Compositions and methods for single well multiple calibration and compensation

Abstract

The present disclosure relates to compositions comprising a plurality of modified particles that allow one-pot (one-pot) calibration, compensation, and spectral unmixing, and methods of use thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 400,039, filed on August 22, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to compositions of matter and methods that allow calibration, compensation, and spectral unmixing in a single well. Background Art

[0004] Flow cytometry, hematology, and image-based cytometry are techniques that allow 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 onto a focused stream of liquid. In one form, multiple detectors are then aimed at the point where the stream passes through the beam: one detector is in line with the beam (e.g., forward scatter or FSC; also known as axial light loss or ALL), and several detectors are perpendicular to the beam (e.g., side scatter or SSC). FSC is typically related to particle volume, and SSC typically depends on the complexity or granularity of the particles (i.e., the shape of the nucleus, the amount and type of cytoplasmic particles, or membrane roughness). Due to these correlations, different specific particle types (i.e., cells, extracellular vesicles) exhibit different FSC and SSC, allowing them to be distinguished from each other.

[0005] These measurements comprise the basis of cytometry analysis. In some forms of analysis, cells are also imaged and descriptive characteristics of the cells are recorded, such as size / shape / volume, and in some cases, when combined with detection reagents, biochemical characteristics are recorded. In other forms of analysis, interferometry, particle tracking analysis, and electrical perturbations are used to measure particle properties. In other forms, cells are labeled with reagents that detect the presence of biomarkers (or nucleic acids) that allow multiple measurements of subject features and characteristics.

[0006] The use of fluorescent molecules (such as fluorophore-labeled antibodies) in flow cytometry is a common way to study cell properties. For clarity, fluorophores and fluorescent dyes are used interchangeably in the instructions. Fluorophores can also be called labels, dyes, or stains. In these types of experiments, labeled antibodies are added to cell samples. The antibodies then bind to specific molecules on the cell surface or inside the cell. Finally, when a laser of the appropriate wavelength illuminates the fluorophore, the fluorescent signal is emitted and detected by the flow cytometer.

[0007] However, when using fluorophores, it can be difficult to describe different target species in detail based solely on corresponding fluorescent signals, which can overlap and obscure the ability to assign meaningful values ​​to each target species. The present disclosure addresses this shortcoming. Summary of the invention

[0008] In one aspect, the present disclosure provides a composition for compensation or spectral unmixing calculation in a single well, the single well comprising a first polymer particle having a first biomarker found on a target cell and a second polymer bead comprising a second biomarker found on the target cell. In another aspect, the present disclosure provides a method for calibrating a cytometry device, the method comprising mixing such a composition with an antibody-fluorophore conjugate specific for each biomarker in a single well in a cytometry device, measuring the fluorescent signal of the mixture, using the known fluorescent signals of the first antibody-fluorophore conjugate and the second antibody-fluorophore conjugate, deconvoluting the fluorescent signals of the first antibody-fluorophore-bound polymer beads and the second antibody-fluorophore-bound polymer beads according to the measured fluorescent signal of the mixture, so as to calculate a compensation or spectral unmixing matrix and calibrate the cytometry device.

[0009] On the one hand, the present disclosure provides a composition for compensation or spectral unmixing calculation in a single well, the single well comprising a first polymer particle and a second polymer bead, the first polymer particle having a first biomarker found on a target cell, the second polymer bead comprising a second biomarker found on the target cell, 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 biomarker and the second biomarker. On the other hand, the present disclosure provides a method for calibrating a cytometry device, the method comprising mixing such a composition in a single well in a cytometry device, measuring the fluorescent signal of the mixture, using the known fluorescent signal properties of the first antibody-fluorophore conjugate and the second antibody-fluorophore conjugate, deconvoluting the measured fluorescent signals of the first antibody-fluorophore-bound polymer beads and the second antibody-fluorophore-bound polymer beads according to the measured fluorescent signal of the mixture, so as to calculate a compensation or spectral unmixing matrix and calibrate the cytometry device.

[0010] In a preferred embodiment, the polymer particles comprise a hydrogel having autofluorescence and other optical properties substantially similar to those of the target cells.

[0011] The present invention allows compensation 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.

[0012] The present disclosure also provides a computational method for performing fluorescence compensation and spectral unmixing using a single reaction vessel, wherein a plurality of separate bead populations are combined with a complete staining set mixture in a single tube. The separate bead populations can be modified a priori with the fluorophores used in a given set, or separate bead populations can be prepared so that they specifically bind to each separate antibody-fluorophore conjugate from the staining set mixture. The present disclosure provides a method for deriving deconvolution data from a single reaction vessel based on the inherent or predetermined fluorescence properties of a single fluorophore / fluorescent dye in order 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 single mixture antibody staining set. The present disclosure also provides a software-driven method for automatically calculating separate compensation and spectral unmixing calculations according to this method.

[0013] In another embodiment, fluorescent features can be conjugated directly to polymer beads. For example, rather than creating a biochemically distinct population of beads in a mixture that specifically binds to individual reagents within a staining set, beads can be labeled a priori with a fluorophore from a staining set (or a combination of fluorophores or pre-bound to an antibody-fluorophore conjugate) so that the beads can be easily deconvoluted for compensation or FMO calculations. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The general concepts of fluorescence compensation and spectral unmixing are demonstrated, highlighting the difference between (A) the traditional method and (B) the one-pot method. The present invention saves a lot of time for high complexity assays (eg, 30-color calculations).

[0015] Figure 2 The traditional compensation bead workflow requiring separate tubes is shown, and why the combined staining panel approach does not work when using existing reagents. Standard compensation beads are bound to the various detection antibodies (depicted as "reagents") in the staining panel. Therefore, the reagents must be separated into separate tubes in order to calculate compensation data (A). If the staining 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).

[0016] Figure 3Depicted are the fluorescence spillover and compensation adjustments required to calculate appropriate emission signal data. In the traditional workflow, each fluorophore-antibody combination must be separated into a separate tube and physically deconvoluted in order to separate the signal for compensation or spectral unmixing calculations. This is largely driven by the fact that the particles used in traditional compensation calculations (whether cells or traditional compensation beads) will indiscriminately bind to most, if not all, antibodies in a given set, and therefore they cannot be separated into distinct events.

[0017] Figure 4 An example of a one-pot compensation or unmixing method of the present disclosure is shown, wherein a separate bead population is pre-modified with a fluorescent dye or a bead population is modified to bind to a separate reagent (antibody-fluorophore conjugate) from a mixed staining set. The biomarker-modified compensation beads will only bind to a single antibody in the reagent mixture, allowing the user to add a complete staining set mixture to a single tube and deconvolute the separate fluorescent signal from the mixture for compensation or spectral unmixing calculations. Separate fluorophore signals can be deconvoluted for compensation or spectral unmixing calculations because each bead will only bind to a specific reagent in the mixture. This significantly simplifies the workflow.

[0018] Figure 5 It is demonstrated how the user can add a pre-aliquoted mixture of selectively bound biomarker beads (e.g., a complete staining set) in a one-pot reaction to computationally deconvolute the individual fluorophore curves to calculate compensation or spectral unmixing. This is achieved by the fact that each individual bead in the mixture will bind to only one antibody from the mixture. The software can then select the local maximum ( Fig.13 ) to selectively isolate curvilinear information, for example, allowing computer-simulated deconvolution to be compared to physically derived deconvolution ( Figure 2 ).

[0019] Figure 6 Shown are the differences between (A) a typical fluorescence minus one (FMO) staining panel workflow and (B) a FMO panel generated using the reagents and methods described in the present disclosure. In a typical workflow, a combination cocktail of antibodies must be generated by the user and then applied to binding particles, typically patient cells. This saves a lot of time for high complexity (e.g., 30-color) assays.

[0020] Figure 7Demonstrates the physical process of generating FMO data for an experiment by generating a combinatorial mixture of the reagents (minus one) used in a staining set. The standard FMO workflow requires the user to laboriously generate a combinatorial mixture of all antibodies (minus one) in a staining set in order to calculate the FMO noise floor. This is driven by the fact that the reagents or cells used for the FMO calculation will bind to all antibodies in the mixture.

