Compositions and methods for single-well multiplex calibration and correction

Abstract

The present disclosure relates to compositions comprising multiple modified particles that enable one-pot calibration, correction, and spectral unmixing, as well as methods for their use. Flow cytometry, hematology, and image-based cytometry are techniques that enable the rapid separation, counting, and characterization of individual particles and are routinely used in clinical and laboratory settings for a variety of applications. The techniques typically rely on directing a beam of light into a focused stream of liquid.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 400,039, filed August 22, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to compositions of matter and methods that enable calibration, correction, and spectral unmixing in a single well. [Background technology]

[0003] Flow cytometry, hematology, and image-based cytometry are techniques that enable the rapid separation, counting, and characterization of individual particles and are routinely used in clinical and laboratory settings for a variety of applications. This technique typically relies on directing a beam of light onto a focused liquid stream. In one form, multiple detectors are then targeted at the point where the stream passes through the light beam: one aligned with the light beam (e.g., forward scatter, or FSC; also known as axial light loss, or ALL) and some perpendicular to it (e.g., side scatter, or SSC). FSC generally correlates with particle volume, while SSC generally depends on particle complexity, or granularity (i.e., nuclear shape, amount and type of cytoplasmic granules, or membrane roughness). As a result of these correlations, different specific particle types (i.e., cells, extracellular vesicles) exhibit different FSC and SSC, allowing them to be distinguished from one another.

[0004] These measurements include the basis of cytometric analysis. In some forms of analysis, cells are also imaged and descriptive characteristics of the cells, such as size / shape / volume, and in some cases, biochemical characteristics when combined with detection reagents, are recorded. Other forms of analysis use interferometry, particle tracking analysis, and electrical perturbation to measure particle properties. In additional forms, cells are labeled with reagents that detect the presence of biomarkers (or nucleic acids), allowing for multiplexed measurement of object features and characteristics.

[0005] The use of fluorescent molecules, such as fluorophore-labeled antibodies, in flow cytometry is a common method for examining cellular characteristics. For clarity, fluorophores and fluorescent dyes are used interchangeably herein. Fluorophores may also be referred to as tags, dyes, or stains. In these types of experiments, labeled antibodies are added to a cell sample. The antibodies then bind to specific molecules on the cell surface or inside the cells. Finally, when laser light of the appropriate wavelength hits the fluorophore, a fluorescent signal is emitted and detected by the flow cytometer.

[0006] However, when using fluorophores, it can be difficult to delineate between different target species based solely on their corresponding fluorescent signals, which may overlap, compromising the ability to assign meaningful values ​​to each target species. The present disclosure addresses this shortcoming. Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a composition for compensation or spectral unmixing calculations in a single well comprising first polymer particles having a first biomarker found on target cells and second polymer beads comprising a second biomarker found on target cells. In another aspect, the present disclosure provides a method of calibrating a cytometry device comprising mixing such a composition in a single well with antibody-fluorophore conjugates specific for each biomarker in the cytometry device, measuring the fluorescent signal of the mixture, deconvolving the fluorescent signals of the first and second antibody-fluorophore-conjugated polymer beads from the measured fluorescent signal of the mixture using the known fluorescent signals of the first and second antibody-fluorophore conjugates to calculate a compensation or spectral unmixing matrix, and calibrating the cytometry device.

[0008] In one aspect, the present disclosure provides a composition for compensation or spectral unmixing calculations in a single well comprising first polymer particles bearing a first biomarker found on target cells and second polymer beads comprising a second biomarker found on target cells, wherein a first antibody-fluorophore conjugate specific for the first biomarker and a second antibody-fluorophore conjugate specific for the second biomarker are pre-bound or pre-conjugated to the first and second biomarkers. In another aspect, the present disclosure provides a method of calibrating a cytometry device comprising mixing such a composition in a single well in a cytometry device, measuring the fluorescent signal of the mixture, deconvolving the measured fluorescent signals of the first and second antibody-fluorophore-bound polymer beads from the measured fluorescent signal of the mixture using known fluorescent signal characteristics of the first and second antibody-fluorophore conjugates to calculate a compensation or spectral unmixing matrix, and calibrating the cytometry device.

[0009] In a preferred embodiment, the polymer particles comprise a hydrogel that substantially resembles the autofluorescence and other optical properties of the target cells.

[0010] The present invention allows correction or spectral unmixing calculations to be performed in a single reaction. The present invention also allows FMO calculations to be performed using a single reagent mixture.

[0011] The present disclosure also provides a computational method for performing fluorescence compensation and spectral unmixing using a single reaction vessel, in which multiple individual bead populations are combined with a complete stain panel mixture in a single tube. The individual bead populations may be pre-modified with the fluorophores used in a given panel, or they may be prepared so that they specifically bind to each individual antibody-fluorophore conjugate from the stain panel mixture. The present disclosure also provides a method for deriving deconvoluted data from a single reaction vessel based on the intrinsic or predetermined fluorescent properties of the individual fluorophores / fluorescent dyes to generate the input data required for compensation or spectral unmixing calculations. The present disclosure also provides a method for deriving FMO control calculations from a one-mix antibody stain panel. The present disclosure also provides a software-driven method for automatically calculating the individual compensation and spectral unmixing calculations from this approach.

[0012] In another embodiment, fluorescent features can be directly conjugated to polymer beads. For example, rather than creating biochemically distinct bead populations in a mixture that specifically bind to individual reagents in a stain panel, beads can be labeled a priori with fluorophores (or combinations of fluorophores, or pre-bound antibody-fluorophore conjugates) from the stain panel so that they can be easily deconvoluted for correction or FMO calculations. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 illustrates the general concept of fluorescence compensation and spectral unmixing, highlighting the differences between (A) traditional and (B) one-pot methods. The present invention provides dramatic time savings for high-complexity assays (e.g., 30-color calculations).

[0014] [Figure 2]Figure 2 illustrates the traditional compensation bead workflow, which requires separate tubes, and why a combined stain panel approach does not work using existing reagents. Standard compensation beads bind to a wide range of detection antibodies (depicted as "reagents") in a stain panel. Therefore, the reagents must be separated into individual tubes to calculate compensation data (A). If the stain panel is combined into a mixture and then added to the compensation beads, multiple fluorophores will be bound to each bead, making deconvolution and compensation or spectral unmixing calculations impossible (B).

[0015] [Figure 3] Figure 3 depicts the fluorescence spillover and compensation adjustments required to calculate suitable emission signal data. In traditional workflows, each fluorophore-antibody combination must be separated into individual tubes and physically deconvolved to separate the signals for compensation or spectral unmixing calculations. This is driven in large part by the fact that the particles used in traditional compensation calculations (whether cells or traditional compensation beads) indiscriminately bind to most, if not all, of the antibodies in a given panel and cannot separate them into distinct events.

[0016] [Figure 4]Figure 4 illustrates an example of the disclosed one-pot compensation or unmixing method in which individual bead populations are pre-modified with fluorescent dyes or bead populations are modified to bind individual reagents (antibody-fluorophore conjugates) from a mixed staining panel. The biomarker-modified compensation beads only bind to a single antibody in the reagent cocktail, allowing the user to add the complete staining panel mixture to a single tube and deconvolute individual fluorescent signals from the mixture for compensation or spectral unmixing calculations. Individual fluorophore signals can be deconvoluted for compensation or spectral unmixing calculations because each bead binds only to a specific reagent in the mixture. This results in a dramatically simplified workflow.

[0017] [Figure 5] Figure 5 illustrates how a user can add a pre-aliquoted mixture of selectively binding biomarker beads (e.g., a full stain panel) in a one-pot reaction to computationally deconvolute the individual fluorophore profiles and calculate corrections or spectral unmixing. This is made possible by the fact that each individual bead in the mixture binds only one antibody from the cocktail. The software then allows selective isolation of profile information by selecting local maxima (Figure 13), for example, allowing in silico deconvolution vs. physical tube-derived deconvolution (Figure 2).

[0018] [Figure 6] Figure 6 illustrates (A) the differences between a typical fluorescence minus one (FMO) staining panel workflow compared to an FMO panel generated using the reagents and approaches described in this disclosure. In a typical workflow, a combinatorial mixture of antibodies must be generated by the user and then applied to binding particles, typically patient cells. This results in dramatic time savings for high-complexity (e.g., 30-color) assays.

[0019] [Figure 7] Figure 7 illustrates the physical process of generating FMO data for an experiment by creating a combinatorial mixture of the reagents (minus one) used in a staining panel. Standard FMO workflows require the user to exhaustively create a combinatorial mixture of all antibodies in the staining panel (minus one) in order to calculate the FMO noise floor. This is driven by the fact that the reagents or cells used for the FMO calculation bind to all antibodies in the cocktail.

[0020] [Figure 8] Figure 8 illustrates the importance of FMO calculations to ensure accurate data generation, especially for dim or poorly expressed biomarkers. In this simplified panel, there are three staining antibodies in the collection, each emitting in a unique channel. Due to fluorescence spillover, the true noise floor or lower limit of detection is highlighted by the dotted line. Any signal below the dotted line cannot be reliably measured because it is generated from orthogonal antibodies in the panel and is not a true biological representation of the presence of the target biomarker for that channel. This is especially important for poorly expressed or "dim" biomarker sets.

[0021] [Figure 9] Figure 9 illustrates a one-reagent mix FMO control workflow in which the user prepares only one master mix of staining antibodies for a staining panel. The user then applies that single mix to a pre-aliquoted combinatorial mix of FMO-conjugated beads, greatly improving operator efficiency and reducing error. The FMO-conjugated beads are pre-aliquoted to contain the full panel (minus one), allowing the user to create only one master mix of reagents, saving considerable time compared to traditional workflows.

[0022] [Figure 10] Figure 10 illustrates a one-mixture FMO control panel and the impact it has on calculating true signal-to-noise, or the noise floor, of staining panels and assays. This configuration allows users to combine combination FMO biomarker beads a priori to create a single cocktail that the user needs to add to each tube of mixed biomarker beads. Each of these mixtures lacks unique biomarker beads and acts like an FMO control. This dramatically accelerates the workflow for calculating FMO controls for complex panels. This also eliminates the need to use actual patient samples or cells for FMO calculations.

[0023] [Figure 11] Figure 11 illustrates a traditional compensation data workflow using six antibodies from the TBNK panel. Typically, individual antibody-fluorophore conjugates must be mixed and coupled with compensation beads in separate tubes to generate the spectra used for compensation or spectral unmixing calculations, as shown (PerCP-Cy5.5, PE-Cy7, APC-Cy7, FITC, PE, APC).

[0024] [Figure 12]Figure 12 depicts six unique antibody-fluorophore conjugates mixed in the same tube and analyzed together. The signal from the mixture will be the same whether traditional compensation beads or the beads of the present invention are used. However, this signal cannot be deconvoluted using traditional compensation beads because each bead binds to all of the reagents in a given mixture. This prevents effective compensation or spectral unmixing calculations. However, when using the compositions and methods of the present invention, the independent signals can be deconvoluted from the mixture and compensation or spectral unmixing calculations can be performed. In this example, a spectral cytometer is used; however, any traditional cytometer can also be used.

[0025] [Figure 13] Figure 13 depicts the process of selecting maxima to deconvolute individual fluorophore signals from a combinatorial mixture of beads described in this disclosure. This allows the data from Figure 12, for example, to be separated into individual channels. For this basic example, the maximum value of each fluorescence channel was used to select individually modified beads.

[0026] [Figure 14] Figure 14 depicts successfully deconvoluted individual fluorophores from a one-pot reaction mixture containing individually modified beads, allowing single-tube correction matrix / spectral unmixing calculations to be performed. Such individual fluorophore signals are indistinguishable from the data generated by the individual separated tubes in Figure 11.

[0027] [Figure 15]FIG. 15 depicts (B) the resulting data from a spectral unmixing TBNK staining panel using the one-pot bead mixture described in this disclosure, which is indistinguishable from (A) the individually separated tubes used for spectral unmixing. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition As used herein, the indefinite articles "a" and "an" and the definite article "the" are intended to include both the singular and the plural unless the context in which they are used clearly indicates otherwise. "At least one" and "one or more" are used interchangeably to mean that the article may include one or more of the listed elements.

[0029] As used herein, the terms "polymeric beads" and "polymeric particles" may be used interchangeably.

[0030] Unless otherwise indicated, it should be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and the like used in the specification and claims can be modified in all instances by the term "about." For example, throughout this application, the term "about" can be used to indicate that a value includes the inherent variation of error for the device or method being used to determine the value or variation present among the sample being measured. Unless otherwise stated or otherwise clear from the context, the term "about" means within more than or less than 10% of the reported numerical value (except where such number would exceed 100% or be less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each recited value in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0031] There is a general trend in the art to increase the number of biophysical characteristics being measured in a single experiment or assay tube. For purposes of this application, the terms assay tube, well, vessel, pot, tube, and reaction vessel are used interchangeably. This is generally referred to as a "stain panel," which is designed to characterize a sample of interest using a set of reagents, typically antibodies recognizing epitopes conjugated to fluorophores. Larger stain panels allow for more "multiplexed" analysis, greater operator efficiency, and reduced sample and analyte requirements for performing complex characterization / phenotyping assays. Exemplary stain panels include optimized multicolor immunofluorescence panels (OMIPs), as outlined by the International Society for Advancement of Cytometry.

