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 on December 16, 2020, entitled "THRESHOLD GATING FOR FLOW CYTOMETRY METHODS", the content of which is hereby incorporated by reference in its entirety.

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

Background Art

[0003] Fluorescence Minus One (FMO) or isotype gating controls for each fluorescent dye - conjugated antibody are industry - standard gating controls for flow cytometry methods used in quality control (QC) situations. The advantage of these gating approaches is that they enable objective gate placement, but these approaches also have drawbacks, including reduced throughput, increased assay complexity (e.g., due to time constraints, failure rates, and sample volume limitations), and increased cost of goods. An improved gating approach is needed. Embodiments are provided that meet such a need.

Summary of the Invention

[0004] Provided herein is a method for determining a static fluorescence threshold gate, comprising (1) measuring cytometric events by flow cytometry for one or more fluorescence signals of a plurality of at least two reference cell samples, wherein the plurality of at least two reference cell samples are (a) a plurality of first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescence signals; and (b) a plurality of second reference cell samples, each of which originates from the same cell source type as the plurality of first reference cell samples, and each of which contains one or more fluorescence signals. A method comprising (2) measuring a plurality of second reference cell samples, each comprising a population of stained cells labeled with at least one of the light signals; and (3) setting a static fluorescence threshold gate for each of one or more fluorescence signals such that the threshold gate for each fluorescence signal is (a) higher fluorescence than the 90th percentile of fluorescence of the plurality of first reference cell samples for each respective fluorescence signal of one or more fluorescence signals; and (b) lower fluorescence than the 10th percentile of fluorescence of the plurality of second reference cell samples for the same respective fluorescence signal.

[0005] Also provided herein is a method for selecting a subset of cytometric events from flow cytometry data, comprising: (1) receiving flow cytometry data comprising a plurality of cytometric events from a test cell sample comprising labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescence signals, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is determined according to the method of claim 1; and (3) for each of the one or more fluorescence signals, identifying a subset of cells from the test sample having a fluorescence signal above the static threshold gate.

[0006] Also provided herein is a method for selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data comprising multiple cytometry events from a test cell sample comprising labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) individually applying a static fluorescence threshold gate to each of the one or more fluorescence signals, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) cytometry events measured by flow cytometry for one or more fluorescence signals for a plurality of at least two reference cell samples, each sorted according to one or more fluorescence signals, wherein the plurality of at least two reference cell samples comprises (i) a plurality of first reference cell samples, each comprising a population of unstained cells not labeled with one or more fluorescence signals; and (ii) a plurality of second reference cell samples, A cytometry event comprising: (b) a cytometry event comprising a plurality of second reference cell samples, each of which originates from the same cell 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 labeled with at least one of one or more fluorescent signals; and (c) applying a static fluorescence threshold gate for each of the one or more fluorescent signals, determined by setting the threshold gate for each fluorescent signal to (i) have fluorescence higher than the 90th percentile of fluorescence of the plurality of first reference cell samples for each respective fluorescent signal of the one or more fluorescent signals; and (ii) have fluorescence lower than the 10th percentile of fluorescence of the plurality of second reference cell samples for the same respective fluorescent signal; and (d) identifying a subset of cells from a test cell sample having fluorescence signals above the static threshold gate for each of the one or more fluorescent signals.

[0007] Also provided herein is a method for applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data comprising multiple cytometry events from a test cell sample comprising labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescence signals, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is determined according to any method for determining a static fluorescence threshold gate disclosed herein.

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

[0009] In any part of such embodiments, each of the test cell samples, as well as each of the first and second reference cell samples, is from the same cell source type. In any part of such embodiments, the cell source type is a cell line. In any part of such embodiments, the cell source type is a primary cell population from a subject. In any part of such embodiments, the cell source type of each of the test cell samples, as well as each of the first and second reference cell samples, is from different subjects. In any part of such embodiments, each of the different subjects has the same or similar disease or condition. In any part of such embodiments, the cell source type is a whole blood sample, an apheresis sample, or a leukocyte apheresis sample. In any part of such embodiments, the cell source type is an enriched population of cells, optionally an enriched population of T cells. In any part of such embodiments, the cell source type is an engineered population of cells containing nucleic acids encoding recombinant proteins, optionally the recombinant proteins introduced into the cell population by gene transfer, optionally transduction. In some of such embodiments, the cell source type is cell therapy.

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

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

[0012] In any part of such embodiments, each of the first and second reference cell samples is a source type of cells from a subject having a disease or condition in a clinical trial. In any part of such embodiments, the source type of cells is autologous cell therapy, and each of the first and second reference cell samples is a sample of cell therapy from a subject in a clinical trial to test the cell therapy. In any part of such embodiments, the cell therapy is T cell therapy, as appropriate, CAR-T cell therapy, TCR-T cell therapy, or TIL therapy. In any part of such embodiments, the cell therapy is NK cell therapy. In any part of such embodiments, the cell therapy is stem cell therapy.

[0013] In any such embodiment, the cell therapy is CAR-T cell therapy. In any such embodiment, the cell therapy is TCR-T cell therapy. In any such embodiment, the cell therapy is TIL therapy.

[0014] In any part 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 any part of such embodiments, the one or more staining reagents include a marker-specific binder and a fluorescent dye capable of emitting one of one or more fluorescent signals. In any part of such embodiments, the marker is a cell surface marker or a survival marker.

[0015] In any part of such embodiments, each of one or more staining reagents comprises a marker-specific binder and a fluorescent dye capable of emitting one of one or more fluorescent signals.

[0016] In any part of 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 any part of 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 any part of such embodiments, the sample is each of a test cell sample and a second reference cell sample.

[0017] In any part of such embodiments, one or more fluorescent signals comprise two or more different fluorescent signals. In any part of such embodiments, cell staining is a multicolor cell staining for labeling cells using two or more different fluorescent signals, where each staining reagent labels a different marker using a different fluorescent signal. In any part of such embodiments, 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. In any part 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 any part of such embodiments, two or more different fluorescent signals are signals emitted by dyes selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

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

[0019] In any part of such embodiments, the cell staining of the test cell sample and the second reference cell sample is performed using the same staining reagent and under the same protocol conditions.

[0020] In any part of such embodiments, the plurality of second reference cell samples includes two sets of samples of the same stained cells. In any part of such embodiments, the plurality of second reference cell samples includes three sets of samples of the same stained cells.

[0021] In any part of such embodiments, the static fluorescence threshold gate for at least one fluorescence signal is fluorescence higher than the 95th percentile of fluorescence among multiple first reference cell samples for the fluorescence signal. In any part of such embodiments, the static fluorescence threshold gate for at least one of one or more fluorescence signals is fluorescence higher than the 97th percentile of fluorescence among multiple first reference cell samples for the fluorescence signal. In any part of such embodiments, the static fluorescence threshold gate for at least one of one or more fluorescence signals is fluorescence higher than the 99th percentile of fluorescence among multiple first reference cell samples for the fluorescence signal.

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

[0023] In any part of such embodiments, if the fluorescence of a population of unstained cells and a population of stained cells differs by more than twofold for the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence among multiple second reference cell samples for the fluorescence signal or less than the overall percentile of fluorescence. In any part of such embodiments, if the fluorescence of a population of unstained cells and a population of stained cells differs by more than fivefold for the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence among multiple second reference cell samples for the fluorescence signal or less than the overall percentile of fluorescence. In any part of such embodiments, if the fluorescence of a population of unstained cells and a population of stained cells differs by more than tenfold for the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence among multiple second reference cell samples for the fluorescence signal or less than the overall percentile of fluorescence.

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

[0025] In some embodiments of any such embodiment, for each of one or more fluorescence signals, the static fluorescence threshold gate is even on the logarithmic flow axis. In some embodiments of any such embodiment, for each of one or more fluorescence signals, the static fluorescence threshold gate is rounded to the nearest power of ten, the nearest power of one hundred, or the nearest power of one thousand on the logarithmic flow axis.

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

[0027] In some embodiments of any such embodiment, the method further comprises identifying a subset of cells from a test cell sample having fluorescence signals that exceed the static fluorescence threshold gate for all of the one or more fluorescence signals.

[0028] In some embodiments of any such embodiment, 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 in the test cell sample. In some embodiments of any such embodiment, the static fluorescence threshold gate for each of the one or more fluorescence signals is set to comprise at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells in the test cell sample.

[0029] In some of the provided embodiments, for an identified subset of cytometry events for at least one of one or more fluorescence signals, the method further includes evaluating the fluorescence intensity of at least one fluorescence signal in the identified subset of cytometry events. In some of the provided embodiments, for each of the identified subsets of cytometry events for each of one or more fluorescence signals, the method further includes evaluating the fluorescence intensity of the fluorescence signal in the identified subset of cytometry events. In some embodiments, the fluorescence intensity is the mean fluorescence intensity.

[0030] In some of the provided embodiments, for an identified subset of cytometry events for at least one of one or more fluorescence signals, the method further includes determining the percentage of the identified subset of cytometry events compared to the total number of cytometry events collected. In some embodiments, for each of the identified subsets of cytometry events for each of one or more fluorescence signals, the method further includes determining the percentage of the identified subset of cytometry events for each fluorescence signal compared to the total number of cytometry events collected.

[0031] In any of the provided embodiments, the method involves a flow cytometer detecting one or more fluorescence signals. calibration Includes. In some embodiments, calibration This is executed at least once a day. [Brief explanation of the drawing]

[0032] [Figure 1A]Figures 1A and 1B depict graphical representations of flow cytometry event analysis. Figure 1A depicts multiple cell populations from different samples, identifying 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 analyses of the negative, unstained cell population (left) and the positive, stained cell population (right), with the mean fluorescence intensity (MFI) and the 95th and 99th percentiles of fluorescence determined for the negative, unstained cell population (left), and the MFI and the 1st and 5th percentiles of fluorescence determined for the positive, stained cell population (right). [Figure 1B] Figures 1A and 1B depict graphical representations of flow cytometry event analysis. Figure 1A depicts multiple cell populations from different samples, identifying 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 analyses of the negative, unstained cell population (left) and the positive, stained cell population (right), with the mean fluorescence intensity (MFI) and the 95th and 99th percentiles of fluorescence determined for the negative, unstained cell population (left), and the MFI and the 1st and 5th percentiles of fluorescence determined for the positive, stained cell population (right). [Figure 2A] Figures 2A and 2B depict the analysis of negative, unstained cell populations (Figure 2A) and positively stained cell populations (Figure 2B) for frequency attributes using AF700 fluorophores. [Figure 2B] Figures 2A and 2B depict the analysis of negative, unstained cell populations (Figure 2A) and positively stained cell populations (Figure 2B) for frequency attributes using AF700 fluorophores. [Figure 3]Figure 3 illustrates the application of static fluorescence gating thresholds to fluorescence minus one (FMO) gating controls for each of the six tested attributes. [Modes for carrying out the invention]

[0033] Provided herein is a method for determining a static fluorescence threshold gate in relation to cell gating in flow cytometry methods. In some embodiments, the method provided provides an automated gating method that can be performed on cells rapidly and efficiently, and in some embodiments, in a high-throughput manner. The method provided is particularly useful for cell quality control evaluation, for example, for the purpose of evaluating certain attributes or characteristics of cells. In some embodiments, the method provided for setting the static gate threshold utilizes a reference cell population that is known or suspected to have similar characteristics or features to 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 method provided is particularly useful in evaluating similar cell samples (e.g., cells of the same source type) in clinical trials or studies or among subjects being treated according to the same or similar treatment regimens. 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 manufactured or generated according to similar protocols or procedures or from similar groups of subjects (e.g., subjects having the same underlying disease or condition).