[0021] Figure 8 Depicts the importance of FMO calculations, especially for unclear or poorly expressed biomarkers, to ensure accurate data generation. In this simplified set, there are three staining antibodies in the set, each of which emits in a unique channel. Due to fluorescence overflow, the true noise floor or detection limit is highlighted by the dotted line. Since the signal is generated by the orthogonal antibodies in the set and is not a true biological representation of the presence of the target biomarker of this channel, any signal below the dotted line cannot be reliably measured. This is especially important for poorly expressed or "dim" biomarker sets.

[0022] Fig. 9 A single reagent mix FMO control workflow is demonstrated, where the user prepares only one master mix of staining antibodies for a staining set. The user then applies this single mix to a pre-aliquoted combined mix of FMO binding beads, greatly improving operator efficiency and reducing errors. The FMO binding beads are pre-aliquoted to contain the entire set (minus one), enabling the user to create only one master mix of reagents, saving a significant amount of time compared to traditional workflows.

[0023] Fig.10 A single mixture FMO control set and its impact on calculating the true signal-to-noise ratio or noise floor of the staining set and assay is demonstrated. In this format, the combined FMO biomarker beads are combined a priori, allowing the user to prepare a single mixture that needs to be added to each tube of mixed biomarker beads. Each of these mixtures lacks a unique biomarker bead that acts like an FMO control. This greatly speeds up the workflow for calculating FMO controls for complex sets. This also eliminates the need to use actual patient samples or cells for FMO calculations.

[0024] Fig.11 A traditional compensation data workflow with 6 antibodies from the TBNK panel is shown. Typically, individual antibody-fluorophore conjugates must be mixed and combined with compensation beads in separate tubes to generate spectra for compensation or spectral unmixing calculations as shown. (PerCP-Cy5.5, PE Cy7, APC-Cy7, FITC, PE, APC).

[0025] Fig.12Depicted are six unique antibody-fluorophore conjugates mixed in the same tube and analyzed together. Whether using conventional compensation beads or the beads of the present invention, the signal from the mixture will be the same. However, this signal cannot be deconvoluted using conventional compensation beads because each bead binds to all 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 from the mixture can be deconvoluted and compensation or spectral unmixing calculations can be performed. In this example, a spectrocytometer is used, but any conventional cytometer can also be used.

[0026] Fig.13 Depicted is the process of selecting the maximum to deconvolute the individual fluorophore signals from the combined mixture of beads described in this disclosure. This allows Fig.12 The data can be separated into individual channels, for example. For this basic example, the maximum value of each fluorescent channel is used to select the individually modified beads.

[0027] Fig.14 Depicts successful deconvolution of individual fluorophore signals from a one-pot reaction mixture containing individually modified beads, enabling single-tube compensation matrix / spectral unmixing calculations to be performed. Such individual fluorophore signals cannot be combined with Fig.11 The data generated by the separate separation tubes are distinguished.

[0028] Fig.15 Depicted are the resulting data from (B) a spectrally unmixed TBNK staining set using a one-pot bead mixture as described in the present disclosure, which is indistinguishable from (A) a single separate tube for spectrally unmixed. DETAILED DESCRIPTION

[0029] definition

[0030] As used herein, the indefinite article "a / 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 indicate that an article may include one or more than one of the listed elements.

[0031] As used herein, the terms "polymer beads" and "polymer particles" may be used interchangeably.

[0032] Unless otherwise noted, it should be understood that the amount, ratio and numerical properties of the expression components, reaction conditions, etc. used in the specification and claims are all considered to be modified by the term "about" in all cases. For example, throughout this application, the term "about" can be used to indicate that the value includes the inherent error variation of the device, method for determining the value, or the variation between the samples measured. Unless otherwise stated or otherwise obvious from the context, the term "about" means within 10% of the reported value (unless such a value will exceed 100% of the possible value or be lower than 0%). When used in conjunction with a range or series of values, unless otherwise stated, the term "about" applies to each value in the values ​​listed in the end points of the range or the series. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0033] The general trend in this area is to increase the quantity of the biophysical characteristics measured in a single experiment or assay tube. For the purpose of this application, the terms assay tube, hole, container, pan, tube and reaction vessel are used interchangeably. This is commonly referred to as a "staining set", which is designed to characterize a sample of interest using a set of reagents, which is typically an antibody that identifies an epitope conjugated to a fluorophore. Larger staining sets allow for higher "multiple" analysis, improve operator efficiency and reduce sample and analyte requirements to run complex characterization / phenotypic analysis. Exemplary staining sets include an optimized multicolor immunofluorescence panel (OMIP) outlined by the International Society for Advancement of Cytometry.

[0034] The complex methods involved in fluorescence detection present researchers with significant hurdles to consider. This includes the hands-on time to physically separate each reagent in a staining panel for individual analyses during compensation control setup, which can involve hours of labor for complex panels. This also increases the potential for operator error in many setups. The additional complexity of flow cytometry, combined with the subsequent design of experimental protocols and detailed analysis involving many fluorophores and fluorescent signals, presents additional barriers to the effective use of flow cytometry. Currently, appropriate consideration of the spectral overlap that results from the use or inclusion of multiple fluorescent materials in different detection systems is a response to these issues as they arise.

[0035] As stain panels increase in complexity, it becomes increasingly important to distinguish fluorescent or spectral signals from one another, often through the use of compensation or spectral unmixing methods, because there is a greater chance that a given set of fluorophores cannot be reliably distinguished from one another. This is known as fluorescence 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 a range of excitation and emission spectra relative to a single wavelength emission spectrum. Compensation therefore becomes increasingly important as stain panels increase in complexity.

[0036] Fluorescence

[0037] A fluorophore is a molecule that is able to fluoresce. In its ground state, the fluorophore molecule is in a relatively low-energy, stable configuration, and it does not fluoresce. When light from an external source hits the fluorophore molecule, the molecule can absorb light energy. If the absorbed energy is sufficient, the molecule reaches an excited state (high energy); this process is called excitation. Depending on the wavelength and energy of the external light source, a fluorophore can reach multiple excited states or energy levels. Since the fluorophore is unstable in a high-energy configuration, it eventually adopts a semi-stable, lowest-energy excited state. The excitation lifetime (the length of time the fluorophore is in an excited state) is very short; the fluorophore rearranges from the semi-stable excited state back to the ground state, and a portion of the excess energy can be released and emitted as light. The emitted light has a lower energy and a longer wavelength than the absorbed light, so the color of the emitted light is different from the color of the light that has been absorbed. Deexcitation returns the fluorophore to its ground state. The fluorophore can absorb light energy again and repeatedly go through the excited state to ground state process.

[0038] Fluorescence spectrum

[0039] Fluorescent dyes absorb light within a certain range of wavelengths, and each dye has a characteristic excitation range. This range of excitation wavelengths is called the fluorescence excitation spectrum, and reflects the range of possible excitation 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 of the fluorophore. Less efficient excitation may occur at wavelengths close to the excitation maximum; however, the intensity of the emitted fluorescence is reduced. Although illumination at the excitation maximum of the fluorophore produces maximum fluorescence output, illumination at lower or higher wavelengths only affects the intensity of the emitted light; the range and overall shape of the emission spectrum remain unchanged.

[0040] Due to changes that may occur during the lifetime of the excitation, the molecules may emit at different wavelengths with each excitation event, but each emission will be within the fluorescence emission spectrum. Although the fluorophore molecules all emit light of the same intensity, the wavelength, and therefore the color of the emitted light, is not uniform. The emission maximum is the wavelength at which the population of molecules fluoresces most intensely. An excited fluorophore may also emit light at a wavelength close to the emission maximum. However, this light will be less intense.

