellular fluorescence for each of the one or more fluorescent signals among a first reference cell sample and a lower boundary of fluorescence based on a lower boundary of cellular fluorescence for each of the one or more fluorescent signals among a second reference cell sample. In some embodiments, the upper and lower boundaries are determined as percentiles of the distribution of fluorescence in the sample, e.g., to maximize inclusion of positive / stained cells and minimize exclusion of negative / unstained cells.

[0035] In some embodiments, the provided method also includes applying a static fluorescence threshold gate to cytometry events from a test population of labeled cells that are labeled (stained) for the same attribute (e.g., marker) using the same one or more fluorescent signals used to set the static gate threshold. In some embodiments, applying a static fluorescence threshold gate can be used to evaluate a feature or characteristic of a desired population of interest. Provided herein is a method of selecting a subset of cytometry events from flow cytometry data, the method comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test population of labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer; (2) applying a static fluorescence threshold gate to each of the one or more fluorescent signals individually; and (3) identifying a subset of cytometry events from the test population of cells that have a fluorescent signal above the static threshold gate for each of the one or more fluorescent signals.

[0036] In some embodiments, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, the method further comprises evaluating the fluorescent intensity of at least one fluorescent signal of the identified subset of cytometry events. In some embodiments, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, the method further comprises evaluating the intensity of the fluorescent signal of the identified subset of cytometry events. In some embodiments, the fluorescent intensity is the mean fluorescent intensity. In some embodiments, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, the method further comprises determining a percentage of the identified subset of cytometry events compared to the total number of collected cytometry events. In some embodiments, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, the method further comprises determining a percentage of the identified subset of cytometry events for each fluorescent signal compared to the total number of collected cytometry events.

[0037] Flow cytometry is a technique used to analyze the physical and chemical properties of particles, such as molecules, analyte-bound beads, or individual cells, in a sheath fluid as they pass through one or more lasers. Most commonly, cell particles are fluorescently labeled and then excited by a laser to emit different wavelengths of light. Flow cytometers are widely used to count cell subsets based on the binding of fluorescent monoclonal antibodies. These instruments are designed to cause cells in suspension to move in a single file through a laser beam for monitoring. The cells are then classified by the way they scatter the incident laser light, which provides information about the size and internal granularity of the cells. Additionally, any fluorescence that the cells may emit allows for the detection of specific antibody binding and, therefore, subsequent cell subset identification. A summary of standard flow cytometry apparatus and methods is provided in U.S. Pat. No. 4,284,412 to Hoffman and Hansen, entitled "Method and Apparatus for Automated Identification and Enumeration of Specified Blood Cell Subclasses," which is incorporated herein by reference.

[0038] In some embodiments, a flow cytometer has five main components. First, a fluid sheath is formed that carries the cells and aligns them so that they can pass through a light beam in single file for sensing. Second, a measurement system, usually a laser beam, which is aligned with the fluid sheath. Third, a detector and an analog-to-digital conversion system that converts analog measurements of measured parameters from the laser beam, such as forward and side scatter light, into digital signals that can be processed by a computing system. Fourth, an amplification system, linear or logarithmic. Finally, a computer system for analysis of the signals. Flow cytometers have the ability to analyze thousands of particles per second and can actively separate and isolate particles with unique physical or chemical properties. The process of collecting data from a sample using flow cytometry is called acquisition. Acquisition is mediated by a computer that is physically connected to the flow cytometer and receives digital or analog signals from the flow cytometer. Modern flow cytometers typically have multiple lasers and detectors built in to detect multiple antibodies or markers on the surface of the particles.

[0039] Flow cytometry devices and other particle analysis devices (e.g., mass cytometers) provide identification and characterization of particles (e.g., cells) based on certain predetermined parameters, such as optical parameters including light scattering and fluorescence. In flow cytometry, for example, particles (e.g., cells) in fluid suspension are passed through a detection region where the particles are exposed to excitation light, typically from one or more lasers, and the light scattering and fluorescence properties of the particles are measured. The particles or their components are typically labeled with one or more fluorescent dyes to facilitate detection. A multiplicity of different particles or components may be detected simultaneously by labeling the different particles or components using spectrally distinct fluorescent dyes. In some implementations, a multiplicity of photodetectors, one for each of the scattering parameters measured and one for each of the distinct dyes detected, are included in the analysis device. The data obtained includes the measured signals for each of the light scattering parameters and the fluorescence emission.

[0040] In some embodiments, the sensors in the detection region are arranged to simultaneously detect multiple different properties, for example, for each of the fluorochromes used, and one or more light scattering properties, such as forward scatter (FCS), side scatter (SSC), etc. For example, parameters measured using a flow cytometer typically include FSC, which is the excitation light scattered by the particle mostly along the forward direction; SSC, which is the excitation light scattered by the particle mostly in the side direction, and the light emitted from each of the fluorochromes in a spectral channel (range of frequencies), referred to as FL1, FL2, etc. These sensors, for example, photodetectors, obtain data about the particles in real time as they pass through the detection region, and transfer the data to a computer-readable medium for data storage.

[0041] In some embodiments, a flow cytometry sample can include a large number of points in a multidimensional space. In some embodiments, the dimensions are anywhere from about 3 to 15, and the number of points (or cytometry events), which usually correspond to cells, is often tens to hundreds of thousands. In aspects of flow cytometry analysis, two of the dimensions usually correspond to the forward and side scatter intensities, which characterize the physical properties (e.g., size and granularity) of the cells. The remaining dimension corresponds to the intensity of the fluorescence of the cells at a given wavelength (color).

[0042] Both flow cytometers and scanning cytometers are commercially available, for example, from BD Biosciences (San Jose, Calif.).Flow cytometry is described, for example, in Landy et al. (eds.), Clinical Flow Cytometry, Annals of the New York Academy of Sciences Volume 677 (1993); Bauer et al. (eds.), Clinical Flow Cytometry: Principles and Applications, Williams & Wilkins (1993); Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1994); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); and Shapiro HM: Practical Flow Cytometry. 4th edition. New York: John Wiley & Sons; 2003 (all incorporated herein by reference). Fluorescence imaging microscopy is described, for example, in Pawley (ed.), Handbook of Biological Confocal Microscopy, 2nd Edition, Plenum Press (1989), incorporated herein by reference.

[0043] In some embodiments, the cytometer may further comprise a means for recording the measured data and for analyzing the data. For example, data storage and analysis may be performed using a computer connected to the detection electronics. For example, data can be stored in a table format, where each row corresponds to the data for one particle and each column corresponds to each of the measured parameters. The use of a standard file format, such as the "FCS" file format, for storing data from a flow cytometer facilitates the analysis of the data using a separate program and / or instrument. Using current analysis methods, data is typically displayed in a two-dimensional (2D) plot for ease of visualization, although other methods may be used to visualize multidimensional data.

[0044] In some embodiments, flow cytometry data plots each event independently and represents the light signal intensity detected in each channel for every event. In some embodiments, a histogram typically plots the intensity detected in a single channel along one axis, and the number of events detected at that intensity is on a separate axis. Multiple events detected at one particular intensity are displayed as spikes on the histogram. In other embodiments, events are plotted as dot plots, which are plots that compare two or three parameters simultaneously on a two- or three-dimensional scatter plot. In a dot plot, each event is represented as a single point on the scatter plot. For a 2D plot, the intensity of two different channels is represented along two axes. For a three-dimensional (3D) plot, the intensity of three different channels is represented along various axes. Events with similar intensities cluster together in the same area on the scatter plot. For example, for dot plot data, large samples often result in heavy clustering of events represented in the same area of ​​the plot. There are many ways to add additional resolution to these areas of the dot plot. For example, a heat map may be used to provide information about event density in a given area of ​​the plot.

[0045] The data obtained may be multidimensional in nature, with each particle corresponding to a point in a multidimensional space defined by the measured parameters. Populations, or clusters, of cells of a certain type are identified based on their correlation to one another in this multidimensional space. Different cell types may be identified by scattering parameters and fluorescent emission resulting from labeling of various cellular proteins with dye-labeled antibodies. Identification of clusters, and thereby populations, may be performed by gating of cells. Typically, a gate corresponding to a subset of particles of interest, e.g., CD4+ lymphocytes in a blood sample, is defined by the user with the aid of software operatively associated with the flow system. The gate then provides the user with a convenient way to select a subset of particles for counting, isolation, or other manipulation. Gating is used to aid in the detection of the large amount of data that may be generated from a sample. Thus, facilitating the creation and manipulation of gates efficiently can aid in increasing the speed and accuracy of understanding the meaning of the results.

[0046] In some embodiments, the gate may be a "threshold" gate, which is a gate for only one optical parameter that defines an open region in multi-dimensional space. In most existing methods, "threshold" gating has been mainly used only for forward light scatter to remove high frequency low level signals caused by debris in the article, e.g., sample, that would overwhelm the processing power of the detection system electronics designed to process the signal generated by the particle of interest. More commonly, "window" gating is used, which usually defines a closed region in multi-dimensional space, e.g., by defining upper and lower boundaries for the signal value. In "window" gating, a region is generated to correspond to the particle or cell type to be counted, sorted, or excluded. In some embodiments, gating is performed on a 2D plot of two parameters, e.g., side scatter (e.g., on the vertical axis) and fluorescence signal (e.g., on the horizontal axis).

[0047] In some embodiments, traditional flow cytometry gating control typically utilizes isotype gating control or fluorochrome-conjugated antibody, which is the industry standard gating control for flow cytometry method, especially in quality control situations.In some aspects, such methods can be advantageous because they allow objective gate placement.On the other hand, such methods are associated with one or more drawbacks, such as reduced throughput per plate, increased assay complexity (e.g., time, failure rate, required sample volume) and increased cost of goods.

[0048] Furthermore, in many existing methods, gating is performed by manually drawing a gate around a population displayed in one or more two-dimensional plots, referred to as "scatter plots" or "dot plots" of cytometric events present in the data. In manual gating, the user collects flow cytometric data (or cytometric events) from one or more channels representing one or more different parameters, e.g., SSC, FSC or different fluorescent signals, on a dot plot. Based on the acquired data, the user draws a gate box that selects a subpopulation of cells for further analysis. The researcher may use one of several tools to draw a geometric gate, e.g., a rectangle, oval, or polygon. The subpopulation of cells within the gate is particularly highlighted on other plots that display information from one or more other alternative channels. Multiple gates can be established for a single scatter plot, and gates can be "stacked" and combined (i.e., a subpopulation of cells gated on channels 1 and 2 can be further gated on channels 3 and 4 to allow for additional specificity and deeper analysis). Although manual plotting is typically used for its flexibility and its intuitiveness, manual sequential gating to extract cell populations of interest is tedious and labor-intensive at larger file sizes. Moreover, the tools may not allow precise gates to be drawn because the data of interest may not be perfectly geometric or easily represented using available tools. Additionally, manual gating may also result in variability between different users who perform manual gating. Therefore, improved methods are needed.

[0049] The provided method is based on generating a fluorescence threshold as a gate for each attribute (e.g., those labeled with a fluorescent signal) in a flow cytometry panel. The provided embodiment with the use of a static gate threshold is particularly advantageous in samples with good resolution between positive and negative staining, sufficient representative reference samples (e.g., clinical samples), and environments with robust fluorescence control of the cytometer and reagents. As demonstrated herein, the provided embodiment with proper setting and use of a static gate threshold excludes background fluorescence, for example, as applied to FMO samples. Additionally, reportables are comparable to current gating methods. Finally, the provided method is precise with low inter-user variability, for example, a coefficient of variation of less than 3% for all evaluated attributes. The provided method thus provides an improved option for evaluating stained cell samples by flow cytometry, especially in environments where rapid and high-throughput quality control implementation is desired.