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

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

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

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

[0038] In some embodiments, a flow cytometer has five main components. First, a fluid sheath is formed to transport cells and align them so that a light beam can pass through them in a line for sensing. Second, a measurement system, typically a laser beam aligned with the fluid sheath. Third, a detector, as well as an analog-to-digital conversion system that converts measured parameters from the laser beam, e.g., analog measurements of forward and side-scattered light, into digital signals that can be processed by a computing system. Fourth, a linear or logarithmic amplification system. Finally, a computer system for analyzing the signal. Flow cytometers have the capability 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 incorporated to detect multiple antibodies or markers on the surface of particles.

[0039] Flow cytometry devices and other particle analyzers (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 a 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. The multiplicity of different particles or components may be detected simultaneously by labeling different particles or components using spectrally distinct fluorescent dyes. In some implementations, the multiplicity of photodetectors is included in the analyzer, one for each of the scattering parameters being measured and one for each of the distinct dyes being detected. The resulting data includes measured signals for each of the light scattering parameters and fluorescence emission.

[0040] In some embodiments, sensors within the detection region are arranged to simultaneously detect multiple different characteristics, for example, each of the fluorescent dyes used, and one or more light scattering characteristics, such as forward scattered light (FCS), side scattered light (SSC), etc. For example, parameters measured using a flow cytometer typically include FSC, which is excitation light scattered by the particle, mostly along the forward direction; SSC, which is excitation light scattered by the particle, mostly in the side direction; and light emitted from each of the fluorescent dyes in the spectral channels (frequency ranges), referred to as FL1, FL2, etc. These sensors, for example, photodetectors, obtain data about particles in real time as the particles 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 contain a large number of points in multidimensional space. In some embodiments, the dimensions are approximately 3 to 15, and the number of points (or cytometry events) that typically correspond to cells is often tens of thousands to hundreds of thousands. In embodiments of flow cytometry analysis, two of the dimensions typically correspond to the intensities of forward and side scattering that characterize the physical properties of the cells (e.g., size and granularity). The remaining dimension corresponds to the intensity of 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 include means for recording and analyzing the measured data. For example, data storage and analysis may be performed using a computer connected to the detection electronic equipment. For example, the data may be stored in tabular format, where each row corresponds to data for one particle and each column corresponds to each of the measured parameters. The use of a standard file format for storing data from the flow cytometer, such as the "FCS" file format, facilitates the analysis of the data using separate programs and / or instruments. Using current analytical methods, the data is typically displayed in two-dimensional (2D) plots for ease of visualization, but other methods may be used to visualize multidimensional data.

[0044] In some embodiments, flow cytometry data plots each event independently, representing 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, with the number of events detected at that intensity on separate axes. Multiple events detected at a particular intensity are displayed as spikes on the histogram. In other embodiments, events are plotted as a dot plot, which is a plot that compares 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 intensities of two different channels are represented along two axes. For a three-dimensional (3D) plot, the intensities of three different channels are 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 clusters of events of varying intensity represented in the same area of ​​the plot. There are many ways to add further decomposition to these areas of the dot plot. For example, a heatmap may be used to provide information about the event density in a given region of a plot.

[0045] The resulting data is multidimensional in nature, and each particle may correspond to a point in a multidimensional space defined by the measured parameters. A population or cluster of a particular type of cell is identified based on their correlation with one another in this multidimensional space. Different cell types can be identified by scattering parameters and fluorescence emission resulting from the labeling of various cell proteins using dye-labeled antibodies. Cluster, and thereby population, identification can be performed by cell gating. 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 assistance of software operationally associated with the flow system. The gate then provides the user with a convenient method for selecting a subset of particles for counting, isolation, or other manipulation. Gating is used to help detect the large amount of data that may be generated from a sample. Therefore, efficiently facilitating the creation and manipulation of gates can help improve the speed and accuracy of understanding what the results mean.

[0046] In some embodiments, the gate may be a “threshold” gate, which is a gate for a single optical parameter that defines an open region in multidimensional space. In most existing methods, “threshold” gating has been used primarily for forward light scattering to remove high-frequency, low-level signals caused by objects, e.g., debris in a sample, which would overwhelm the processing power of the detection system electronics designed to handle signals generated by particles of interest. More commonly, “window” gating is used, which defines a region that is usually closed in multidimensional space, for example, by defining upper and lower boundaries for signal values. In “window” gating, regions are generated to correspond to particle or cell types to be counted, sorted, or excluded. In some embodiments, gating is performed on a 2D plot of two parameters, e.g., side scattering (e.g., on the vertical axis) and fluorescence signal (e.g., on the horizontal axis).

[0047] In some embodiments, traditional flow cytometry gating controls typically utilize isotype gating controls or fluorescent dye conjugated antibodies, which are industry-standard gating controls for flow cytometry methods, particularly in quality control settings. In some embodiments, such methods may be advantageous because they allow for objective gate placement. On the other hand, such methods are associated with one or more disadvantages, such as reduced throughput per plate, increased assay complexity (e.g., time, failure rate, required sample volume), and increased product cost.

[0048] Furthermore, in many existing methods, gating is performed by manually drawing gates around populations displayed in one or more two-dimensional plots, referred to as “scatter plots” or “dot plots” of cytometry events present in the data. In manual gating, the user collects flow cytometry data (or cytometry events) from one or more channels representing one or more different parameters on the dot plot, e.g., SSC, FSC, or different fluorescence signals. Based on the acquired data, the user draws gate boxes to select subpopulations of cells for further analysis. Researchers may use one of several tools to draw geometric gates, e.g., rectangles, ellipses, or polygons. Subpopulations of cells within a gate are particularly highlighted on other plots displaying 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., subpopulations of cells gated for channels 1 and 2 may be further gated for channels 3 and 4 to allow for further specificity and deeper analysis). While manual plotting is typically used due to its flexibility and intuitiveness, manual sequential gating for extracting cell populations of interest is tedious and labor-intensive with larger file sizes. Furthermore, since the data of interest may not be fully geometric or easily represented using available tools, the tools may not allow for the drawing of precise gates. In addition, manual gating can also result in variability among different users performing the manual gating. Therefore, an improved method is needed.

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

[0050] The technologies described herein may be implemented in hardware, software, firmware, or any combination thereof. Such technologies may be implemented in any of the following devices, such as general-purpose computers, wireless communication devices, or integrated circuit devices having multiple applications, 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 operating logic devices. When implemented in software, the technology may be realized by a computer-readable data storage medium containing program code that, when executed, includes instructions to 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 also include packaging materials. Computer-readable media may include memory or data storage media, such as random access memory (RAM), such as 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. Computer-readable media may also be non-temporary storage media. The technology may additionally, or alternatively, be implemented, at least in part, in computer-readable communication media, such as propagating signals or waves, that transmit or communicate program code in the form of instructions or data structures, and that are accessible, read, and / or executable 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 circuits. Such processors may be configured to perform any of the techniques described herein. The general-purpose processor may be a microprocessor, but alternatively, the processor may be any conventional processor, controller, microcontroller, or state machine. The 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 combined with a DSP core, or any other such configuration. Thus, the term “processor” as used herein may mean any of the above structures, any combination thereof, or any other structure or device suitable for implementing the techniques described herein. In addition, in some embodiments, the functionality described herein may be provided within a dedicated software module or hardware module configured for encoding and decoding, or it may be incorporated into a combined video encoder-decoder (CODEC).

[0052] All publications referenced in this application, including patent documents, scientific articles, and databases, are incorporated by reference in whole for all purposes to the same extent that each individual document is incorporated by reference individually. If any definitions set forth herein contradict or otherwise contradict any definitions set forth herein in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall prevail over the definitions incorporated herein by reference.

[0053] Section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described herein.

[0054] I. Flow cytometry and data acquisition In some embodiments, the method provided herein includes receiving flow cytometry data. In some embodiments, the method includes receiving flow cytometry data comprising multiple cytometry events from a test population of labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer. In some embodiments, the test population of cells is or contains a sample, e.g., a biological sample. In the embodiments provided, the test population of cells is stained with one or more fluorescence signals to label one or more attributes of the cell sample and evaluated by flow cytometry to collect cytometry events. The cytometry events may 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 acquisition involves a number of principles, such as fluid systems, optics and detection, signal processing, and electrostatic cell sorting. In some embodiments, flow cytometry is used to measure the properties of individual particles from a sample, such as individual cells.

[0056] In some embodiments, flow cytometry data acquisition begins with the collection of flow cytometry cell samples. Flow cytometry protocols involving direct or indirect staining of the cell samples may be used in the acquisition of flow cytometry cell samples. Direct staining flow cytometry protocols are one of the most common staining methods, in which live or fixed cells are incubated with directly labeled antibodies against cell surface antigens. Indirect staining flow cytometry is used when directly labeled antibodies are unavailable 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 is known to those skilled in the art, other variations of staining are possible; for example, intracellular staining is used when the antigen is not present on the cell surface.

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

[0058] In some embodiments, the sample is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-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 organs, and / or cells derived therefrom. Samples include autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0059] In some cases, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. In some embodiments, the sample contains lymphocytes including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some embodiments, it contains cells other than erythrocytes and platelets.

[0060] In some embodiments, blood cells collected from the subject are washed, for example, to remove the plasma fraction and to place the cells in a suitable buffer or culture 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 achieved using 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 achieved by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are washed with various biocompatible buffers, e.g., Ca 2+ / Mg 2+ The cells are resuspended in free PBS. In certain embodiments, components of the blood cell sample are removed, and the cells are directly resuspended in the culture medium.

[0061] In some embodiments, the preparation method includes a step for freezing the cells before staining, for example, for cryopreservation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a cryopreservation solution after a washing step to remove, for example, plasma and platelets. Any of the various known cryopreservation solutions and parameters may be used in some embodiments. In some embodiments, the cells are encapsulated in the medium and / or solution at concentrations of 12.5% ​​or about 12.5%, 12.0% or about 12.0%, 11.5% or about 11.5%, 11.0% or about 11.0%, 10.5% or about 10.5%, 10.0% or about 10.0%, 9.5% or about 9.5%, 9.0% or about 9.0%, 8.5% or about 8.5%, 8.0% or about 8.0%, 7.5% or about 7.5%, 7.0% or about 7. The product is frozen, for example, cryopreserved or cryopreserved, with a final concentration of DMSO of 0%, 6.5% or approximately 6.5%, 6.0% or approximately 6.0%, 5.5% or approximately 5.5%, or 5.0% or approximately 5.0%, or from 1% or approximately 1% to 15% or approximately 15%, from 6% or approximately 6% to 12% or approximately 12%, from 5% or approximately 5% to 10% or approximately 10%, or from 6% or approximately 6% to 8% or approximately 8%. In certain embodiments, cells are frozen, for example, cryopreserved or cryopreserved, with final concentrations of HSA in the medium and / or solution of 5.0% 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%, or 0.1% to -5%, 0.25% to 4%, 0.5% to 2%, or 1% to 2%. One example involves the use of PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA).In some embodiments, this is then diluted 1:1 with culture medium, resulting in final concentrations of DMSO and HSA of 10% and 4%, respectively. In some embodiments, the cells are then frozen to -80°C or approximately -80°C at a rate of 1°C / min or approximately 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 cell staining. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to lyse or remove cells sensitive to a particular reagent, for example, to remove undesirable components, to concentrate desired components. In some examples, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity to and / or resistance to a particular component. In some embodiments, the method includes density-based cell separation methods, such as lysing of erythrocytes and preparation of leukocytes from peripheral blood by centrifugation through a Percoll or Ficoll gradient.