[0041] 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 peaks of varying intensities throughout the spectrum. When using a broadband white light source, it is necessary to filter the desired wavelengths required for excitation; this is typically done using optical filters. Optical filters selectively allow certain wavelengths of light to pass while blocking undesired wavelengths. Bandpass excitation filters transmit a narrow range of wavelengths 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 the sample. High-output light emitting diodes (LEDs) offer selective wavelengths, low cost and energy consumption, and long life. Monochromatic LEDs are ideal for low-cost instrumentation, where they can be combined with a simple long-pass filter that blocks LED excitation and allows transmission of the dye signal. However, the range of wavelengths emitted from each LED is still relatively wide, and filters may also need to be used to narrow the bandwidth. More information on fluorescence spectroscopy can be found in the Journal of the American Society of Fluorescence Spectroscopy. Handbook - A Guide to Fluorescent Probes and Labeling Technologies Handbook—A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety.

[0042] Fluorescence detection

[0043] In traditional or conventional flow cytometers and other instruments that employ multiple photodetectors to detect multiple dyes, the 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 specific photodetector or photomultiplier tube (PMT) is limited to a predetermined range of wavelengths, sometimes referred to as a detection channel. The detection channels and dyes are selected so that the peak of the emission spectrum of each dye is within the frequency range of different detection channels, e.g., each detection channel primarily detects emission from a single dye. However, due to the breadth of the emission spectrum of fluorescent dyes, typically the dye will fluoresce in more than one detection channel, and therefore, the measurement of dye fluorescence is not independent. The emission of one dye in a detection channel intended for detection of other dyes is referred to by many terms, such as spillover, spectral overlap, and crosstalk.

[0044] Spectral flow cytometry is a technique based on conventional flow cytometry, in which the spectrometer and multi-channel detector (e.g., charge-coupled device (CCD)) are replaced by the traditional mirrors, optical filters, and PMTs in conventional systems. In spectral flow cytometry, fluorescence is collected directly or through optical fibers and displayed as a spectrometer, where the entire light signal is dispersed and displayed as a high-resolution spectrum on the CCD or coupled into one or more multi-channel detectors for detection.

[0045] In order to correctly interpret the data, the fluorescence recorded from one fluorescent source must be distinguished from the fluorescence recorded from other fluorescent sources. Therefore, an ideal fluorophore has a fluorescence emission spectrum with very intense narrow peaks that are well separated from all other emission peaks. However, typical fluorophores have broad emission peaks that can overlap or spill over. Such overlap or spillover can compromise data and analysis.

[0046] Background fluorescence, which may originate from endogenous sample components (autofluorescence) or from unbound or non-specifically bound reagents, may impair fluorescence detection. Detection of autofluorescence can be minimized by selecting filters that reduce transmission or detection of autofluorescence, but by doing so, overall fluorescence intensity detection is compromised. Full-spectrum flow cytometers will detect autofluorescence.

[0047] calibration

[0048] Most assays require some form of calibration and setup to ensure accurate performance when analyzing biological samples. Examples of calibration include setting the gain and voltage for detection, measuring inter-laser fall delays, and ensuring linearity of detected fluorescence signals. In many cases, ideal calibration and setup reagents look and act similar to the biological particles being analyzed. This helps ensure similar performance in diagnostic instruments while not introducing artifacts into the measurement process.

[0049] Most synthetic or polymer products used for cell analysis are made of polystyrene, which is an opaque polymer with a high refractive index that typically has a fixed forward scattering spectrum and a side scattering spectrum based on the particle diameter. This high refractive index is different from biological particles such as cells and extracellular vesicles, which are translucent and allow optical resolution and measurement of internal features such as organelles in the case of cells. In particular, extracellular vesicles typically have a low refractive index relative to their diameter. Polystyrene is also hydrophobic and has a high elastic modulus, both of which also distinguish it from cells and biomaterials. In some forms, silicon dioxide (SiO2) is used as a substitute for biological particles. Although the refractive index of silicon dioxide is closer to that of typical extracellular vesicles, there are still substantial differences in the optical response, requiring interpolation when used as a calibration reagent.

[0050] These properties together make polystyrene and silica particles less than ideal for generating controls and calibration reagents for bioparticle measurements. Therefore, many practitioners use cell materials or biologically derived particles (e.g., lipid nanoparticles) as process and reference controls before measuring samples. Cells and biomaterials also have other disadvantages, including poor stability, limited and complex supply chains, high batch-to-batch variability, and high production costs. In addition, the materials typically have strict cold chain handling requirements, which limits the scope and application of the control.

[0051] Compensation and spectral unmixing

[0052] Another example of calibration includes fluorescence compensation, where the excitation and emission spectra of a given fluorophore are different from a potentially overlapping set of fluorophores used in the same set of experiments. Another example of calibration includes spectral unmixing, where the spectral response of a given fluorophore (sometimes referred to as the emission or fingerprint or signature or pattern of the fluorophore) is different from a potentially overlapping set of fluorophores used in the same set of experiments. For conventional cytometers, the process is called "compensation", while for spectral cytometers, it is called "spectral unmixing". Although compensation and spectral unmixing share the same conceptual goal, they are based on different mathematical calculations.

[0053] Ideally, when a dye is used in an experiment, its emission spectrum will be narrow enough so that the fluorescence from this dye is detected by only a single detector in the instrument. In practice, due to the wide emission spectra of available fluorescent dyes, the dye used may emit a large amount of fluorescence in 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 the data so that the values ​​from a single detector come from separate dyes. In order to separate these signals or compensate for overlapping emissions, a percentage of each overlapping emission is subtracted from the target emission. Traditionally, this compensation is performed by the instrument during acquisition. However, modern instruments are able to store data in an uncompensated form, and compensation can be applied by the 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.

[0054] Compensation involves creating two matrices. The spillover matrix represents the percentage of signal from a given channel that spills over into adjacent channels. The compensation matrix used to correct for spillover is the inverse of the spillover matrix.

[0055] In an embodiment, the target parameter is a parameter of a detection signal (possibly from multiple sources). The source parameter is the main parameter that the signal is expected to enter, but the dye may also (possibly) infiltrate (bleeding) multiple targets. In other words, the percentage of the source that "bleeds" into the target is subtracted. In the example given above, parameter 2 is the target, and parameter 1 is the source. The source and target family are referred to as compensation definitions. The compensation definition describes all the ways in which the fluorescence from different channels interacts with each other under a given set of conditions and is equivalent to a single compensation matrix. Typically, the instrument user will set the gain of all channels at the beginning of the experiment, and use these settings during the experiment. Therefore, the compensation definition will apply to the entire experiment.

[0056] Using a spectral cytometry instrument, a continuous high-resolution spectrum is collected for each event in the sample. The spectrum is the sum of the spectra of all dyes present in the event of interest. Spectral unmixing is the process of transforming data to determine the contribution of each dye to the total signal. Several mathematical models can be used to perform spectral unmixing calculations, including the Ordinary Least Square method. The Ordinary Least Square method assumes a linear contribution of a constant reference spectrum to the mixture spectrum of an unknown sample. In turn, the calculation allows the contribution of each spectrum (i.e., each dye) to be estimated.

[0057] Ordinary least squares uses a linear factorization algorithm to solve the following equation:

[0058] Y=AC+E

[0059] For simplicity, the random measurement error term "E" can be initially ignored, and the remaining terms can be of interest, where Y is the measured spectrum matrix (i.e., a 1-column matrix containing the spectra of the events of interest), A is the reference spectrum matrix (i.e., an n-column matrix containing the spectra of each reference dye), and C is the concentration matrix (i.e., a 1-row matrix containing the contribution of each dye to the total measured spectrum). Given a series of reference spectra (A) and measured spectra (Y), this method allows estimation of term C, and therefore allows estimation of the contribution of each dye to the total signal intensity of the event of interest.

[0060] As used herein, the term "compensation" refers to correcting the emission signal to accurately estimate the fluorescence signal of a given fluorophore. As used herein, the term "spectral unmixing" refers to separating the emission spectra to accurately estimate the spectral signal of a given fluorophore. Both compensation and spectral unmixing involve removing spillover signals from other fluorophores. Spectral unmixing processes data from more detectors than compensation.