[0032] The complex methodology involved in fluorescence detection presents significant hurdles for researchers to consider. This includes the operational time required to physically separate each reagent in a staining panel for individual analysis during a correction control setup, which can involve several hours of labor for a complex panel. This also increases the likelihood of operator error in many settings. The added complexity of flow cytometry, combined with the consequent design of experimental protocols and detailed analyses involving multiple fluorophores and fluorescent signals, presents additional obstacles to its efficient use. Proper consideration of the spectral overlap resulting from the use or inclusion of multiple fluorescent materials in different detection systems is currently being addressed to address these issues as they arise.

[0033] As the complexity of a stain panel increases, it becomes more important to distinguish fluorescent or spectral signals from one another, typically through compensation or spectral unmixing methods, because there is a greater likelihood that a given set of fluorophores will not be reliably distinguishable from one another. This is known as fluorescent or spectral overlap / spillover / crosstalk and can confound interpretation of results. From a first-principles perspective, this effect is driven by the fact that most fluorescent reagents have broad excitation and emission spectra versus single-wavelength emission profiles. Therefore, compensation becomes increasingly important as the complexity of a stain panel increases.

[0034] fluorescence A fluorophore is a molecule capable of emitting fluorescence. In its ground state, a fluorophore molecule is in a relatively low-energy, stable configuration; it does not fluoresce. When light from an external source strikes a fluorophore molecule, the molecule can absorb the light energy. If enough energy is absorbed, the molecule reaches an excited (high-energy) state, a process known as excitation. Depending on the wavelength and energy of the external light source, there are multiple excited states or energy levels that a fluorophore can achieve. Because a fluorophore is unstable in its high-energy configuration, it eventually adopts its lowest-energy excited state, which is semistable. The excited lifetime (the length of time a fluorophore remains in an excited state) is very short, and the fluorophore can reconfigure from the semistable excited state back to the ground state, releasing some of the excess energy and emitting it as light. The emitted light is lower in energy and longer in wavelength than the absorbed light; thus, the color of the emitted light is different from the color of the absorbed light. Deexcitation returns the fluorophore to its ground state. The fluorophore can absorb light energy again and go through the excited to ground state process repeatedly.

[0035] Fluorescence spectrum Fluorescent dyes absorb light over a range of wavelengths, and every dye has a characteristic excitation range. This range of excitation wavelengths is referred to as the fluorescence excitation spectrum and reflects the range of possible excited states that the dye can achieve. Certain wavelengths within this range are more effective for excitation than others. Fluorophores are most efficiently excited by light of a specific wavelength. This wavelength is the excitation maximum for the fluorophore. Less efficient excitation can occur at wavelengths close to the excitation maximum; however, the intensity of the emitted fluorescence is reduced. Irradiation at the excitation maximum of a fluorophore produces maximum fluorescence output, while irradiation at lower or higher wavelengths only affects the intensity of the emitted light; the range and overall shape of the emission profile remain unchanged.

[0036] Due to changes that may occur during the excitation lifetime, the molecules may emit at different wavelengths with each excitation event, but each emission will be within the fluorescence emission spectrum. Fluorophore molecules all emit light with the same intensity, but the wavelength, and therefore the color, of the emitted light is not uniform. The emission maximum is the wavelength at which a population of molecules fluoresces most strongly. Excited fluorophores can also emit light at wavelengths close to the emission maximum; however, this light is less intense.

[0037] Different types of light sources are used to excite fluorophores. Common sources include broadband sources, such as mercury arc lamps and tungsten-halogen lamps. These lamps produce white light with varying intensity peaks across the spectrum. When using a broadband white light source, it is necessary to filter the desired wavelengths required for excitation; this is most often done using optical filters. Optical filters selectively allow certain wavelengths of light to pass while blocking undesired wavelengths. Bandpass excitation filters transmit narrow wavelength ranges and can be used for selective excitation. Laser excitation sources can also be used. Lasers provide well-defined, selective, and high-intensity wavelength peaks, allowing for more selective illumination of samples. High-power light-emitting diodes (LEDs) offer selective wavelengths, low cost, low energy consumption, and long lifetimes. Monochromatic LEDs are ideal for low-cost instruments, as they can be combined with simple long-pass filters that block the LED excitation and allow the dye signal to pass. However, the range of wavelengths emitted from each LED is still relatively broad and may require the use of filters to narrow the bandwidth. Further information regarding fluorescence spectra can be found in The MolecularProbes® Handbook - A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety.

[0038] Fluorescence detection In traditional or conventional flow cytometers and other instruments that use multiple photodetectors to detect multiple dyes, collected light is typically separated into specific wavelength ranges by a system of frequency-dependent filters and dichroic mirrors, so that the light detected by a particular photodetector or photomultiplier tube (PMT) is limited to a predetermined wavelength range, sometimes referred to as a detection channel. The detection channels and dyes are selected so that the peak of each dye's emission spectrum falls within the frequency range of a different detection channel; for example, each detection channel detects emission primarily from a single dye. However, due to the breadth of a fluorescent dye's emission spectrum, a dye typically fluoresces in more than one detection channel, and thus, measurements of dye fluorescence are not independent. The emission of one dye in a detection channel intended for the detection of another dye is referred to by a number of terms, such as spillover, spectral overlap, and crosstalk.

[0039] Spectral flow cytometry is a technology based on conventional flow cytometry in which spectroscopy and multichannel detectors (e.g., charge-coupled devices (CCDs)) replace the traditional mirrors, optical filters, and PMTs in conventional systems. In a spectral flow cytometer, fluorescent light is collected and displayed, either directly or through optical fibers, as a spectrometer, where the total light signal is dispersed and displayed as a high-resolution spectrum on a CCD or coupled to one or more multichannel detectors for detection.

[0040] For proper data interpretation, the fluorescence light recorded from one fluorescence source must be distinguished from that recorded from other fluorescence sources. For that reason, an ideal fluorophore has a very intense, narrow-peaked fluorescence emission profile that is well separated from all other emission peaks. However, typical fluorophores have broad emission peaks that can overlap or spill over. This overlap or spillover can corrupt the data and analysis.

[0041] Background fluorescence, which may originate from endogenous sample components (autofluorescence) or from unbound or nonspecifically bound reagents, can impair fluorescence detection. Detection of autofluorescence can be minimized by selecting filters that reduce the transmission or detection of autofluorescence, however, doing so impairs overall fluorescence intensity detection. Full-spectrum flow cytometers detect autofluorescence.

[0042] calibration Most assays require some form of calibration and setup to ensure accurate performance when analyzing biological samples. Examples of calibration include setting gain and voltage for detection, measuring inter-laser drop delays, and ensuring linearity in detecting fluorescent signals. In many instances, the ideal calibration and setup reagent would look and act like the biological particles being analyzed. This helps ensure similar performance in diagnostic equipment while not introducing artifacts into the measurement process.

[0043] Most synthetic or polymeric products used in cellular analysis are made of polystyrene, an opaque polymer with a high refractive index that generally has fixed forward and side scattering profiles based on the particle's diameter. This high refractive index distinguishes it from biological particles, such as cells and extracellular vesicles, which are translucent and allow for optical resolution and measurement of internal features, such as organelles in the case of cells. Extracellular vesicles, in particular, often have a low refractive index compared to their diameter. Polystyrene is also hydrophobic and has a high elastic modulus, two characteristics that distinguish it from cellular and biological materials. In some forms, silica (SiO2) is used as a substitute for biological particles. While the refractive index of silica more closely matches that of typical extracellular vesicles, there is still a substantial difference in optical response, requiring interpolation when used as a calibration reagent.

[0044] Taken together, these characteristics make polystyrene and silica particles less than ideal for creating control and calibration reagents for biological particle measurements. As a result, many physicians use cellular materials or biologically derived particles (e.g., lipid nanoparticles) as process and reference controls before measuring samples. Cellular and biological materials suffer from other drawbacks, including poor stability, limited and complex supply chains, high batch-to-batch variability, and high production costs. Additionally, the materials typically have strict cold-chain handling requirements, limiting the scope and application of controls.

[0045] Correction and Spectral Unmixing Another example of calibration includes fluorescence compensation, where the excitation and emission spectra of a given fluorophore are differentiated from potentially overlapping sets of fluorophores used in the same set of experiments. An additional example of calibration includes spectral unmixing, where the spectral response of a given fluorophore (sometimes referred to as the fluorophore's emission, fingerprint, signature, or pattern) is differentiated from potentially overlapping sets of fluorophores used in the same set of experiments. In traditional cytometers, the process is referred to as "compensation," while in spectral cytometers, it is termed spectral unmixing. While compensation and spectral unmixing share the same conceptual goal, they are based on different mathematical calculations.

[0046] Ideally, when a dye is used in an experiment, its emission spectrum is narrow enough that fluorescence from that dye is detected by only a single detector in the instrument. In practice, due to the broad emission spectra of available fluorescent dyes, the dye being used is likely to emit significant amounts of fluorescence at several different detectors. In other words, the light reaching a given detector is composed of signals from multiple fluorescent dyes. Compensation is the process of transforming data so that values ​​from a single detector appear to originate from individual dyes. To separate these signals, or to compensate for overlapping emissions, a percentage of each overlapping emission is subtracted from the target emission. Traditionally, this compensation was performed by the instrument during acquisition. However, modern instruments are capable of storing data in uncompensated form, allowing the compensation to be applied by analysis software. In an exemplary calculation, the compensated parameter 2 fluorescence is equal to the observed parameter 2 fluorescence minus 5% of the observed parameter 1 fluorescence.

[0047] The compensation involves creating two matrices: the spillover matrix, which represents the percentage of signal from a given channel that spills into adjacent channels, and the compensation matrix, which is used to compensate for spillover, is the inverse of the spillover matrix.

[0048] In embodiments, the target parameter is the parameter that is detecting the signal (potentially from multiple sources). The source parameter is the primary parameter where we want the signal to enter, but the dye is also (potentially) spilling into multiple targets. In other words, we subtract the percentage of the source that is "spilling" into the target. In the example given above, parameter 2 is the target and parameter 1 is the source. A family of sources and targets is called a compensation definition. A compensation definition describes all the ways in which fluorescence from different channels affect each other under a given set of conditions and is equivalent to a single compensation metric. Typically, an instrument user sets the gains for all channels at the start of an experiment and uses these settings for the duration of the experiment. In this way, the compensation definition will apply for the entire experiment.

[0049] Using a spectral cytometry instrument, a continuous, high-resolution optical spectrum is collected for each event in a sample. The spectrum is the sum of the spectra resulting from all dyes present in the event of interest. Spectral unmixing is the process of transforming the data to determine the contribution of each dye to the total signal. Several mathematical models can be used to perform spectral unmixing calculations, including ordinary least squares. Ordinary least squares assumes a linear contribution of a constant reference spectrum to the mixture spectrum of the unknown sample. The calculation then allows for an estimation of the contribution of each spectrum (i.e., each dye).

[0050] Ordinary least squares uses a linear decomposition algorithm to solve the equations. Y=AC+E

[0051] For simplicity's sake, the term "E" represents random measurement error and can be initially ignored, focusing on the remaining term, where Y is the measured spectra matrix (i.e., a one-column matrix containing the spectrum of the event of interest), A is the reference spectra matrix (i.e., an n-column matrix containing the spectra of each reference dye), and C is the concentration matrix (i.e., a one-row matrix containing the contribution of each dye to the total measured spectrum). Given a set of reference spectra (A) and measured spectra (Y), this method allows estimation of the term C, and thus the contribution of each dye to the total signal intensity of the event of interest.

[0052] As used herein, the term "compensation" refers to the modification of emission signals to accurately estimate the fluorescence signal for a given fluorophore. As used herein, the term "spectral unmixing" refers to separating emission spectra to accurately estimate the spectral signal for a given fluorophore. Both compensation and spectral unmixing are directed at removing spillover signals from other fluorophores. Spectral unmixing processes data from more detectors than compensation.

[0053] Methods of compensation and spectral unmixing, as introduced above, are known in the art, and include adjusting the signal measured by each photodetector by an amount calculated to compensate for contributions from dyes other than the primary dye being detected. Examples in the field of flow cytometry include Bagwell et al., 1993, "Fluorescence Spectral Overlap Compensation for any Number of Flow Cytometer Parameters", Ann. NYAcad. Sci. 677:167-184; Roederer et al., 1997, "Eight Color, 10-Parameter Flow Cytometry to Elucidate Complex Leukocyte Hetrogeneity", Cytometry 29:328-339; and Bigos et al., 1999, Cytometry 36:36-45; Verwer, 2002, BD FACSDiVa® Option for the BD FACSVersion SE Flow Cytometer White Paper, and U.S. Patent No. 6,897,954, each of which is incorporated herein by reference. WinList™ (Verity Software House, Topsham, ME), Orfeo ToolBox (CNES), FCS Express (De Novo Software, Pasadena, CA), and FlowJo 5.7.2 software (Tree Star, Inc., Ashland, OR) are stand-alone software packages that allow software correction on stored data files produced by flow cytometers.