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

[0064] Any markers known or suspected to be expressed on the cell type of interest in the sample can be targeted by a staining reagent. Exemplary markers include those present on lymphocyte populations, e.g., T cells. In some embodiments, the sample may be a whole blood sample. In some embodiments, the sample may be an apheresis sample. In some embodiments, the sample may be a leukocyte apheresis 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, and 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 a population. In some embodiments, the polynucleotide encoding the marker may be introduced into cells 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 TCR. In some embodiments, the staining reagent for a CAR may be an antibody directed to the antigen-binding domain (e.g., an anti-idiotype antibody) or an antibody directed to the spacer region of the extracellular domain of the 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) co-expressed in cells with another recombinant protein. Transduction markers or surrogate markers can be used to detect cells into which a polynucleotide, e.g., a polynucleotide encoding the recombinant protein, has been introduced. In some embodiments, the transduction marker can indicate or confirm the modification of the cell. In some embodiments, the surrogate marker is a protein co-expressed on the cell surface with a recombinant receptor, e.g., a CAR. In certain embodiments, such a surrogate marker is a surface protein modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide encoding the recombinant protein (e.g., the recombinant receptor). In some embodiments, the nucleic acid sequence encoding the recombinant protein may be operably linked to the nucleic acid sequence encoding the marker, or to a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome skipping, e.g., a 2A sequence, e.g., T2A, P2A, E2A, or F2A, which may be separated by an internal ribosome entry site (IRES). Exogenous marker genes may be used, in some cases, to enable the detection or selection of cells in association with manipulated cells, and also, in some cases, to promote cell suicide.

[0067] Exemplary surrogate markers may include cleaved cell surface polypeptides, e.g., non-functional cleaved forms that do not transmit or have the ability to transmit signals normally transmitted by signaling or full-length cell surface polypeptides, and / or do not or have the ability to internalize them. Exemplary cleaved cell surface polypeptides include cleaved growth factors or other receptors, e.g., cleaved human epidermal growth factor receptor 2 (tHER2), cleaved 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 that can be used to identify or select cells engineered with the EGFRt construct and recombinant receptor, e.g., a chimeric antigen receptor (CAR), and / or to exclude or isolate 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, includes CD34, NGFR, CD19, or cleaved CD19, e.g., cleaved non-human CD19, or whole or partial (e.g., cleaved form) of the epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably ligated to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A. For example, the marker, and optionally the linker sequence, may be any of those disclosed in PCT International Publication No. 2014031687. For example, the marker may be cleaved EGFR (tEGFR), which may be ligated to a linker sequence, e.g., a T2A cleavable linker sequence.

[0068] In some embodiments, the surrogate marker is a variant of or includes a fluorescent protein, e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), e.g., super-fold GFP, red fluorescent protein (RFP), e.g., 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 species variants, monomer variants, and codon-optimized and / or enhanced variants of the fluorescent protein. In some embodiments, the marker is an enzyme, e.g., luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), or the same. Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or their variants.

[0069] In some embodiments, the marker is a cell health marker. Non-limiting examples of cell health markers include caspase cleavage products, e.g., chromogenic substrates, c-PARP, cleaved cytokeratin 18, cleaved caspases, cleaved caspase 3, cytochrome C, apoptosis-inducing factors (AIFs), apoptosis-inhibiting factors (IAPs) family members, as well as other molecules, e.g., anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL), BH3-only apoptosis-sensitizing factors (PUMA, NOXA, Bim, Bad), and Bcl-2 family members including pro-apoptotic proteins (Bad, Bax) (see below), p53, c-myc proto-oncogenes, APO-1 / Fas / CD95, growth-stimulating 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 of apoptosis. Living cells can be stained with Annexin V, which binds to phosphatidylserine, normally located inside the cell but moved to the cell surface when the cell is undergoing apoptosis or other forms of cell death.

[0070] In some embodiments, the staining reagent is a fluorophore dye, which is a survival dye selective to dead cells, emitting fluorescence at a specific wavelength when bound to dead cells. In some embodiments, the survival dye is an amine-reactive dye, such as an NHS-active ester covalently bonded to the dye (which may include succinimidyl esters, sulfosuccinimidyl esters, tetrafluorophenyl esters, or sulfodichlorophenol esters). Such survival dyes enable cell survival detection based on the reaction of the fluorescently reactive dye with primary amines, such as lysine residues in cellular proteins. Since these dyes cannot penetrate the membrane of living cells, only in living cells are cell surface proteins available to react with the dye, resulting in dimmer staining. However, in dead cells, the reactive dye can penetrate the damaged membrane of the dead cell to stain both internal and external amines, potentially resulting in stronger staining. In some embodiments, the covalently bonded dye may be Alexa Fluor 405, Pacific Blue, Alexa Fluor 430, Fluorescein, Alexa Fluor 488, Texas Red, or Alexa Fluor 594. In some embodiments, fluorophore dyes may be defined as green, red, blue, violet, cyan, or yellow. Green and red fluorescent dyes are excited by a 488 nm laser, violet, cyan, and yellow dyes require a 405 nm excitation source with different emission wavelengths, blue fluorescent reactive dyes require UV excitation, and finally infrared and near-infrared dyes are excited at 633 / 635 nm. The availability of amine-reactive dyes with different fluorescent dyes that can be excited at different wavelengths allows for the selection of a suitable reagent combination for any configuration of the 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 comprises one or more fluorescent markers, which may be attached, for example, by chemical conjugation, to a binder capable of binding specifically to the markers. In some embodiments, the binder is a protein. In some embodiments, the binder is an antibody or an antigen-binding fragment. The fluorescent markers may be conjugated to a binder, such as an antibody, by any method known in the art.

[0073] As is well known in the art, “antibody” is an Ig molecule having the ability to specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the immunoglobulin (Ig) molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also their fragments, e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins containing an antibody moiety having an antigen-binding fragment of the required specificity, chimeric antibodies, nanobodies, and any other modified configurations of immunoglobulin molecules containing an antigen-binding site or fragment (epitope recognition site) of the required specificity. Minibodies containing scFv conjugated to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, 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; International Publication No. 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] The terms "specifically binding" or "preferentially binding" (as used interchangeably herein) to a marker, such as an antibody, are well understood in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular marker target more frequently, more rapidly, for a longer duration, and / or with higher affinity than with an alternative marker. An antibody specifically or preferentially binds to a target if it binds with higher affinity, avidity, more readily, and / or for 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 may include) exclusive binding. Methods for determining such specific or preferential binding are also well known in the art, such as immunoassays.

[0075] In the embodiments provided, a binder, such as an antibody, is conjugated to a fluorescent marker, such as 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-dichlorofluorescien, 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, and 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) This includes, but is not limited to, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800.

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

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

[0078] In some embodiments, the cell attribute is a low-frequency attribute. In some embodiments, a low-frequency attribute is an attribute detectable in less than 5% of the sample, for example, less than 5% of the cells in the sample's cell population. In some embodiments, for a population of cells presumed to be healthy, the low-frequency attribute is Live / Dead (cell survival). In some embodiments, for a population of T-cell enriched cells, the low-frequency attribute may be a B-cell marker, such as CD19, CD20, CD22, and / or CD138.

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

[0080] In some embodiments, the fluorescent marker includes emission detection means, which advantageously emits light of at least a fluorescence wavelength. In some embodiments, one or more fluorescent markers are selected such that the emission wavelength spectrum of each fluorescent marker of the one or more fluorescent markers is distinguishable from the excitation wavelength spectra of the other fluorescent markers of the one or more fluorescent markers. Different fluorescent markers may be excited by light of the same or different wavelengths. Preferably, the emission wavelengths are 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, fluorescent markers, for example, one or more staining reagents, such as antibodies, conjugated, are selected to minimize energy transfer between them, for example, 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 using a single wavelength or multiple wavelengths, but detection is performed in regions where the peak emission spectra do not overlap. In some embodiments, one or more of the fluorescent markers may be excited by a single or the same wavelength of light, but different wavelengths of light are emitted from them.

[0082] In some embodiments, each of 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 using multiple markers, such as multiple staining reagents for cell surface markers. In some embodiments, this results in cells labeled with multiple markers, allowing for the characterization of a well-defined subset of cells.

[0084] In some embodiments, cell staining involves incubation with an antibody or binding partner that specifically binds to such markers, followed in some embodiments by a washing step and separation of cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner. In some embodiments of such processing, a certain volume of cells is mixed with a certain amount of the desired staining reagent and incubated under conditions for cell staining. In some embodiments, staining or labeling is performed at a temperature of 0°C to 25°C, for example, 4°C or about 4°C. In some embodiments, staining or labeling is performed for longer than 5 minutes, typically longer than 15 minutes. In some embodiments, staining or labeling is performed for 15 minutes to 6 hours, for example, 30 minutes to 2 hours. In some embodiments, staining or labeling is performed 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 these. In some embodiments, labeling with one or more staining reagents is performed simultaneously. In some embodiments, one or more washing steps are performed prior to introducing the sample into the flow cytometer for analysis.

[0085] In some embodiments, the cell sample is 1 × 10⁶ to allow the cells to pass through the flow cytometer for reading. 6 ~1 × 10 7 It is prepared by suspending single cells at a density of cells / ml. In some embodiments, this cell concentrate is called a fluid sheath. In some embodiments, the fluid sheath affects the rate of flow sorting, which typically proceeds at about 2,000 to 20,000 cells per second. Fluid sheaths of cell samples are typically made from phosphate-buffered saline solution, but other solutions are available as are known and understood to those skilled in the art.

[0086] In some embodiments, the sample is introduced into a flow cytometer. When the sample enters the flow cytometer, the 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 fluid system using the application of hydraulic pressure. This stream is then passed through one or more beams of light scattering or fluorescence emission. A laser typically serves as the light source in the flow cytometer. The laser produces light of a single wavelength, which, once in contact with the cell sample, produces forward scattered light as an indicator of cell size, lateral scattered light as an indicator of cell complexity, and fluorescence, also emitted laterally 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 at an offset of 0–20 degrees 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 excitation line is called lateral scattered light and fluorescence signal. Both forward and side-scattered light are unique to any particle passing through the flow cytometer, and all combinations of these can be used to distinguish particles in a cell sample. Fluorescence channels are typically designated by names such as FL1, FL2, FL3, depending on the number of channels in the instrument. Each fluorescence channel is fitted with a barrier filter to detect a specific dye of selection while filtering out everything else. The channel in which the specific dye is primarily detectable may be referred to as the primary fluorescence channel, while other fluorescence channels may be called secondary channels. The scattering and fluorescence emission signals are converted into electron pulses, which are processed by the flow cytometry engine and displayed on a graphical user interface (GUI).

[0087] II. Static Gating Thresholds In some embodiments, the method provided includes applying a static fluorescence threshold gate individually to each of one or more fluorescence signals present in the flow cytometry data collected from a test sample, as described, for example, in Section I.

[0088] In some embodiments, a static fluorescence threshold gate for each of the one or more fluorescence signals is determined by (a) cytometry events measured by flow cytometry for one or more fluorescence signals for multiple reference populations of cells each sorted according to one or more fluorescence signals; and (b) setting a static fluorescence threshold gate for each of the one or more fluorescence signals.

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

[0090] A. Setting static gating thresholds Provided herein is a method for determining a static fluorescence threshold gate, comprising: (1) measuring cytometric events by flow cytometry for one or more fluorescence signals for a plurality of at least two reference cell samples, wherein the plurality of at least two reference cell samples comprises: (a) a first plurality of reference cell samples, each comprising a population of unstained cells not labeled with one or more fluorescence signals; and (b) a second plurality of reference cell samples, each of which comprises a second plurality of reference cell samples, each of which comprises the same cell source type as the first plurality of reference cell samples, and each of which comprises a population of stained cells labeled with at least one of the one or more fluorescence signals; and (2) setting a static fluorescence threshold gate for each of the one or more fluorescence signals.