[0050] The techniques described herein may be implemented in hardware, software, firmware, or any combination thereof. Such techniques may be implemented in a variety of devices, such as general purpose computers, wireless communication devices, or integrated circuit devices with multiple uses, including applications in wireless communication device handsets and other devices. Any features described as modules or components may be implemented together in an integrated logic device, or separately as separate but cooperatively operable logic devices. When implemented in software, the techniques may be realized, at least in part, by a computer-readable data storage medium that includes program code that includes instructions that, when executed, perform one or more of the methods described above. The computer-readable data storage medium may form part of a computer program product, which may include packaging materials. The computer-readable medium may include memory or data storage media, such as random access memory (RAM), e.g., synchronous dynamic random access memory (SDRAM), read-only memory (ROM), non-volatile random access memory (NVRAM), electrically erasable programmable read-only memory (EEPROM), FLASH memory, and magnetic or optical data storage media. The computer-readable medium may be a non-transitory storage medium. The techniques may additionally or alternatively be embodied, at least in part, in a computer-readable communication medium, e.g., a propagated signal or wave, that transmits or communicates program code in the form of instructions or data structures and that can be accessed, read, and / or executed by a computer.

[0051] The program code may be executed by one or more processors, which may include, for example, one or more digital signal processors (DSPs), general-purpose microprocessors, application specific integrated circuits (ASICs), field programmable logic arrays (FPGAs), or other equivalent integrated or discrete logic circuitry. Such processors may be configured to perform any of the techniques described in this disclosure. A general-purpose processor may be a microprocessor, but alternatively, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, for example, a combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors in combination with a DSP core, or any other such configuration. Thus, the term "processor" as used herein may refer to any of the above structures, any combination of the above structures, or any other structure or apparatus suitable for implementing the techniques described herein. Additionally, in some aspects, the functionality described herein may be provided in dedicated software or hardware modules configured for encoding and decoding, or may be incorporated into a combined video encoder-decoder (CODEC).

[0052] All publications, including patent documents, scientific articles and databases, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was individually incorporated by reference. In the event that the definitions set forth herein are opposite or otherwise inconsistent with the definitions set forth in the patents, applications, application publications and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0054] I. Flow Cytometry and Data Acquisition In some embodiments, the method provided herein comprises receiving flow cytometry data. In some embodiments, the method comprises receiving flow cytometry data comprising a plurality of cytometry events from a test population of labeled cells sorted according to one or more fluorescent signals measured by a flow cytometer. In some embodiments, the test population of cells is or is included in a sample, e.g., a biological sample. In the embodiments provided, the test population of cells is stained with one or more fluorescent signals to label one or more attributes of the cell sample, and evaluated by flow cytometry to collect cytometry events. The cytometry events can be analyzed by setting a static gate threshold as described herein, including by the method described in Section II.

[0055] In some embodiments, flow cytometry data collection involves multiple principles, such as fluidics, optics and detection, signal processing, and electrostatic cell sorting. In some embodiments, flow cytometry is used to measure properties of individual particles, such as individual cells, from a sample.

[0056] In some embodiments, flow cytometry data collection begins with the collection of a flow cytometry cell sample. In the acquisition of a flow cytometry cell sample, a flow cytometry protocol with direct or indirect staining of the cell sample can be used. The direct staining flow cytometry protocol is one of the most common staining methods, in which live or fixed cells are incubated with a directly labeled antibody against a cell surface antigen. Indirect staining flow cytometry is used when a directly labeled antibody is not available or when the goal is to amplify a signal already obtained. In indirect staining flow cytometry, cells are stained with a primary antibody against the antigen of interest and visualized using a labeled secondary antibody that recognizes the first primary antibody. As known to those skilled in the art, other variants of staining are possible, for example, intracellular staining is used when the antigen is not present on the cell surface.

[0057] In some embodiments, the cells for staining (e.g., test sample) may be cells from a cell line or a biological sample. Any biological sample containing cells of interest may be obtained for staining. In some embodiments, the biological sample is obtained from or derived from a subject, such as a subject having a particular disease or condition, or in need of therapy (e.g., cell therapy), or to which a therapeutic treatment (e.g., cell therapy) is administered. In some aspects, the subject is a human, such as a patient in need of a particular therapeutic intervention. Thus, the cells are, in some embodiments, primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, including processed samples derived therefrom.

[0058] In some embodiments, the sample is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0059] In some instances, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0060] In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some embodiments, the washing step is accomplished in a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter) according to the manufacturer's instructions. In some embodiments, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed with various biocompatible buffers, such as Ca, HCl ... 2+ / Mg 2+ In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0061] In some embodiments, the preparation method includes a step for freezing, e.g., cryopreserving, the cells prior to staining. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters may be used in some aspects. In some embodiments, the cells are suspended in a medium and / or solution at or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, or about 7.5%. The mixture is frozen, e.g., cryofrozen or cryopreserved, with a final concentration of 0%, 6.5% or about 6.5%, 6.0% or about 6.0%, 5.5% or about 5.5%, or 5.0% or about 5.0% DMSO, or from 1% or about 1% to 15% or about 15%, 6% or about 6% to 12% or about 12%, 5% or about 5% to 10% or about 10%, or 6% or about 6% to 8% or about 8% DMSO. In certain embodiments, the cells are frozen, e.g., cryofrozen or cryopreserved, with a final concentration of at or about 5.0%, 4.5% or about 4.5%, 4.0% or about 4.0%, 3.5% or about 3.5%, 3.0% or about 3.0%, 2.5% or about 2.5%, 2.0% or about 2.0%, 1.5% or about 1.5%, 1.25% or about 1.25%, 1.0% or about 1.0%, 0.75% or about 0.75%, 0.5% or about 0.5%, or 0.25% or about 0.25% HSA, or 0.1%-5%, 0.25%-4%, 0.5%-2%, or 1%-2% HSA in the medium and / or solution. One example involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium.In some embodiments, this is then diluted 1:1 with culture medium resulting in final concentrations of 10% and 4% DMSO and HSA, respectively. In some embodiments, the cells are then frozen to or about -80°C at a rate of or about 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank.

[0062] In some embodiments, one or more preparation and / or non-affinity-based cell separation steps may be performed prior to staining of cells. In some cases, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove undesired components, to concentrate desired components, to lyse or remove cells that are sensitive to a particular reagent. In some cases, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to a particular component. In some embodiments, the method includes a density-based cell separation method, such as preparation of white blood cells from peripheral blood by lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0063] In some embodiments, the cells of the cell population are labeled with one or more fluorescent markers (e.g., one or more fluorophores) that generate a fluorescent signal that can be measured by a flow cytometer. In some embodiments, at least a portion of the obtained sample is subjected to cell staining by incubation of the cells in the sample with one or more staining reagents, each of which contains a fluorescent signal or marker (e.g., a fluorophore). The staining reagent can be any reagent for characterizing, selecting, or isolating a particular cell type or subtype of cells. In some embodiments, the one or more staining reagents are immunoaffinity-based reagents, such as antibodies. In some embodiments, the staining reagent stains cells based on the cellular expression or expression level of one or more markers, for example, based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, the marker is a cell surface marker.

[0064] Any marker known or suspected to be expressed on the cell type of interest in the sample can be targeted by the staining reagent. Exemplary markers include markers present on lymphocyte populations, such as T cells. In some embodiments, the sample may be a whole blood sample. In some embodiments, the sample is an apheresis sample. In some embodiments, the sample is a leukapheresis sample. In some embodiments, the cell surface markers may include one or more surface markers CD3, CD4, CD8, CD45, CD45RA, CD45RO, CD28, CD62L, CCR7, CD27, CD95, CD127. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind to such markers.

[0065] In some embodiments, the marker may be a marker for a recombinant protein known to be expressed on or by one or more cells in the population. In some embodiments, a polynucleotide encoding the marker may be introduced into the cell by a cell transformation method, such as cell transfection or transduction. In some embodiments, the marker is a recombinant receptor, such as a chimeric antigen receptor (CAR) or a TCR. In some embodiments, the staining reagent for a CAR may be an antibody directed against the antigen-binding domain (e.g., an anti-idiotypic antibody) or an antibody directed against the spacer region of the extracellular domain of a CAR (e.g., an immunoglobulin hinge region). In some embodiments, the staining reagent for a TCR may be a major histocompatibility complex (MHC) tetramer.

[0066] In some embodiments, the marker is a recombinant protein that is a surrogate marker (also called a transduction marker) that is co-expressed with another recombinant protein in a cell. A transduction marker or a surrogate marker can be used to detect cells that have been introduced with a polynucleotide, such as a polynucleotide that encodes a recombinant protein. In some embodiments, a transduction marker can indicate or support the modification of a cell. In some embodiments, a surrogate marker is a protein that is co-expressed on the cell surface with a recombinant receptor, such as a CAR. In certain embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, a surrogate marker is encoded on the same polynucleotide that encodes a recombinant protein (e.g., a recombinant receptor). In some embodiments, the nucleic acid sequence encoding the recombinant protein is operably linked to a nucleic acid sequence encoding a marker, which may be separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, such as T2A, P2A, E2A, or F2A. Exogenous marker genes may be utilized in some cases to allow detection or selection of cells in the context of engineered cells, and also in some cases to promote cell suicide.

[0067] Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and do not transmit or have no ability to transmit signals or signals normally transmitted by full-length forms of cell surface polypeptides, and / or do not internalize or have no ability to do so. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (EGFRt) or prostate specific membrane antigen (PSMA), or modified forms thereof. EGFRt may contain an epitope recognized by the antibody cetuximab [Erbitux®], or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with EGFRt constructs and recombinant receptors, such as chimeric antigen receptors (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434. In some embodiments, the marker, e.g., surrogate marker, comprises all or a portion (e.g., a truncated form) of CD34, NGFR, CD19 or a truncated CD19, e.g., a truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, e.g., a cleavable linker sequence, e.g., T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in PCT Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), which may be linked to a linker sequence, e.g., a T2A cleavable linker sequence.

[0068] In some embodiments, the surrogate marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP, red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or mutants thereof.

[0069] In some embodiments, the marker is a cell health marker. Non-limiting examples of cell health markers include caspase cleavage products, such as chromogenic substrates, c-PARP, cleaved cytokeratin 18, cleaved caspase, cleaved caspase 3, cytochrome C, apoptosis-inducing factor (AIF), inhibitor of apoptosis (IAP) family members, as well as other molecules, such as anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL), BH3-only apoptosis sensitizers (PUMA, NOXA, Bim, Bad), and Bcl-2 family members (see below), including pro-apoptotic proteins (Bad, Bax), p53, c-myc proto-oncogene, APO-1 / Fas / CD95, growth stimulatory genes, or tumor suppressor genes, mitochondrial membrane dyes, Annexin-V, 7-AAD, Amine Aqua, trypan blue, propidium iodide, or other viability dyes. In some embodiments, the marker is Annex V, a marker for apoptosis. Live cells can be stained with Annexin V, which binds to phosphatidylserine, which is normally intracellular but is translocated to the cell surface when cells are undergoing apoptosis or other forms of cell death.

[0070] In some embodiments, the staining reagent is a fluorophore dye, which is a viability dye selective for dead cells that emits fluorescence of a specific wavelength when bound to dead cells. In some embodiments, the viability dye is an amine-reactive dye, such as an NHS active ester (which may include succinimidyl ester, sulfosuccinimidyl ester, tetrafluorophenyl ester, or sulfodichlorophenol ester) covalently attached to the dye. Such viability dyes allow cell viability detection based on the reaction of the fluorescent reactive dye with primary amines, such as lysine residues in cellular proteins. These dyes cannot penetrate the membrane of live cells, so only for live cells are cell surface proteins available to react with the dye, resulting in dim staining. However, in dead cells, the reactive dye can penetrate the damaged membrane of dead cells to stain both internal and external amines, resulting in stronger staining. In some embodiments, the covalently attached dye may be Alexa Fluor 405, Pacific Blue, Alexa Fluor 430, Fluorescein, Alexa Fluor 488, Texas Red, Alexa Fluor 594. In some embodiments, the fluorophore dyes can be defined as green, red, blue, violet, cyan or yellow. The green and red fluorescent dyes are excited by a 488 nm laser, the violet, cyan and yellow dyes require a 405 nm excitation source with different emission wavelengths, the blue fluorescent reactive dyes require UV excitation, and finally the infrared and near infrared dyes are excited at 633 / 635 nm. The availability of amine reactive dyes with different fluorophores excitable at different wavelengths allows one to select a suitable reagent combination for any configuration of flow cytometer used.