[0054] Typically, the amount of fluorescence compensation required is determined experimentally using compensation control beads (coupled to a single antibody-fluorophore conjugate) or monochromatic particles stained with one of the fluorophores used in the assay. The fluorescent signal of each bead is measured in each of the channels, which directly provides a measure of signal overlap in each of the channels. One method for measuring the signal overlap of fluorescently labeled antibody reagents (e.g., detection reagents) in each of the detection channels is to use BD™ CompBeads compensation particles (BD Biosciences, San Jose, CA). Anti-Ig antibody-coated particles are combined with fluorescently labeled antibody reagents and captured on the surface of the beads to produce fluorescently labeled particles. The signal overlap of the dyes is determined by measuring the emission of the labeled particles in each of the detection channels. Measurements are typically made relative to the emission spectrum from unlabeled particles. This process becomes more difficult as the number of fluorophores used in the assay increases, resulting in more signal overlap. There is a need for methods and compositions that can improve spectral unmixing and compensation (and thereby improve resolution) in multiparameter flow cytometry.

[0055] In simplified form, the percentage of fluorescence is subtracted from one channel measuring a fluorophore from a second channel measuring the fluorescence of a second (or multiple) fluorophores, so that the contribution of incidental fluorescence is removed. All fluorophore combinations that exhibit spectral overlap must be corrected. To determine the amount of correction required to correct the fluorescence data, single-color samples (either aliquots of cell samples or microspheres conjugated to all of the antibodies in a staining panel, stained separately with each fluorophore in individual tubes) are utilized and analyzed with experimental samples, which are typically then stained with multiple fluorophores.

[0056] Compensation is typically performed by using modified polystyrene beads that bind to the detection antibody, often via the Fc region. In this configuration, the compensation beads indiscriminately bind most, if not all, of the antibodies used in the experiment, allowing the operator to measure the fluorescence / spectral profile of a given antibody-fluorophore / fluorochrome conjugate when they are measured in isolation in individual tubes.

[0057] In its current form, the operator must dispense individual tubes of beads and separately add individual antibodies from the stain panel to each tube to deconvolute individual signals for fluorescence compensation or spectral unmixing. As the complexity of stain panels increases, this process can sometimes take hours to complete and is highly prone to operator / user error due to the number of pipetting steps required.

[0058] Similarly, fluorescence minus one (FMO) controls are equally important for determining true signal to noise for a given biomarker / channel, especially when there are different expression levels for a given biomarker in a multiplexed assay. In these instances, a combination cocktail of all antibodies used in a given panel minus one must be mixed and bound to target cells to determine the noise floor, or lower limit of detection, for a given assay and staining panel. Simply put, due to spillover effects, the measured amount of fluorescence in a particular channel may not represent the biological measurement of the biomarker in that channel. Instead, it often represents accidental fluorescence spillover from adjacent channels measuring other biomarkers. FMO controls are crucial for determining true biological signal from noise. This is especially important for poorly expressed or "dim" biomarkers with very low signal intensity. This process is extremely time-consuming, involves combinatorial mixing of antibodies for the panel into individual tubes, and is more often dependent on the actual cells being assayed. In situations where there is limited cellular material (e.g., primary cells from patients or rare diseases), FMO controls become excessively difficult to perform accurately.

[0059] Thus, there is a need in the art for synthetic compositions that allow for more efficient and less error-prone calibration and FMO process control for proper setup of analytical devices for multiplexed cytometric analysis.

[0060] Referring now to the drawings, as shown in Figures 1 and 2, compensation is typically performed by separating individual fluorophores / reagents into different tubes containing compensation beads designed to bind to common regions, such as Fc regions, of the antibody reagents used in the staining panel. Individual fluorophores must be physically separated in the tubes to deconvolute the fluorescent signals from the antibody-fluorophore conjugates used in the staining panel because each individual compensation bead binds to multiple different antibodies used in the staining panel. As a result, an individual compensation bead may have different fluorophores attached when it is added to a mixture of reagents with different fluorophores. For example, an individual compensation bead may have anti-CD4-FITC and anti-CD8-Texas Red both attached to such an individual compensation bead. This prevents compensation or spectral unmixing from being effectively calculated. Therefore, each bead and detection antibody combination must be physically separated to deconvolute the signal associated with a given fluorophore in the staining panel. This process is extremely labor-intensive when working with complex panels and can require hours of operator time. It is also a significant source of operator error in large-scale diagnostic settings, such as reference laboratories. While this lengthy, labor-intensive process is crucial for ensuring accurate assay performance, it significantly increases the costs of clinical trials and research pipelines. As an alternative to modified polystyrene beads, users often use cellular materials as a substitute for calibration and unmixing setups. Biologically derived calibration products are unstable and suffer from batch-to-batch variability, introducing noise into measurements and causing discrepancies in the interpretation of diagnostic data. Furthermore, both the standard calibration beads and cells used in the assay typically bind most, if not all, of the reagents used in the staining panel, requiring the separation of individual tubes for calibration and unmixing calculations, as described above.

[0061] Thus, the present disclosure provides compositions comprising hydrogel or polymer particles, modified to bind to individual antibodies in a staining panel but not others. Alternatively, the particles may also be pre-modified with the same fluorophores (or antibody-fluorophore conjugates) used in the staining panel to achieve the same effect. Individual beads containing biomarkers representing the complete repertoire of the staining panel are then combined a priori in a single tube. In one embodiment, the pre-modified fluorophore (or antibody-fluorophore conjugate) beads are used directly in an automated deconvolution and correction or unmixing process, in which the signals of individual beads can be isolated using expected fluorescence or spectral maxima, generating data that can be used in the correction or unmixing calculations. In another embodiment, users can add multiple beads designed to individually bind to biomarkers recognized by antibodies in the staining panel in a one-pot, single-tube reaction across their entire staining panel to generate the same data using the same deconvolution process.

[0062] The present disclosure also provides a method for combining individually modified beads in a way that allows users to perform FMO controls using a single mixture of antibodies. Traditionally, for FMO calculations, it is useful to generate a combinatorial mixture of all antibodies used in a staining panel minus one to determine the noise floor of a given assay and determine true signal to noise. In reality, antibodies in a staining panel will produce some overlapping signals in different, unintended channels. If cells are stained with a mixture lacking a specific antibody-fluorophore for a given channel, any signal in that channel represents a false-positive noise floor or true biological lower limit of detection, which can be determined using FMO controls. This process is extremely labor-intensive when performing high-complexity staining assays because the complete combinatorial matrix of FMO staining-antibody cocktails must be prepared by the user, which can take several hours to complete for a complex panel and can lead to user error and extensive operator cost and fatigue. This disclosure provides a method for performing FMO calculations, allowing users to add a single complete mixture of a staining panel to a tube already containing individually modified beads, minus one bead-epitope type for a given panel. By providing tubes containing multiple individually modified beads, minus one, users can significantly simplify the process of setting up FMO controls by allowing them to use one antibody cocktail for the entire process, reducing operator error, saving time, and saving costs.

[0063] Also provided is a method for deconvolving single-well compensation, unmixing, or FMO control reactions, the method comprising: a) analyzing single-well data to find fluorescence maxima corresponding to individual fluorophores in a staining panel; b) deconvoluting individually modified beads using local maxima as cutoffs; and c) performing compensation, unmixing, or FMO calculations using the deconvoluted data from individual bead populations, thereby calibrating a cytometry device for analysis of a target biological object.

[0064] Hydrogels and Polymers The particles of the present disclosure may comprise a hydrogel or hydrophobic polymer. A hydrogel is a material that contains a three-dimensional network of polymers that allows it to swell in the presence of water and shrink in the absence (or reduction in the amount) of water, but does not dissolve in water.

[0065] In another embodiment, the polymer particles can include a non-polystyrenic material, such as PLGA, etc. In other embodiments, the polymer particles are produced using polystyrene and latex.

[0066] The hydrogels provided herein are synthesized in the form of beads / particles by polymerizing one or more of the monomers provided herein. The synthesis is carried out to form individual hydrogel particles. In one embodiment, the monomeric materials (monomers) are polymerized to form homopolymers. However, in other embodiments, copolymers of different monomeric units (i.e., comonomers) are synthesized and used in the methods provided herein. The monomers or comonomers used in the methods and compositions described herein, in one embodiment, are or include bifunctional monomers (comonomers are used). In one embodiment, the hydrogel is synthesized in the presence of a crosslinker. In a further embodiment, the hydrogel is synthesized in the presence of a polymerization initiator.

[0067] The amount of monomer can be varied by the user of the present invention to obtain particular optical properties, for example, those that are substantially similar to those of a target cell. In one embodiment, the monomer component (i.e., monomer, comonomer, bifunctional monomer, or combinations thereof, e.g., bis / acrylamide, allylamine, or other comonomers that provide chemical functionality for secondary labels / conjugates or alginates, in various crosslinking ratios, is present at about 10 weight percent to about 95 weight percent of the hydrogel. In further embodiments, the monomer component is present at about 15 weight percent to about 90 weight percent of the hydrogel, or at about 20 weight percent to about 90 weight percent of the hydrogel.

[0068] Examples of various monomers and crosslinking chemistries available for use with the present invention are provided in the Thermo Scientific Crosslinking Technical Handbook entitled "Simple Molecular Bond Crosslinking Techniques" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf, the disclosure of which is incorporated by reference in its entirety and for all purposes). For example, hydrazine (e.g., with NHS ester compounds) or EDC coupling reactions (e.g., with maleimide compounds) can be used to construct hydrogels of the present invention.

[0069] In one embodiment, the monomer for use with the hydrogels provided herein is lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, derivatized versions thereof, or combinations thereof.

[0070] In one embodiment, one or more of the following monomers are used herein to form the hydrogels of the present invention: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, or combinations thereof.

[0071] In another embodiment, one or more of the following monomers are used herein to form the tunable hydrogels: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate. , pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate Acrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenyl N-methylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, as described in U.S. Pat. No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0072] The passive optical properties of the polymer beads may be adjusted to mimic the passive optical properties of target cells. Exemplary target cells are included in the non-exhaustive list in Table 1.

[0073] In embodiments, each polymeric bead comprises less than 10%, 20%, 30%, or 40% polystyrene by hydrated or dehydrated volume. Depending on their composition, the hydrated and dehydrated volumes of the polymeric beads may be the same.

[0074] In embodiments, the hydrogel or polymer particles are functionalized to mimic one or more optical properties of target cells or labeled target cells. In embodiments, the hydrogel or polymer particles include one or more high refractive index molecules. In embodiments, the hydrogel or polymer particles include multiple high refractive index molecules. In embodiments, the high refractive index molecules enable mimicking of SSCs of target cells. In embodiments, the high refractive index molecules are selected from one or more of colloidal silica, alkyl acrylates, alkyl methacrylates, or combinations thereof. In embodiments, the high refractive index molecules are alkyl acrylates, alkyl methacrylates, or both. In embodiments, the alkyl acrylates or alkyl methacrylates contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group. In embodiments, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, 2-ethylhexyl, heptyl, or octyl. In embodiments, the alkyl group is branched. In embodiments, the alkyl group is linear.

[0075] The three primary modes of deconvolution for flow cytometry are the two passive optical properties of polymer particles (FSC, which corresponds to refractive index, or RI, and SSC) and biomarkers present on the surface of a given cell type. Thus, compositions of the disclosed polymer particles, or polymer beads, that enable them to mimic a specific cell type with respect to these three modes are useful for providing synthetic, robust calibrators for flow cytometry.

[0076] In one embodiment, the RI of the disclosed polymer beads is greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0077] In another embodiment, the RI of the disclosed polymeric beads is from about 1.10 to about 3.0, or from about 1.15 to about 3.0, or from about 1.20 to about 3.0, or from about 1.25 to about 3.0, or from about 1.30 to about 3.0, or from about 1.35 to about 3.0, or from about 1.4 to about 3.0, or from about 1.45 to about 3.0, or from about 1.50 to about 3.0, or from about 1.6 to about 3.0, or from about 1.7 to about 3.0, or from about 1.8 to about 3.0, or from about 1.9 to about 3.0, or from about 2.0 to about 3.0.

[0078] In some embodiments, the RI of the disclosed polymeric beads is less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0079] The SSC of the disclosed polymer beads is most meaningfully measured in comparison to that of target cells. In some embodiments, the disclosed polymer beads have an SSC within 30%, 25%, 20%, 15%, 10%, 5%, or 1% of that of target cells, as measured by a cytometry device.

[0080] In one embodiment, the SSC of the polymer beads is adjusted by incorporating a high refractive index molecule(s) into the polymer beads. In one embodiment, the high refractive index molecule is provided in the polymer beads, and in further embodiments, the high refractive index molecule is colloidal silica, alkyl acrylate, alkyl methacrylate, or a combination thereof. Thus, in some embodiments, the polymer beads of the present disclosure comprise alkyl acrylate and / or alkyl methacrylate. The concentration of the monomer in one embodiment is adjusted to further adjust the refractive index of the polymer beads.

[0081] The alkyl acrylate or alkyl methacrylate can contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, 2-ethylhexyl, heptyl, or octyl groups, etc. The alkyl group can be branched or linear.

[0082] High refractive index molecules also include vinylarenes, such as styrene and methylstyrene, which can optionally be substituted on the aromatic ring with alkyl groups, such as methyl, ethyl, or tert-butyl, or with halogens, such as chlorostyrene.