[0091] In some embodiments, the cell source type of a reference cell sample is a population of cells from any biological sample containing 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, e.g., having a particular disease or condition, or requiring treatment (e.g., cell therapy), or being administered therapeutic treatment (e.g., cell therapy). In some embodiments, the subject is a human, e.g., a patient requiring a particular therapeutic intervention. Thus, the cell source type in some embodiments is primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject. Biological samples may be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissues, and organ samples, and include processed samples derived therefrom.

[0092] In some embodiments, the sample is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-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 organs, and / or cells derived therefrom. Samples include autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0093] In some cases, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. In some embodiments, the sample contains lymphocytes including T cells, monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some embodiments, it contains cells other than erythrocytes and platelets.

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

[0095] In some embodiments, the cell source type is an enriched population of cells of a particular type or subtype, in which such cells are selected from a starting cell sample (e.g., an apheresis or leukocyte apheresis sample), for example, by positive or negative selection of cells from the sample. Among the T cell populations that may 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 produced by enrichment or depletion of cells based on a particular subtype or a particular surface marker expression profile. For example, among the subtypes of T cells that may be enriched and / or selected (e.g., CD4+ or CD8+ T cells), some are defined by their potential for function, activation state, maturation, differentiation, proliferation, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the T cells and / or CD4+ and / or CD8+ T cell subtypes and subpopulations that can be enriched, isolated and / or selected are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, e.g., stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, e.g., 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 T cell populations enriched and / or selected from a sample in a cell sample provided herein are positive for one or more specific markers, such as a surface marker (marker+), or express it at a high level (marker). high ) or negative for one or more markers (marker-) or expressing them at a relatively low level (marker low ) cells. In some cases, such markers 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 (i.e., positively selected) for cells that are positive for or express CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L at high surface levels, and / or depleted (e.g., negatively selected) for cells that are positive for or express CD45RA at high surface levels. In some embodiments, cells are enriched or depleted for cells that are positive for or express CD122, CD95, CD25, CD27, and / or IL7-Rα (CD127) at high surface levels. In some cases, CD8+ T cells are enriched with cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L.

[0097] In some embodiments, the CD4+ T cell population and the CD8+ T cell subpopulation are, for example, subpopulations enriched with respect to 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 cell source type is a population of engineered cells into which nucleic acids encoding recombinant proteins have been introduced, for example, by gene transfer (e.g., transduction). Examples of recombinant proteins to be expressed include recombinant receptors, e.g., chimeric antigen receptors (CARs) or T cell receptors (TCRs). Various methods for introducing genetically engineered components, e.g., recombinant receptors, e.g., CARs or TCRs, are well known and may be used with the provided methods and cell samples. Exemplary methods include methods for introducing nucleic acids encoding recombinant proteins, including viral transduction, e.g., retroviral or lentiviral transduction, transposons, and electroporation. In some embodiments, the genetic engineering involves introducing nucleic acids encoding recombinant or engineered components into a cell-containing composition, e.g., by retroviral transduction, transfection, or transformation. The introduction of nucleic acid molecules encoding recombinant proteins, e.g., recombinant receptors, into cells may be carried out using any of a number of known vectors. Such vectors include viral and nonviral systems, including lentiviral and gamma-retroviral systems, as well as transposon-based systems, e.g., PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include methods for the transfer of receptor-encoding nucleic acids, including viral transduction, such as retroviral or lentiviral transduction, transposons, and electroporation.

[0100] In some embodiments, the primary cell samples used as the cell source type for each of the multiple reference cell samples are from different subjects (e.g., patients), each different from the subject (e.g., patient) from which the cell source type was obtained (or may 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 cell source type may be a sample from a subject (e.g., patient) having a disease or condition for which it is desired to perform flow cytometry analysis on the subject's cells prior to or in the course of 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 cell source type of the cells being tested is the cells of a cell therapy administered to the subject (e.g., patient). In some embodiments, the cell therapy is CAR cell therapy (e.g., CAR T cell therapy), TCR cell therapy, tumor-infiltrating lymphocyte (TIL) cell therapy, natural killer (NK) cell therapy, or stem cell therapy. With respect to the subject being treated, the cells may be homogeneous and / or autologous. In some embodiments, the cell therapy is 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 individually generated from multiple different subjects in a clinical trial, while the test cell sample is a cell therapy sample generated from a test subject (e.g., a patient) identified or approved for treatment using a particular cell therapy. In some embodiments, the method provided may be performed in connection with the quality control or characterization of a manufactured, e.g., manipulated, cell therapy prior to its introduction into the subject.

[0101] In certain embodiments, the source type of cells in a reference cell sample is known or suspected to contain similar attributes or characteristics to the test cell sample that is to be analyzed by the provided method using a static gating threshold. Therefore, in the provided embodiments, the test cell sample, as well as the first and second reference cell samples, are from the same cell source. For example, if the test cell sample is a leukocyte apheresis cell sample, the cell source in the reference cell sample is also a leukocyte apheresis cell sample. In some embodiments, where the cells are primary cells from a subject, the cell sources of the reference cell sample and the test cell sample may also be obtained from or originate from a subject having similar attributes or characteristics. For example, in some embodiments, the test cell sample originates from a subject with a disease, and the reference cell population sample originates 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 carcinoma. In some embodiments, the test cell sample is a population of cells that have been manipulated, for example, by gene transfer (e.g., transduction) or gene editing procedures, or by exposure to or incubation in the presence of modifiers, such as activators, cytokines, immunomodulators, small molecule compounds, or other agents that can alter one or more characteristics or properties of cells. In such embodiments, the reference cell sample is also a population of cells that have been similarly manipulated under the same conditions as the test cell sample (or optionally, a plan for the test cell sample).

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

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

[0104] In some embodiments, the second reference sample is subjected to a labeling procedure by individually contacting each of the multiple cell samples of the second reference cell sample with one or more staining reagents. 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 the cells with one or more fluorescent signals. In some embodiments, the staining reagents may be any reagents described in Section I. In some embodiments, the cell staining procedure may be performed using any method described in Section I. In the provided embodiments, a binder specific to a particular marker and dye is selected so that the staining of cells with the staining reagents 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 clear separation between positively stained and negatively stained samples.

[0105] In the embodiments provided, cell staining of each of the test cell sample and the second reference cell sample is performed using the same staining reagent and under the same protocol conditions. The reference “same” does not mean that the staining reagent or protocol is identical in all respects, and it is understood that some variation between reagents or in the execution of the protocol is to be expected. It is within the level of the art to ensure that procedures are taken to minimize variation and to ensure that the procedures are as similar as possible. For example, it is not necessary that the staining be performed using the exact same lot or batch of staining reagent, such as those 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 preceding lots or batches of the same staining reagent. In some embodiments, cell staining is performed using staining reagents that are known to have the same protein sequence or chemical structure, containing 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 using multiple staining reagents for multiple markers, such as cell surface markers. In some embodiments, this results in labeled cells using multiple markers, allowing for the characterization of a well-defined subset of cells. Therefore, in some embodiments, cell staining is a multicolor cell staining for labeling cells using two or more different fluorescent signals, where each staining reagent labels a different marker using a different fluorescent signal. In some embodiments, the two or more different fluorescent signals include 2 to 10 fluorescent signals. In some embodiments, cell staining is performed to label cells using two different fluorescent signals. In some embodiments, cell staining is performed to label cells using three different fluorescent signals. In some embodiments, cell staining is performed to label cells using four different fluorescent signals. In some embodiments, cell staining is performed to label cells using five different fluorescent signals. In some embodiments, cell staining is performed to label cells using six different fluorescent signals. In some embodiments, cell staining is performed to label cells using seven different fluorescent signals. In some embodiments, cell staining is performed to label cells using eight different fluorescent signals. In some embodiments, cell staining is performed to label cells using nine different fluorescent signals. In some embodiments, cell staining is performed to label cells using ten different fluorescent signals. In some embodiments, each of two or more different fluorescent signals has a different emission spectrum. In some embodiments, each of two or more different fluorescent signals has a peak emission spectrum of a fluorescent signal that does not overlap with one or more other fluorescent signals in cell staining. In some embodiments, cell staining is performed using a staining reagent in which two or more different fluorescent signals are emitted by dyes 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, followed in some embodiments by a washing step and separation of cells bound to the staining reagent from cells not bound to the antibody or binding partner. In some embodiments of such a process, a certain volume of cells is mixed with a certain amount of the desired staining reagent and incubated under conditions for cell staining. In some embodiments, staining or labeling is performed at a temperature of 0°C to 25°C, for example, 4°C or about 4°C. In some embodiments, staining or labeling is performed for longer than 5 minutes, typically longer than 15 minutes. In some embodiments, staining or labeling is performed for 15 minutes to 6 hours, for example, 30 minutes to 2 hours. In some embodiments, staining or labeling is performed for any value between, 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 of these values. In some embodiments, labeling with one or more staining reagents is performed simultaneously. In some embodiments, one or more washing steps are performed prior to introducing the sample into the flow cytometer for analysis.

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

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

[0110] In some embodiments, cell staining can be repeated multiple times, for example in two or three sets, for each of the source types of cells to be stained for labeling using one or more fluorescent signals, resulting in two or three sets of stained samples. In some embodiments, the plurality of second reference cell samples includes two sets of samples of the same stained cells. In some embodiments, the plurality of second reference cell samples includes three sets of samples of the same stained cells.

[0111] In some embodiments, prior to flowing the sample through the flow cytometer, the flow cytometer is aligned, compensated and calibration One or more of the following will be performed: alignment, compensation, and calibration In addition to ensuring that the equipment operates at its maximum efficiency, it also ensures that reproducibility is achieved so that data obtained over time or using various equipment is equivalent.

[0112] In some embodiments, alignment is the process of adjusting and focusing various optical and electrical components so that the scattered and fluorescent signals are tuned to their highest intensity and densest distribution, i.e., the lowest coefficient of variation (CV) of the distribution. The components of the flow cytometer to be aligned include lasers, lenses, mirrors, barrier filters, and PMTs.

[0113] In some cases, multifluorescence analysis, i.e., analysis using two or more fluorescent dyes simultaneously, can be performed on a flow cytometer. However, for accurate analysis, it is necessary to adjust the electrical compensation circuit in the flow cytometer so that any fluorescence emission overlapping with other fluorescence channels can be subtracted from such other channels. Compensation is a process that electronically removes residual signals from the fluorescent dye in secondary fluorescence channels resulting from spectral overlap that is not removed by the barrier filters for each channel. Flow cytometer using appropriate standards calibration This ensures that results from the sample are equivalent over time and across different instruments. calibration In order for it to be independent of specific equipment and equipment settings, calibration The excitation and emission spectra of the standard and the sample being measured must be equivalent, and each measurement must be performed under the same instrument settings. calibration The method is publicly known and is described, for example, in U.S. Patent Nos. 4,714,682; 4,767,206; 4,774,189; and 5,620,842, as well as in PCT International Publication No. 1991000509. In some embodiments, calibration The method involves aligning the fluorescence channel of the flow cytometer and calibration Both blank and fluorescently labeled microbeads can be used for this purpose. For example, U.S. Patent Nos. 4,774,189 and 4,767,206 describe the use of such microbeads in flow cytometry. calibration The method is described. In some embodiments, for example, as described in U.S. Patent No. 6,008,052, calibration The standard used for this purpose mimics the cells to be studied in the sample.

[0114] In some embodiments, fluorescence calibrationThis ensures the accuracy and reproducibility of the method provided. In some embodiments, to obtain accurate and reproducible results, the flow cytometer is used for alignment and calibration It must be done. If operating with one or more fluorescent dyes, the instrument will also require compensation for the photomultiplier tube (PMT).