[0061] As introduced above, methods of compensation and spectral unmixing are known in the art.Such methods involve adjusting the signal measured by each photodetector by an amount calculated to compensate for the contribution from dyes other than the primary dye to be 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. NY Acad. 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 FACS DiVa for BD FACSVantage SE Flow Cytometer TM Option White Paper (BD FACS DiVa TM Option for the BD FACSVantage SE Flow Cytometer White Paper), and U.S. Patent No. 6,897,954, each of which is incorporated herein by reference. WinList TM (Verity Software House, Topsham, Me.), Orfeo ToolBox (CNES), FCS Express (De Novo Software, Pasadena, California), and FlowJo 5.7.2 software (Tree Star, Inc., Ashland, Oreg.) are stand-alone software packages that allow software compensation of stored data files generated by flow cytometers.

[0062] Typically, the amount of fluorescence compensation required is determined experimentally using compensation control beads (bound to a single antibody-fluorophore conjugate) or single-color particles dyed with one of the fluorescent dyes used in the assay. The fluorescence signal of each bead is measured in each of the channels, which directly provides a measure of the signal overlap in each of the channels. One method of measuring the signal overlap of a fluorescently labeled antibody reagent (e.g., detection reagent) in each of the detection channels is to use a BD TM CompBeads compensation particles (BD Biosciences, San Jose, Calif.). Particles coated with anti-Ig antibodies are combined with fluorescently labeled antibody reagents captured on the surface of the beads to produce particles labeled with fluorescent dyes. The signal overlap of the dyes is determined by measuring the emission of the labeled particles in each of the detection channels. The measurement is usually performed relative to the emission spectrum from the unlabeled particles. As the number of fluorophores used in the assay increases, this process becomes more challenging, resulting in more signal overlap. Methods and compositions that can improve spectral unmixing and compensation (and thereby improve resolution) in multiparameter flow cytometry are needed.

[0063] In simplified form, a percentage of fluorescence is subtracted from one channel measuring a fluorophore from a second channel measuring fluorescence of a second (or more) fluorophore, so that the contribution of incidental fluorescence is removed. Each fluorophore combination that exhibits spectral overlap must be compensated. To determine the amount of compensation required to correct the fluorescence data, a single color sample is utilized (aliquots of a cell sample or microspheres bound to all antibodies in a staining set are stained with each fluorophore in separate tubes) and analyzed with experimental samples, which are typically then stained with multiple fluorophores.

[0064] Compensation is typically performed by using polystyrene beads that have been modified so that they bind to the detection antibody, typically through the Fc region. In this format, the compensation beads will bind indiscriminately to most, if not all, of the antibodies used in the experiment, allowing the operator to individually measure the fluorescence / spectral profile of a given antibody-fluorophore / fluorochrome conjugate in a separate tube.

[0065] In its current form, the operator must aliquot tubes of individual beads and add individual antibodies from the staining panel to each tube separately in order to deconvolute the individual signals for fluorescence compensation or spectral unmixing. As staining panels increase in complexity, this process can sometimes take hours to complete and is highly susceptible to operator / user error due to the number of pipetting steps required.

[0066] Similarly, fluorescence minus one (FMO) controls are equally important for determining the true signal-to-noise ratio of a given biomarker / channel, especially when there are different expression levels of a given biomarker in a multiplex assay. In these cases, a combination mixture of all antibodies (minus one) used in a given set must be mixed and combined with target cells to determine the noise floor or detection limit of a given assay and staining set. In short, due to the spillover effect, the amount of fluorescence measured in a specific channel may not represent the biological measurement result of the biomarker in this channel. On the contrary, it usually represents the unexpected fluorescence spillover from adjacent channels measuring other biomarkers. In order to determine the true biological signal based on the noise, FMO controls are essential. This is especially important for poorly expressed or "dim" biomarkers with extremely low signal intensity. This process can be very time-consuming, involving mixing the antibody combination of the set into a separate tube, and more often relying on the actual cells being measured. In the case of limited cell material (e.g., primary cells from patients or rare diseases), FMO controls become very difficult to perform accurately.

[0067] Therefore, there is a need in the art for synthetic compositions that allow for more efficient and less error-prone compensation and FMO process control in order to properly set up analytical devices for multiplex cytometry analysis.

[0068] Now refer to the accompanying drawings, Figure 1 and Figure 2As shown, compensation is usually performed by separating individual fluorophores / reagents into different tubes containing compensation beads, which are designed to bind to common regions (such as Fc regions) of antibody reagents used in staining kits, for example. Individual fluorophores must be physically separated in the tubes in order to deconvolve the fluorescent signal from the antibody-fluorophore conjugates used in the staining kit, because each individual compensation bead will bind to a variety of different antibodies used in the staining kit. Therefore, if individual compensation beads are added to a mixture of reagents with different fluorophores, the individual compensation beads can be connected to different fluorophores. For example, individual compensation beads can have anti-CD4-FITC and anti-CD8-Texas Red, both of which are connected to such individual compensation beads. This will prevent effective calculation of compensation or spectral unmixing. Therefore, each bead and detection antibody combination must be physically separated in order to deconvolve the signal associated with a given fluorophore within the staining kit. When using complex kits, this process is very labor-intensive and may require hours of operating time. In addition, in large diagnostic settings such as reference laboratories, this is a key source of operator error. This lengthy and labor-intensive process, while essential for ensuring accurate assay performance, greatly increases the cost of clinical trials and research processes. As an alternative to modified polystyrene beads, users can often use cell material as a substitute for compensation and unmixing settings. Biologically derived calibration products are unstable and have batch-to-batch variability, thereby introducing noise into the measurement results and causing differences in the interpretation of diagnostic data. In addition, both the standard compensation beads and cells used in the assay are typically combined with most, if not all, of the reagents used in the staining set, thereby requiring separate tubes to be used for compensation and unmixing calculations as described above.

[0069] Therefore, the present disclosure provides compositions comprising hydrogel or polymer particles, wherein the particles have been modified to bind to the individual antibodies in the staining set, but not to other antibodies. Alternatively, particles can also be pre-modified with the same fluorophore (or antibody-fluorophore conjugate) used in the staining set to achieve the same effect. Next, a priori, the individual beads of the biomarkers comprising the complete library representing the staining set are combined into a single tube. In one form, the pre-modified fluorophore (or antibody-fluorophore conjugate) beads are directly used for automatic deconvolution and compensation or unmixing processes, wherein the signals of the individual beads can be separated using expected fluorescence or spectral maxima, thereby generating data that can be used for compensation or unmixing calculations. In another form, the user can add its staining set as a whole to a large number of beads designed to be combined with the biomarkers identified by the antibodies in the staining set in a one-pot single-tube reaction individually, so as to generate the same data using the same deconvolution process.

[0070] The present disclosure also provides a method for combining individually modified beads in a manner that allows a user to perform FMO controls using a single mixture of antibodies. Traditionally, for FMO calculations, it is useful to generate a combination mixture of all antibodies (minus one) used in a staining set to determine the noise floor of a given assay and determine the true signal-to-noise ratio. In fact, the antibodies in the staining set will produce some overlapping signals in different unexpected channels. When cells are stained with a mixture lacking a specific antibody-fluorophore for a given channel, any signal in this channel represents a false positive noise floor or a true biological detection lower limit, which can be determined using FMO controls. This process is extremely labor-intensive when performing high-complexity staining assays, because a complete combination matrix of FMO staining antibody mixtures must be prepared by the user, which may require hours to complete a complex set, resulting in user errors and a large amount of operator costs and fatigue. The present disclosure provides a method for performing FMO calculations, wherein a user can add a single complete mixture of a staining set to a tube already containing individually modified beads (minus one bead\epitope type for a given set). By providing tubes containing multiple individually modified beads (minus one), users can greatly simplify the process of setting up FMO controls by enabling them to use one antibody cocktail for the entire process, thereby reducing operator error, saving time, and saving costs.