[0071] In some embodiments, the viability dye is propidium iodide, DRAQ7, 7-AAD, eBioscience Fixable Viability Dye eFluor® 455UV, eBioscience Fixable Viability Dye eFluor® 450, eBioscience Fixable Viability Dye eFluor® 506, eBioscience Fixable Viability Dye eFluor® 520, eBioscience Fixable Viability Dye eFluor® 660, eBioscience Fixable Viability Dye eFluor® 780, BioLegend Zombie Aqua™, BioLegend Zombie NIR™, BioLegend Zombie Red™, BioLegend Zombie Violet™, BioLegend Zombie UV™, or BioLegend Zombie Yellow™.

[0072] In some embodiments, the staining reagent is composed of one or more fluorescent markers that may be attached, for example, by chemical conjugation, to a binding agent that can bind, for example, specifically bind, to the marker. In some embodiments, the binding agent is a protein. In some embodiments, the binding agent is an antibody or an antigen-binding fragment. The fluorescent marker may be conjugated to the binding agent, for example, an antibody, by any method known in the art.

[0073] As is well known in the art, an "antibody" is an immunoglobulin (Ig) molecule that has the ability to specifically bind to a target, such as carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule.As used herein, the term includes intact polyclonal or monoclonal antibodies, as well as fragments thereof, such as dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising antibody portions with antigen-binding fragments of required specificity, chimeric antibodies, nanobodies, and any other modified configurations of immunoglobulin molecules that comprise antigen-binding sites or fragments (epitope recognition sites) of required specificity.Minibodies comprising scFv joined to CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, e.g., Ward, ES et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0074] A binding agent, such as an antibody, that "specifically binds" or "preferentially binds" to a marker (used interchangeably herein) is a term well understood in the art. A molecule is said to exhibit "specific binding" or "preferential binding" when it reacts or associates more frequently, more rapidly, with a longer duration, and / or with a higher affinity with a particular marker target than with alternative markers. An antibody specifically binds or preferentially binds to a target when it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. It is also understood by reading this definition that specific or preferential binding does not necessarily require (but can include) exclusive binding. Methods for determining such specific or preferential binding are also well known in the art, such as immunoassays.

[0075] In provided embodiments, the binding agent, e.g., an antibody, is conjugated to a fluorescent marker, e.g., a fluorophore. For example, cells may be incubated with one or more fluorescently labeled antibodies. In some embodiments, any fluorescent marker or fluorophore suitable for use with flow cytometry analysis may be used. Some non-limiting examples of fluorescent markers include fluorescent proteins (e.g., GFP, YFP, RFP), fluorescent moieties (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor (AF)], nucleic acid stains (e.g., 4',6-diamidino-2-phenylindole (DAPI), SYT016, propidium iodide (PI)], cell membrane stains (e.g., FMI-43), cell functional dyes (e.g., Fluo-4, Indo-1), and synthetic dyes (e.g., Brilliant Violet (BV)].Exemplary fluorophores include hydroxycoumarin, Cascade Blue, Dylight 405 Pacific Orange, Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2,7-Dichlorofluorescein, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 532, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700(AF700), Cy5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, PE-Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, BV421, BV510, BV570, BV605, BV650, BV711, BV750, BV785, Qdot (registered trademark) 525, Qdot (registered trademark) Examples of suitable fluorophores include, but are not limited to, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800.

[0076] In some embodiments, the one or more fluorescent markers include one or more fluorescent markers that detect a cellular attribute. The cellular attribute may be any marker that is an attribute of a particular cell or cell type. In some embodiments, each cellular attribute is selected from the group consisting of CD3, CD4, CD8, CD19, CD45, and Live / Dead.

[0077] In some embodiments, the cell attribute is a frequency attribute. A frequency attribute is an attribute that is detectable in at least 5% or more of a sample, e.g., at least 5% or more of the cells in a population of cells of a sample. In some embodiments, for a sample that contains a population of T cells, e.g., that is enriched for T cells, the frequency attribute can include CD3, CD4, CD8, and / or CD45.

[0078] In some embodiments, the cell attribute is a low frequency attribute. A low frequency attribute is an attribute that is detectable in less than 5% of a sample, for example, less than 5% of cells in a population of cells of a sample. In some embodiments, for a population of cells that are deemed healthy, the low frequency attribute is Live / Dead. In some embodiments, for a population of cells enriched for T cells, the low frequency attribute may be a B cell marker, for example, CD19, CD20, CD22, and / or CD138.

[0079] In some embodiments, the one or more fluorescent markers include one or more fluorescent markers each generating a fluorescent signal that is measured by a flow cytometer, and thus in some embodiments, each of the one or more fluorescent signals detects a cellular attribute recognized by the fluorescent marker or stain.

[0080] In some embodiments, the fluorescent marker comprises an emission detection means, which advantageously emits light at least at a fluorescent wavelength. In some embodiments, the one or more fluorescent markers are selected such that the emission wavelength spectrum of each of the one or more fluorescent markers is distinguishable from the excitation wavelength spectrum of other fluorescent markers of the one or more fluorescent markers. The different fluorescent markers may be excitable by light of the same wavelength or different wavelengths. Preferably, the emission wavelength is different for each of the one or more fluorescent markers.

[0081] In some embodiments, multicolor staining or labeling is performed using multiple fluorophores, and multiple staining reagents for different markers (e.g., cell surface markers) are incubated with cells. In some embodiments, for example, fluorescent markers, conjugated to one or more staining reagents, e.g., antibodies, are selected to minimize energy transfer between them, e.g., to avoid or minimize overlapping emission and absorption spectra. In some embodiments, each fluorescent marker has a different emission spectrum. In some embodiments, multiple fluorescent markers may be excited with a single wavelength or multiple wavelengths, but detection is performed in a region where the peak emission spectra do not overlap. In some embodiments, excitation of one or more of the fluorescent markers may be done with a single or same wavelength of light, but different wavelengths of light are emitted from them.

[0082] In some embodiments, each of the one or more fluorescent markers individually comprises a fluorophore selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0083] In some embodiments, cells are stained with multiple markers, e.g., multiple staining reagents for cell surface markers. In some embodiments, this results in cells labeled with multiple markers, allowing characterization of well-defined subsets of cells.

[0084] In some embodiments, cell staining involves incubation with an antibody or binding partner that specifically binds to such marker, which in some embodiments is followed by a washing step and separation of cells bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of the desired staining reagent and incubated under conditions for staining of the cells. In some embodiments, the staining or labeling is carried out at a temperature between 0°C and 25°C, for example at or about 4°C. In some embodiments, the staining or labeling is carried out for more than 5 minutes, typically more than 15 minutes. In some embodiments, the staining or labeling is carried out for 15 minutes to 6 hours, for example 30 minutes to 2 hours. In some embodiments, the staining or labeling is carried out for, for example, at or about 15 minutes, 30 minutes or about 30 minutes, 1 hour or about 1 hour, 1.5 hours or about 1.5 hours, 2 hours or about 2 hours, 2.5 hours or about 2.5 hours, 3 hours or about 3 hours, or any value between any of the above. In some embodiments, labeling with one or more staining reagents is performed simultaneously, hi some embodiments, one or more washing steps are performed prior to introducing the sample into a flow cytometer for analysis.

[0085] In some embodiments, the cell sample is prepared by dissolving 1×10 cells in water to allow the cells to pass through a flow cytometer for reading. 6 ~1×10 7 A cell sample is prepared by suspending single cells at a density of 1000 to 20000 cells / ml. In some embodiments, this concentration of cells is referred to as a fluid sheath. In some embodiments, the fluid sheath affects the speed of flow sorting, which typically proceeds at about 2,000 to 20,000 cells per second. The fluid sheath of the cell sample is typically made from a phosphate buffered saline solution, although other solutions are available as known and understood by those of skill in the art.

[0086] In some embodiments, a sample is introduced into a flow cytometer. As the sample enters the flow cytometer, particles are randomly distributed in the three-dimensional space of the cell sample. The cell sample is typically narrowed into a single stream through a fluidic system using the application of hydraulic pressure. This stream is then passed through one or more beams of light scattering or fluorescent emission. A laser typically serves as the light source in a flow cytometer. The laser produces a single wavelength of light that, once in contact with the cell sample, produces light scattered in the forward direction as an indication of cell size, light scattered in the side direction as an indication of cell complexity, and fluorescent light that is also emitted in the side direction in proportion to the relative amount of a particular cell marker. A photomultiplier tube (PMT) or photodiode receives the light reflected from the cells. In some embodiments, light originating from a 0-20 degree offset from the laser excitation line is known as forward scattered light. In some embodiments, light measured at an angle of about 90 degrees to the laser's excitation line is referred to as side scattered light and fluorescent signal. Both forward and side scatter light are unique for every particle passing through the flow cytometer, and a combination of all can be used to distinguish particles in a cell sample. Fluorescence channels are usually designated by the designation FL1, FL2, FL3, etc., depending on the number of channels in the instrument. Each fluorescence channel is equipped with a barrier filter to detect a selected specific dye while filtering out all others. The channel in which a specific dye is primarily detectable may be referred to as its primary fluorescence channel, and the other fluorescence channels may be referred to as secondary channels. The scatter and fluorescence emission signals are converted into electronic pulses that are processed by the flow cytometry engine and displayed on a graphical user interface, or "GUI."

[0087] II. Static Gating Threshold In some embodiments, the methods provided include applying a static fluorescence threshold gate individually to each of one or more fluorescent signals present in flow cytometry data collected from a test sample, e.g., as described in Section I.

[0088] In some embodiments, a 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 populations of cells, each sorted according to the one or more fluorescent signals; and (b) setting a static fluorescence threshold gate for each of the one or more fluorescent signals.

[0089] In some embodiments, a static fluorescence threshold gate for each of the one or more fluorescent signals is determined from (a) measuring cytometry events by flow cytometry for the one or more fluorescent signals for a plurality of at least two reference populations of cells; and (b) setting a static fluorescence threshold gate for each of the one or more fluorescent signals.

[0090] A. Setting the static gating threshold Provided herein is 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 at least two reference cell samples including: (a) a first plurality of reference cell samples, each comprising a population of unstained cells that are not labeled with one or more fluorescent signals; and (b) a second plurality of reference cell samples, each of the second plurality of reference cell samples derived from the same cellular source type as the first plurality of reference cell samples, and each of the second plurality of reference cell samples comprising at least one population of stained cells 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.

[0091] In some embodiments, the source type of cells of the reference cell sample is a population of cells from any biological sample that contains cells of interest for staining. In some embodiments, the cells may be cells from a cell line or a biological sample. In some embodiments, the biological sample may include primary cells obtained from or derived from a subject, such as a subject having a particular disease or condition or in need of treatment (e.g., involving cell therapy) or to which a therapeutic treatment (e.g., cell therapy) is administered. In some aspects, the subject is a human subject, such as a patient in need of a particular therapeutic intervention. Thus, the source type of cells is, in some embodiments, primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or samples that have been processed. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, including processed samples derived therefrom.

[0092] In some embodiments, the sample is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0093] In some instances, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0094] In some embodiments, the source type of cells may be a primary cell population isolated from a subject. In some embodiments, the isolated cell population typically includes a population of cells, such as blood or blood-derived cells, such as hematopoietic cells, leukocytes, peripheral blood mononuclear cells (PBMCs), and / or cells of the immune system, such as cells of innate or adaptive immunity, such as myeloid or lymphoid cells, such as lymphocytes, typically T cells and / or NK cells.

[0095] In some embodiments, the source type of cells is an enriched population of cells of a particular type or subtype, in which such cells have been selected from a starting cell sample (e.g., an apheresis or leukapheresis sample), for example, by positive or negative selection of cells from the sample. Among the T cell populations that can be enriched and / or selected are populations of CD3+ T cells, CD4+ cells, CD8+ cells, and subpopulations of CD4+ and / or CD8+ T cells, including subpopulations of T cells generated by enrichment or depletion of cells of a particular subtype or based on a particular surface marker expression profile. For example, among the subtypes of T cells (e.g., CD4+ or CD8+ T cells) that can be enriched and / or selected are those defined by function, activation state, maturation, differentiation, expansion, recirculation, localization, and / or persistence potential, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the T cells and / or subtypes and subpopulations of CD4+ and / or CD8+ T cells that may be enriched, isolated and / or selected are naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0096] In some embodiments, one or more of the T cell populations enriched and / or selected from the cell samples provided herein are positive for (marker+) or express high levels of one or more particular markers, e.g., surface markers (marker+). high ), or are negative for one or more markers (marker-) or express them at relatively low levels (marker low ) cells. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (e.g., CD8+ cells or T cells, e.g., CD3+ cells) are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L (i.e., positively selected) and / or depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negatively selected). In some embodiments, cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Rα (CD127). In some instances, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and positive for CD62L.