[0115] In some embodiments, each of the cell samples, for example, each of several first and second reference cell samples, is introduced individually into the flow cytometer. Both forward and side-scattered light are unique for any particle passing through the flow cytometer, and all combinations of these can be used to distinguish particles in the cell sample. In some embodiments, the forward, side-scattered, and emitted light signals are converted into electron pulses, which are processed by a 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, flow cytometry data from each of a plurality of first reference cell samples and each of a plurality of second reference cell samples are used to set a static threshold gate. In some embodiments, setting a static fluorescence threshold gate includes setting a static fluorescence threshold gate for each of one or more fluorescence signals. In some embodiments, the static threshold gate is a fixed threshold selected to distinguish negative staining from positive staining for each of one or more fluorescence signals (see Figure 1A). In some embodiments, for each of one or more fluorescence signals, the static gate threshold is set by (1) determining the upper fluorescence boundary of the fluorescence signal from each of the unstained samples from the plurality of first reference samples; (2) determining the lower fluorescence boundary of the fluorescence signal from each of the stained samples from the plurality of second reference samples; and (3) setting a fixed or static threshold for each fluorescence signal as a threshold gate for the fluorescence signal. In some embodiments, determining the upper boundary of each fluorescence signal may be determined as an upper percentile (e.g., 90th percentile or higher) according to the distribution of fluorescence signals in the unstained samples from the plurality of first reference samples. In some embodiments, determining the lower boundary of each fluorescence signal may be determined as a lower percentile (e.g., the 10th percentile or lower) according to the distribution of fluorescence signals in stained samples from multiple second reference samples.

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

[0118] Therefore, among those provided herein is a method for determining a static fluorescence threshold gate, comprising (1) measuring cytometric events by flow cytometry for one or more fluorescence signals for a plurality of at least two reference cell samples, wherein the plurality of at least two reference cell samples are (a) a plurality of first reference cell samples, each comprising a population of unstained cells not labeled with one or more fluorescence signals; and (b) a plurality of second reference cell samples, each of which is derived from the same cell source type as the plurality of first reference cell samples, There is a method comprising (2) measuring a plurality of second reference cell samples, which include a population of stained cells labeled with at least one of one or more fluorescent signals; and (2) setting a static fluorescence threshold gate for each of the one or more fluorescent signals, wherein each fluorescence signal threshold gate is set such that (a) it is higher than the 90th percentile of fluorescence of a plurality of first reference populations of cells for each respective fluorescence signal of the one or more fluorescent signals; and (b) it is lower than the 10th percentile of fluorescence of a plurality of second reference populations of cells for the same respective fluorescence signal.

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

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

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

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

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

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

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

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

[0127] In some embodiments, a static fluorescence threshold gate for at least one of one or more fluorescence signals is fluorescence above the 90th percentile of fluorescence among multiple cells in a first reference population for each fluorescence signal, and fluorescence below the 10th percentile of fluorescence among multiple cells in a second reference population for the same fluorescence signal.

[0128] In some embodiments, a static fluorescence threshold gate for at least one of one or more fluorescence signals is fluorescence above the 95th percentile of fluorescence among multiple cells in a first reference population for each fluorescence signal, and fluorescence below the 5th percentile of fluorescence among multiple cells in a second reference population for the same fluorescence signal.

[0129] In some embodiments, a static fluorescence threshold gate for at least one of one or more fluorescence signals is fluorescence above the 99th percentile of fluorescence among multiple cells in a first reference population for each fluorescence signal, and fluorescence below the 1st percentile of fluorescence among multiple cells in a second reference population for the same fluorescence signal.

[0130] Selecting a specific static fluorescence threshold gate for a particular fluorescence signal is within the realm of the art. Factors to consider in selecting a static fluorescence threshold gate include the acceptable degree of false positive or false negative in the stained sample. 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 many positive populations as possible, with the exception of 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 positive and negative / unstained fluorescence (e.g., below the 1st percentile or below the overall percentile among multiples in a second reference population). In some embodiments, a wide range in fluorescence of a fluorescence signal exists when the fluorescence-positive and unstained / negative populations of cells differ by more than twofold, e.g., threefold, fourfold, fivefold, sixfold, sevenfold, eightfold, ninefold, tenfold or greater multiples. In some embodiments, a wide range in fluorescence of a fluorescence signal exists when the fluorescence-positive and unstained / negative populations of cells differ by more than fivefold. In some embodiments, a broad range of fluorescence in the fluorescence signal exists when the fluorescence-positive and unstained / negative populations of cells differ by more than a 10-fold.

[0131] In some embodiments, for low-frequency attributes (e.g., those where the frequency of cells positive for an attribute is expected to be less than 5% among a population of cells), an additional mean fluorescence intensity (MFI) analysis may be used to aid in threshold determination. In such examples, the stated percentile information (e.g., the 99th and 1st percentiles of fluorescence, respectively, between multiple samples of a first reference sample and multiple samples of a second reference sample) is useful and helps determine population boundaries or margins, but in some cases it may be distorted by rare events or outliers in a population that may have a small number of events to potentially start with for low-frequency attributes. MFI is a median measurement and is less susceptible to distortion from outliers. For this reason, in some embodiments, MFI may be used as a tertiary analysis. In some embodiments, MFI may 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 boundaries of the difference populations are determined based on percentiles (e.g., the 99th percentile of fluorescence between the first reference cell samples and the 1st percentile of fluorescence between the second reference cell samples), a threshold can be set to separate the two populations. Setting a threshold considering MFI for a particular fluorescence signal is within the realm of the art.

[0132] In some embodiments, the set fluorescence, determined to be the static fluorescence gate threshold according to any of the provided embodiments, may be rounded to an even number on the logarithmic flow axis. In some embodiments, selecting an even number may optimize the workflow. In some embodiments, for each of one or more fluorescence signals, the static fluorescence threshold gate is rounded to the nearest tens digit, nearest hundreds digit, or nearest thousands digit on the logarithmic flow axis.

[0133] B. Application of static gating threshold and cell identification In some embodiments, the provided method includes applying a static fluorescence threshold gate to flow cytometry data, which includes multiple cytometry events from a test population, for each of the one or more fluorescence signals described herein, for example, in Section II, Part A.

[0134] Among the methods provided herein is a method for applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data including multiple cytometry events from a test population of labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescence signals according to one of the provided methods for setting a static fluorescence threshold gate.

[0135] For example, provided herein is a method for applying a static fluorescence threshold gate using flow cytometry, comprising: (1) receiving flow cytometry data comprising multiple cytometry events from a test population of labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) applying a static fluorescence threshold gate individually to each of the one or more fluorescence signals, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) a cytometry event measured by flow cytometry for one or more fluorescence signals for a plurality of at least two reference populations of cells sorted according to one or more fluorescence signals, wherein the plurality of at least two reference populations is (i) a population of unstained cells that are not labeled with one or more fluorescence signals A method comprising (i) a plurality of first reference populations of cells comprising; and (ii) a plurality of second reference populations of cells, each of which comprises at least one population of stained cells, each of which comprises a plurality of first reference populations of cells, and each comprises a plurality of second reference populations of cells, which comprises at least one population of stained cells labeled with at least one of 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 to be set such that for each fluorescent signal, (i) the fluorescence is higher than the 90th percentile of the fluorescence of the plurality of first reference populations of cells for each of the one or more fluorescent signals; and (ii) the fluorescence is also lower than the 10th percentile of the fluorescence of the plurality of second reference populations of cells for the same respective fluorescent signal.

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

[0137] Also provided herein is a method for selecting a subset of cytometry events from flow cytometry data, comprising: (1) receiving flow cytometry data comprising multiple cytometry events from a test population of labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) individually applying a static fluorescence threshold gate to each of the one or more fluorescence signals, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) cytometry events measured by flow cytometry for one or more fluorescence signals for a plurality of at least two reference populations of cells sorted according to one or more fluorescence signals, wherein the plurality of at least two reference populations comprises (i) a plurality of first reference populations of cells each comprising a population of unstained cells not labeled with one or more fluorescence signals; and (ii) a plurality of second reference populations of cells, the second reference population of cells A cytometry event comprising: (b) setting a static fluorescence threshold gate for each of the one or more fluorescence signals, determined from setting the threshold gate, such that for each fluorescence signal it is (i) higher than the 90th percentile of fluorescence in the first reference population of cells for each of the one or more fluorescence signals; and (ii) lower than the 10th percentile of fluorescence in the second reference population of cells for each of the same fluorescence signal; and (3) identifying a subset of cytometry events from a test population of cells having fluorescence signals above the static threshold gate for each of the one or more fluorescence signals.

[0138] In some embodiments of the provided method, the fluorescence features or characteristics of an identified subset of cells are determined for at least one of the fluorescence signals. In some embodiments, the fluorescence features or characteristics of an identified subset of cells are determined for each of one or more fluorescence signals.

[0139] In some embodiments, for an identified subset of cytometry events for at least one of one or more fluorescence signals, the method further includes evaluating the fluorescence intensity of at least one fluorescence signal in the identified subset of cytometry events. In some embodiments, for each of the identified subsets of cytometry events for each of one or more fluorescence signals, the method further includes evaluating the fluorescence intensity of the fluorescence signal in the identified subset of cytometry events. In some embodiments, the fluorescence intensity is the mean fluorescence intensity.

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

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

[0142] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context otherwise explicitly specifies. For example, "a" or "an" means "at least one" or "one or more." The embodiments and variations described herein are understood to include embodiments and variations that "consist of" and / or "essentially consist of."

[0143] The terms “at least one” and “one or more” can be understood to include any integer greater than or equal to 1, i.e., 1, 2, 3, 4, [...] etc. The term “plural” (plural things) can 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" relating 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" relating to a group of elements may be used herein to mean one of the listed elements, one plural of the listed elements, a plural of individual listed elements, or a selection of multiple plurals from the listed elements.

[0145] Throughout this disclosure, various aspects of the claimed subject matter are presented in the form of scopes. It should be understood that descriptions in the form of scopes are merely for convenience and conciseness and should not be interpreted as a firm limitation on the scope of the claimed subject matter. Therefore, a scope description should be considered to have individual numerical values ​​within that scope, in addition to all possible partial scopes specifically disclosed. For example, where a range of values ​​is provided, it should be understood that each intervening value between the upper and lower limits of that range, and any other described or intervening values ​​within the described range, are included in the claimed subject matter. These smaller upper and lower limits may be independently included in the smaller range, also included in the claimed subject matter, and subject to any particularly excluded limits within the described scope. If the described scope includes one or both limits, the scope excluding either or both of those included limits is also included in the claimed subject matter. This applies regardless of the breadth of the scope.

[0146] The term “about” as used herein refers to the normal range of error for each readily known value. References to “about” values ​​or parameters herein include (and describe) embodiments directed toward that value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

[0147] As used herein, “event” or “cytometry event” refers to data measured by a flow cytometer from a single particle, such as a cell or synthetic particle. Typically, data measured from a single particle includes a number of parameters, including one or more light scattering parameters and at least one fluorescence signal parameter. Thus, each event is represented as a vector of parameter measurements, and each measured parameter corresponds to one dimension in the data space.

[0148] A "fluorescent marker" or "fluorescence marker" refers to a fluorescent marker containing a fluorophore that has the ability to absorb energy in a wavelength range and emit energy in wavelength ranges other than the absorption range. Therefore, it refers to a fluorescent compound that can emit light when excited by light. It is understood that the term "fluorescent marker" or its variations may be used interchangeably with the term "fluorophore". The term "excitation wavelength" refers to the wavelength range in which the fluorophore absorbs energy. The term "emission wavelength" refers to the wavelength range in which the fluorophore emits energy or fluorescence.