[0083] In some embodiments, the FSC is adjusted by adjusting the percentage of monomer present in the composition, thereby varying the water content present during polymer bead formation. In one embodiment, a monomer and a comonomer are used, and the ratio of the monomer and comonomer is adjusted to vary the forward scattering properties of the polymer beads.

[0084] For example, the ratio of monomer to comonomer can be used to tailor the elasticity (i.e., Young's modulus) of the polymeric beads to be substantially similar to that of the target cells. The ratio of monomer to comonomer can vary the Young's modulus for the polymeric beads, which can range from 0.2 kilopascals (kPa) to 400 kPa, based on the elasticity of the target cells. The elasticity (e.g., softness or hardness) of the polymeric beads can affect the function of the target cells with which they interact.

[0085] The FSC of the disclosed polymer beads is most meaningfully measured in comparison to the FSC of target cells. In some embodiments, the disclosed polymer beads have an FSC within 30%, 25%, 20%, 15%, 10%, 5%, or 1% of that of target cells, as measured by a cytometry device.

[0086] FSC is related to particle volume and can thus be adjusted by varying particle diameter, as described herein. It has been observed that larger objects generally refract more light than smaller objects, leading to a higher forward scatter signal (and vice versa). Thus, in one embodiment, particle diameter is varied to adjust the FSC properties of polymer beads. For example, polymer bead diameter is varied in one embodiment by utilizing larger microfluidic channels during particle formation.

[0087] SSCs can be engineered by encapsulating nanoparticles within polymer beads to mimic organelles in target cells. In some embodiments, the polymer beads of the present disclosure comprise one or more types of nanoparticles selected from the group consisting of polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. While not wishing to be bound by theory, the ability to selectively tune both the forward and side scattering of polymer beads, as described herein, enables a robust platform to mimic a wide range of cell types.

[0088] Biomarkers In embodiments, the hydrogel or polymer particles comprise a cell surface marker, an epitope binding region of a cell surface marker, or a combination thereof.

[0089] The polymer particles of the present disclosure may be of any shape, including, but not limited to, spherical, non-spherical, elongated, cubic, cuboidal, conical, and cylindrical. In some embodiments, the hydrogel particles of the present disclosure have material modulus properties (e.g., elasticity) that more closely resemble those of target cells compared to polystyrene beads of the same diameter. The polymer particles of the present disclosure may also mimic extracellular vesicles, viruses, virus-like particles, spheroids, organoids, or any other biological target of interest.

[0090] Hydrogel or polymer particles can be functionalized, allowing them to mimic the optical properties of labeled biological particles. Functionalization can be mediated by compounds containing free amine groups, such as allylamine, which can be incorporated into the hydrogel particles during the formation process. The polymer particles of the present invention can be functionalized with any biomarker, polypeptide, peptide, protein, epitope, or antigen known in the art, including, but not limited to: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-gamma, CD20, TCR gamma / delta, TNF alpha, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR V alpha 7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR V delta 2, TIGIT, CD1c, CD2, CD7, CD8a, CD15, CD32, CD103, CD107a, CD141, CD158, CD159c, IL-13, IL-21, KLRG1, TIM-3, CCR5, CD5, CD33, CD45.2, CD80, CD159a (NKG2a), CD244, CD272, CD278, CD337, Granzyme B, Ig lambda light chain, IgA, IL-17A, streptavidin, TCR V delta 1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, Fc epsilon R1 alpha, IL-22, Lag-3, MIP-1 beta, perforin, TCR V gamma 9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Ig kappa light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21 R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC class II (IA / IE), MHC II, TCR alpha / beta, TCR beta, TCR V alpha 24, Akt(pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, CD16 / 32, CD41a, CD3 epsilon, CD8b, CD11b / c, CD16 / CD32, CD23, CD29, CD43, CD45.1, CD48, CD49b, CD49d, CD66, CD68, CD71, CD85k, CD93, CD99, CD106, CD122, CD133, CD1 34, CD146, CD150, CD158b, CD158b1 / b2, j, CD158e, CD166, CD169, CD184, CD200, CD200 R, CD235a, CD267, CD268, CD273, CD274, CD317, CD324, CD326, CD328, CD336, CD357, CD366, DDR2, eFluor 780 Fix Viability, EGF receptor, EGFR (pY845), EOMES, EphA2, ERK1 / 2 (pT202 / pY204), F4 / 80, FCRL5, Flt-3, FVS575V, FVS700, Granzyme A, HER2 / ErbB2, Hes1, Hoechst (33342), ICAM-1, IFN-alpha, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1 RAcP, IL-6, IL-10, IL-12, IL-17, integrin alpha 4 beta 7, isotype Ctrl, KLRC1, KLRC2, Live / Dead Fix Aqua, Ly-6A / Ly-6E, Ly-6G / Ly-6C, mannose receptor, MDR1, Met(pY1234 / pY1235), MMP-9, NGF receptor p75, ORAI1, ORAI2, ORAI3, p53, P2RY12, PARP, cleavage, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCR delta, TCR delta / gamma, TCR V alpha 24 J alpha 18, TCR V beta 11, TCR V gamma 1.1, TCR V gamma 2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1.

[0091] The hydrogel particles, in one embodiment, are functionalized with one or more cell surface markers (see, e.g., Tables 1, 2, and 3), or fragments thereof, e.g., their extracellular portions in the case of transmembrane proteins, by attaching one or more cell surface markers, their extracellular portions, or ligand-binding regions to the particles via free amine, free carboxyl, and / or free hydroxyl groups present on the surface of the hydrogel particles. Functionalization of hydrogel particles with dyes or cell surface molecules can also occur through linkers, e.g., streptavidin / biotin conjugates.

[0092] Depending on the target cell, individual hydrogel particles can be derivatized with one or more cell surface markers, or fragments thereof, e.g., their extracellular portions in the case of transmembrane proteins, to further mimic the structural characteristics of the target cell. Tables 4 and 7-8 are provided below and show non-limiting lists of cell surface markers that can be used to derivatize hydrogel particles, depending on the target cell. While cell surface markers are provided, it is understood that portions of the cell surface marker, e.g., receptor-binding portions, ligand-binding portions, or extracellular portions of the marker, can be used to derivatize hydrogel particles (with free functional groups, as described above). [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13]

[0093] Hydrogels and other polymeric particles are known in the art and are described in US Pat. Nos. 9,915,598 and 10,753,846, which are incorporated herein by reference in their entireties.

[0094] Fluorophores The present invention may use any fluorophore known in the art, including fluorescent dyes, tags, and stains listed in the Molecular Probes® Handbook—A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety. Tags include surface-enhanced Raman scattering (SERS) tags. In embodiments, hydrogel or polymer particles can be functionalized with fluorophores through covalent interactions, non-covalent interactions, or a combination thereof. In embodiments, hydrogel or polymer particles can be functionalized with fluorophores through biomarker or antibody mediation, or by direct conjugation, for example, via an amine-reactive fluorophore. As above, functionalization can be facilitated by free amine groups, such as allylamine, which can be incorporated into the hydrogel particles during the formation process.

[0095] In embodiments, the fluorophore or fluorescent dye is selected from the group consisting of peridinin chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5), phycoerythrin-cyanine 7 (PE-Cy7), allophycocyanin-cyanine 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, 5-(and-6)-carboxyeosin, 5-carboxyfluorescein, 6-carboxyfluorescein, 5-(and-6)-carboxyfluorescein, S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether, -alanine-carboxamide, or succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 6-carboxyfluorescein succinimidyl ester, 5 ... diethyl ester, 5-(and -6)-carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotriazinyl)aminofluorescein, 2',7'-difluorofluorescein, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, 6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester, 6-(fluorescein-5-(and -6)-carboxamido)hexanoic acid or succinimidyl ester, fluorescein-S-EX succinimidyl ester, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, OregonGreen® 488 carboxylic acid or succinimidyl ester, Oregon Green® 488 isothiocyanate, Oregon Green® 488-X succinimidyl ester, Oregon Green® 500 carboxylic acid, Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt, Oregon Green® 514 carboxylic acid, Oregon Green® 514 carboxylic acid or succinimidyl ester, Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride salt,Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester, Rhodamine Green™-X succinimidyl ester or hydrochloride, RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester, bis-(4-carboxypiperidinyl)sulfonarhodamine or di(succinimidyl ester), 5-(and-6) carboxynaphthofluorescein, 5-(and-6) carboxynaphthofluorescein succinimidyl ester, 5-carboxyrhodamine 6G hydrochloride, 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester, 6-carboxyrhodamine 6G succinimidyl ester, 5-(and-6)-carboxyrhodamine 6G succinimidyl ester, 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester bis-(diisopropylethylammonium) salt, 5-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine Rhodamine, 5-(and -6)-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimidyl ester, 6-carboxytetramethylrhodamine succinimidyl ester, 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester, 6-carboxy-X-rhodamine, 5-carboxyl-X-rhodamine succinimidyl ester, 6-carboxy-X-rhodamine succinimidyl ester, 5-(and -6)-carboxy-X-rhodamine succinimidyl ester, 5-carboxy-X-rhodamine triethylammonium salt, Lissamine™ rhodamine B sulfonyl chloride, malachite green, isothiocyanate, NANOGOLD® mono(sulfosuccinimidyl ester), QSY® 21 carboxylic acid or succinimidyl ester, QSY® 7 carboxylic acid or succinimidyl ester, Rhodamine Red™-X succinimidyl ester, 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid, succinimidyl ester, tetramethylrhodamine-5-isothiocyanate,Tetramethylrhodamine-6-isothiocyanate, tetramethylrhodamine-5-(and -6)-isothiocyanate, Texas Red® sulfonyl, Texas Red® sulfonyl chloride, Texas Red®-X STP ester or sodium salt, Texas Red®-X succinimidyl ester, Texas Red®-X succinimidyl ester, X-rhodamine-5-(and -6) isothiocyanate, commercially available BODIPY® dyes from Invitrogen, including but not limited to BODIPY® FL, BODIPY® TMR STP ester, BODIPY® TR-X STP ester, BODIPY® 630 / 650-X STPester, BODIPY® 650 / 665-X STP esters include 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3-propanoic acid succinimidyl ester, pionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3propionic acid, sulfosuccinimidyl ester or sodium salt, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)amino)hexanoic acid, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester,N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt, 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid succinimidyl ester, 4,4- Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, 4,4-difluoro-5-(4-phenyl-1,3 butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene Indacene-3-propionic acid succinimidyl ester, 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-1,3,5,7-tetramethyl- 4-bora-3a,4diaza-s-indacene-8-propionic acid, 4,4-difluoro-1,3,5,7-tetramethyl-4bora-3a,4a-diaza-sindacen-8-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacen-3-propionic acid succinimidyl ester, 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4diazas-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester,and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, Alexa Fluor dyes commercially available from Invitrogen, including but not limited to Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 647 carboxylic acid, Alexa Fluor® 660 carboxylic acid, Alexa Fluor® 680 carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech, including but not limited to Cy3 NHS ester, Cy5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester, and / or any conjugation or derivative thereof.

[0096] In embodiments, the hydrogel or polymer particles may comprise from 1 to about 20 fluorescent dyes, from 1 to about 10 fluorescent dyes, or from 1 to about 5 fluorescent dyes. In embodiments, the hydrogel or polymer particles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 fluorescent dyes, inclusive of the endpoints, and all values ​​and subranges therebetween.

[0097] In embodiments, the hydrogel or polymer particles comprise "rainbow particles." Rainbow particles comprise a mixture of fluorophores. In embodiments, the rainbow particles comprise 1 to about 20 fluorophores, 1 to about 10 fluorophores, or 1 to about 5 fluorophores. In embodiments, the hydrogel or polymer particles comprise rainbow particles with 1, 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 fluorophores, including the endpoints and all values ​​and subranges therebetween. In embodiments, the user selects the wavelength at which to excite the rainbow particles depending on the fluorophores being investigated. Rainbow particles are commercially available in a variety of diameters (e.g., catalog numbers RCP20-5 (4 colors), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)) from, for example, BD Biosciences (catalog numbers 556298 (mid-range FL1 fluorescence), 556286 (6 colors, 3.0-3.4 µm), 556288 (6 colors, 6.0-6.4 µm), 559123 (8 colors)), and Spherotech.

[0098] A non-exhaustive list of fluorophores suitable for use with the present invention is provided in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15]

[0099] In embodiments, the hydrogel or polymer particles comprise a scattering control additive. In embodiments, the scattering control additive comprises polymeric nanoparticles. In embodiments, the polymeric nanoparticles comprise polystyrene. In embodiments, the scattering control additive comprises a comonomer. In embodiments, the scattering control additive comprises a suspension of nanoparticles.

[0100] In embodiments, the hydrogel or polymer particles are chemically functionalized hydrogel or polymer particles. In embodiments, the hydrogel comprises free amine groups. In embodiments, the hydrogel beads comprise allylamine. In embodiments, the hydrogel or polymer particles comprise biotin. In embodiments, the hydrogel or polymer particles comprise streptavidin. In embodiments, the hydrogel or polymer particles comprise avidin. In embodiments, the chemically functionalized hydrogel or polymer particles comprise amine groups, carboxyl groups, hydroxyl groups, or combinations thereof. In embodiments, the hydrogel or polymer particles comprise multiple bifunctional monomers for functionalizing the hydrogel or polymer particles with different chemicals and / or molecules.