[0071] 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 set; b) using the local maximum as a cutoff to deconvolute the individually modified beads; and c) using the deconvoluted data from the individual bead populations to perform compensation, unmixing or FMO calculations, thereby calibrating the cytometry device for analysis of a target biological object.

[0072] Hydrogels and polymers

[0073] The particles of the present disclosure may comprise a hydrogel or a hydrophobic polymer. A hydrogel is a material comprising a three-dimensional network of macromolecules that allows it to swell in the presence of water, shrink in the absence of water (or by reducing the amount of water) but not dissolve in water.

[0074] In another aspect, the polymer particles can comprise non-polystyrene based materials, such as PLGA. In other aspects, the polymer particles are generated using polystyrene and latex.

[0075] The hydrogel in the form of beads / particles provided herein is synthesized by polymerizing one or more of the monomers provided herein.Synthesize to form independent hydrogel particles.In one embodiment, the monomer material (monomer) is polymerized to form a homopolymer.However, in another embodiment, a copolymer of different monomer units (i.e., comonomers) is synthesized and used in the method provided herein.In one embodiment, the monomer or comonomer used in the method and composition described herein is a difunctional monomer or includes a difunctional monomer (wherein the comonomer is adopted).In one embodiment, the hydrogel is synthesized in the presence of a crosslinking agent.In another embodiment, the hydrogel is synthesized in the presence of a polymerization initiator.

[0076] The amount of monomers can be varied by the user of the present invention, for example, to obtain specific optical properties that are substantially similar to the optical properties of target cells. In one embodiment, the monomer component (i.e., monomer, comonomer, bifunctional monomer, or combination thereof), for example, various crosslinking ratios of bis / acrylamide, allylamine, or other comonomers or alginic acid that provide chemical functionality for secondary labeling / conjugation is present at about 10% to about 95% by weight of the hydrogel. In a further embodiment, the monomer component is present at about 15% to about 90% by weight of the hydrogel or about 20% to about 90% by weight of the hydrogel.

[0077] Examples of various monomers and crosslinking chemistries that can be used in the present invention are provided in the section entitled "Easy molecular bonding crosslinking technology" in the Thermo Scientific Crosslinking Technical Handbook (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf), the disclosure of which is incorporated by reference in its entirety 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 the hydrogels of the present invention.

[0078] In one embodiment, the monomer used 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-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, derivatives thereof, or combinations thereof.

[0079] 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, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, or a combination thereof.

[0080] In another embodiment, one or more of the following monomers are used herein to form a tunable hydrogel: 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, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzyl Acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenyl methacrylamide, N-(4-methylphenyl) methacrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl) acrylamide, N-triphenyl methacrylamide, N-(4-hydroxyphenyl) acrylamide, N,N-methylphenyl acrylamide, N,N-phenylphenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methylphenyl) methacrylamide

[00136] The invention also includes but is not limited to methyl methacrylamide, ...

[0081] The passive optical properties of the polymer beads can be tuned to mimic the passive optical properties of target cells. Exemplary target cells are included in the non-exhaustive list in Table 1.

[0082] In embodiments, each polymer bead comprises less than 10%, 20%, 30% or 40% polystyrene by hydrated or dehydrated volume.Depending on its composition, the hydrated and dehydrated volumes of the polymer bead may be the same.

[0083] In an embodiment, the hydrogel or polymer particles are functionalized to simulate one or more optical properties of a target cell or a labeled target cell. In an embodiment, the hydrogel or polymer particles include one or more high refractive index molecules. In an embodiment, the hydrogel or polymer particles include multiple high refractive index molecules. In an embodiment, the high refractive index molecules enable the simulation of the SSC of the target cell. In an embodiment, the high refractive index molecules are selected from one or more of the following: colloidal silica, alkyl acrylates, alkyl methacrylates, or a combination thereof. In an embodiment, the high refractive index molecules are alkyl acrylates, alkyl methacrylates, or both. In an embodiment, alkyl acrylates or alkyl methacrylates contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group. In an embodiment, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, 2-ethylhexyl, heptyl or octyl. In an embodiment, the alkyl group is branched. In an embodiment, the alkyl group is linear.

[0084] The three main deconvolution modes of flow cytometry are two passive optical properties of the polymer particles (FSC, corresponding to the refractive index (or RI), and SSC) and biomarkers present on the surface of a given cell type. Therefore, compositions that allow the polymer particles or polymer beads of the present disclosure to mimic a specific cell type with respect to these three modes can be used to provide synthetic, robust calibrants for flow cytometry.

[0085] 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.

[0086] In another embodiment, the RI of the disclosed polymer beads is about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0087] In some embodiments, the RI of the disclosed polymer 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.

[0088] The SSC of the disclosed polymer beads is most meaningfully measured compared to the SSC of the target cells. In some embodiments, the SSC of the disclosed polymer beads is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the SSC of the target cells as measured by a cytometry device.

[0089] In one embodiment, the SSC of the polymer beads is adjusted by incorporating a high refractive index molecule (or a plurality thereof) into the polymer beads. In one embodiment, the high refractive index molecule is provided in the polymer beads, and in a further embodiment, the high refractive index molecule is colloidal silica, an alkyl acrylate, an alkyl methacrylate, or a combination thereof. Thus, in some embodiments, the polymer beads of the present disclosure comprise an alkyl acrylate and / or an alkyl methacrylate. In one embodiment, the concentration of the monomer is adjusted to further adjust the refractive index of the polymer beads.

[0090] The alkyl acrylate or alkyl methacrylate may 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. The alkyl group may be branched or straight chain.

[0091] High refractive index molecules may also include vinyl aromatics, such as styrene and methylstyrene, optionally substituted with alkyl groups, such as methyl, ethyl or tert-butyl, or with halogens, such as chlorostyrene.

[0092] In some embodiments, the FSC is adjusted by adjusting the percentage of monomer present in the composition, thereby changing the water content present during the formation of the polymer beads. In one embodiment, where monomers and comonomers are employed, the ratio of monomers and comonomers is adjusted to change the forward scattering properties of the polymer beads.

[0093] For example, the ratio of monomers and comonomers can be used to adjust the elasticity (i.e., Young's Modulus) of the polymer beads to be substantially similar to the elasticity of the target cells. Based on the elasticity of the target cells, the ratio of monomers and comonomers can be varied. The Young's Modulus of the polymer beads can be in the range of 0.2 kilopascals (kPa) to 400 kPa. The elasticity (e.g., softness or hardness) of the polymer beads can affect the function of the target cells interacting with the polymer beads.

[0094] The FSC of the disclosed polymer beads is the most meaningful measurement compared to the FSC of the target cells. In some embodiments, the FSC of the disclosed polymer beads is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the FSC of the target cells, as measured by a cytometry device.

[0095] FSC is related to particle volume, and thus can be adjusted by changing the particle diameter, as described herein. Generally, it has been observed that large objects refract more light than small objects, resulting in a high forward scattering signal (and vice versa). Therefore, in one embodiment, the particle diameter is changed to adjust the FSC properties of the polymer beads. For example, in one embodiment, the polymer bead diameter is changed by utilizing a larger microfluidic channel during particle formation.

[0096] 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 polymethylmethacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. Without wishing to be bound by theory, the ability to selectively tune both forward scattering and side scattering of polymer beads as described herein allows a robust platform to simulate a large number of cell types.

[0097] Biomarkers

[0098] In embodiments, the hydrogel or polymer particle comprises a cell surface marker, an epitope binding region of a cell surface marker, or a combination thereof.

[0099] The polymer particles of the present disclosure can have any shape, including but not limited to spherical, non-spherical, elongated, cubic, cuboid, cone, and cylinder. In some embodiments, the hydrogel particles of the present disclosure have material modulus properties (e.g., elasticity) that are closer to those of target cells than polystyrene beads of the same diameter. The polymer particles of the present disclosure can also simulate extracellular vesicles, viruses, virus-like particles, spheroids, organoids, or any other biological target of interest.