[0149] As used herein, “fluorescence” or “fluorescence intensity” are interchangeable terms and refer to the output of a detection system that measures fluorescence intensity from the fluorescence emission sample intensity of the emission of a particular fluorescent signal, e.g., from a fluorescent marker, e.g., a fluorophore. Fluorescence intensity is the amount of light (photons) emitted by a fluorescent marker after the fluorescent marker has absorbed light or other electromagnetic radiation.

[0150] As used herein, “Average Fluorescence Intensity” or “MFI” refers to the average fluorescence intensity in a particular fluorescence channel.

[0151] As used herein, “gate” refers to a set of boundary points that identify a subset of data of interest. In cytometry, a gate can define a boundary for a particular group of events of interest. In some embodiments, a gate may be a window surrounding 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, “threshold gate” refers to a gate set at a specific fluorescence intensity that separates cells from a cell population emitting fluorescence signals or fluoresces above that specific fluorescence intensity from cells from a cell population emitting fluorescence signals or fluoresces below that specific fluorescence intensity. In some embodiments, it defines an open region in multidimensional space.

[0153] As used herein, “static threshold gate” refers to a fixed or unchanging threshold gate for a given fluorescence signal that can be applied to multiple stained or labeled samples. In some embodiments, the static threshold gate is set for each attribute (e.g., a marker) that is labeled with a fluorescence signal in a flow cytometry assay, such as a multicolor flow cytometry assay.

[0154] As used herein, “labeled” means having a detectable label attached, such as a fluorescent marker or stain. For example, cells in a cell population may 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 in which a given percentage of the score in its frequency distribution falls below that score.

[0156] As used herein, a “enriched” population of cells refers to one or more specific cell types or subsets or cell populations subjected to enrichment, isolation, or selection steps to increase the number or percentage of cell types or populations compared to, for example, the percentage in the starting population of cells. Therefore, it refers to increasing the percentage or frequency of such cells, or compared to other cell types, by, for example, positive selection based on markers expressed by the population or cells, or negative selection based on markers not present on the cell populations or cells to be depleted. The term does not require the complete removal of other cells, cell types, or populations from the composition, nor does it require that the cells thus enriched be present in 100% or near 100% of the enriched composition. In some embodiments, an enriched population of cells contains more of a particular cell type or subset than 50%, 60%, 70%, 80%, 90%, 95%, or higher percentages. For example, a cell-enriched population may be a T cell population containing more T cells (e.g., CD3+ cells) or their CD4+ or CD8+ subsets than 50%, 60%, 70%, 80%, 90%, 95%, or higher. 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, selection of one of the CD4+ or CD8+ populations enriches either the CD4+ or CD8+ population for the said population, but in some cases may also include some residual or small percentage of other unselected cells, which may still be present in the enriched population.

[0157] As used herein, the statement that a cell or population of cells is “positive” for a particular marker means the detectable presence of a particular marker, typically a surface marker, on or within the cell. When referring to a surface marker, the term means the presence of a surface expression that can be detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detection of said antibody, wherein the stain is detectable by flow cytometry at a level substantially higher than that detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to that for cells known to be positive for the marker, and / or at a level substantially higher than that for cells known to be negative for the marker.

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

[0159] As used herein, “subject” means a mammal, such as a human or other animal, and is typically a human.

[0160] IV. Exemplary Embodiments The embodiments provided include: 1. A method for determining a static fluorescence threshold gate, (1) Measuring cytometric events by flow cytometry for one or more fluorescence signals from multiple reference cell samples of at least two reference cell samples, (a) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (b) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. including, The measurement of the aforementioned; and (2) A static fluorescence threshold gate for each of the one or more fluorescence signals, and for each fluorescence signal, the threshold gate is (a) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (b) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The method, including the method described above. 2. A method for selecting a subset of cytometry events from flow cytometry data, (1) To receive flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is determined according to the method of Embodiment 1; and (3) For each of the one or more fluorescence signals, identify a subset of cytometry events that have a fluorescence signal exceeding the static threshold gate. The method, including the method described above. 3. A method for selecting a subset of cytometry events from flow cytometry data, (1) To receive flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) A cytometry event measured by flow cytometry for one or more fluorescent signals of a plurality of reference cell samples, each selected according to one or more fluorescent signals, wherein the plurality of at least two reference cell samples is (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. The cytometry events, including the aforementioned events; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, and for each fluorescence signal, the threshold gate is (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The application of the foregoing is determined from; and (3) For each of the one or more fluorescence signals, identify a subset of cytometry events that have a fluorescence signal exceeding the static threshold gate. The method, including the method described above. 4. A method for applying a static fluorescence threshold gate using flow cytometry, (1) Receiving flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is determined in accordance with the method described in Embodiment 1. The method, including the method described above. 5. A method for applying a static fluorescence threshold gate using flow cytometry, (1) To receive flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) A cytometry event measured by flow cytometry for one or more fluorescent signals of a plurality of reference cell samples, each selected according to one or more fluorescent signals, wherein the plurality of at least two reference cell samples is (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. The cytometry events, including the aforementioned events; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, and for each fluorescence signal, the threshold gate is (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of first reference cell samples o; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The application of the above is determined from The method, including the method described above. 6. The method according to any one of Embodiments 1 to 5, wherein each of the test cell samples and the plurality of first and second reference cell samples are from the same cell source type. 7. The method according to any one of embodiments 1 to 6, wherein the cell source type is a cell line. 8. The method according to any one of embodiments 1 to 6, wherein the cell source type is a primary cell population from the subject. 9. The method according to Embodiment 8, wherein the source type of cells for each of the test cell samples, and for each of the plurality of the first and second reference cell samples, is from different subjects. 10. The method according to Embodiment 9, wherein each different subject has the same or similar disease or condition. 11. The method according to any one of Embodiments 1 to 10, wherein the cell source type is a whole blood sample, an apheresis sample, or a leukocyte apheresis sample. 12. The method according to any one of Embodiments 1 to 11, wherein the cell source type is a cell-enriched population, optionally a T cell-enriched population. 13. The method according to any one of Embodiments 1 to 12, wherein the cell source type is an engineered population of cells containing nucleic acids encoding recombinant proteins, and the recombinant proteins are introduced into the population of cells by gene transfer, or by transduction. 14. The method according to any one of Embodiments 1 to 13, wherein the cell source type is cell therapy. 15. The method according to any one of Embodiments 1 to 14, 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 according to any one of Embodiments 1 to 15, 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 according to any one of Embodiments 1 to 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 according to any one of Embodiments 1 to 16, wherein the cell source type is 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 to test the cell therapy. 19. The method according to 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 according to Embodiment 14 or Embodiment 18, wherein the cell therapy is NK cell therapy. 21. The method according to Embodiment 14 or Embodiment 18, wherein the cell therapy is 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 is subjected to cell staining using one or more staining reagents for labeling the cells with the one or more fluorescent signals. 23. The method according to Embodiment 22, wherein the one or more staining reagents include a marker-specific binder and a fluorescent dye capable of releasing one of the one or more fluorescent signals. 24. The method according to Embodiment 23, wherein the marker is a cell surface marker or a survival marker. 25. The method according to 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 cells in the sample. 26. The method according to 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 according to any one of Embodiments 1 to 26, wherein the one or more fluorescent signals include two or more different fluorescent signals. 28. The method according to any one of Embodiments 22 to 27, wherein the cell staining is a polychromatic cell staining for labeling the cells using two or more different fluorescent signals, and each staining reagent labels a different marker using a different fluorescent signal. 29. The method according to Embodiment 27 or Embodiment 28, wherein the two or more different fluorescent signals include 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 according to any one of embodiments 27 to 29, wherein each of the two or more different fluorescence signals has a different emission spectrum, and / or the peak emission spectra of each fluorescence signal do not overlap. 31. The method according to any one 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 one of embodiments 22 to 31, wherein the cell staining of each of the test cell sample and the second reference cell sample is performed using the same staining reagent and under the same protocol conditions. 33. The method according to any one of Embodiments 1 to 32, wherein the plurality of second reference cell samples includes a set of two samples of the same stained cells. 34. The method according to any one of Embodiments 1 to 32, wherein the plurality of second reference cell samples includes a triplicate of the same stained cells. 35. The method according to any one of embodiments 1 to 34, wherein the static fluorescence threshold gate for at least one fluorescence signal is higher than the 95th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal. 36. The method according to any one of embodiments 1 to 34, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is higher than the 97th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal. 37. The method according to any one of embodiments 1 to 34, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is higher than the 99th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal. 38. The method according to any one of embodiments 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is a fluorescence lower than the fifth percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal. 39. The method according to any one of embodiments 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is a fluorescence lower than the third percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal. 40. The method according to any one of embodiments 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is a fluorescence lower than the first percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal. 41. The method according to any one of embodiments 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and the fluorescence of the said population of stained cells differ by more than twofold, the setting of the static fluorescence is made below the first percentile of fluorescence or below the overall percentile of fluorescence among the multiple second reference cell samples for the fluorescence signal. 42. The method according to any one of embodiments 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and the fluorescence of the said population of stained cells differ by more than 5 times, the setting of the static fluorescence is made below the first percentile of fluorescence or below the overall percentile of fluorescence among the multiple second reference cell samples for the fluorescence signal. 43. The method according to any one of embodiments 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and the fluorescence of the said population of stained cells differ by more than a 10-fold difference in the fluorescence signal, the setting of the static fluorescence is made below the first percentile of fluorescence or below the overall percentile of fluorescence among the multiple second reference cell samples for the fluorescence signal. 44. The method according to any one of Embodiments 1 to 43, further comprising setting the static fluorescence threshold gate for low-frequency attributes to identify the mean fluorescence intensity (MFI) for one or more of the one or more fluorescence signals. 45. The method according to any one of embodiments 1 to 44, wherein for each of the one or more fluorescence signals, the static fluorescence threshold gate is an even number on the logarithmic flow axis. 46. ​​The method according to any one of embodiments 1 to 45, wherein for each of the one or more fluorescence signals, the static fluorescence threshold gate is rounded to the nearest tens digit, the nearest hundreds digit, or the nearest thousands digit on the logarithmic flow axis. 47. The method according to any one of embodiments 2, 3, and 6-46, further comprising identifying a subset of cells from the test population of cells having fluorescence signals exceeding the static threshold gate for at least two of the one or more fluorescence signals, at least three of the one or more fluorescence signals, at least four of the one or more fluorescence signals, at least five of the one or more fluorescence signals, at least six of the one or more fluorescence signals, or at least seven or more of the one or more fluorescence signals. 48. The method according to any one of embodiments 2, 3, and 6-47, further comprising identifying a subset of cells from the test population of cells having fluorescence signals above the static threshold gate for all of the one or more fluorescence signals. 49. The method according to 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 according to any one of Embodiments 1 to 49, wherein the static fluorescence threshold gate is set for each of the one or more fluorescence signals to include at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the population of stained cells in the test sample. 51. The method according to any one of embodiments 2, 3, and 6-50, further comprising evaluating the fluorescence intensity of the at least one fluorescence signal of the identified subset of cytometry events for at least one of the one or more fluorescence signals. 52. The method according to any one of embodiments 2, 3, and 6-50, further comprising evaluating the intensity of the fluorescence signal of the identified subset of cytometry events for each of the one or more fluorescence signals. 53. The method according to Embodiment 51 or Embodiment 52, wherein the fluorescence intensity is the average fluorescence intensity. 54. The method according to any one of embodiments 2, 3, and 6-53, further comprising determining the percentage of the identified subset of cytometry events for at least one of the one or more fluorescence signals compared to the total number of cytometry events collected. 55. The method according to any one of embodiments 2, 3, and 6-53, further comprising determining the percentage of the identified subset of cytometry events for each of the one or more fluorescence signals compared to the total number of cytometry events collected. 56. The one or more fluorescence signals obtained by the flow cytometer calibration The method according to any one of embodiments 1 to 55, including the method described above. 57. The above calibration The method according to embodiment 56, wherein the method is performed at least once a day. [Examples]

[0161] V. 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 cytometry analysis Fluorescence Minus One (FMO) or isotype gating controls for each fluorescent dye conjugate antibody are industry-standard gating controls for flow cytometry methods used in quality control (QC) settings. While the advantage of these gating approaches is that they allow for objective gate placement, they also have drawbacks, including reduced throughput, increased assay complexity (e.g., due to time constraints, failure rates, and sample volume limitations), and increased product costs. To address these drawbacks, static gating thresholds for flow cytometry analysis were developed.