[0097] In some embodiments, a CD4+ T cell population and a CD8+ T cell subpopulation, eg, a subpopulation enriched for central memory (TCM) cells.

[0098] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0099] In some embodiments, the source type of cells is a population of engineered cells into which a nucleic acid encoding a recombinant protein has been introduced, for example, by gene transfer (e.g., transduction). Examples of recombinant proteins to be expressed include recombinant receptors, for example, chimeric antigen receptors (CARs) or T cell receptors (TCRs). A variety of methods for the introduction of engineered components, for example, recombinant receptors, for example, CARs or TCRs, are well known and may be used with the provided methods and cell samples. Exemplary methods include methods for the transfer of nucleic acids encoding recombinant proteins, including via viral transduction, for example, retroviral or lentiviral transduction, transposons, and electroporation. In some embodiments, genetic engineering involves the introduction of a nucleic acid encoding a recombinant or engineered component into a composition containing cells, for example, by retroviral transduction, transfection, or transformation. The introduction of a nucleic acid molecule encoding a recombinant protein, for example, a recombinant receptor, into a cell may be carried out using any of a number of known vectors. Such vectors include viral and non-viral systems, including lentiviral and gamma retroviral systems, as well as transposon-based systems, for example, PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include methods for transfer of nucleic acid encoding the receptor, including via viral transduction, eg, retroviral or lentiviral transduction, transposons, and electroporation.

[0100] In some embodiments, the primary cell sample used as the source type of cells for each of the multiple reference cell samples is from a different subject (e.g., patient), each different from the subject (e.g., patient) from which the source type of cells was obtained (or will be obtained) for the test cell sample. In some embodiments, each different subject (e.g., patient) has the same or similar disease or condition. For example, each different subject has the same type of cancer. In some cases, the source type of cells may be a sample from a subject (e.g., patient) with a disease or condition for which it is desired to perform flow cytometry analysis on the subject's cells prior to or during a particular therapy. In some cases, the reference cell sample may be a sample from a subject from a clinical trial for testing a particular therapy. In some embodiments, the source type of cells to be tested is the cells of a cell therapy administered to a subject (e.g., patient). In some embodiments, the cell therapy is a CAR cell therapy (e.g., CAR T cell), a TCR cell therapy, a tumor infiltrating lymphocyte (TIL) cell therapy, a natural killer (NK) cell therapy, or a stem cell therapy. The cells may be allogeneic and / or autologous with respect to the subject being treated. In some embodiments, the cell therapy is an autologous cell therapy. In some embodiments, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs) or cells differentiated therefrom. In some cases, the reference cell sample may be a cell therapy sample generated individually from multiple different subjects in a clinical trial, and the test cell sample is a cell therapy sample generated from a test subject (e.g., a patient) identified or approved for treatment with a particular cell therapy. In some embodiments, the provided methods may be performed in the context of quality control or characterization of a manufactured, e.g., engineered, cell therapy prior to introduction into a subject.

[0101] In certain embodiments, the source type of cells in the reference cell sample is a cell that is known or suspected to contain similar attributes or features as the test cell sample that is desired to be analyzed by the provided method using a static gating threshold. Thus, in the provided embodiments, the test cell sample and the first and second reference cell samples are from the same cell source. For example, when the test cell sample is a leukapheresis cell sample, the source of cells in the reference cell sample is also a leukapheresis cell sample. In some embodiments, in the case where the cells are primary cells from a subject, the source of cells of the reference cell sample and the test cell sample may also be obtained from or derived from a subject with similar attributes or features. For example, in some embodiments, the test cell sample is derived from a subject with a disease, and the reference cell population sample is derived from another control subject with the same or similar disease. In some embodiments, the disease is cancer, or a specific type or subtype of cancer, such as lymphoma, leukemia, myeloma, or solid tumor cancer. In some embodiments, the test cell sample is a population of cells that has been manipulated, for example, by gene transfer (e.g., transduction) or gene editing procedures, or by exposure or incubation in the presence of a modulator, such as an activator, cytokine, immunomodulator, small molecule compound, or other agent that can alter one or more characteristics or properties of the cells. In such embodiments, the reference cell sample is also a population of cells similarly manipulated under the same conditions as the test cell sample (or optionally the plan for the test cell sample).

[0102] In some embodiments, the plurality of first reference cell samples comprises more than two cell samples. In some embodiments, the plurality of first reference cell samples comprises more than 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cell samples. In some embodiments, the plurality of first reference cell samples comprises 100-500 cell samples, such as 100-400 cell samples, 100-300 cell samples, 100-200 cell samples, 200-500 cell samples, 200-400 cell samples, 200-300 cell samples, 300-500 cell samples, 300-400 cell samples, or 400-500 cell samples. In some embodiments, each of the first reference cell samples is an unlabeled population of cells, such that the cells are not stained or exposed with any fluorescent marker, and thus the cells are not labeled with any fluorescent signal.In some embodiments, the plurality of first cell samples represents a population of unstained cells that can serve as background control to account for negative staining or autofluorescence of cells in the sample.

[0103] In some embodiments, the plurality of second reference cell samples comprises more than two cell samples. In some embodiments, the plurality of second reference cell samples comprises more than 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cell samples. In some embodiments, the plurality of second reference cell samples comprises 100-500 cell samples, such as 100-400 cell samples, 100-300 cell samples, 100-200 cell samples, 200-500 cell samples, 200-400 cell samples, 200-300 cell samples, 300-500 cell samples, 300-400 cell samples, or 400-500 cell samples. In some embodiments, a portion of the same source of cells as the first reference cell sample (unlabeled or unstained cell sample) is used as the starting source of cells for each of the second reference cell samples. Thus, in the embodiments provided, each of the plurality of the second reference population of cells is derived from one of the plurality of the first reference population of cells.

[0104] In some embodiments, the second reference sample is subjected to a labeling procedure by contacting each of the plurality of cell samples of the second reference cell sample with one or more staining reagents individually. For the implementation of the provided method, each of the test sample and the second reference sample is subjected to cell staining using one or more staining reagents to label cells with one or more fluorescent signals. In some embodiments, the staining reagent can be any reagent described in Section I. In some embodiments, the cell staining procedure can be carried out using any method described in Section I. In the provided embodiment, a binding agent specific for a particular marker and dye is selected such that staining of cells with the staining reagent results in a clear separation between a positive population and a negative / unstained population for a particular marker attribute. For example, it is understood that the provided method may not be suitable for attributes that are not suitable for a clear separation between positively stained samples and negatively stained samples.

[0105] In the embodiments provided, the cell staining of each of the test cell sample and the second reference cell sample is performed using the same staining reagent and using the same protocol conditions. It is understood that the reference "same" does not mean that the staining reagent or protocol is identical in all respects, and some variation between reagents or in the implementation of the protocol is expected. It is within the level of the art to ensure that variations are minimized and steps are taken to ensure that the procedures are as similar as possible. For example, it is not necessary that the staining is performed using the exact same lot or batch of staining reagent, for example, as available from a commercial supplier. Typically, each manufactured batch or lot of staining reagent is characterized so that its mean fluorescence intensity (MFI) matches one or more previous lots or batches of the same staining reagent. In some embodiments, the cell staining is performed using staining reagents that are known to have the same protein sequence or chemical structure, including the same fluorophore type. Typically, the staining reagents are reagents obtained from the same commercial vendor or supplier, for example, sold under the same catalog number.

[0106] In some embodiments, cells are stained with multiple markers, e.g. multiple staining reagents for cell surface markers. In some embodiments, this results in cells labeled with multiple markers, allowing characterization of well-defined subsets of cells. Thus, in some embodiments, the cell staining is a multicolor cell staining for labeling cells with two or more different fluorescent signals, each staining reagent for labeling a different marker with a different fluorescent signal. In some embodiments, the two or more different fluorescent signals include 2-10 fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with two different fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with three different fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with four different fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with five different fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with six different fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with seven different fluorescent signals. In some embodiments, the cell staining is performed for labeling cells with eight different fluorescent signals. In some embodiments, cell staining is performed to label cells with 9 different fluorescent signals. In some embodiments, cell staining is performed to label cells with 10 different fluorescent signals. In some embodiments, each of the two or more different fluorescent signals has a different emission spectrum. In some embodiments, each of the two or more different fluorescent signals has a peak emission spectrum of fluorescent signal that does not overlap with one or more other fluorescent signals in the cell staining. In some embodiments, cell staining is performed using a staining reagent in which the two or more different fluorescent signals are emitted by a dye selected from PE-Cy7, APC, AF700, BV421, Aqua, or BV605.

[0107] In some embodiments, cell staining involves incubation with a staining reagent that specifically binds to such markers, such as an antibody or binding partner conjugated to a fluorophore dye, which in some embodiments is followed by a washing step and separation of cells bound to the staining reagent from cells that are not bound to the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of the desired staining reagent and incubated under conditions for staining of the cells. In some embodiments, the staining or labeling is carried out at a temperature between 0°C and 25°C, such as at or about 4°C. In some embodiments, the staining or labeling is carried out for more than 5 minutes, typically more than 15 minutes. In some embodiments, the staining or labeling is carried out for 15 minutes to 6 hours, such as 30 minutes to 2 hours. In some embodiments, staining or labeling is carried out for, for example, 15 minutes or about 15 minutes, 30 minutes or about 30 minutes, 1 hour or about 1 hour, 1.5 hours or about 1.5 hours, 2 hours or about 2 hours, 2.5 hours or about 2.5 hours, 3 hours or about 3 hours, or any value between any of the above.In some embodiments, labeling with one or more staining reagents is carried out simultaneously.In some embodiments, one or more washing steps are carried out prior to introducing the sample into a flow cytometer for analysis.

[0108] In some embodiments, each of the cell samples, e.g., each of the first and second plurality of reference cell samples, is 1×10 6 to allow the cells to pass through a flow cytometer for reading. 6 ~1×10 7 Cell samples are individually prepared by suspending single cells at a density of 1000 to 20000 cells / ml. In some embodiments, this concentration of cells is referred to as the fluid sheath. In some embodiments, the fluid sheath affects the speed of flow sorting, which typically proceeds at about 2,000 to 20,000 cells per second. The fluid sheath of the cell sample is typically made from a phosphate buffered saline solution, although other solutions are available as known and understood by those of skill in the art.

[0109] In some embodiments, flow cytometry was performed on a single cell sample for each of the plurality of first reference cell samples.

[0110] In some embodiments, cell staining can be repeated multiple times, for example in duplicate or triplicate, for each of the source types of cells stained for labeling with one or more fluorescent signals, resulting in duplicate or triplicate stained samples.In some embodiments, the plurality of second reference cell samples comprises duplicate samples of the same stained cells.In some embodiments, the plurality of second reference cell samples comprises triplicate samples of the same stained cells.

[0111] In some embodiments, prior to running a sample through the flow cytometer, one or more of alignment, compensation, and calibration of the flow cytometer are performed. Alignment, compensation, and calibration ensure that the instrument is operating at its maximum efficiency as well as achieving reproducibility so that data obtained over time or with different instruments are comparable.

[0112] In some embodiments, alignment is the process of adjusting and focusing the various optical and electrical components such that the scatter and fluorescence signals are tuned to their highest intensity and tightest distribution, i.e., lowest distribution coefficient of variation (CV). Flow cytometer components that are aligned include lasers, lenses, mirrors, barrier filters, and PMTs.