[0100] The hydrogel or polymer particles may be functionalized, allowing them to mimic the optical properties of labeled biological particles. Functionalization may be mediated by compounds containing free amine groups, such as allylamine, which may 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-γ, CD20, TCRγ / δ, TNFα, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR Vα7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR Vδ2, 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, Igλ light chain, IgA, IL-17A, streptavidin, TCR Vδ1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, FcεR1α, IL-22, Lag-3, MIP-1β, perforin, TCR Vγ9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Igκ light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC class II (IA / IE), MHC II, TCRα / β, TCRβ, TCR Vα24, Akt (pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cd16 / 32, cd41a, CD3ε, 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 780Fix 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-α, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1RAcP, IL-6, IL-10, IL-12, IL-17, Integrin α4β7, Isotype control, 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, cleaved, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCRδ, TCRδ / γ, TCR Vα24Jα18, TCR Vβ11, TCR Vγ1.1, TCR Vγ2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1.

[0101] In one embodiment, the hydrogel particles are functionalized with one or more cell surface markers (see, e.g., Table 1, Table 2, and Table 3) or fragments thereof (e.g., the extracellular portion thereof in the case of transmembrane proteins), e.g., by attaching one or more cell surface markers, extracellular portions, or ligand binding regions thereof to the particles via free amines, free carboxyls, and / or free hydroxyls present on the surface of the hydrogel particles. Functionalization of the hydrogel particles with dyes or cell surface molecules can also occur via linkers, e.g., streptavidin / biotin conjugates.

[0102] Depending on the target cell, individual hydrogel particles can be derivatized with one or more cell surface markers or fragments thereof (e.g., the extracellular portion thereof in the case of a transmembrane protein) to further mimic the structural properties of the target cell. Table 4 and Tables 7-8 provided below list a non-limiting list of cell surface markers that can be used to derive hydrogel particles, depending on the target cell. Although cell surface markers are provided, it should be understood that a portion of a cell surface marker, e.g., a receptor binding portion, a ligand binding portion, or an extracellular portion of a marker, can be used to derive hydrogel particles (at a free functional group, as described above).

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] Hydrogels and other polymer 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 entirety.

[0122] Fluorophore

[0123] The present invention can use any fluorophore known in the art, including Handbook - A Guide to Fluorescent Probes and Labeling Technologies Handbook - A Guide to Fluorescent Probes and Labeling Technologies)" is incorporated herein by reference in its entirety. Labels include surface enhanced Raman scattering (SERS) labels. In an embodiment, the hydrogel or polymer particles can be functionalized with a fluorophore through covalent interactions, non-covalent interactions, or a combination thereof. In an embodiment, the hydrogel or polymer particles can be functionalized with a fluorophore mediated by a biomarker or antibody or through direct conjugation of, for example, an amine-reactive fluorophore. Similar to the above, functionalization can be promoted by free amine groups (such as allylamine), which can be incorporated into the hydrogel particles during the formation process.

[0124] In embodiments, the fluorophore or fluorescent dye is selected from one or more of the following: Peridinium 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt ; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas -XSTP ester or sodium salt; Texas -X succinimidyl ester; Texas -X succinimidyl ester; X-rhodamine-5-(and -6) isothiocyanate, commercially available from Invitrogen dye,

[0125] Including but not limited to FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-Diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7 -Dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester 6-(4,4-Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid, succinimidyl ester; 4,4-Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid, succinimidyl ester; 4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid, succinimidyl ester; 6-(4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid, succinimidyl ester; 3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid Succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s Dicyclopentaphenone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 carboxylic acid; and Alexa 680 carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech, including but not limited to Cy3NHS ester; Cy5NHS ester; Cy5.5 NHS ester; Cy7 NHS ester, and / or any conjugate or derivative thereof.

[0126] In embodiments, the hydrogel or polymer particle may comprise 1 to about 20 fluorescent dyes, 1 to about 10 fluorescent dyes, or 1 to about 5 fluorescent dyes. In embodiments, the hydrogel or polymer particle comprises 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, including all values ​​and subranges therebetween, including endpoints.

[0127] In an embodiment, the hydrogel or polymer particles comprise "rainbow particles". The rainbow particles contain a mixture of fluorophores. In an embodiment, the rainbow particles comprise 1 to about 20 fluorophores, 1 to about 10 fluorophores, or 1 to about 5 fluorophores. In an embodiment, the hydrogel or polymer particles comprise rainbow particles having 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 all values ​​and subranges therebetween, including the endpoints. In an embodiment, the user selects the wavelength used to excite the rainbow particles, depending on the fluorophores being interrogated. Rainbow particles are commercially available, for example, from BD Biosciences (Catalog Nos. 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 in various diameters (e.g., Catalog Nos. RCP20-5 (4 colors), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)).

[0128] A non-exhaustive list of fluorophores suitable for use with the present invention is provided in Table 4 below.

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] In an embodiment, the hydrogel or polymer particles comprise a scattering modulating additive. In an embodiment, the scattering modulating additive comprises polymer nanoparticles. In an embodiment, the polymer nanoparticles comprise polystyrene. In an embodiment, the scattering modulating additive comprises a comonomer. In an embodiment, the scattering modulating additive comprises a suspension of nanoparticles.

[0146] In an embodiment, the hydrogel or polymer particle is a chemically functionalized hydrogel or polymer particle. In an embodiment, the hydrogel comprises free amine groups. In an embodiment, the hydrogel beads comprise allylamine. In an embodiment, the hydrogel or polymer particle comprises biotin. In an embodiment, the hydrogel or polymer particle comprises streptavidin. In an embodiment, the hydrogel or polymer particle comprises avidin. In an embodiment, the chemically functionalized hydrogel or polymer particle comprises amine groups, carboxyl groups, hydroxyl groups, or a combination thereof. In an embodiment, the hydrogel or polymer particle comprises a plurality of bifunctional monomers to allow the hydrogel or polymer particle to be functionalized with different chemicals and / or molecules.

[0147] Composition

[0148] The compositions of the present disclosure may comprise a population of polymer beads. In an embodiment, the polymer bead population may comprise a fluorophore, a biomarker, etc. In an embodiment, the polymer bead population may comprise at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 polymer bead populations. In an embodiment, each bead population comprises a fluorophore, a biomarker, etc.

[0149] In embodiments, each bead population may comprise a single fluorophore. In embodiments, each bead population may comprise different fluorophores. 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.

[0150] For example, in one embodiment, the first polymer bead population comprises a first fluorophore and the second polymer bead population comprises a second fluorophore. The first fluorophore and the second fluorophore can each be selected from the fluorophores outlined above and can be the same or different. In addition, the final polymer bead population does not comprise any fluorophore.

[0151] In an embodiment, each bead population may contain a single biomarker. In an embodiment, each bead population may contain different biomarkers. For example, in one embodiment, the first polymer bead population contains a first biomarker, and the second polymer bead population contains a second biomarker. The first biomarker and the second biomarker may each be selected from the biomarkers outlined above, and may be the same or different. In addition, the final polymer bead population does not contain any biomarker.

[0152] Example

[0153] Example 1 - Deconvolution using separate tubes

[0154] Representing the TBNK staining set, six sets of antibody-fluorophore conjugates were prepared and directly linked to polymer particles. The following fluorophores were used: PerCP-Cy5.5, PE Cy7, APC-Cy7, FITC, PE, APC.

[0155] Six separate sample tubes were prepared with 100 uL of phosphate buffered saline (PBS). Approximately 25,000 beads from each group 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 acquired on a Cytek Northern Lights using normal acquisition parameters. TM Analyze on a flow cytometer. Then, measure the unstained / negative control in the same manner. Fig.11 Individual fluorophore signals for each sample tube are shown.

[0156] Example 2 - One-pot analysis and compensation

[0157] The six polymer bead pools from Example 1 were combined into a single tube and acquired on a Cytek Northern Lights using conventional acquisition parameters. TM , and the combined spectrum ( Fig.12 ). In this example, the individual bead-antibody-fluorophore particles contain only one type of fluorophore uniquely per bead.