[0101] composition Compositions of the present disclosure may include a population of polymeric beads. In embodiments, the population of polymeric beads may include a fluorophore, a biomarker, and / or the like. In embodiments, the population of polymeric beads may include up to 5, up to 10, up to 12, up to 18, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 populations of polymeric beads. In embodiments, each bead population includes a fluorophore, a biomarker, and / or the like.

[0102] In embodiments, each bead population may comprise a single fluorophore. In embodiments, each bead population may comprise a different fluorophore. In embodiments, each fluorophore emits fluorescence at one, two, three, four, five, six, seven, eight, or nine wavelengths. In embodiments, each fluorophore has a diameter between about 500 nm and about 10 μm.

[0103] For example, in embodiments, a first population of polymeric beads comprises a first fluorophore and a second population of polymeric beads comprises a second fluorophore. The first and second fluorophores may each be selected from the fluorophores outlined above and may be the same or different. Furthermore, the final population of polymeric beads does not comprise any fluorophore.

[0104] In embodiments, each bead population may comprise a single biomarker. In embodiments, each bead population may comprise a different biomarker. For example, in embodiments, a first population of polymeric beads comprises a first biomarker and a second population of polymeric beads comprises a second biomarker. The first and second biomarkers may each be selected from the biomarkers outlined above and may be the same or different. Furthermore, the final population of polymeric beads does not comprise any biomarkers. [Example]

[0105] Example 1 - Deconvolution using separate tubes Shown in the TBNK staining panel, six sets of antibody-fluorophore conjugates were prepared and directly attached to polymer particles. The following fluorophores were used: PerCP-Cy5.5, PE-Cy7, APC-Cy7, FITC, PE, and APC.

[0106] Six separate sample tubes were prepared using 100 μL of phosphate-buffered saline (PBS). Approximately 25,000 beads from each set were added to each such tube. For clarity, each sample tube contained only one set of antibody-fluorophore conjugates. A seventh sample tube was prepared as an unstained / negative control. Each sample tube was vortexed to resuspend the particles and analyzed on a Cytek North Lights™ flow cytometer using routine acquisition parameters. The unstained / negative control was then measured in the same manner. Figure 11 illustrates the individual fluorophore signals for each sample tube.

[0107] Example 2 - One-pot analysis and calibration Six sets of polymer beads from Example 1 were combined into a single tube and analyzed on a Cytek North Lights™ using routine acquisition parameters, resulting in a combined spectrum ( FIG. 12 ). In this example, each individual bead-antibody-fluorophore particle uniquely contained only one type of fluorophore per bead.

[0108] The known fluorescence signal of each antibody-fluorophore conjugate was used to deconvolute the individual fluorophore signals from each polymer-bound antibody-fluorophore. Specifically, the fluorescence maximum associated with a given antibody-fluorophore conjugate from the stained antibody mixture was used to select a subset of beads bound to that particular antibody-fluorophore conjugate (Figure 13). This allowed for deconvolution of the total fluorescence signal for each fluorophore from the mixed reaction. The results of such deconvolution are shown in Figure 14. The individual spectra seen in Figure 14 were indistinguishable from those generated from separate tubes (Figure 11) and can be used for downstream correction or spectral unmixing calculations with performance equivalent to that of individual, physically separated tubes (Figure 15). The resulting unmixed TBNK staining panel using the one-pot polymer particle method described in this disclosure was indistinguishable from the staining panel using separate tubes described in Example 1. See Figure 15.

[0109] Example 3 - One-pot correction using biomarker-modified beads Six sets of polymer beads, each containing a single biomarker from an exemplary TBNK panel (CD3, CD16, CD56, CD45, CD4, CD19, CD8), were added to a single tube containing 100 μL of PBS. The entire panel of antibody-fluorophore conjugates from the TBNK panel was then added to the same single reaction tube and vortexed to resuspend the mixture. The mixture was then incubated under light protection and analyzed on a calibrated flow cytometer using routine acquisition parameters, resulting in a combined spectrum. In this example, each biomarker-modified bead binds only to a specific antibody-fluorophore from the staining panel mixture, generating a uniformly labeled set of beads.

[0110] The known fluorescence signal of each antibody-fluorophore conjugate is used to deconvolute the individual fluorophore signals from each polymer-bound antibody-fluorophore. Specifically, the fluorescence maximum associated with a given antibody-fluorophore conjugate from the stained antibody mixture is used to select a subset of beads bound to that particular antibody-fluorophore conjugate. This allows for deconvolution of the total fluorescence signal for each individual fluorophore from the mixture reaction for use in correction or spectral unmixing calculations.

[0111] Example 4 - One-pot correction using pre-modified beads The six sets of beads from Example 1 can be pre-modified with a suitable fluorophore, as opposed to an antibody-fluorophore conjugate, to achieve the same effect.

[0112] Example 5 - Streamlined FMO correction using a single biomarker bead All possible combinations of six sets of beads from Example 3, minus one biomarker-fluorophore channel, were added to individual tubes to represent the FMO staining control set. For clarity, exemplary sets include: (CD16, CD56, CD45, CD4, CD19, CD8), (CD3, CD56, CD45, CD4, CD19, CD8), (CD3, CD16, CD45, CD4, CD19, CD8), (CD3, CD16, CD56, CD4, CD19, CD8), (CD3, CD16, CD56, CD45, CD19, CD8), (CD3, CD16, CD56, CD45, CD19, CD8), (CD3, CD16, CD56, CD45, CD4, CD8), and (CD3, CD16, CD56, CD45, CD4, CD19). In this product format, a single master mix of the TBNK staining panel can be added to all tubes to generate the FMO control matrix. Specifically, adding a single cocktail of anti-CD3-FITC, anti-CD16-PE, anti-CD56-PE, anti-CD45-PerCP Cy5.5, anti-CD4 PE-Cy7, anti-CD19-APC, and anti-CD8-APC Cy7 to each of the premixed FMO tubes described in this disclosure would allow users to generate a complete FMO panel. In each of these examples, the polymer particles in the tube bind all but one of the antibody-fluorophore conjugates in the complete TBNK panel, simulating a traditional FMO approach in a highly streamlined product format. In contrast, using traditional methods would require users to generate a combinatorial cocktail of antibodies in every combination to achieve the same result, because all of the antibodies could be bound to cells or traditional compensation beads used for FMO calculations.

[0113] Example 6 - Streamlined FMO correction using a single biomarker bead The set of beads in Example 5 can be pre-modified with a suitable fluorophore, or antibody-fluorophore conjugate combination, to achieve the same effect.

[0114] Further Numbered Embodiments Further embodiments of the present invention are provided in the numbered embodiments below:

[0115] Embodiment 1. A composition comprising (i) a first population of polymeric beads comprising a first fluorophore, and (ii) a second population of polymeric beads comprising a second fluorophore.

[0116] Embodiment 2. 2. The composition of embodiment 1, comprising up to 5, up to 10, up to 12, up to 18, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 populations of polymeric beads, each bead population comprising a fluorophore, and wherein the fluorophores for each population of beads are different.

[0117] Embodiment 3. 2. The composition of embodiment 1, wherein the first fluorophore and the second fluorophore are different fluorophores.

[0118] Embodiment 3.1. 4. The composition of any one of embodiments 1 to 3, wherein each population of polymer beads comprises only a single type of fluorophore.

[0119] Embodiment 4. The composition of any one of embodiments 1-3.1, further comprising a final population of polymeric beads that do not contain any fluorophores.

[0120] Embodiment 4.1. 5. The composition of any one of embodiments 1-4, wherein each fluorophore is independently selected from those listed in Table 4.

[0121] Embodiment 5. Each fluorophore is a peridinin chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5), phycoerythrin-cyanine 7 (PE Cy7), allophycocyanin-cyanin 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, 5-(and -6)-carboxyeosin, 5-carboxyfluorescein, 6-carboxyfluorescein, 5-(and -6)-carboxyfluorescein, S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether, -alanine-carboxamide, or succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 6-carboxyfluorescein succinimidyl ester, 5-(and -6)-carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotriazinyl)aminofluorescein, 2',7'-Difluorofluorescein, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, 6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester, 6-(fluorescein-5-(and -6)-carboxamido)hexanoic acid or succinimidyl ester, fluorescein-S-EX succinimidyl ester, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, OregonGreen® 488 carboxylic acid or succinimidyl ester, Oregon Green® 488 isothiocyanate, Oregon Green® 488-X succinimidyl ester, Oregon Green® 500 carboxylic acid, Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt, Oregon Green® 514 carboxylic acid, Oregon Green® 514 carboxylic acid or succinimidyl ester, Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride, Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester, Rhodamine Green™-X succinimidyl ester or hydrochloride, RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester, bis-(4-carboxypiperidinyl)sulfonarhodamine or di(succinimidyl ester), 5-(and-6) carboxynaphthofluorescein, 5-(and-6) carboxynaphthofluorescein succinimidyl ester, 5-carboxyrhodamine 6G hydrochloride, 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester, 6-carboxyrhodamine 6G succinimidyl ester, 5-(and-6)-carboxyrhodamine 6G succinimidyl ester, 5-carboxy-2',4',5',7'-Tetrabromosulfonefluorescein succinimidyl ester bis-(diisopropylethylammonium) salt, 5-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, 5-(and -6)-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimidyl ester, 6-carboxytetramethylrhodamine succinimidyl ester, 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester, 6-carboxy-X-rhodamine, 5-carboxyl-X-rhodamine succinimidyl ester Rhodamine succinimidyl ester, 6-carboxy-X-rhodamine succinimidyl ester, 5-(and -6)-carboxy-X-rhodamine succinimidyl ester, 5-carboxy-X-rhodamine triethylammonium salt, Lissamine™ rhodamine B sulfonyl chloride, Malachite Green, isothiocyanate, NANOGOLD® mono (sulfosuccinimidyl ester), QSY® 21 carboxylic acid or succinimidyl ester, QSY® 7 carboxylic acid or succinimidyl ester, Rhodamine Red™-X succinimidyl ester, 6-(tetramethylrhodamine-5-(and -6)-carboxamido)hexanoic acid, succinimidyl ester, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, tetramethylrhodamine-5-(and -6)-isothiocyanate, Texas Red® sulfonyl, Texas Red® sulfonyl chloride, Texas Red®-X STP ester or sodium salt, Texas Red®-X succinimidyl ester, Texas Red®-X succinimidyl ester, X-rhodamine-5-(and -6) isothiocyanate, BODIPY® FL, BODIPY® TMR STP ester, BODIPY® TR-X STP ester, BODIPY® 630 / 650-X STPester, BODIPY® 650 / 665-X STP ester, 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene Indacene-3 propionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3 propionic acid, sulfosuccinimidyl ester or sodium salt, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, Methyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt, 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid , 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, 4,4-difluoro-5-(4-phenyl-1,3 butadienyl)-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid Fluoro-1,3,5,7-tetramethyl-4-bora-3a,4diaza-s-indacene-8-propionic acid, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacen-8-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacen-3-propionic acid succinimidyl ester, 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4diazas-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester, and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,The composition of any one of embodiments 1 to 4, wherein the carboxylic acid is independently selected from any one of 4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 64 7 carboxylic acid, Alexa Fluor® 660 carboxylic acid, Alexa Fluor® 680 carboxylic acid, Cy3 NHS ester, Cy 5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester.

[0122] Embodiment 6. 6. The composition of any one of embodiments 1-5, wherein each fluorophore emits fluorescence at one, two, three, four, five, six, seven, eight, or nine wavelengths.

[0123] Embodiment 7. 7. The composition of any one of embodiments 1 to 6, wherein each fluorophore has a diameter between about 500 nm and about 10 μm.

[0124] Embodiment 8. 8. The composition of any one of embodiments 1-7, wherein the polymeric beads comprise less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0125] Embodiment 8.1. 8. The composition of any one of embodiments 1-7, wherein the polymeric beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dry volume.

[0126] Embodiment 9. The composition of any one of embodiments 1-8.1, wherein the polymer beads are hydrogel beads.

[0127] Embodiment 10. 10. The composition of embodiment 9, wherein the hydrogel comprises a monomer.

[0128] Embodiment 11. The monomers are hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl meth ... Phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate Acrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,11. The composition of embodiment 10, wherein the alkyl group is N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthylacrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenylphenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methylphenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethyl methacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N'-phenylphenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0129] Embodiment 12. 12. The composition of any one of embodiments 1 to 11, wherein the polymer beads exhibit at least one optical property substantially similar to an optical property of a target cell.

[0130] Embodiment 12.1. 12. The composition of any one of the preceding embodiments, wherein at least one population of polymeric beads exhibits at least one optical property that differs from the corresponding optical property of another population of polymeric beads within the composition.

[0131] Embodiment 13. The composition of embodiment 12 or 12.1, wherein the at least one optical property is side scattering.

[0132] Embodiment 14. The composition of embodiment 12 or 12.1, wherein the at least one optical property is forward scattering.

[0133] Embodiment 15. The composition of embodiment 12 or 12.1, wherein the at least one optical property comprises side scattering and forward scattering.

[0134] Embodiment 16. 16. The composition of any one of embodiments 12-15, wherein each target cell is independently selected from any one of a T cell, a B cell, and a natural killer cell.