[0113] In some cases, multi-fluorescence analysis, i.e., analysis using two or more fluorochromes simultaneously, can be performed on a flow cytometer. However, to perform accurate analysis, it is necessary to adjust the electronic compensation circuitry in the flow cytometer so that any fluorescence emission that overlaps other fluorescence channels can be subtracted from such other channels. Compensation is the process of electronically removing residual signals from fluorochromes in secondary fluorescence channels that result from spectral overlap that is not removed by the barrier filters for each channel. Calibration of a flow cytometer with proper standards ensures that results from samples are comparable over time and between different instruments. For calibration of the intensity of the fluorescent signal to be independent of the specific instrument and instrument settings, the excitation and emission spectra of the calibration standards and the sample being measured must be comparable, and measurements in each must be performed under the same instrument settings. The method of calibration of flow cytometer is known, and is described, for example, in U.S. Patent No. 4,714,682; U.S. Patent No. 4,767,206; U.S. Patent No. 4,774,189; and U.S. Patent No. 5,620,842 and PCT Application WO1991000509.In some embodiments, calibration method uses both blank and fluorescently labeled microbeads that can be used to align and calibrate the fluorescent channel of flow cytometer.For example, U.S. Patent No. 4,774,189 and U.S. Patent No. 4,767,206 describe the method of calibrating flow cytometer using such microbeads.In some embodiments, the standard used for calibration mimics the cells to be studied in sample, as described, for example, in U.S. Patent No. 6,008,052.

[0114] In some embodiments, fluorescence calibration ensures the accuracy and reproducibility of the provided method. In some embodiments, to obtain accurate and reproducible results, the flow cytometer must be aligned and calibrated. When working with more than one fluorescent dye, the instrument also requires compensation for the fluorescence photomultiplier tube (PMT).

[0115] In some embodiments, each of the cell samples, for example each of the first and second reference cell samples, is introduced into the flow cytometer individually. Both forward and side scattered light are unique for every particle passing through the flow cytometer, and the combination of all can be used to distinguish particles in the cell sample. In some embodiments, the forward scattered light, side scattered light and emitted light signals are converted into electronic pulses, which are processed by the flow cytometry engine and provided as flow cytometry data. In some embodiments, the flow cytometry data is displayed on a graphical user interface "GUI".

[0116] In some embodiments, the flow cytometry data from each of the first plurality of reference cell samples and each of the second plurality of reference cell samples is used to set a static threshold gate. In some embodiments, setting a static fluorescence threshold gate comprises setting a static fluorescence threshold gate for each of one or more fluorescent signals. In some embodiments, the static threshold gate is a fixed threshold selected to distinguish positive staining from negative staining for each of one or more fluorescent signals (see FIG. 1A). In some embodiments, for each of one or more fluorescent signals, the static gate threshold is set by (1) determining an upper fluorescence boundary of the fluorescent signal from each of the unstained samples from the first plurality of reference samples; (2) determining a lower fluorescence boundary of the fluorescent signal from each of the stained samples from the second plurality of reference samples; and (3) setting a fixed or static threshold for each fluorescent signal as a threshold gate for the fluorescent signal. In some embodiments, determining the upper boundary of each of the fluorescent signals can be determined as an upper percentile (e.g., 90th percentile or higher) according to the distribution of the fluorescent signals in the unstained samples from the first plurality of reference samples. In some embodiments, determining the lower boundary of each of the fluorescent signals may be determined as a lower percentile (e.g., the 10th percentile or lower) according to the distribution of fluorescent signals in stained samples from a plurality of second reference samples.

[0117] In some embodiments, setting a static fluorescence threshold gate for each of the one or more fluorescent signals is done such that for each fluorescent signal the threshold gate is (i) higher than the 90th percentile of the fluorescence of the plurality of members of a first reference population of cells for each respective fluorescent signal of each of the one or more fluorescent signals; and (ii) lower than the 10th percentile of the fluorescence of the plurality of members of a second reference population of cells for the same respective fluorescent signal.

[0118] As such, provided herein is a method of determining a static fluorescence threshold gate, 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 at least two reference cell samples comprising: (a) a first plurality of reference cell samples, each comprising a population of unstained cells that are not labeled with one or more fluorescent signals; and (b) a second plurality of reference cell samples, each of the second plurality of reference cell samples derived from the same cellular source type as the first plurality of reference cell samples, and each of the second plurality of reference cell samples comprising: and (2) setting a static fluorescence threshold gate for each of the one or more fluorescent signals, wherein each fluorescent signal threshold gate is set such that: (a) the fluorescence of the plurality of first reference populations of cells for each respective fluorescent signal of each of the one or more fluorescent signals is higher than the 90th percentile; and (b) the fluorescence of the plurality of second reference populations of cells for the same respective fluorescent signal is lower than the 10th percentile.

[0119] In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is the fluorescence of the fluorescent signal that is higher than the 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, or 99th percentile of the fluorescence among the plurality of the first reference population of cells for each fluorescent signal. In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is the fluorescence of the fluorescent signal that is higher than the 90th, 91st, 92nd, 93rd, 94th, or 95th percentile of the fluorescence among the plurality of the first reference population of cells for each fluorescent signal.

[0120] In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is the fluorescence of the fluorescent signal higher than the 95th, 96th, 97th, 98th, or 99th percentile of the fluorescence among a plurality of the first reference population of cells for the respective fluorescent signal.

[0121] In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is a fluorescence of the fluorescent signal that is lower than the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd or 1st percentile of the fluorescence among the plurality of the second reference population of cells for each fluorescent signal. In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is a fluorescence of the fluorescent signal that is lower than the 5th, 4th, 3rd, 2nd or 1st percentile of the fluorescence among the plurality of the second reference population of cells for the same respective fluorescent signal.

[0122] In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is a fluorescence higher than the 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, or 99th percentile of the fluorescence among a plurality of a first reference population of cells for the respective fluorescent signal; and a fluorescence of the fluorescent signal lower than the 10th, 9th, 8th, 7th, 6th, 5th, 4th, 3rd, 2nd, or 1st percentile of the fluorescence among a plurality of a second reference population of cells for the same respective fluorescent signal.

[0123] In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is a fluorescence higher than the 90th, 91st, 92nd, 93rd, 94th, or 95th percentile of the fluorescence among a plurality of a first reference population of cells for the respective fluorescent signal; and a fluorescence of the fluorescent signal lower than the 5th, 4th, 3rd, 2nd, or 1st percentile of the fluorescence among a plurality of a second reference population of cells for the same respective fluorescent signal.

[0124] In some embodiments, for each fluorescent signal, the static fluorescence threshold gate is a fluorescence higher than the 95th, 96th, 97th, 98th, or 99th percentile of the fluorescence among a plurality of a first reference population of cells for the respective fluorescent signal; and a fluorescence signal lower than the 5th, 4th, 3rd, 2nd, or 1st percentile of the fluorescence among a plurality of a second reference population of cells for the same respective fluorescent signal.

[0125] In some embodiments, the static fluorescence threshold gate for at least one of the fluorescent signals is a fluorescence higher than the 95th percentile of the fluorescence among the plurality of the first reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 96th percentile of the fluorescence among the plurality of the first reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 97th percentile of the fluorescence among the plurality of the first reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 98th percentile of the fluorescence among the plurality of the first reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 99th percentile of the fluorescence among the plurality of the first reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is approximately a full percentile of fluorescence among the plurality of first reference cell samples for the fluorescent signal or higher.

[0126] In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a lower fluorescence than the 5th percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a lower fluorescence than the 4th percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a lower fluorescence than the 3rd percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a lower fluorescence than the 2nd percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a lower fluorescence than the 1st percentile of the fluorescence among the plurality of second reference cell samples for the fluorescent signal. In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than the total percentile of fluorescence among the plurality of second reference cell samples for the fluorescent signal.

[0127] In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 90th percentile of fluorescence among a plurality of a first reference population of cells for the respective fluorescent signal and a fluorescence lower than the 10th percentile of fluorescence among a plurality of a second reference population of cells for the same respective fluorescent signal.

[0128] In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 95th percentile of fluorescence among a plurality of a first reference population of cells for the respective fluorescent signal and a fluorescence lower than the 5th percentile of fluorescence among a plurality of a second reference population of cells for the same respective fluorescent signal.

[0129] In some embodiments, the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence higher than the 99th percentile of fluorescence among a plurality of a first reference population of cells for the respective fluorescent signal and a fluorescence lower than the 1st percentile of fluorescence among a plurality of a second reference population of cells for the same respective fluorescent signal.

[0130] It is within the level of the art to select a particular static fluorescence threshold gate for a particular fluorescent signal. Factors to consider in selecting a static fluorescence threshold gate include the degree of false positives or false negatives that are acceptable in stained samples. In certain embodiments, the static fluorescence threshold gate is selected to completely exclude the unstained or negative population. In some embodiments, the static fluorescence threshold gate is selected to include as much of the positive population as possible, excluding statistical outliers in some cases. In some embodiments, the static fluorescence gate threshold is set conservatively in the positive population when there is a wide range between the positive fluorescence and the negative / unstained fluorescence (e.g., lower than 1 percentile or lower than all percentiles among a plurality of the second reference population). In some embodiments, a wide range in the fluorescence of the fluorescent signal exists when the fluorescent positive and unstained / negative populations of cells differ by more than 2-fold, for example, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more. In some embodiments, a wide range in the fluorescence of the fluorescent signal exists when the fluorescent positive and unstained / negative populations of cells differ by more than 5-fold. In some embodiments, a wide range in fluorescence of the fluorescent signal is present when the fluorescent positive and unstained / negative populations of cells differ by more than 10-fold.

[0131] In some embodiments, for low frequency attributes (e.g., those where the frequency of cells positive for the attribute among a population of cells is expected to be lower than 5%), additional mean fluorescence intensity (MFI) analysis can be used to assist in threshold determination. In such an example, the percentile information described (e.g., the 99th and 1st percentiles of fluorescence among a plurality of first and second reference samples, respectively) is informative and helps determine the boundaries or edges of the population, but in some cases can be distorted by rare events or outliers in a population that potentially has a small number of events to begin with for low frequency attributes. MFI is a median measurement and is less susceptible to distortion from outliers. Therefore, in some embodiments, MFI can be used as a tertiary analysis. In some embodiments, MFI can be used to identify the center or densest part of a low frequency population. In some embodiments, once the centers of the positive / stained and negative / unstained populations are determined based on MFI, and the edge of the difference population is determined based on percentiles (e.g., the 99th percentile of fluorescence between the first reference cell sample and the 1st percentile of fluorescence between the second reference cell sample), a threshold can be set to separate the two populations. Setting a threshold value taking into account the MFI for a particular fluorescent signal is within the level of skill of a person skilled in the art.

[0132] In some embodiments, the set fluorescence determined to be the static fluorescence gate threshold according to any of the embodiments provided can be rounded to an even number on the logarithmic flow axis. In some embodiments, selecting an even number can optimize workflow. In some embodiments, for each of one or more fluorescent signals, the static fluorescence threshold gate is rounded to the nearest 10, 100, or 1,000 on the logarithmic flow axis.

[0133] B. Application of a static gating threshold and identification of cells In some embodiments, the methods provided include applying a static fluorescence threshold gate for each of one or more fluorescent signals described herein, e.g., in Section II, Part A, to flow cytometry data comprising a plurality of cytometry events from a test population.

[0134] Among the methods provided herein are methods of applying a static fluorescence threshold gate using flow cytometry, the method including: (1) receiving flow cytometry data including a plurality of cytometry events from a test population of 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 according to any of the methods provided for setting a static fluorescence threshold gate.

[0135] For example, provided herein is a method of applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test population of 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 comprises: (a) cytometry events measured by flow cytometry for one or more fluorescent signals for a plurality of at least two reference populations of cells each sorted according to the one or more fluorescent signals, wherein the plurality of at least two reference populations comprises: (i) a population of unlabeled, unstained cells each sorted according to the one or more fluorescent signals; and (ii) a second reference population of cells, each of the plurality of second reference population of cells derived from one of the plurality of first reference population of cells, and each of the plurality of second reference population of cells comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals; and (b) applying a static fluorescence threshold gate for each of the one or more fluorescent signals, which is determined from setting, for each fluorescent signal, a gate that is (i) higher than the 90th percentile of the fluorescence of the plurality of first reference population of cells for the respective fluorescent signal of each of the one or more fluorescent signals; and (ii) lower than the 10th percentile of the fluorescence of the plurality of second reference population of cells for the same respective fluorescent signal.