[0158] The individual fluorophore signals from each polymer-bound antibody-fluorophore were deconvoluted using the known fluorescence signal of each antibody-fluorophore conjugate. 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 this specific antibody-fluorophore conjugate ( Fig.13 ). This allows the complete fluorescence signal of the individual fluorophores to be deconvoluted from the mixed reaction. The results of this deconvolution are shown in Fig.14middle. Fig.14 The individual spectra seen in the image are similar to the spectra generated from the individual tubes ( Fig.11 ) are indistinguishable and can be used for downstream compensation or spectral unmixing calculations with performance comparable to that of separate physically isolated tubes ( Fig.15 ). The resulting unmixed TBNK staining set using the one-pot polymer particle method described in this disclosure was indistinguishable from the staining set using the separate tubes described in Example 1. Fig.15 .

[0159] Example 3 - One-pot compensation using biomarker modified beads

[0160] Six polymer beads, each containing a single biomarker (CD3, CD16, CD56, CD45, CD4, CD19, CD8) from the example TBNK set, were added to a single tube containing 100uL of PBS. Next, a complete set of antibody-fluorophore conjugates from the TBNK set was added to the same single reaction tube and vortexed to resuspend the mixture. After the mixture was incubated under light protection, it was analyzed on a calibrated flow cytometer using conventional acquisition parameters to obtain a combined spectrum. In this example, individual biomarker-modified beads were combined only with specific antibodies-fluorophores from the staining set mixture to produce a uniformly labeled bead set.

[0161] The individual fluorophore signals from each polymer-bound antibody-fluorophore are deconvoluted using the known fluorescence signal of each antibody-fluorophore conjugate. 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 this specific antibody-fluorophore conjugate. This allows the complete fluorescence signal of the individual fluorophores to be deconvoluted from the mixed reaction for compensation or spectral unmixing calculations.

[0162] Example 4 - One-pot compensation with pre-modified beads

[0163] Compared to the antibody-fluorophore conjugates, the six bead sets from Example 1 can be pre-modified with appropriate fluorophores to achieve the same effect.

[0164] Example 5 - Simplifying FMO compensation using single biomarker beads

[0165] All possible combinations of the six sets of beads from Example 3 (minus one biomarker-fluorophore channel) were added to separate tubes to represent the FMO staining control set. For clarity, the example set includes the following: (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, CD4, CD8), and (CD3, CD16, CD56, CD45, CD4, CD19). In this product format, a single master mix of the TBNK staining set can be added to all tubes to generate the FMO control matrix. Specifically, adding a single mixture 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 the present disclosure will allow the user to generate a complete FMO set. In each of these cases, the polymer particles in the tube will be combined with all of the antibody-fluorophore conjugates except one of the antibody-fluorophore conjugates in the complete TBNK set, thereby simulating the traditional FMO method in a greatly simplified product form. In contrast, using traditional methods will require the user to generate a combination mixture of antibodies in all combinations in order to achieve the same result, because all antibodies will be combined with cells or traditional compensation beads for FMO calculations.

[0166] Example 6 - Simplifying FMO compensation using single biomarker beads

[0167] The bead collection in Example 5 can be pre-modified with an appropriate fluorophore or antibody-fluorophore conjugate combination to achieve the same effect.

[0168] Additional numbered embodiments

[0169] Further embodiments of the invention are provided in the following numbered embodiments:

[0170] Embodiment 1. A composition comprising (i) a first population of polymer beads comprising a first fluorophore, and (ii) a second population of polymer beads comprising a second fluorophore.

[0171] Embodiment 2. A composition according to embodiment 1, comprising at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each bead population comprises a fluorophore, and wherein the fluorophore of each bead population is different.

[0172] Embodiment 3. The composition of embodiment 1, wherein the first fluorophore and the second fluorophore are different fluorophores.

[0173] Embodiment 3.1. The composition of any of Embodiments 1 to 3, wherein each population of polymer beads comprises only a single type of fluorophore.

[0174] Embodiment 4. The composition of any one of embodiments 1 to 3.1, further comprising a final polymer bead population that does not comprise any fluorophore.

[0175] Embodiment 4.1. The composition of any one of Embodiments 1 to 4, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

[0176] Embodiment 5. The composition of any one of embodiments 1 to 4, wherein each fluorophore is independently selected from any one of the following: Peridinium chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5); Phycoerythrin-cyanine 7 (PECy7); 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-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine 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; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas -X STP ester or sodium salt; Texas -X succinimidyl ester; Texas -X succinimidyl ester; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-Diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7 -Dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl 4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone- 3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid Succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0177] Embodiment 6. The composition of any one of embodiments 1 to 5, wherein each fluorophore emits fluorescence at one, two, three, four, five, six, seven, eight, or nine wavelengths.

[0178] Embodiment 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.

[0179] Embodiment 8. The composition of any one of embodiments 1 to 7, wherein the polymer beads comprise less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0180] Embodiment 8.1. The composition of any of Embodiments 1 to 7, wherein the polymer beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dehydrated volume.

[0181] Embodiment 9. The composition of any one of embodiments 1 to 8.1, wherein the polymer beads are hydrogel beads.

[0182] Embodiment 10. The composition of embodiment 9, wherein the hydrogel comprises a monomer.

[0183] Embodiment 11. The composition of embodiment 10, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methacrylic acid Methoxy poly(ethylene glycol) ester, methacrylic acid, sodium methacrylate, glyceryl 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, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide , N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine or a combination thereof.

[0184] Embodiment 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.

[0185] Embodiment 12.1. The composition of any of Embodiments 1 to 11, at least one population of polymer beads exhibiting at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

[0186] Embodiment 13. The composition of Embodiment 12 or 12.1, wherein the at least one optical property is side scattering.

[0187] Embodiment 14. The composition of Embodiment 12 or 12.1, wherein the at least one optical property is forward scattering.

[0188] Embodiment 15. The composition of Embodiment 12 or 12.1, wherein the at least one optical property comprises side scatter and forward scatter.

[0189] Embodiment 16. The composition of any one of embodiments 12 to 15, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

[0190] Embodiment 17. A composition according to any one of embodiments 1 to 16, further comprising one or more of the following: (iii) a third polymer bead population, wherein the third polymer bead population comprises a third fluorophore, (iv) a fourth polymer bead population, wherein the fourth polymer bead population comprises a fourth fluorophore, (v) a fifth polymer bead population, wherein the fifth polymer bead population comprises a fifth fluorophore, and / or (vi) a sixth polymer bead population, wherein the sixth polymer bead population comprises a sixth fluorophore.

[0191] Embodiment 17.1. A composition according to Embodiment 17, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

[0192] Embodiment 18. The composition of embodiment 17, wherein the first fluorophore, the second fluorophore, the third fluorophore, the fourth fluorophore, the fifth fluorophore and the sixth fluorophore are independently selected from the group consisting of: Peridinium chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5); Phycoerythrin-cyanine 7 (PE-Cy5.5); 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TMCarboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas -X STP ester or sodium salt; Texas -X succinimidyl ester; Texas -X succinimidyl ester; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-Diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7 -Dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl 4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone- 3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid Succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0193] Embodiment 18.1. 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.

[0194] Embodiment 18.2. The composition of Embodiment 18.1, wherein the epitope is a biomarker contained in the polymer bead.

[0195] Embodiment 18.3. A composition according to Embodiment 18.1 or 18.2, wherein the fluorophore is a commercially available antibody-label conjugate.

[0196] Embodiment 18.4. A composition according to Embodiment 18.2 or 18.3, wherein the biomarker is selected from the biomarkers listed in Tables 1-3 of the present specification.

[0197] Embodiment 19. A method for calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (i) using the cytometry device to measure the fluorescence signal of a composition according to any one of Embodiments 1 to 18.3, (ii) deconvolving the fluorescence signal from each polymer bead population of the composition to calculate a compensation or spectral unmixing matrix, and (iii) using the compensation or spectral unmixing matrix to calibrate the cytometry device.