[0135] Embodiment 17. 17. The composition of any one of embodiments 1-16, further comprising one or more of: (iii) a third population of polymeric beads comprising a third fluorophore; (iv) a fourth population of polymeric beads comprising a fourth fluorophore; (v) a fifth population of polymeric beads comprising a fifth fluorophore; and / or (vi) a sixth population of polymeric beads comprising a sixth fluorophore.

[0136] Embodiment 17.1. 18. The composition of embodiment 17, wherein each fluorophore is independently selected from those listed in Table 4.

[0137] Embodiment 18. The first, second, third, fourth, fifth, and sixth fluorophores were Peridinin Chlorophyll Protein-Cyanine 5.5 dye (PerCP-Cy5.5), Phycoerythrin-Cyanine 7 (PE Cy7), allophycocyanin-cyanin 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, 5-(and -6)-carboxyeosin, 5-carboxyfluorescein, 6-carboxyfluorescein, 5-(and -6)-carboxyfluorescein, S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether, -alanine-carboxamide, or succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 6-carboxyfluorescein succinimidyl ester, 5-(and -6)-carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotriazinyl)aminofluorescein, 2',7'-Difluorofluorescein, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, 6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester, 6-(fluorescein-5-(and -6)-carboxamido)hexanoic acid or succinimidyl ester, fluorescein-S-EX succinimidyl ester, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, OregonGreen® 488 carboxylic acid or succinimidyl ester, Oregon Green® 488 isothiocyanate, Oregon Green® 488-X succinimidyl ester, Oregon Green® 500 carboxylic acid, Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt, Oregon Green® 514 carboxylic acid, Oregon Green® 514 carboxylic acid or succinimidyl ester, Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride, Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester, Rhodamine Green™-X succinimidyl ester or hydrochloride, RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester, bis-(4-carboxypiperidinyl)sulfonarhodamine or di(succinimidyl ester), 5-(and-6) carboxynaphthofluorescein, 5-(and-6) carboxynaphthofluorescein succinimidyl ester, 5-carboxyrhodamine 6G hydrochloride, 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester, 6-carboxyrhodamine 6G succinimidyl ester, 5-(and-6)-carboxyrhodamine 6G succinimidyl ester, 5-carboxy-2',4',5',7'-Tetrabromosulfonefluorescein succinimidyl ester bis-(diisopropylethylammonium) salt, 5-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, 5-(and -6)-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimidyl ester, 6-carboxytetramethylrhodamine succinimidyl ester, 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester, 6-carboxy-X-rhodamine, 5-carboxyl-X-rhodamine succinimidyl ester Rhodamine succinimidyl ester, 6-carboxy-X-rhodamine succinimidyl ester, 5-(and -6)-carboxy-X-rhodamine succinimidyl ester, 5-carboxy-X-rhodamine triethylammonium salt, Lissamine™ rhodamine B sulfonyl chloride, Malachite Green, isothiocyanate, NANOGOLD® mono (sulfosuccinimidyl ester), QSY® 21 carboxylic acid or succinimidyl ester, QSY® 7 carboxylic acid or succinimidyl ester, Rhodamine Red™-X succinimidyl ester, 6-(tetramethylrhodamine-5-(and -6)-carboxamido)hexanoic acid, succinimidyl ester, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, tetramethylrhodamine-5-(and -6)-isothiocyanate, Texas Red® sulfonyl, Texas Red® sulfonyl chloride, Texas Red®-X STP ester or sodium salt, Texas Red®-X succinimidyl ester, Texas Red®-X succinimidyl ester, X-rhodamine-5-(and -6) isothiocyanate, BODIPY® FL, BODIPY® TMR STP ester, BODIPY® TR-X STP ester, BODIPY® 630 / 650-X STPester, BODIPY® 650 / 665-X STP ester, 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene Indacene-3 propionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3 propionic acid, sulfosuccinimidyl ester or sodium salt, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, Methyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt, 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid , 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, 4,4-difluoro-5-(4-phenyl-1,3 butadienyl)-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid Fluoro-1,3,5,7-tetramethyl-4-bora-3a,4diaza-s-indacene-8-propionic acid, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacen-8-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacen-3-propionic acid succinimidyl ester, 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4diazas-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester, and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,18. The composition of embodiment 17, wherein the carboxylic acid is independently selected from the group consisting of 4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 64 7 carboxylic acid, Alexa Fluor® 660 carboxylic acid, Alexa Fluor® 680 carboxylic acid, Cy3 NHS ester, Cy 5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester.

[0138] Embodiment 18.1. 19. The composition of any one of embodiments 1 to 18, wherein the fluorophore is conjugated to an antibody or fragment thereof that binds to an epitope within the polymer bead.

[0139] Embodiment 18.2. The composition of embodiment 18.1, wherein the epitope is a biomarker contained in a polymer bead.

[0140] Embodiment 18.3. The composition of embodiment 18.1 or 18.2, wherein the fluorophore is a commercially available antibody-labeled conjugate.

[0141] Embodiment 18.4. The composition of embodiment 18.2 or 18.3, wherein the biomarker is selected from those listed in Tables 1-3 herein.

[0142] Embodiment 19. 18. A method of calibrating a cytometry device for compensation or spectral unmixing, comprising: (i) measuring a fluorescent signal of the composition of any one of embodiments 1 to 18.3 using a cytometry device; (ii) deconvolving the fluorescent signal from each polymer bead population of the composition to calculate a compensation or spectral unmixing matrix; and (iii) calibrating the cytometry device using the compensation or spectral unmixing matrix.

[0143] Embodiment 19.1. A method of calibrating a cytometry device for compensation or spectral unmixing, comprising: (A) using a cytometry device to measure a fluorescent signal of a composition comprising (i) a first population of polymeric beads comprising a first fluorophore and (ii) a second population of polymeric beads comprising a second fluorophore; (B) deconvoluting the fluorescent signal from each polymeric bead population of the composition to calculate a compensation or spectral unmixing matrix; and (C) calibrating the cytometry device using the compensation or spectral unmixing matrix.

[0144] Embodiment 19.2. The method of embodiment 19 or 19.1, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the fluorescence emission maximum.

[0145] Embodiment 19.3. The method of embodiment 19 or 19.1, wherein the fluorescent signal from each population of polymer beads is deconvoluted based on the optical properties of each population of polymer beads.

[0146] Embodiment 19.4. 19. The method of any one of embodiments 19-19.3, wherein the measured composition comprises up to 5, up to 10, up to 12, up to 18, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 populations of polymeric beads, each bead population comprising a fluorophore, and the fluorophores for each population of beads are different.

[0147] Embodiment 19.5. The method of any one of embodiments 19 to 19.4, wherein the first fluorophore and the second fluorophore are different fluorophores.

[0148] Embodiment 19.6. The method of any one of embodiments 19 to 19.4, wherein each population of polymer beads contains only a single fluorophore.

[0149] Embodiment 19.7. The method according to any one of embodiments 19 to 19.6, wherein the measured composition comprises a final population of polymer beads that does not contain any fluorophore.

[0150] Embodiment 19.7.1. The method of any one of embodiments 19.19.6, wherein each fluorophore is independently selected from those listed in Table 4.

[0151] Embodiment 19.8. Each fluorophore is a peridinin chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5), phycoerythrin-cyanine 7 (PE Cy7), allophycocyanin-cyanin 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, 5-(and -6)-carboxyeosin, 5-carboxyfluorescein, 6-carboxyfluorescein, 5-(and -6)-carboxyfluorescein, S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether, -alanine-carboxamide, or succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 6-carboxyfluorescein succinimidyl ester, 5-(and -6)-carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotriazinyl)aminofluorescein, 2',7'-Difluorofluorescein, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, 6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester, 6-(fluorescein-5-(and -6)-carboxamido)hexanoic acid or succinimidyl ester, fluorescein-S-EX succinimidyl ester, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, OregonGreen® 488 carboxylic acid or succinimidyl ester, Oregon Green® 488 isothiocyanate, Oregon Green® 488-X succinimidyl ester, Oregon Green® 500 carboxylic acid, Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt, Oregon Green® 514 carboxylic acid, Oregon Green® 514 carboxylic acid or succinimidyl ester, Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride, Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester, Rhodamine Green™-X succinimidyl ester or hydrochloride, RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester, bis-(4-carboxypiperidinyl)sulfonarhodamine or di(succinimidyl ester), 5-(and-6) carboxynaphthofluorescein, 5-(and-6) carboxynaphthofluorescein succinimidyl ester, 5-carboxyrhodamine 6G hydrochloride, 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester, 6-carboxyrhodamine 6G succinimidyl ester, 5-(and-6)-carboxyrhodamine 6G succinimidyl ester, 5-carboxy-2',4',5',7'-Tetrabromosulfonefluorescein succinimidyl ester bis-(diisopropylethylammonium) salt, 5-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, 5-(and -6)-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimidyl ester, 6-carboxytetramethylrhodamine succinimidyl ester, 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester, 6-carboxy-X-rhodamine, 5-carboxyl-X-rhodamine succinimidyl ester Rhodamine succinimidyl ester, 6-carboxy-X-rhodamine succinimidyl ester, 5-(and -6)-carboxy-X-rhodamine succinimidyl ester, 5-carboxy-X-rhodamine triethylammonium salt, Lissamine™ rhodamine B sulfonyl chloride, Malachite Green, isothiocyanate, NANOGOLD® mono (sulfosuccinimidyl ester), QSY® 21 carboxylic acid or succinimidyl ester, QSY® 7 carboxylic acid or succinimidyl ester, Rhodamine Red™-X succinimidyl ester, 6-(tetramethylrhodamine-5-(and -6)-carboxamido)hexanoic acid, succinimidyl ester, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, tetramethylrhodamine-5-(and -6)-isothiocyanate, Texas Red® sulfonyl, Texas Red® sulfonyl chloride, Texas Red®-X STP ester or sodium salt, Texas Red®-X succinimidyl ester, Texas Red®-X succinimidyl ester, X-rhodamine-5-(and -6) isothiocyanate, BODIPY® FL, BODIPY® TMR STP ester, BODIPY® TR-X STP ester, BODIPY® 630 / 650-X STPester, BODIPY® 650 / 665-X STP ester, 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene Indacene-3 propionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3 propionic acid, sulfosuccinimidyl ester or sodium salt, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, Methyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt, 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid , 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, 4,4-difluoro-5-(4-phenyl-1,3 butadienyl)-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid Fluoro-1,3,5,7-tetramethyl-4-bora-3a,4diaza-s-indacene-8-propionic acid, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacen-8-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacen-3-propionic acid succinimidyl ester, 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4diazas-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester, and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,The method of any one of embodiments 19 to 19.7, wherein the carboxylic acid is independently selected from the group consisting of 4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 64 7 carboxylic acid, Alexa Fluor® 660 carboxylic acid, Alexa Fluor® 680 carboxylic acid, Cy3 NHS ester, Cy 5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester.

[0152] Embodiment 19.9. The method of any one of embodiments 19 to 19.8, wherein each fluorophore emits fluorescence at 1, 2, 3, 4, 5, 6, 7, 8, or 9 wavelengths.

[0153] Embodiment 19.10. The method of any one of embodiments 19 to 19.9, wherein each fluorophore has a diameter between about 500 nm and about 10 μm.

[0154] Embodiment 19.11. The method of any one of embodiments 19-19.10, wherein the polymeric beads comprise less than 10%, 20%, 30%, 40% polystyrene by hydrated volume.

[0155] Embodiment 19.11.1. The method of any one of embodiments 19-19.10, wherein the polymeric beads comprise less than 10%, 20%, 30%, 40% polystyrene by dry volume.

[0156] Embodiment 19.12. The method of any one of embodiments 19 to 19.11.1, wherein the polymer beads are hydrogel beads.

[0157] Embodiment 19.13. The method of embodiment 19.12, wherein the hydrogel comprises a monomer.

[0158] Embodiment 19.14. The monomers are hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl meth ... Phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate Acrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,The method of embodiment 19.13, wherein the alkyl group is N-dibenzyl methacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0159] Embodiment 19.15. The method according to any one of embodiments 19 to 19.7, wherein the polymer beads exhibit at least one optical property substantially similar to the optical property of the target cells.

[0160] Embodiment 19.16. The method of any one of embodiments 19-19.15, wherein at least one population of polymeric beads exhibits at least one optical property that differs from the corresponding optical property of another population of polymeric beads within the composition.

[0161] Embodiment 19.17. The method of embodiment 19.15 or 19.16, wherein the at least one optical property is side scattering.

[0162] Embodiment 19.18. The method of embodiment 19.15 or 19.16, wherein at least one optical property is forward scattering.

[0163] Embodiment 19.19. The method of embodiment 19.15 or 19.16, wherein the at least one optical property comprises side scattering and forward scattering.

[0164] Embodiments 19.20. The method of embodiment 19.15 or 19.17-19.19, wherein each target cell is independently selected from any one of T cells, B cells, and natural killer cells.

[0165] Embodiment 19.21. The method of any one of embodiments 19.1 to 19.20, wherein the measured composition comprises one or more of: (iii) a third population of polymeric beads comprising a third fluorophore; (iv) a fourth population of polymeric beads comprising a fourth fluorophore; (v) a fifth population of polymeric beads comprising a fifth fluorophore; and / or (vi) a sixth population of polymeric beads comprising a sixth fluorophore.