[0136] In some embodiments, the methods provided further include identifying, for each of the one or more fluorescent signals, a subset of cytometry events from the test population of cells having a fluorescent signal above a static threshold gate.

[0137] Also provided herein is a method of selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test population of 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 comprises: (a) a plurality of first reference populations of cells each comprising a population of unstained cells that are not labeled with the one or more fluorescent signals; and (ii) a plurality of second reference populations of cells each comprising a population of unstained cells that are not labeled with the one or more fluorescent signals; and (b) applying a static fluorescence threshold gate for each of the one or more fluorescent signals, the static fluorescence threshold gate being determined from setting such that, for each fluorescent signal, it is (i) higher than the 90th percentile of the fluorescence of the first reference population of cells for each respective fluorescent signal of each of the one or more fluorescent signals; and (ii) lower than the 10th percentile of the fluorescence of the second reference population of cells for the same respective fluorescent signal; and (3) identifying, for each of the one or more fluorescent signals, a subset of cytometry events from the test population of cells having a fluorescent signal above the static threshold gate.

[0138] In some embodiments of the provided methods, a fluorescent feature or characteristic of the identified subset of cells for at least one of the fluorescent signals is determined. In some embodiments, a fluorescent feature or characteristic of the identified subset of cells for each of the one or more fluorescent signals is determined.

[0139] In some embodiments, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, the method further comprises evaluating the fluorescent intensity of at least one fluorescent signal of the identified subset of cytometry events.In some embodiments, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, the method further comprises evaluating the intensity of the fluorescent signal of the identified subset of cytometry events.In some embodiments, the fluorescent intensity is the average fluorescent intensity.

[0140] In some embodiments, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, the method further comprises determining a percentage of the identified subset of cytometry events compared to the total number of collected cytometry events. In some embodiments, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, the method further comprises determining a percentage of the identified subset of cytometry events for the respective fluorescent signal compared to the total number of collected cytometry events.

[0141] III. Definition Unless otherwise defined, all technical terms, notations, and other scientific, technical, or scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having commonly understood meanings have been defined herein for clarity and / or quick reference, and the inclusion of such definitions herein should not be interpreted as necessarily representing a substantial departure from what is commonly understood in the art.

[0142] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that the embodiments and variations described herein include "consisting of" and / or "consisting essentially of" embodiments and variations.

[0143] The terms "at least one" and "one or more" may be understood to include any integer greater than or equal to 1, i.e., 1, 2, 3, 4, [...], etc. The term "plurality" may be understood to include any integer greater than or equal to 2, i.e., 2, 3, 4, 5, [...], etc.

[0144] The phrase "at least one of" with respect to a group of elements may be used herein to mean at least one element from a group of elements. For example, the phrase "at least one of" with respect to a group of elements may be used herein to mean the selection of one of the listed elements, a plurality of one of the listed elements, a plurality of each of the listed elements, or a plurality of a plurality of the listed elements.

[0145] Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to have individual numerical values ​​within that range, as well as all possible subranges specifically disclosed. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within the stated range, is encompassed by the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed by the claimed subject matter, subject to any specifically excluded limits in the stated ranges. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed by the claimed subject matter. This applies regardless of the breadth of the range.

[0146] The term "about" as used herein refers to the normal error range for each value that is easily known. Reference herein to "about" a value or parameter includes (and describes) an embodiment directed to the value or parameter itself. For example, a description that refers to "about X" includes the description of "X".

[0147] As used herein, "event" or "cytometry event" refers to the data measured by a flow cytometer from a single particle, such as a cell or a synthetic particle. Typically, the data measured from a single particle includes multiple parameters, including one or more light scattering parameters and at least one fluorescent signal parameter. Therefore, each event is represented as a vector of parameter measurements, with each measured parameter corresponding to one dimension of the data space.

[0148] "Fluorescent marker" or "fluorescence marker" refers to a fluorescent marker, including a fluorophore, that has the ability to absorb energy in a wavelength range and emit energy in a wavelength range other than the absorption range. As such, it refers to a fluorescent compound that can emit light when excited by light. It is understood that the term "fluorescent marker" or variations thereof can be used interchangeably with the term "fluorophore." The term "excitation wavelength" refers to the range of wavelengths at which a fluorophore absorbs energy. The term "emission wavelength" refers to the range of wavelengths at which a fluorophore emits energy or fluorescence.

[0149] As used herein, "fluorescence" or "fluorescence intensity" are terms that may be used interchangeably and refer to the output of a detection system that measures the fluorescence brightness from a fluorescence emission sample intensity of a specific fluorescent signal emission, e.g., emitted from a fluorescent marker, e.g., a fluorophore. Fluorescence intensity is the amount of light (photons) emitted by a fluorescent marker after it absorbs light or other electromagnetic radiation.

[0150] As used herein, "mean fluorescence intensity" or "MFI" refers to the average of the fluorescence intensity in a particular fluorescence channel.

[0151] As used herein, a "gate" refers to a set of boundary points that identify a subset of data of interest. In cytometry, a gate may demarcate a group of events of particular interest. In some embodiments, a gate may be a window that encompasses a particular event. In other embodiments, a gate may be a "threshold gate." As used herein, "gating" refers to the process of defining a gate for a given set of data.

[0152] As used herein, a "threshold gate" refers to a gate that is set at a particular fluorescence intensity and separates cells of a cell population that emit a fluorescence signal or intensity above that particular intensity from cells of the cell population that emit a fluorescence signal or intensity below that particular intensity, and thus in some embodiments, defines an open region in a multidimensional space.

[0153] As used herein, a "static threshold gate" refers to a fixed or non-changing threshold gate for a given fluorescent signal that can be applied to multiple stained or labeled samples. In some embodiments, a static threshold gate is set for each attribute (e.g., marker) that is labeled with a fluorescent signal in a flow cytometry assay, e.g., a multi-color flow cytometry assay.

[0154] As used herein, "labeled" refers to a state in which a detectable label, such as a fluorescent marker or stain, is attached.For example, the cells of a cell population can be labeled with one or more fluorescent markers so that one or more fluorescent signals can be measured by a flow cytometer.

[0155] As used herein, "percentile" refers to a score below which a given percentage of the scores in its frequency distribution fall.

[0156] As used herein, the reference to an "enriched" population of cells refers to one or more specific cell types or subsets or cell populations that have been subjected to an enrichment, isolation or selection process to increase the number or percentage of the cell type or population, for example, compared to the percentage in the starting population of cells.Thus, it refers to increasing the percentage or frequency of such cells, or compared to other cell types, for example, by positive selection based on the marker expressed by the population or cell, or by negative selection based on the marker that is not present on the cell population or cell to be depleted.The term does not require the complete removal of other cells, cell types, or populations from the composition, and does not require that the cells that are so enriched are present at or near 100% in the enriched composition.In some embodiments, the enriched population of cells contains more than 50%, 60%, 70%, 80%, 90%, 95% or more of a specific cell type or subset. For example, the enriched population of cells may be an enriched T cell population that contains more than 50%, 60%, 70%, 80%, 90%, 95% or more of T cells (e.g., CD3+ cells) or CD4+ or CD8+ subsets thereof. Reference to positive selection or enrichment for a particular type of cell, e.g., cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells. For example, in some embodiments, the selection of one of the CD4+ or CD8+ populations enriches for said populations for either the CD4+ or CD8+ population, but may also contain some residual or small percentage of other unselected cells, which may in some cases include the other of the CD4 or CD8 population that still exists in the enriched population.

[0157] As used herein, the statement that a cell or a group of cells is "positive" for a particular marker refers to the detectable presence of a particular marker, typically a surface marker, on or in the cell.When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, and the staining is detectable by flow cytometry at a level substantially higher than the staining detected by carrying out the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to the level for cells known to be positive for the marker, and / or at a level substantially higher than the level for cells known to be negative for the marker.

[0158] As used herein, the statement that a cell or a group of cells is "negative" for a particular marker refers to the absence of the substantial detectable presence of a particular marker, typically a surface marker, on or in the cell.When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, and the staining is not detected by flow cytometry at a level substantially higher than the staining detected by carrying out the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than the level for cells known to be positive for the marker, and / or at a level substantially similar to the level for cells known to be negative for the marker.

[0159] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human.