[0163] Fluorescence thresholds for six attributes were determined using flow cytometry (Table E1). 344 clinical lots of samples were used. Each clinical sample originated from a manufacturing lot of a drug product containing CAR T cells. Negative staining analysis was performed on unstained samples in a single series, and positive staining analysis was performed on stained samples in a triple series (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] Using analysis of positively stained replicas, the lower boundary of positive fluorescence was characterized for each attribute. For positively stained samples, this included determining the 1st percentile of fluorescence, which is considered the minimum lower boundary of the stained fluorescence; determining the 5th percentile of fluorescence, which is considered the secondary lower boundary of the stained fluorescence; and, in some cases, determining the MFI (Figure 1B, right).

[0167] MFI was used to identify the center or densest part of a cell population, which can be particularly useful when analyzing low-frequency attributes where the reliability of the fluorescence percentile in isolation may be distorted, for example, by outliers in cell populations with a relatively small number of fluorescent cells (either positively stained or negatively stained cell populations). The centers of the negatively stained 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 percentiles of fluorescence for the negatively stained cell population, or the 1st and / or 5th percentiles of fluorescence for the positively stained cell population. This allowed for setting thresholds for separating the negatively stained cell population from the positively stained cell population. MFI analysis was not necessary when analyzing dense and robust cell populations, e.g., high-frequency attributes, but was used as an additional way to characterize the fluorescence properties of sparse cell populations, 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 AF700 fluorophores. As shown in Figure 2A, the maximum 95th percentile for unstained or negative fluorescence had a relative fluorescence intensity of 62.9, and the maximum 99th percentile for unstained or negative fluorescence had a relative fluorescence intensity of 173.00. As shown in Figure 2B, the 1st percentile for fluorescence in positively stained samples had a relative fluorescence intensity of 3,597.0, and the 5th percentile for fluorescence in positively stained samples had a relative fluorescence intensity of 5,324.0. Based on this data, a static fluorescence threshold of 3,000 was set for this attribute.

[0169] The following fluorescence thresholds were established for each attribute that distinguishes positive staining from unstained or negatively stained samples.

[0170] For frequency attributes (those occurring in ≥5% of the sample) that may be referred to as high-frequency attributes, fluorescence thresholds were established by (1) excluding unstained or negative populations by setting a fluorescence threshold above the maximum 99th percentile for unstained or negative populations, (2) including as many positive populations as possible while excluding statistical outliers, (3) setting a conservative threshold for positive populations when there is a wide range between positive and negative or unstained fluorescence, and (4) rounding the threshold to an even number on the logarithmic flow axis to optimize the workflow. By applying this criterion to frequency attributes using the analyses shown in Figures 2A and 2B, a fluorescence threshold of 3,000 was established. To include as many positive populations as possible while excluding statistical outliers, the fluorescence threshold for positive populations for each attribute was set below the minimum 5th percentile whenever possible, and this was done for each frequency attribute analyzed.

[0171] For low-frequency attributes (frequency of ≤5% in the sample), fluorescence thresholds were established by (1) excluding unstained or negative populations by setting a fluorescence threshold above the maximum 99th percentile for unstained or negative populations, (2) including as many positive populations as possible while excluding statistical outliers, (3) using MFI analysis to assist in threshold determination, and (4) rounding thresholds to an even number on the logarithmic flow axis to optimize the workflow. To include as many positive populations as possible while excluding statistical outliers, the fluorescence threshold for the positive population for each attribute was set below the minimum 5th percentile whenever possible. For attribute 5, the dye (Aqua, as shown in Table E2 below) had a high level of background, so the minimum 5th percentile value of 1506.3 was rounded upward to the nearest overall scale marker of 2000 in order to preferentially include the positive population over the unstained or negative population.

[0172] MFI analysis involved characterizing unstained or negative and positive populations to identify the mean fluorescence intensity or the 50th percentile of intensity, which is typically where the center and densest parts of the population are located on the fluorescence scale. MFI analysis was then used to assist in determining the fluorescence threshold / boundary for each attribute.

[0173] Table E2 below summarizes the unstained, negative-stained, and positive-stained analyses for each of the six reported attributes, providing established fluorescence thresholds for each attribute. Attributes 1-3 and 6 are frequency attributes, while attributes 4 and 5 are low-frequency attributes.

[0174] [Table 2]

[0175] The fluorescence thresholds for low-frequency attributes 4 and 5 were determined by MFI, partly due to distortions in the 1st and 5th percentile values ​​in positive staining analysis.

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

[0177] [Table 3]

[0178] To evaluate the accuracy of static fluorescence threshold gating, accuracy based on inter-laborator coefficient of variation (CV) percentage (%) was assessed among three workers for 97 patient sample lots across four frequency attributes (attributes 1-3 and 6).

[0179] As shown in Table E4, static fluorescence threshold gating was precise between operators, and a CV of less than 3% was observed for all attributes evaluated.

[0180] [Table 4]

[0181] The Lin coincident correlation test was used to characterize the correlation between static fluorescence threshold gating reporting targets and reporting targets. The results of the coincident correlation test are detailed in Table E5 below.

[0182] [Table 5]

[0183] To evaluate background fluorescence using static fluorescence threshold gating applied to FMO samples, static fluorescence threshold gating was applied to three development samples, each having FMO samples stained using each of the same six attributes and then analyzed using appropriate fluorescence channels. Background percentages are reported as a percentage 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 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 static fluorescence threshold gating for each of the six attributes was effective in excluding background staining.

[0184] [Table 6]

[0185] The present invention is not intended to be limited to specific disclosed embodiments, for example, provided to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teaching herein. Such modifications are implementable without deviating from the true scope and spirit of the disclosure and are intended to fall within the scope of the disclosure. This disclosure provides, for example, the following: [Section 1] A method for determining a static fluorescence threshold gate, (1) Measuring cytometric events by flow cytometry for one or more fluorescence signals from multiple reference cell samples of at least two reference cell samples, (a) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (b) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. including, The measurement of the aforementioned; and (2) A static fluorescence threshold gate for each of the one or more fluorescence signals, and for each fluorescence signal, the threshold gate is (a) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (b) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The method, including the method described above. [Section 2] A method for selecting a subset of cytometry events from flow cytometry data, (1) To receive flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is determined in accordance with the method described in item 1; and (3) For each of the one or more fluorescence signals, identify a subset of cytometry events from the test sample that have a fluorescence signal exceeding the static threshold gate. The method, including the method described above. [Section 3] A method for selecting a subset of cytometry events from flow cytometry data, (1) To receive flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) A cytometry event measured by flow cytometry for one or more fluorescent signals of a plurality of reference cell samples, each selected according to one or more fluorescent signals, wherein the plurality of at least two reference cell samples is (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. The cytometry events, including the aforementioned events; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, and for each fluorescence signal, the threshold gate is (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up as follows The application of the foregoing is determined from; and (3) For each of the one or more fluorescence signals, identify a subset of cytometry events from the test cell sample that have a fluorescence signal exceeding the static threshold gate. The method, including the method described above. [Section 4] A method for applying a static fluorescence threshold gate using flow cytometry, (1) Receiving flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is determined in accordance with the method described in item 1. The method, including the method described above. [Section 5] A method for applying a static fluorescence threshold gate using flow cytometry, (1) To receive flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) A cytometry event measured by flow cytometry for one or more fluorescent signals of a plurality of reference cell samples, each selected according to one or more fluorescent signals, wherein the plurality of at least two reference cell samples is (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. The cytometry events, including the aforementioned events; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, and for each fluorescence signal, the threshold gate is (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The application of the above is determined from The method, including the method described above. [Section 6] The method according to any one of items 1 to 5, wherein each of the test cell samples and the plurality of first and second reference cell samples are from the same cell source type. [Section 7] The method according to any one of items 1 to 6, wherein the source type of the cells is a cell line. [Section 8] The method according to any of items 1 to 6, wherein the cell source type is a primary cell population from the subject. [Section 9] The method according to item 8, wherein the source type of cells in each of the test cell samples and each of the multiple first and second reference cell samples is from different sources. [Section 10] The method according to paragraph 9, wherein each different subject has the same or similar disease or condition. [Section 11] The method according to any one of items 1 to 10, wherein the cell source type is a whole blood sample, an apheresis sample, or a leukocyte apheresis sample. [Section 12] The method according to any one of items 1 to 11, wherein the cell source type is a cell-enriched population, optionally a T cell-enriched population. [Section 13] The method according to any one of claims 1 to 12, wherein the cell source type is an engineered population of cells containing nucleic acids encoding recombinant proteins, and the recombinant proteins are introduced into the population of cells by gene transfer, or by transduction. [Section 14] The method described in any of sections 1 to 13, wherein the cell source type is cell therapy. [Section 15] The method according to any one of claims 1 to 14, 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. [Section 16] The method according to any one of claims 1 to 15, 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. [Section 17] The method according to any one of items 1 to 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. [Section 18] The method according to any one of claims 1 to 16, wherein the cell source type is 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 to test the cell therapy. [Section 19] The method according to any one of claims 14 to 18, wherein the cell therapy is T cell therapy, CAR-T cell therapy, TCR-T cell therapy, or TIL therapy, as appropriate. [Section 20] The method according to any one of items 14 to 18, wherein the cell therapy is NK cell therapy. [Section 21] The method according to any one of items 14 to 18, wherein the cell therapy is stem cell therapy. [Section 22] The method according to any one of claims 1 to 21, wherein 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 the cells with the one or more fluorescent signals. [Section 23] The method according to claim 22, wherein each of the one or more staining reagents comprises a marker-specific binder and a fluorescent dye capable of emitting one of the one or more fluorescent signals. [Section 24] The method according to item 23, wherein the marker is a cell surface marker or a survival marker. [Section 25] The method according to claim 23 or 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 cells in each of the test cell sample and the second reference cell sample. [Section 26] The method according to any one of items 23 to 25, wherein at least one marker is a low-frequency attribute 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. [Section 27] The method according to any one of claims 1 to 26, wherein the one or more fluorescent signals include two or more different fluorescent signals. [Section 28] The method according to any one of items 22 to 27, wherein the cell staining is a multicolor cell staining for labeling the cells using two or more different fluorescent signals, and each staining reagent labels a different marker using a different fluorescent signal. [Section 29] The method according to claim 27 or 28, wherein the two or more distinct fluorescent signals include 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. [Section 30] The method according to any one of claims 27 to 29, wherein each of the two or more different fluorescence signals has a different emission spectrum, and / or the peak emission spectra of each fluorescence signal do not overlap. [Section 31] The method according to any one of claims 27 to 30, wherein each of the two or more distinct fluorescent signals is a signal emitted by a dye selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605. [Section 32] The method according to any one of sections 22 to 31, wherein the cell staining of each of the test cell sample and the second reference cell sample is performed using the same staining reagent and under the same protocol conditions. [Section 33] The method according to any one of claims 1 to 32, wherein the plurality of second reference cell samples includes two sets of samples of the same stained cells. [Section 34] The method according to any one of claims 1 to 32, wherein the plurality of the second reference cell sample comprises a triplicate sample of the same stained cells. [Section 35] The method according to any one of claims 1 to 34, wherein the static fluorescence threshold gate for at least one fluorescence signal is higher fluorescence than the 95th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal. [Section 36] The method according to any one of claims 1 to 34, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is higher fluorescence than the 97th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal. [Section 37] The method according to any one of claims 1 to 34, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is higher fluorescence than the 99th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal. [Section 38] The method according to any one of claims 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is a fluorescence lower than the fifth percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal. [Section 39] The method according to any one of claims 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is a fluorescence lower than the third percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal. [Section 40] The method according to any one of claims 1 to 37, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is a fluorescence lower than the first percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal. [Section 41] The method according to any one of claims 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and a population of stained cells differ by more than twofold for the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence or less than the overall percentile of fluorescence for the second reference cell sample among the plurality for the fluorescence signal. [Section 42] The method according to any one of claims 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and a population of stained cells differ by more than a 5-fold difference for the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence or less than the overall percentile of fluorescence for the second reference cell sample among the plurality for the fluorescence signal. [Section 43] The method according to any one of claims 1 to 37 and 40, wherein if the fluorescence of a population of unstained cells and a population of stained cells differ by more than a 10-fold difference for the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence or less than the overall percentile of fluorescence for the second reference cell sample among the plurality for the fluorescence signal. [Section 44] The method according to any one of claims 1 to 43, wherein, with respect to low-frequency attributes, the setting of the static fluorescence threshold gate further comprises identifying the mean fluorescence intensity (MFI) for one or more of the one or more fluorescence signals. [Section 45] The method according to any one of items 1 to 44, wherein for each of the one or more fluorescence signals, the static fluorescence threshold gate is even on the logarithmic flow axis. [Section 46] The method according to any one of items 1 to 45, wherein for each of the one or more fluorescence signals, the static fluorescence threshold gate is rounded to the nearest tens digit, the nearest hundreds digit, or the nearest thousands digit on the logarithmic flow axis. [Section 47] The method of any one of claims 2 to 46, further comprising identifying a subset of cells from the test cell sample having fluorescence signals that exceed the static fluorescence threshold gate for at least two of the one or more fluorescence signals, at least three of the one or more fluorescence signals, at least four of the one or more fluorescence signals, at least five of the one or more fluorescence signals, at least six of the one or more fluorescence signals, or at least seven or more of the one or more fluorescence signals. [Section 48] The method according to any one of claims 2 to 47, further comprising identifying a subset of cells from the test cell sample having fluorescence signals that exceed the static fluorescence threshold gate for all of the one or more fluorescence signals. [Section 49] The method according to any one of claims 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 in the test cell sample. [Section 50] The method according to any one of claims 1 to 49, wherein the static fluorescence threshold gate for each of the one or more fluorescence 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 in the test cell sample. [Section 51] The method according to any one of claims 2, 3, and 6-50, further comprising evaluating the fluorescence intensity of the at least one fluorescence signal of the identified subset of cytometry events for at least one of the one or more fluorescence signals. [Section 52] The method according to any one of claims 2, 3, and 6-50, further comprising evaluating the intensity of the fluorescence signal of the identified subset of cytometry events for each of the one or more fluorescence signals. [Section 53] The method according to claim 51 or claim 52, wherein the fluorescence intensity is the average fluorescence intensity. [Section 54] The method according to any one of claims 2, 3, and 6-53, further comprising determining the percentage of the identified subset of cytometry events for at least one of the one or more fluorescence signals compared to the total number of cytometry events collected. [Section 55] The method according to any one of claims 2, 3, and 6-53, further comprising determining the percentage of the identified subset of cytometry events for each of the one or more fluorescence signals compared to the total number of cytometry events collected. [Section 56] The method according to any one of claims 1 to 55, comprising calibration of the one or more fluorescence signals using the flow cytometer. [Section 57] The method according to paragraph 56, wherein the calibration is performed at least once a day.