[0166] Embodiment 19.22. The method of embodiment 19.2, wherein the first, second, third, fourth, fifth, and sixth fluorophores are independently selected from the group consisting of PerCP-Cy5.5, PE Cy7, APC-Cy7, FITC, PE, and APC.

[0167] Embodiment 20. 1. A method of calibrating a cytometry device for compensation or spectral unmixing, comprising: (A) providing a composition comprising (i) a first population of polymeric beads comprising a first fluorophore and (ii) a second population of polymeric beads comprising a second fluorophore; (B) measuring a fluorescent signal of the composition using a cytometry device; (C) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix; and (D) calibrating the cytometry device using the compensation or spectral unmixing matrix.

[0168] Embodiment 20.1. 21. The method of any one of embodiments 19-20, wherein each population of polymer beads contains a sufficiently high content of fluorophores to produce a fluorescent signal at least as strong as the fluorescent signal from the cell population analyzed via the cytometry device.

[0169] Embodiment 21. A composition comprising (i) a first population of polymer beads comprising a first biomarker and (ii) a second population of polymer beads comprising a second biomarker.

[0170] Embodiment 22. 22. The composition of embodiment 21, comprising a population of up to 5, up to 10, up to 12, up to 18, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 polymeric beads, each bead population comprising a biomarker, and wherein the biomarkers for each bead population are different.

[0171] Embodiment 23. 23. The composition of embodiment 21 or 22, wherein each population of polymer beads comprises a different biomarker.

[0172] Embodiment 23.1. 24. The composition of any one of embodiments 21 to 23, wherein each population of polymer beads comprises only a single biomarker.

[0173] Embodiment 24. The composition of any one of embodiments 21 to 23.1, comprising a population of fluorophore-free beads.

[0174] Embodiment 25. 25. The composition of any one of embodiments 21-24, comprising a population of beads that do not contain biomarkers.

[0175] Embodiment 26. 26. The composition of any one of embodiments 21-25, wherein the polymeric beads comprise less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0176] Embodiment 26.1. 26. The composition of any one of embodiments 21-25, wherein the polymeric beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dry volume.

[0177] Embodiment 27. 27. The composition of any one of embodiments 21-26, wherein the polymer beads are hydrogel beads.

[0178] Embodiment 27.1. 28. The composition of any one of embodiments 21 to 27, wherein the biomarker is a polypeptide.

[0179] Embodiment 27.2. The composition of any one of embodiments 21 to 27.1, wherein the biomarker is an epitope for a fluorescent dye.

[0180] Embodiment 27.3. The composition of any one of embodiments 21 to 27.2, wherein each of the first and second populations of polymeric beads each comprises a different fluorophore.

[0181] Embodiment 27.4. The composition of any one of embodiments 21 to 27.3, wherein the biomarker is an epitope for an antibody.

[0182] Embodiment 27.5. The composition of embodiment 27.4, wherein the antibody is configured to bind to a fluorescent dye or to bind to a secondary antibody-fluorophore conjugate.

[0183] Embodiment 27.6. The composition of any one of embodiments 21 to 27.5, wherein the fluorophore is conjugated to an antibody or fragment thereof that binds to an epitope within the polymer bead.

[0184] Embodiment 27.7. The composition of embodiment 27.6, wherein the epitope is a biomarker contained in a polymer bead.

[0185] Embodiment 27.8. The composition of embodiment 27.6 or 27.7, wherein the fluorophore is a commercially available antibody-labeled conjugate.

[0186] Embodiment 27.9. The composition of any one of embodiments 27.6 to 27.8, wherein the biomarker is selected from those listed in Tables 1 to 3 herein.

[0187] Embodiment 28. Each biomarker was identified as CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-gamma, CD20, TCR gamma / delta, TNF alpha, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, and TCR V alpha 7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR V delta 2, TIGIT, CD1c, CD2, CD7, CD8a, CD15, CD32, CD103, CD107a, CD141, CD158, CD159c, IL-13, IL-21, KLRG1, TIM-3, CCR5, CD5, CD33, CD45.2, CD80, CD159a (NKG2a), CD244, CD272, CD278, CD337, granzyme B, Ig lambda light chain, IgA, IL-17A, streptavidin, TCR V delta 1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, Fc epsilon R1 alpha, IL-22, Lag-3, MIP-1 beta, perforin, TCR V gamma 9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Ig kappa light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21 R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC class II (IA / IE), MHC II, TCR alpha / beta, TCR beta, TCR V alpha 24, Akt(pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cd16 / 32, cd41a, CD3 epsilon, CD8b, CD11b / c, CD16 / CD32, CD23, CD29, CD43, CD45.1, CD48, CD49b, CD49d, CD66, CD68, CD71, CD85k, CD93, CD99, CD106, CD122, CD133, CD1 34, CD146, CD150, CD158b, CD158b1 / b2, j, CD158e, CD166, CD169, CD184, CD200, CD200 R, CD235a, CD267, CD268, CD273, CD274, CD317, CD324, CD326, CD328, CD336, CD357, CD366, DDR2, eFluor 780 Fix Viability, EGF receptor, EGFR (pY845), EOMES, EphA2, ERK1 / 2 (pT202 / pY204), F4 / 80, FCRL5, Flt-3, FVS575V, FVS700, Granzyme A, HER2 / ErbB2, Hes1, Hoechst (33342), ICAM-1, IFN-alpha, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1 RAcP, IL-6, IL-10, IL-12, IL-17, integrin alpha 4 beta 7, isotype Ctrl, KLRC1, KLRC2, Live / Dead Fix 27. The method of any one of embodiments 21-27.2, wherein the antibody is independently selected from any one of Aqua, Ly-6A / Ly-6E, Ly-6G / Ly-6C, mannose receptor, MDR1, Met(pY1234 / pY1235), MMP-9, NGF receptor p75, ORAI1, ORAI2, ORAI3, p53, P2RY12, PARP, cleavage, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCR delta, TCR delta / gamma, TCR V alpha 24 J alpha 18, TCR V beta 11, TCR V gamma 1.1, TCR V gamma 2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1.

[0188] Embodiment 29. 29. The composition of any one of embodiments 27-28, wherein the hydrogel comprises polyacrylamide.

[0189] Embodiment 30. 30. The composition of any one of embodiments 21-29, comprising: (iii) a third population of beads comprising a third biomarker; (iv) a fourth population of beads comprising a fourth biomarker; (v) a fifth population of beads comprising a fifth biomarker; and (vi) a sixth population of beads comprising a sixth biomarker.

[0190] Embodiment 31. 31. The composition of embodiment 30, wherein the first, second, third, fourth, fifth, and sixth biomarkers are independently selected from the group consisting of CD3, CD16, CD56, CD45, CD4, CD19, and CD8.

[0191] Embodiment 32. 32. The composition of embodiment 30 or 31, wherein each population of polymeric beads comprises a different fluorophore.

[0192] Embodiment 33. 28. The composition of embodiment 27, wherein the hydrogel comprises a matrix comprising the monomer.

[0193] Embodiment 34. The monomers are hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl meth ... Phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate Acrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,34. The composition of embodiment 33, wherein the alkyl acrylate is N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthylacrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenylphenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methylphenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethyl methacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N'-phenylphenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0194] Embodiment 35. 35. The composition of any one of embodiments 21 to 34, wherein the polymer beads exhibit at least one optical property substantially similar to an optical property of a target cell.

[0195] Embodiment 35.1. 35. The composition of any one of embodiments 21-34, wherein at least one population of polymeric beads exhibits at least one optical property that differs from the corresponding optical property of another population of polymeric beads within the composition.

[0196] Embodiment 36. The composition of embodiment 35 or 35.1, wherein the at least one optical property is side scattering.

[0197] Embodiment 37. The composition of embodiment 35 or 35.1, wherein at least one optical property is forward scattering.

[0198] Embodiment 38. The composition of embodiment 35 or 35.1, wherein the at least one optical property comprises side scattering and forward scattering.

[0199] Embodiment 39. 36. The composition of embodiment 35, wherein each target cell is independently selected from any one of a T cell, a B cell, and a natural killer cell.

[0200] Embodiment 40. A method of calibrating a cytometry device for compensation or spectral unmixing, comprising: (i) measuring the fluorescent signal of a composition described in any one of embodiments 21 to 39 using a cytometry device; (ii) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix; and (iii) calibrating the cytometry device using the compensation or spectral unmixing matrix.

[0201] Embodiment 40.1. A method of calibrating a cytometry device for compensation or spectral unmixing, comprising: (A) using a cytometry device to measure a fluorescent signal of a composition comprising (i) a first population of polymeric beads comprising a first biomarker and (ii) a second population of polymeric beads comprising a second biomarker; (B) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix; and (C) calibrating the cytometry device using the compensation or spectral unmixing matrix.

[0202] Embodiment 40.2. The method of embodiment 40.1, wherein each population of polymer beads comprises a different fluorophore.

[0203] Embodiment 41. 1. A method for producing a multicolor compensation control for a cytometry device, the method comprising: (A) contacting in a single reaction a composition comprising (i) a first population of polymer beads comprising a first biomarker and (ii) a second population of polymer beads comprising a second biomarker with a plurality of fluorescent dyes, wherein each dye in the plurality of fluorescent dyes comprises a fluorophore with a different excitation or emission spectrum from the fluorophores in all other dyes in the plurality of fluorescent dyes, and each dye binds to biomarkers on only a single population of polymer beads in the composition, wherein each population of polymer beads is bound to no more than one fluorescent dye, thereby producing a multicolor compensation control.

[0204] Embodiment 41.1. The method of any one of embodiments 40.1 to 41, wherein each biomarker in the population of polymer beads comprises an antigen configured to selectively bind to a fluorescent dye therein.

[0205] Embodiment 41.2. The method of any one of embodiments 41 to 41.1, wherein the plurality of fluorescent dyes are secondary antibody-fluorophore conjugates, and each biomarker in the population of polymer beads comprises an antigen configured to selectively bind to the secondary antibody-fluorophore conjugate.

[0206] Embodiment 42. The method of any one of embodiments 41 to 41.2, wherein each dye in the plurality of fluorescent dyes comprises an antibody-fluorophore conjugate.

[0207] Embodiment 42.1. 43. The method of any one of embodiments 41-42, wherein each fluorophore is independently selected from those listed in Table 4.

[0208] Embodiment 43. Each fluorophore is a peridinin chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5), phycoerythrin-cyanine 7 (PE Cy7), allophycocyanin-cyanin 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, 5-(and -6)-carboxyeosin, 5-carboxyfluorescein, 6-carboxyfluorescein, 5-(and -6)-carboxyfluorescein, S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether, -alanine-carboxamide, or succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 6-carboxyfluorescein succinimidyl ester, 5-(and -6)-carboxyfluorescein succinimidyl ester, 5-(4,6-dichlorotriazinyl)aminofluorescein, 2',7'-Difluorofluorescein, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, 6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester, 6-(fluorescein-5-(and -6)-carboxamido)hexanoic acid or succinimidyl ester, fluorescein-S-EX succinimidyl ester, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, OregonGreen® 488 carboxylic acid or succinimidyl ester, Oregon Green® 488 isothiocyanate, Oregon Green® 488-X succinimidyl ester, Oregon Green® 500 carboxylic acid, Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt, Oregon Green® 514 carboxylic acid, Oregon Green® 514 carboxylic acid or succinimidyl ester, Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride, Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester, Rhodamine Green™-X succinimidyl ester or hydrochloride, RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester, bis-(4-carboxypiperidinyl)sulfonarhodamine or di(succinimidyl ester), 5-(and-6) carboxynaphthofluorescein, 5-(and-6) carboxynaphthofluorescein succinimidyl ester, 5-carboxyrhodamine 6G hydrochloride, 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester, 6-carboxyrhodamine 6G succinimidyl ester, 5-(and-6)-carboxyrhodamine 6G succinimidyl ester, 5-carboxy-2',4',5',7'-Tetrabromosulfonefluorescein succinimidyl ester bis-(diisopropylethylammonium) salt, 5-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, 5-(and -6)-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimidyl ester, 6-carboxytetramethylrhodamine succinimidyl ester, 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester, 6-carboxy-X-rhodamine, 5-carboxyl-X-rhodamine succinimidyl ester Rhodamine succinimidyl ester, 6-carboxy-X-rhodamine succinimidyl ester, 5-(and -6)-carboxy-X-rhodamine succinimidyl ester, 5-carboxy-X-rhodamine triethylammonium salt, Lissamine™ rhodamine B sulfonyl chloride, Malachite Green, isothiocyanate, NANOGOLD® mono (sulfosuccinimidyl ester), QSY® 21 carboxylic acid or succinimidyl ester, QSY® 7 carboxylic acid or succinimidyl ester, Rhodamine Red™-X succinimidyl ester, 6-(tetramethylrhodamine-5-(and -6)-carboxamido)hexanoic acid, succinimidyl ester, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, tetramethylrhodamine-5-(and -6)-isothiocyanate, Texas Red® sulfonyl, Texas Red® sulfonyl chloride, Texas Red®-X STP ester or sodium salt, Texas Red®-X succinimidyl ester, Texas Red®-X succinimidyl ester, X-rhodamine-5-(and -6) isothiocyanate, BODIPY® FL, BODIPY® TMR STP ester, BODIPY® TR-X STP ester, BODIPY® 630 / 650-X STPester, BODIPY® 650 / 665-X STP ester, 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene Indacene-3 propionic acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5,7-dimefhyl-4-bora-3a,4a-diaza-s-indacene-3 propionic acid, sulfosuccinimidyl ester or sodium salt, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid, Methyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt, 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid , 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester, 4,4-difluoro-5-(4-phenyl-1,3 butadienyl)-4-bora-3a,4a-Diaza-s-indacene-3-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester, 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid Fluoro-1,3,5,7-tetramethyl-4-bora-3a,4diaza-s-indacene-8-propionic acid, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacen-8-propionic acid succinimidyl ester, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacen-3-propionic acid succinimidyl ester, 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4diazas-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester, and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,The method of any one of embodiments 40.1 to 42.1, wherein the carboxylic acid is independently selected from one of: 4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 64 7 carboxylic acid, Alexa Fluor® 660 carboxylic acid, Alexa Fluor® 680 carboxylic acid, Cy3 NHS ester, Cy 5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester.