[0160] IV. Illustrative Embodiments Provided embodiments include: 1. A method for determining a static fluorescence threshold gate, comprising: (1) measuring cytometry events by flow cytometry for one or more fluorescent signals for a plurality of at least two reference cell samples, wherein the plurality of at least two reference cell samples comprises: (a) a plurality of first reference cell samples, each of which comprises a population of unstained cells that are not labeled with said one or more fluorescent signals; and (b) a plurality of second reference cell samples, each of the plurality of second reference cell samples being derived from the same cellular source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals. Including, said measuring; and (2) establishing a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal being: (a) a fluorescence greater than a 90th percentile of the fluorescence of said plurality of said first reference cell sample for each respective fluorescent signal of said one or more fluorescent signals; (b) a fluorescence that is less than a tenth percentile of the fluorescence of said plurality of said second reference cell sample for the same respective fluorescent signal. Set it as follows: The method comprising: 2. A method for selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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 embodiment 1; and (3) for each of said one or more fluorescent signals, identifying a subset of cytometry events having a fluorescent signal above said static threshold gate. The method comprising: 3. A method for selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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 comprises: (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, wherein the plurality of at least two reference cell samples comprises: (i) a plurality of first reference cell samples, each of which comprises a population of unstained cells that are not labeled with said one or more fluorescent signals; and (ii) a plurality of second reference cell samples, each of said plurality of second reference cell samples being derived from the same cellular source type as said plurality of first reference cell samples, and each of said plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of said one or more fluorescent signals. The cytometry event comprises: (b) establishing a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal comprising: (i) a fluorescence greater than a 90th percentile of the fluorescence of said plurality of said first reference cell sample for each respective fluorescent signal of said one or more fluorescent signals; (ii) a fluorescence that is less than a tenth percentile of the fluorescence of said plurality of said second reference cell sample for the same respective fluorescent signal. Set it as follows: said applying being determined from; and (3) for each of said one or more fluorescent signals, identifying a subset of cytometry events having a fluorescent signal above said static threshold gate. The method comprising: 4. A method for applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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 embodiment 1. The method comprising: 5. A method for applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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 comprises: (a) cytometry events measured by flow cytometry for said one or more fluorescent signals for a plurality of at least two reference cell samples each sorted according to said one or more fluorescent signals, said plurality of at least two reference cell samples comprising: (i) a plurality of first reference cell samples, each of which comprises a population of unstained cells that are not labeled with said one or more fluorescent signals; and (ii) a plurality of second reference cell samples, each of the plurality of second reference cell samples being derived from the same cellular source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals. The cytometry event comprises: (b) establishing a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal comprising: (i) a fluorescence that is greater than a 90th percentile of the fluorescence of said plurality of first reference cell sample o for each respective fluorescence signal of said one or more fluorescence signals; (ii) a fluorescence that is less than a tenth percentile of the fluorescence of said plurality of said second reference cell sample for the same respective fluorescent signal. Set it as follows: The applying is determined from The method comprising: 6. The method of any one of embodiments 1-5, wherein each of the plurality of test cell samples and the first and second reference cell samples are from the same cellular source type. 7. The method of any one of embodiments 1 to 6, wherein the source type of the cells is a cell line. 8. The method of any one of embodiments 1-6, wherein the source type of the cells is a primary cell population from a subject. 9. The method of embodiment 8, wherein the source type of cells of each of said test cell samples and each of said plurality of said first and second reference cell samples are from different subjects. 10. The method of embodiment 9, wherein each different subject has the same or a similar disease or condition. 11. The method of any of the preceding claims, wherein the source type of the cells is a whole blood sample, an apheresis sample, or a leukapheresis sample. 12. The method of any of the preceding embodiments, wherein the source type of the cells is an enriched population of cells, optionally an enriched population of T cells. 13. The method of any of the preceding claims, wherein the source type of cells is an engineered population of cells comprising a nucleic acid encoding a recombinant protein, and optionally said recombinant protein has been introduced into said population of cells by gene transfer, optionally by transduction. 14. The method of any one of embodiments 1 to 13, wherein the source type of the cells is a cell therapy. 15. The method of any of the preceding claims, wherein the plurality of first reference cell samples comprises more than 2, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cell samples. 16. The method of any of the preceding claims, wherein the plurality of second reference cell samples comprises more than 2, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cell samples. 17. The method of any of embodiments 1-16, wherein each of the plurality of first and second reference cell samples is a source type of cells from a subject having a disease or condition in a clinical trial. 18. The method of any one of embodiments 1 to 16, wherein the source type of the cells is an autologous cell therapy, and each of the first and second reference cell samples is a sample of the cell therapy from a subject in a clinical trial for testing the cell therapy. 19. The method of embodiment 14 or embodiment 18, wherein the cell therapy is T cell therapy, optionally CAR-T cell therapy, TCR-T cell therapy, or TIL therapy. 20. The method of embodiment 14 or embodiment 18, wherein the cell therapy is NK cell therapy. 21. The method of embodiment 14 or embodiment 18, wherein the cell therapy is a stem cell therapy. 22. The method according to any one of embodiments 1 to 21, wherein each of the test cell sample and the second reference cell sample has been subjected to cell staining using one or more staining reagents for labeling the cells with the one or more fluorescent signals. 23. The method of embodiment 22, wherein the one or more staining reagents comprise a binding agent specific for a marker and a fluorescent dye capable of emitting one of the one or more fluorescent signals. 24. The method of embodiment 23, wherein the marker is a cell surface marker or a survival marker. 25. The method of embodiment 23 or embodiment 24, wherein at least one marker is a frequency attribute that is expressed or suspected to be expressed in at least 5% or more of the cells in the sample. 26. The method of embodiment 23 or embodiment 24, wherein at least one marker is a low frequency attribute that is expressed or suspected to be expressed in less than 5% of cells in the sample. 27. The method of any of embodiments 1-26, wherein the one or more fluorescent signals comprise two or more different fluorescent signals. 28. A method according to any of embodiments 22 to 27, wherein the cell staining is a multi-color cell staining for labeling the cells with two or more different fluorescent signals, each staining reagent labeling a different marker with a different fluorescent signal. 29. The method of embodiment 27 or embodiment 28, wherein the two or more different fluorescent signals comprise 2 to 10 fluorescent signals, optionally 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, or 6 or about 6 fluorescent signals. 30. The method of any of embodiments 27-29, wherein each of the two or more different fluorescent signals has a different emission spectrum and / or the peak emission spectra of each fluorescent signal do not overlap. 31. The method of any of embodiments 27 to 30, wherein the two or more different fluorescent signals are signals emitted by a dye selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605. 32. The method according to any of embodiments 22 to 31, wherein the cell staining of each of the test cell sample and the second reference cell sample is carried out with the same staining reagent and using the same protocol conditions. 33. The method of any of embodiments 1-32, wherein said plurality of second reference cell samples comprises duplicate samples of the same stained cells. 34. The method of any of the preceding claims, wherein said plurality of second reference cell samples comprises triplicate samples of the same stained cells. 35. The method of any of embodiments 1 to 34, wherein the static fluorescence threshold gate for at least one fluorescent signal is a fluorescence greater than the 95th percentile of the fluorescence among the plurality of the first reference cell sample for the fluorescent signal. 36. The method of any of embodiments 1 to 34, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence greater than the 97th percentile of fluorescence among the plurality of the first reference cell sample for the fluorescent signal. 37. The method of any of embodiments 1 to 34, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence greater than the 99th percentile of fluorescence among the plurality of the first reference cell sample for the fluorescent signal. 38. The method of any of the preceding claims, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than the 5th percentile of the fluorescence among the plurality of the second reference cell sample for the fluorescent signal. 39. The method of any of the preceding claims, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than the third percentile of the fluorescence among the plurality of the second reference cell sample for the fluorescent signal. 40. The method of any of embodiments 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than 1 percentile of the fluorescence among the plurality of the second reference cell sample for the fluorescent signal. 41. The method of any one of embodiments 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and the population of stained cells for the fluorescent signal differs by more than two-fold, setting the static fluorescence is done at less than one percentile of the fluorescence or less than all percentiles of the fluorescence between the plurality of second reference cell samples for the fluorescent signal. 42. The method of any one of embodiments 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and the population of stained cells for the fluorescent signal differs by more than 5-fold, setting the static fluorescence is done at less than 1 percentile of the fluorescence or less than all percentiles of the fluorescence between the plurality of second reference cell samples for the fluorescent signal. 43. The method of any one of embodiments 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and the population of stained cells for the fluorescent signal differs by more than 10-fold, setting the static fluorescence is done at less than 1 percentile of fluorescence or less than all percentiles of fluorescence between the plurality of second reference cell samples for the fluorescent signal. 44. The method of any one of embodiments 1 to 43, wherein for low frequency attributes, setting the static fluorescence threshold gate further comprises identifying a mean fluorescence intensity (MFI) for one or more of the one or more fluorescent signals. 45. A method according to any one of embodiments 1 to 44, wherein for each of the one or more fluorescent signals, the static fluorescence threshold gate is an even number on the logarithmic flow axis. 46. ​​The method of any one of embodiments 1 to 45, wherein for each of the one or more fluorescent signals, the static fluorescence threshold gate is rounded to the nearest 10, rounded to the nearest 100, or rounded to the nearest 1,000 on a logarithmic flow axis. 47. The method of any one of embodiments 2, 3, and 6-46, further comprising identifying a subset of cells from said test population of cells having fluorescent signals above said static threshold gate for at least two of said one or more fluorescent signals, at least three of said one or more fluorescent signals, at least four of said one or more fluorescent signals, at least five of said one or more fluorescent signals, at least six of said one or more fluorescent signals, or at least seven or more of said one or more fluorescent signals. 48. The method of any one of embodiments 2, 3 and 6 to 47, further comprising identifying a subset of cells from said test population of cells having a fluorescent signal above said static threshold gate for all of said one or more fluorescent signals. 49. The method of any one of embodiments 1 to 47, wherein the static fluorescence threshold gate comprises at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells of the test sample. 50. The method of any one of embodiments 1 to 49, wherein the setting of a static fluorescence threshold gate for each of the one or more fluorescent signals is set to include at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells of the test sample. 51. The method of any of embodiments 2, 3 and 6 to 50, further comprising, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, evaluating the fluorescent intensity of the at least one fluorescent signal of the identified subset of cytometry events. 52. The method of any of embodiments 2, 3 and 6 to 50, further comprising, for each of the identified subsets of cytometry events for each of the one or more fluorescent signals, evaluating the intensity of the fluorescent signal of the identified subset of cytometry events. 53. The method of embodiment 51 or embodiment 52, wherein the fluorescence intensity is a mean fluorescence intensity. 54. The method of any of embodiments 2, 3 and 6 to 53, further comprising determining, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, a percentage of the identified subset of cytometry events compared to the total number of cytometry events collected. 55. The method of any of embodiments 2, 3 and 6 to 53, further comprising, for each of the identified subsets of cytometry events for each of the one or more fluorescent signals, determining a percentage of the identified subset of cytometry events for the respective fluorescent signal compared to the total number of cytometry events collected. 56. A method according to any of the preceding claims, comprising correcting the one or more fluorescent signals by the flow cytometer. 57. The method of embodiment 56, wherein the softening is performed at least once a day. EXAMPLES

[0161] V. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [Example 1]

[0162] Development of static gating thresholds for flow cytometric analysis Fluorescence Minus One (FMO) or isotype gating controls for each fluorochrome-conjugated antibody are the industry standard gating controls for flow cytometry methods used in quality control (QC) situations. The advantage of these gating approaches is that they allow for objective gate placement, but they also have drawbacks, including reduced throughput, increased assay complexity (e.g., due to time constraints, failure rates, and limited sample volume), and increased cost of goods. To address these drawbacks, static gating thresholds for flow cytometry analysis were developed.

[0163] Flow cytometry was used to determine the fluorescence thresholds for six attributes (Table E1). 344 clinical lots of samples were used. Each clinical sample was derived from a manufactured lot of drug product containing CAR T cells. Negative staining analysis was performed in duplicate on unstained samples, and positive staining analysis was performed in triplicate on stained samples (Table E1). Table E1.

[0164] [Table 1]

[0165] Fluorescence expression was characterized using Flowjo analysis software (v10.6). Analysis of unstained samples was used to characterize the upper boundary of unstained or negative fluorescence for each attribute. For unstained or negative samples, this included determining the 99th percentile of fluorescence considered to be the maximum upper boundary of unstained or negative fluorescence; determining the 95th percentile of fluorescence considered to be the secondary upper boundary of unstained or negative fluorescence; and, in some cases, determining the mean fluorescence intensity (MFI) (Figure 1B, left).

[0166] Analysis of positively stained replicates was used to characterize the lower boundary of positive fluorescence for each attribute. For positively stained samples, this included determining the 1st percentile of fluorescence considered as the minimal lower boundary of stained fluorescence; determining the 5th percentile of fluorescence considered as the secondary lower boundary of stained fluorescence; and, in some cases, determining the MFI (FIG. 1B, right).

[0167] MFI was used to identify the center or densest part of the cell population, which may be particularly useful when analyzing low-frequency attributes, where the confidence in the percentile of fluorescence alone may be distorted, for example, by outliers in cell populations (either positively or negatively stained cell populations) with a relatively small number of cells that exhibit fluorescence. The centers of the negatively and positively stained cell populations were determined based on MFI, and the edges of the cell populations were then determined by identifying the 99th and / or 95th percentile of fluorescence for the negatively stained cell population, or the 1st and / or 5th percentile of fluorescence for the positively stained cell population. This allowed setting a threshold value for separating the negatively stained cell population from the positively stained cell population. While MFI analysis was not necessary when analyzing dense and robust populations of cells, e.g., high-frequency attributes, MFI analysis was used as an additional way to characterize the fluorescence properties of sparse populations of cells, e.g., low-frequency attributes.

[0168] Figures 2A and 2B show examples of negative or unstained analysis (Figure 2A) and positive stained analysis (Figure 2B) for a representative attribute using the AF700 fluorophore. As shown in Figure 2A, the maximum of the 95th percentile for unstained or negative fluorescence had a relative fluorescence intensity of 62.9, and the maximum of the 99th percentile for unstained or negative fluorescence had a relative fluorescence intensity of 173.00. As shown in Figure 2B, the 1st percentile of fluorescence for positively stained samples had a relative fluorescence intensity of 3,597.0, and the 5th percentile of fluorescence for positively stained samples had a relative fluorescence intensity of 5,324.0. Based on this data, a static fluorescence threshold for this attribute was set at 3,000.

[0169] Fluorescence thresholds for each attribute that distinguish positive staining from no staining or negative staining were then established.

[0170] For frequency attributes (frequency ≥ 5% in samples), sometimes referred to as high frequency attributes, the fluorescence threshold was established by: (1) excluding the unstained or negative population by setting a fluorescence threshold above the maximum of the 99th percentile for the unstained or negative population; (2) including as much of the positive population as possible while excluding statistical outliers; (3) conservatively placing the threshold for the positive population when there is a wide range between positive and negative or unstained fluorescence; and (3) rounding the threshold to an even number on the logarithmic flow axis to optimize the workflow. By applying this criterion to the frequency attributes using the analysis shown in Figures 2A and 2B, the fluorescence threshold was established at 3,000. Whenever possible, the fluorescence threshold was set below the minimum 5th percentile for the positive population for each attribute in order to include as much of the positive population as possible while excluding statistical outliers, and this was done for each of the frequency attributes analyzed.

[0171] For low frequency attributes (frequency ≦5% in samples), fluorescence thresholds were established by (1) excluding unstained or negative populations by setting a fluorescence threshold above the maximum of the 99th percentile for unstained or negative populations, (2) including as much of the positive population as possible while excluding statistical outliers, (3) using MFI analysis to aid in threshold determination, and (3) rounding the threshold to even numbers on the log-flow axis to optimize the workflow. Whenever possible, fluorescence thresholds were set below the minimum 5th percentile for the positive population for each attribute in order to include as much of the positive population as possible while excluding statistical outliers. Because the dye for attribute 5 (Aqua, as shown in Table E2 below) had a high level of background, the minimum 5th percentile value of 1506.3 was rounded upward to the nearest 2000 whole-scale marker in order to preferentially include the positive population over the unstained or negative populations.