Claims

1. A method for selecting a subset of cytometry events from flow cytometry data, (1) Receiving flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) Measuring cytometric events by flow cytometry for one or more fluorescence signals of multiple reference cell samples of at least two reference cell samples, wherein the multiple reference cell samples of at least two reference cell samples are (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as one of the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. Including the measurement of the foregoing; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, the threshold gate for each fluorescence signal (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up as follows The application of the above is determined according to a method including; and (3) For each of the one or more fluorescence signals, identify a subset of cytometry events from the test sample that have a fluorescence signal exceeding the static threshold gate. The method, including the method described above.

2. A method for selecting a subset of cytometry events from flow cytometry data, (1) Receiving flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) A cytometry event measured by flow cytometry for one or more fluorescent signals of a plurality of at least two reference cell samples, each selected according to the one or more fluorescent signals, wherein the plurality of at least two reference cell samples is (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as one of the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. The cytometry events including; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, the threshold gate for each fluorescence signal (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up as follows The application of the foregoing is determined from; and (3) For each of the one or more fluorescence signals, identify a subset of cytometry events from the test cell sample that have a fluorescence signal exceeding the static threshold gate. The method, including the method described above.

3. A method for applying a static fluorescence threshold gate using flow cytometry, (1) Receiving flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; and (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) Measuring cytometric events by flow cytometry for one or more fluorescence signals of multiple reference cell samples of at least two reference cell samples, wherein the multiple reference cell samples of at least two reference cell samples are (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as one of the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. Including the measurement of the foregoing; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, the threshold gate for each fluorescence signal (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The application of the above is determined according to a method including the following The method, including the method described above.

4. A method for applying a static fluorescence threshold gate using flow cytometry, (1) Receiving flow cytometry data including multiple cytometry events from a test cell sample containing labeled cells sorted according to one or more fluorescence signals measured by a flow cytometer; (2) Applying a static fluorescence threshold gate to each of the one or more fluorescence signals individually, wherein the static fluorescence threshold gate for each of the one or more fluorescence signals is (a) A cytometry event measured by flow cytometry for one or more fluorescent signals of a plurality of at least two reference cell samples, each selected according to the one or more fluorescent signals, wherein the plurality of at least two reference cell samples is (i) Multiple first reference cell samples, each containing a population of unstained cells not labeled with one or more fluorescent signals; and (ii) A plurality of second reference cell samples, each of which the plurality of second reference cell samples originates from the same cell source type as one of the plurality of first reference cell samples, and each of the plurality of second reference cell samples includes at least one population of stained cells labeled with at least one of the one or more fluorescent signals. The cytometry events including; and (b) A static fluorescence threshold gate for each of the one or more fluorescence signals, the threshold gate for each fluorescence signal (i) Each of the one or more fluorescent signals has fluorescence that is higher than the 90th percentile of the fluorescence of the plurality of objects in the first reference cell sample; (ii) For each of the same fluorescence signals, the fluorescence is lower than the 10th percentile of the fluorescence of the plurality of objects in the second reference cell sample. Set it up that way The application of the above is determined from The method, including the method described above.

5. The method according to any one of claims 1 to 4, wherein each of the test cell samples and the plurality of first and second reference cell samples are from the same cell source type.

6. The method according to any one of claims 1 to 5, wherein the source type of the cells is a cell line.

7. The method according to any one of claims 1 to 5, wherein the cell source type is a primary cell population from a subject.

8. The method according to claim 7, wherein the source type of cells in each of the test cell samples and each of the plurality of the first and second reference cell samples is from different sources.

9. The method according to claim 8, wherein each different subject has the same or similar disease or condition.

10. The method according to any one of claims 1 to 9, wherein the cell source type is a whole blood sample, an apheresis sample, or a leukocyte apheresis sample.

11. The method according to any one of claims 1 to 10, wherein the cell source type is an enriched population of cells.

12. The method according to any one of claims 1 to 11, wherein the cell source type is an engineered population of cells containing nucleic acids encoding recombinant proteins.

13. The method according to any one of claims 1 to 12, wherein the cell source type is cell therapy.

14. The method according to any one of claims 1 to 13, 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.

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

16. The method according to any one of claims 13 to 15, wherein the cell therapy is T cell therapy.

17. The method according to claim 16, wherein the T cell therapy is CAR-T cell therapy, TCR-T cell therapy, or TIL therapy.

18. The method according to any one of claims 13 to 15, wherein the cell therapy is NK cell therapy.

19. The method according to any one of claims 13 to 15, wherein the cell therapy is stem cell therapy.

20. The method according to any one of claims 1 to 19, wherein 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 the cells with the one or more fluorescent signals.

21. The method according to claim 20, wherein each of the one or more staining reagents comprises a marker-specific binder and a fluorescent dye capable of releasing one of the one or more fluorescent signals.

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

23. The method according to claim 21 or 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 cells in each of the test cell sample and the second reference cell sample.

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

26. The method according to any one of claims 20 to 25, wherein the cell staining is a multicolor cell staining for labeling the cells using two or more different fluorescent signals, and each staining reagent labels a different marker using a different fluorescent signal.

27. The method according to claim 25 or 26, wherein each of the two or more different fluorescence signals has a different emission spectrum, and / or the peak emission spectra of each fluorescence 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 according to any one of claims 24 to 28, wherein 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.

30. The method according to any one of claims 1 to 29, wherein the plurality of second reference cell samples includes a pair of samples of the same stained cells, and the plurality of second reference cell samples includes a triple of samples of the same stained cells.

31. The method according to any one of claims 1 to 30, wherein the static fluorescence threshold gate for at least one of the one or more fluorescence signals is higher fluorescence than the 99th percentile of fluorescence among the plurality of first reference cell samples for the fluorescence signal.

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

33. The method according to any one of claims 1 to 32, wherein if the fluorescence of a population of unstained cells and a population of stained cells differ by more than a 10-fold in relation to the fluorescence signal, the static fluorescence threshold gate is set to less than the first percentile of fluorescence or less than the overall percentile of fluorescence among the plurality of second reference cell samples for the fluorescence signal.

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

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

36. The method according to any one of claims 1 to 35, wherein for each of the one or more fluorescence signals, 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 in the test cell sample.

37. For each of the one or more fluorescence signals, the static fluorescence threshold gate is: Each fluorescence signal is higher than the 95th percentile of the fluorescence of the plurality of substances in the first reference cell sample; For each of the same fluorescence signals, the fluorescence is lower than the 5th percentile of the fluorescence of the multiple objects in the second reference cell sample. The method according to any one of claims 1 to 36.

38. For each of the one or more fluorescence signals, the static fluorescence threshold gate is: Each fluorescence signal is higher than the 99th percentile of the fluorescence of the plurality of substances in the first reference cell sample; For each of the same fluorescence signals, the fluorescence is lower than the first percentile of the fluorescence of the multiple objects in the second reference cell sample. The method according to any one of claims 1 to 37.

39. The method according to any one of claims 1, 2, and 5 to 36, further comprising evaluating the intensity of the fluorescence signal of the identified subset of cytometry events for each of the one or more fluorescence signals.

40. The method according to claim 39, wherein the fluorescence intensity is the average fluorescence intensity.

41. The method according to any one of claims 1, 2, and 5 to 40, further comprising determining the percentage of the identified subset of cytometry events for at least one of the one or more fluorescence signals compared to the total number of cytometry events collected.

42. The method according to any one of claims 1, 2, and 5-40, further comprising determining the percentage of the identified subset of cytometry events for each of the one or more fluorescence signals compared to the total number of cytometry events collected.

43. The method according to any one of claims 1 to 42, comprising calibration of the one or more fluorescence signals using the flow cytometer.

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