[0209] Embodiment 44. The method of any one of embodiments 40.1-43, wherein the composition comprises up to 5, up to 10, up to 12, up to 18, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 populations of polymeric beads, each bead population comprising a biomarker, and the biomarkers for each bead population are different.

[0210] Embodiment 45. The method of any one of embodiments 40.1 to 44, wherein each population of polymer beads comprises a different biomarker.

[0211] Embodiment 46. The method of any one of embodiments 40.1 to 45, wherein each population of polymer beads contains only a single biomarker.

[0212] Embodiment 47. The method of any one of embodiments 40.1 to 46, comprising a population of beads that do not contain a fluorophore or that lack an epitope for association with an antibody-fluorophore conjugate.

[0213] Embodiment 48. The method of any one of embodiments 40.1 to 47, comprising a population of beads that do not contain biomarkers.

[0214] Embodiment 49. The method of any one of embodiments 40.1 to 48, wherein the polymeric beads comprise less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0215] Embodiment 50. The method of any one of embodiments 40.1 to 48, wherein the polymeric beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dehydrated volume.

[0216] Embodiment 51. The method of any one of embodiments 40.1 to 50, wherein the polymer beads are hydrogel beads.

[0217] Embodiment 52. The method of any one of embodiments 40.1 to 51, wherein the biomarker is a polypeptide.

[0218] Embodiment 53. The method of any one of embodiments 40.1 to 52, wherein the biomarker is an epitope for a fluorescent dye.

[0219] Embodiment 54. The method of any one of embodiments 40.1 to 53, wherein each of the first and second populations of polymer beads each comprises a different fluorophore.

[0220] Embodiment 55. The method of any one of embodiments 40.1 to 54, wherein the biomarker is an epitope for an antibody.

[0221] Embodiment 56. The method of any one of embodiments 40.1 to 55, wherein the antibody is configured to bind to a fluorescent dye or configured to bind to a secondary antibody-fluorophore conjugate.

[0222] Embodiment 57. The method according to any one of embodiments 40.1 to 56, wherein the fluorophore is conjugated to an antibody or fragment thereof that binds to an epitope within the polymer bead.

[0223] Embodiment 58. 58. The method of embodiment 57, wherein the epitope is a biomarker contained in a polymer bead.

[0224] Embodiment 59. The method of any one of embodiments 40.1 to 58, wherein the fluorophore is a commercially available antibody-labeled conjugate.

[0225] Embodiment 60. The method of any one of embodiments 40.1-59, wherein the biomarkers are selected from those listed in Tables 1-3 herein.

[0226] Embodiment 61. Each biomarker was identified as CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-gamma, CD20, TCR gamma / delta, TNF alpha, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, and TCR V alpha 7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR V delta 2, TIGIT, CD1c, CD2, CD7, CD8a, CD15, CD32, CD103, CD107a, CD141, CD158, CD159c, IL-13, IL-21, KLRG1, TIM-3, CCR5, CD5, CD33, CD45.2, CD80, CD159a (NKG2a), CD244, CD272, CD278, CD337, granzyme B, Ig lambda light chain, IgA, IL-17A, streptavidin, TCR V delta 1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, Fc epsilon R1 alpha, IL-22, Lag-3, MIP-1 beta, perforin, TCR V gamma 9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Ig kappa light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21 R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC class II (IA / IE), MHC II, TCR alpha / beta, TCR beta, TCR V alpha 24, Akt(pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cd16 / 32, cd41a, CD3 epsilon, CD8b, CD11b / c, CD16 / CD32, CD23, CD29, CD43, CD45.1, CD48, CD49b, CD49d, CD66, CD68, CD71, CD85k, CD93, CD99, CD106, CD122, CD133, CD1 34, CD146, CD150, CD158b, CD158b1 / b2, j, CD158e, CD166, CD169, CD184, CD200, CD200 R, CD235a, CD267, CD268, CD273, CD274, CD317, CD324, CD326, CD328, CD336, CD357, CD366, DDR2, eFluor 780 Fix Viability, EGF receptor, EGFR (pY845), EOMES, EphA2, ERK1 / 2 (pT202 / pY204), F4 / 80, FCRL5, Flt-3, FVS575V, FVS700, Granzyme A, HER2 / ErbB2, Hes1, Hoechst (33342), ICAM-1, IFN-alpha, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1 RAcP, IL-6, IL-10, IL-12, IL-17, integrin alpha 4 beta 7, isotype Ctrl, KLRC1, KLRC2, Live / Dead Fix The method of any one of embodiments 40.1-59, wherein the antibody is independently selected from any one of Aqua, Ly-6A / Ly-6E, Ly-6G / Ly-6C, mannose receptor, MDR1, Met(pY1234 / pY1235), MMP-9, NGF receptor p75, ORAI1, ORAI2, ORAI3, p53, P2RY12, PARP, cleavage, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCR delta, TCR delta / gamma, TCR V alpha 24 J alpha 18, TCR V beta 11, TCR V gamma 1.1, TCR V gamma 2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1.

[0227] Embodiment 62. 62. The method of any one of embodiments 51-61, wherein the hydrogel comprises polyacrylamide.

[0228] Embodiment 63. The method of any one of embodiments 40.1 to 62, comprising (iii) a third population of beads comprising a third biomarker, (iv) a fourth population of beads comprising a fourth biomarker, (v) a fifth population of beads comprising a fifth biomarker, and (vi) a sixth population of beads comprising a sixth biomarker.

[0229] Embodiment 64. 64. The method of embodiment 63, wherein the first, second, third, fourth, fifth, and sixth biomarkers are independently selected from the group consisting of CD3, CD16, CD56, CD45, CD4, CD19, and CD8.

[0230] Embodiment 65. The method of any one of embodiments 40.1 to 64, wherein each population of polymer beads comprises a different fluorophore.

[0231] Embodiment 66. 66. The method of any one of embodiments 51 to 65, wherein the hydrogel comprises a matrix comprising a monomer.

[0232] Embodiment 67. The monomers are hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl meth ... Phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate Acrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,67. The method of embodiment 66, wherein the alkyl group is N-dibenzyl methacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0233] Embodiment 68. The method according to any one of embodiments 40.1 to 67, wherein the polymer beads exhibit at least one optical property substantially similar to the optical property of the target cells.

[0234] Embodiment 69. The method of any one of embodiments 40.1 to 68, wherein at least one population of polymeric beads exhibits at least one optical property that differs from the corresponding optical property of another population of polymeric beads within the composition.

[0235] Embodiment 70. 70. The method of embodiment 68 or 69, wherein the at least one optical property is side scattering.

[0236] Embodiment 71. 70. The method of any one of embodiments 68-69, wherein at least one optical property is forward scattering.

[0237] Embodiment 72. 70. The method of any one of embodiments 68-69, wherein the at least one optical property comprises side scattering and forward scattering.

[0238] Embodiment 73. The method of any one of embodiments 40.1 to 72, wherein each target cell is independently selected from any one of a T cell, a B cell, and a natural killer cell.

[0239] Embodiment 74. 1. A method of calibrating a cytometry device for compensation or spectral unmixing, comprising: (i) providing a composition comprising (a) a first population of polymeric beads comprising a first biomarker and (b) a second population of polymeric beads comprising a second biomarker, each of the first and second populations of polymeric beads comprising a different fluorophore; (ii) contacting the composition with at least two populations of antibodies or fragments thereof, each population of antibodies capable of binding to only one of the biomarkers in the composition, wherein the antibodies or fragments thereof are conjugated to a fluorophore capable of generating a fluorescent signal; (iii) measuring the fluorescent signal of the composition using a cytometry device; (iv) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix; and (v) calibrating the cytometry device using the compensation or spectral unmixing matrix.

[0240] Embodiment 75. 75. The method of any one of embodiments 40 to 74, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the fluorescence emission maximum.

[0241] Embodiment 76. 75. The method of any one of embodiments 40 to 74, wherein the fluorescent signal from each population of polymer beads is deconvoluted based on the optical properties of each population of polymer beads. * * * * * * * *

[0242] All documents, patents, patent applications, publications, product descriptions, and protocols cited throughout this application are incorporated by reference in their entirety for all purposes. This document expressly incorporates the following U.S. and PCT patent applications in their entirety for all purposes: US2022 / 0178810, US2020 / 0400546, US2021 / 0341469, US2021 / 0231552, US2020 / 0400546, PCT / US2023 / 06668, and PCT / US2023 / 067893.

[0243] The embodiments illustrated and discussed herein are intended solely to teach those skilled in the art the best way known to the inventors to make and use the invention of this disclosure. Modifications and variations of the above-described embodiments of this disclosure are possible without departing from the spirit of the invention, as will be understood by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention of this disclosure may be practiced otherwise than as specifically described.

Claims

1. A method for calibrating a cytometry device for correction or spectral unmixing, (i) A step of measuring the fluorescence signal of a composition comprising (a) a first group of polymer beads containing a first fluorophore and (b) a second group of polymer beads containing a second fluorophore, using a cytometry device. (ii) A step of deconvolving a fluorescent signal from each polymer bead group of the composition, (iii) A step of calculating a correction or spectral unmixing matrix, and (iv) Calibrating the cytometry device using the correction or spectral unmixing matrix, The above method, including.

2. The method according to claim 1, wherein a first group of polymer beads and a second group of polymer beads each independently exhibit at least one optical property that is substantially similar to the optical properties of a target cell.

3. The composition according to claim 2, wherein at least one group of polymer beads exhibits at least one optical property that is different from the corresponding optical property of another group of polymer beads in the composition.

4. The method according to claim 2, wherein at least one optical property is side scattering, forward scattering, or both.

5. The method according to claim 1, wherein the step of calculating a correction or spectral unmixing matrix includes performing a least squares calculation.

6. The method according to claim 1, comprising the step of deconvolving a fluorescence signal from each polymer bead group based on the maximum fluorescence emission of each group, the optical properties of each group, or a combination thereof.

7. (v) The method according to claim 1, comprising the step of inserting a biological cell into a cytometry device, wherein the biological cell comprises a first fluorophore and / or a second fluorophore attached to the cell.

8. The method according to claim 7, comprising the steps of (vi) measuring fluorescence from a first fluorophore and / or a second fluorophore attached to a cell, and normalizing the measured fluorescence based on the correction or spectral unmixing of step (iii).

9. The method according to claim 1, wherein the first fluorophore and the second fluorophore exhibit spectral overlap in their emission spectra.

10. The method according to claim 9, wherein calculating a correction or spectral unmixing matrix includes determining the amount of spectral overlap between a first fluorophore and a second fluorophore.

11. The method according to claim 1, wherein the first fluorophore and / or the second fluorophore are linked to a biomarker on the surface of a hydrogel bead.

12. The method according to claim 1, wherein the first fluorophore and the second fluorophore of the composition are different fluorophores.

13. The method according to claim 1, wherein each group of polymer beads in the composition comprises only a single fluorophore.

14. The method according to claim 1, wherein the composition comprises a third group of polymer beads that do not contain fluorophores.

15. The method according to claim 1, wherein the polymer beads in the composition are hydrogel beads.

16. Hydrogel beads include hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2 - Phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate Tacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,The method according to claim 15, comprising a monomer selected from the group consisting of N-dibenzylmethacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

17. The composition is (c) A third group of polymer beads containing a third fluorophore, (d) A fourth group of polymer beads containing a fourth fluorophore, (e) A fifth group of polymer beads containing a fifth fluorophore, and / or (f) The method according to claim 1, comprising one or more sixth groups of polymer beads containing a sixth fluorophore.

18. The composition is (g) The seventh group of polymer beads that do not contain fluorophores, The method according to claim 17, including the method described in claim 17.

19. The method according to claim 17, wherein each group of polymer beads comprises a single fluorophore.

20. A composition, (i) A first group of polymer beads containing a first fluorophore, and (ii) comprising a second group of polymer beads containing a second fluorophore, The composition wherein a first group of polymer beads and a second group of polymer beads each independently exhibit at least one optical property that is substantially similar to the optical properties of a target cell.

Images