[0172] MFI analysis involved characterization of the unstained or negative and positive populations to identify the mean fluorescence intensity or the 50th percentile of intensity, which is typically where the middle and densest part of the population is on the fluorescence scale. MFI analysis was then used to help determine the fluorescence threshold / boundaries for each attribute.

[0173] Table E2 below summarizes the unstained or negative staining analysis and the positive staining analysis for each of the six reportable attributes and provides the established fluorescence thresholds for each attribute. Attributes 1-3 and 6 are frequent attributes, while attributes 4 and 5 are low frequency attributes.

[0174] [Table 2]

[0175] Fluorescence thresholds for low frequency attributes 4 and 5 were determined in part by MFI due to skewness of the 1st and 5th percentile values ​​in the positive staining analysis.

[0176] To determine whether the static fluorescence threshold gates are comparable to current cluster gating practices, FMO controls for each attribute were analyzed using fluorescence thresholds for frequency attributes, including attributes 1, 2, and 6. FMO-gated attributes that were not threshold-gated were included in this evaluation to determine whether upstream gating changes with the fluorescence threshold would affect the reported values. Table E3 summarizes the results from the comparative analysis. The static fluorescence threshold gates for each of the frequency attributes were below the frequencies used to set the FMO-guided gates (Figure 3; Table E3). As shown in Table E3, the static fluorescence threshold gates for attributes 1, 2, and 6 were shown to be comparable to the current gating method using FMO analysis. This demonstrates that the static fluorescence threshold gating adequately excludes background fluorescence when applied to FMO samples, and the reported values ​​are comparable to the current gating method of FMO analysis.

[0177] [Table 3]

[0178] To assess the accuracy of static fluorescence threshold gating, precision based on interoperator coefficient of variation (CV) percentage (%) was assessed among three operators in 97 patient sample lots for four frequency attributes (attributes 1–3 and 6).

[0179] As shown in Table E4, static fluorescence threshold gating was precise between operators, with <3% CV observed for all attributes evaluated.

[0180] [Table 4]

[0181] Lin's concordance correlation test was used to characterize correlations between static fluorescence threshold gating reportables and reportable attributes. The concordance correlation results are detailed in Table E5 below.

[0182] [Table 5]

[0183] To assess background fluorescence using static fluorescence threshold gating applied to the FMO samples, static fluorescence threshold gating was applied to three development samples, each with an FMO sample stained using each of the same six attributes and then analyzed using the appropriate fluorescence channel. Background percentages were reported as a percent of the parent population. As previously determined (data not provided), the maximum acceptable background for FMO evaluation was 1.0% of the parent population for all populations except for population 4, which had a maximum acceptable background of 0.2%. FMO comparison results showing the maximum background for each population are provided in Table E6 below. As shown in Table E6, the maximum background for each population was acceptable as it was well below the maximum acceptable background percentage of 1%. This demonstrates that the static fluorescence threshold gates for each of the six attributes were effective in eliminating background staining.

[0184] [Table 6]

[0185] The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the descriptions and teachings herein. Such variations can be made without departing from the true scope and spirit of the present disclosure and are intended to fall within the scope of the present disclosure.

Claims

1. 1. A method for determining a static fluorescence threshold gate, 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 at least two reference cell samples comprising: (a) a plurality of first reference cell samples, each of which comprises a population of unstained cells that are not labeled with one or more fluorescent signals; and (b) a plurality of second reference cell samples, each of the plurality of second reference cell samples being derived from the same cellular source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals. Including, said measuring; and (2) establishing a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal being: (a) a fluorescence greater than a 90th percentile of the fluorescence of the plurality of the first reference cell sample for each respective fluorescent signal of the one or more fluorescent signals; (b) a fluorescence that is less than a tenth percentile of the fluorescence of said plurality of said second reference cell sample for the same respective fluorescent signal. Set it as follows: The method comprising:

2. 1. A method for selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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, the static fluorescence threshold gate for each of the one or more fluorescent signals being 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 having a fluorescent signal above the static threshold gate. The method comprising:

3. 1. A method for selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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, the static fluorescence threshold gate for each of the one or more fluorescent signals comprising: (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, wherein the plurality of at least two reference cell samples comprises: (i) a plurality of first reference cell samples, each of which comprises a population of unstained cells that are not labeled with said one or more fluorescent signals; and (ii) a plurality of second reference cell samples, each of the plurality of second reference cell samples being derived from the same cellular source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals. The cytometry event comprises: (b) establishing a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal comprising: (i) a fluorescence greater than a 90th percentile of the fluorescence of the plurality of first reference cell samples for each respective fluorescent signal of the one or more fluorescent signals; (ii) a fluorescence that is less than a tenth percentile of the fluorescence of said plurality of said second reference cell sample for the same respective fluorescent signal. Set it to said applying being determined from; and (3) for each of the one or more fluorescent signals, identifying a subset of cytometry events from the test cell sample having a fluorescent signal above the static threshold gate. The method comprising:

4. 1. A method of applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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, the static fluorescence threshold gate for each of the one or more fluorescent signals being determined according to the method of claim 1. The method comprising:

5. 1. A method of applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including a plurality of cytometry events from a test cell sample including 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, the static fluorescence threshold gate for each of the one or more fluorescent signals comprising: (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, wherein the plurality of at least two reference cell samples comprises: (i) a plurality of first reference cell samples, each of which comprises a population of unstained cells that are not labeled with said one or more fluorescent signals; and (ii) a plurality of second reference cell samples, each of the plurality of second reference cell samples being derived from the same cellular source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples comprising at least one population of stained cells that are labeled with at least one of the one or more fluorescent signals. The cytometry event comprises: (b) establishing a static fluorescence threshold gate for each of the one or more fluorescent signals, the threshold gate for each fluorescent signal comprising: (i) a fluorescence greater than a 90th percentile of the fluorescence of the plurality of first reference cell samples for each respective fluorescent signal of the one or more fluorescent signals; (ii) a fluorescence that is less than a tenth percentile of the fluorescence of said plurality of said second reference cell sample for the same respective fluorescent signal. Set it as follows: The applying is determined from The method comprising:

6. The method of any of claims 1 to 5, wherein each of the plurality of test cell samples and the first and second reference cell samples are from the same cellular source type.

7. The method of any one of claims 1 to 6, wherein the source type of the cells is a cell line.

8. The method of any one of claims 1 to 6, wherein the source type of the cells is a primary cell population from a subject.

9. 9. The method of claim 8, wherein the source type of cells of each of the test cell samples and each of the plurality of the first and second reference cell samples are from different subjects.

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

11. The method according to any of claims 1 to 10, wherein the source type of the cells is a whole blood sample, an apheresis sample, or a leukapheresis sample.

12. The method of any one of claims 1 to 11, wherein the source type of cells is an enriched population of cells, optionally an enriched population of T cells.

13. 13. The method of any of claims 1 to 12, wherein the source type of cells is an engineered population of cells comprising a nucleic acid encoding a recombinant protein, optionally said recombinant protein having been introduced into said population of cells by gene transfer, optionally by transduction.

14. The method according to any one of claims 1 to 13, wherein the source type of the cells is cell therapy.

15. The method of any of claims 1-14, wherein each of said plurality of first and second reference cell samples is of a source type of cells from a subject having a disease or condition in a clinical trial.

16. 15. The method of any of claims 1-14, wherein the source type of cells is an autologous cell therapy, and each of the first and second reference cell samples is a sample of the cell therapy from a subject in a clinical trial for testing the cell therapy.

17. The method of any of claims 14 to 16, wherein the cell therapy is T cell therapy, optionally CAR-T cell therapy, TCR-T cell therapy, or TIL therapy.

18. The method according to any one of claims 14 to 16, wherein the cell therapy is NK cell therapy.

19. The method according to any one of claims 14 to 16, wherein the cell therapy is a stem cell therapy.

20. 20. The method of any of claims 1 to 19, wherein each of the test cell sample and the second reference cell sample has been subjected to cell staining with one or more staining reagents for labeling the cells with the one or more fluorescent signals.

21. 21. The method of claim 20, wherein each of the one or more staining reagents comprises a binding agent specific for a marker and a fluorescent dye capable of emitting one of the one or more fluorescent signals.

22. The method of claim 21 , wherein the marker is a cell surface marker or a survival marker.

23. 23. The method of claim 21 or claim 22, wherein at least one marker is a frequency attribute that is expressed or suspected to be expressed in at least 5% or more of the cells in each of the test cell sample and the second reference cell sample.

24. 24. The method of any of claims 21 to 23, wherein at least one marker is a low frequency attribute that is expressed or suspected to be expressed in less than 5% of cells in each of the test cell sample and the second reference cell sample.

25. The method of any of claims 1 to 24, wherein the one or more fluorescent signals comprise two or more different fluorescent signals.

26. 26. The method of any of claims 20 to 25, wherein the cell staining is a multi-color cell staining for labeling the cells with two or more different fluorescent signals, each staining reagent labeling a different marker with a different fluorescent signal.

27. 27. The method of claim 25 or claim 26, wherein each of the two or more different fluorescent signals has a different emission spectrum and / or the peak emission spectra of each fluorescent signal do not overlap.

28. The method according to any one of claims 25 to 27, wherein each of the two or more different fluorescent signals is a signal emitted by a dye selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

29. The method of any of claims 24 to 28, wherein the cell staining of each of the test cell sample and the second reference cell sample is carried out with the same staining reagent and using the same protocol conditions.

30. 30. The method of any of claims 1-29, wherein the plurality of second reference cell samples comprises duplicate samples of the same stained cells, and wherein the plurality of second reference cell samples comprises triplicate samples of the same stained cells.

31. 31. The method of any of claims 1-30, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence greater than a 99th percentile of fluorescence among the plurality of the first reference cell sample for the fluorescent signal.

32. 32. The method of any of claims 1-31, wherein the static fluorescence threshold gate for at least one of the one or more fluorescent signals is a fluorescence lower than one percentile of fluorescence among the plurality of the second reference cell sample for the fluorescent signal.

33. 33. The method of any of claims 1 to 31 and 32, wherein the static fluorescence threshold gate is set below 1 percentile of fluorescence or below all percentiles of fluorescence among the plurality of second reference cell samples for the fluorescent signal if the fluorescence of a population of unstained cells and a population of stained cells for the fluorescent signal differs by more than 10-fold.

34. 34. The method of any of claims 1-33, wherein for low frequency attributes, the setting of the static fluorescence threshold gate further comprises identifying a mean fluorescence intensity (MFI) for one or more of the one or more fluorescent signals.

35. 35. The method of any of claims 1-34, wherein for each of the one or more fluorescent signals, the static fluorescence threshold gate is an even number on a logarithmic flow axis, and for each of the one or more fluorescent signals, the static fluorescence threshold gate is rounded to the nearest tens, rounded to the nearest hundreds, or rounded to the nearest thousandth on the logarithmic flow axis.

36. 36. The method of any of claims 1-35, wherein the static fluorescence threshold gate comprises at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells of the test cell sample.

37. 37. The method of any of claims 2, 3, and 6-36, further comprising, for each of the identified subsets of cytometry events for each of the one or more fluorescent signals, evaluating an intensity of the fluorescent signal of the identified subset of cytometry events.

38. 38. The method of claim 37, wherein the fluorescence intensity is a mean fluorescence intensity.

39. 39. The method of any of claims 2, 3, and 6-38, further comprising, for the identified subset of cytometry events for at least one of the one or more fluorescent signals, determining a percentage of the identified subset of cytometry events compared to a total number of collected cytometry events.

40. 39. The method of any of claims 2, 3, and 6-38, further comprising, for each of the identified subset of cytometry events for each of the one or more fluorescent signals, determining a percentage of the identified subset of cytometry events for the respective fluorescent signal compared to a total number of cytometry events collected.

41. The method of any of claims 1 to 40, comprising detection of said one or more fluorescent signals by said flow cytometer.