[0198] Example 19.1. A method for calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (A) using the cytometry device to measure a fluorescence signal of a composition, the composition comprising (i) a first polymer bead population, the first polymer bead population comprising a first fluorophore, and (ii) a second polymer bead population, the second polymer bead population comprising a second fluorophore, (B) deconvolving the fluorescence signal from each polymer bead population of the composition to calculate a compensation or spectral unmixing matrix, and (C) using the compensation or spectral unmixing matrix to calibrate the cytometry device.

[0199] 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.

[0200] Embodiment 19.3. The method of Embodiment 19 or 19.1, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the optical properties of each polymer bead population.

[0201] Embodiment 19.4. A method according to any one of Embodiments 19 to 19.3, wherein the measured composition comprises at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 polymer bead populations, wherein each bead population comprises a fluorophore, and wherein the fluorophore of each bead population is different.

[0202] 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.

[0203] Embodiment 19.6. The method of any one of Embodiments 19 to 19.4, wherein each population of polymer beads comprises only a single fluorophore.

[0204] Embodiment 19.7. The method of any one of Embodiments 19 to 19.6, wherein the measured composition comprises a final polymer bead population that does not comprise any fluorophores.

[0205] Embodiment 19.7.1. The method of any of Embodiments 19.19.6, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

[0206] Embodiment 19.8. A method according to any one of embodiments 19 to 19.7, wherein each fluorophore is independently selected from any one of the following: Peridinium 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreenTM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas -X STP ester or sodium salt; Texas -X succinimidyl ester; Texas -X succinimidyl ester; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-Diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7 -Dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl 4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone- 3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid Succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3NHS ester; Cy 5NHS ester; Cy5.5NHS ester; and Cy7 NHS ester.

[0207] Embodiment 19.9. The method of any one of Embodiments 19 to 19.8, wherein each fluorophore emits fluorescence at one, two, three, four, five, six, seven, eight, or nine wavelengths.

[0208] 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.

[0209] Embodiment 19.11. The method of any of Embodiments 19 to 19.10, wherein the polymer beads comprise less than 10%, 20%, 30%, 40% polystyrene by hydrated volume.

[0210] Embodiment 19.11.1. The method of any of Embodiments 19 to 19.10 wherein the polymer beads comprise less than 10%, 20%, 30%, 40% polystyrene by dehydrated volume.

[0211] Embodiment 19.12. The method of any one of Embodiments 19 to 19.11.1, wherein the polymer beads are hydrogel beads.

[0212] Embodiment 19.13. The method of Embodiment 19.12, wherein the hydrogel comprises monomers.

[0213] Embodiment 19.14. A method according to Embodiment 19.13, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methyl Methoxypoly(ethylene glycol) acrylate, methacrylic acid, sodium methacrylate, glyceryl 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 Ester, 2-naphthyl acrylate, 2-naphthyl methacrylate, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide amine, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, 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.

[0214] Embodiment 19.15. The method of any one of Embodiments 19 to 19.7, wherein the polymer beads exhibit at least one optical property substantially similar to an optical property of a target cell.

[0215] Embodiment 19.16. The method of any of Embodiments 19 to 19.15, wherein at least one population of polymer beads exhibits at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

[0216] Embodiment 19.17. A method according to Embodiment 19.15 or 19.16, wherein the at least one optical property is side scatter.

[0217] Embodiment 19.18. A method according to Embodiment 19.15 or 19.16, wherein the at least one optical property is forward scattering.

[0218] Embodiment 19.19. The method of Embodiment 19.15 or 19.16, wherein the at least one optical property comprises side scatter and forward scatter.

[0219] Embodiment 19.20. The method of any one of Embodiments 19.15 and 19.17 to 19.19, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

[0220] Embodiment 19.21. A method according to any one of Embodiments 19.1 to 19.20, wherein the measured composition comprises one or more of the following: (iii) a third polymer bead population, wherein the third polymer bead population comprises a third fluorophore, (iv) a fourth polymer bead population, wherein the fourth polymer bead population comprises a fourth fluorophore, (v) a fifth polymer bead population, wherein the fifth polymer bead population comprises a fifth fluorophore, and / or (vi) a sixth polymer bead population, wherein the sixth polymer bead population comprises a sixth fluorophore.

[0221] Embodiment 19.22. A method according to Embodiment 19.2, wherein the first fluorophore, the second fluorophore, the third fluorophore, the fourth fluorophore, the fifth fluorophore and the sixth fluorophore are independently selected from the group consisting of: PerCP-Cy5.5, PE Cy7, APC-Cy7, FITC, PE and APC.

[0222] Example 20. A method for calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (A) providing a composition comprising (i) a first polymer bead population, the first polymer bead population comprising a first fluorophore, and (ii) a second polymer bead population, the second polymer bead population comprising a second fluorophore, (B) measuring a fluorescence signal of the composition using the cytometry device, (C) deconvolving the fluorescence signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix, and (D) using the compensation or spectral unmixing matrix to calibrate the cytometry device.

[0223] Embodiment 20.1. A method according to any one of Embodiments 19 to 20, wherein each population of polymer beads contains a sufficiently high fluorophore content to generate a fluorescent signal at least as strong as the fluorescent signal of the cell population to be analyzed by the cytometry device.

[0224] 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.

[0225] Embodiment 22. A composition according to embodiment 21, comprising at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each bead population comprises a biomarker, and wherein the biomarker of each bead population is different.

[0226] Embodiment 23. The composition of embodiment 21 or 22, wherein each population of polymer beads comprises a different biomarker.

[0227] Embodiment 23.1. The composition of any one of Embodiments 21 to 23, wherein each population of polymer beads comprises only a single biomarker.

[0228] Embodiment 24. The composition of any one of Embodiments 21 to 23.1, comprising a population of beads that do not comprise a fluorophore.

[0229] Embodiment 25. The composition of any one of embodiments 21 to 24, comprising a population of beads that do not comprise a biomarker.

[0230] Embodiment 26. The composition of any one of embodiments 21 to 25, wherein the polymer beads comprise less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0231] Embodiment 26.1. The composition of any of Embodiments 21 to 25, wherein the polymer beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dehydrated volume.

[0232] Embodiment 27. The composition of any one of embodiments 21 to 26, wherein the polymer beads are hydrogel beads.

[0233] Embodiment 27.1. The composition of any one of Embodiments 21 to 27, wherein the biomarker is a polypeptide.

[0234] Embodiment 27.2. The composition of any one of Embodiments 21 to 27.1, wherein the biomarker is an epitope of a fluorescent dye.

[0235] Embodiment 27.3. The composition of any of Embodiments 21 to 27.2, wherein each of the first population of polymer beads and the second population of polymer beads each comprises a different fluorophore.

[0236] Embodiment 27.4. The composition of any one of Embodiments 21 to 27.3, wherein the biomarker is an epitope of an antibody.

[0237] Embodiment 27.5. A composition according to Embodiment 27.4, wherein the antibody is configured to be conjugated to a fluorescent dye or is configured to be conjugated to a secondary antibody-fluorophore conjugate.

[0238] 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.

[0239] Embodiment 27.7. The composition of Embodiment 27.6, wherein the epitope is the biomarker contained in the polymer bead.

[0240] Embodiment 27.8. A composition according to Embodiment 27.6 or 27.7, wherein the fluorophore is a commercially available antibody-label conjugate.

[0241] Embodiment 27.9. A composition according to any one of Embodiments 27.6 to 27.8, wherein the biomarker is selected from the biomarkers listed in Tables 1-3 of the present specification.

[0242] Embodiment 28. A composition according to any one of embodiments 21 to 27.2, wherein each biomarker is independently selected from any one of the following: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-γ, CD20, TCRγ / δ, TNFα, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR Vα7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR Vδ2, 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, Igλ light chain, IgA, IL-17A, streptavidin, TCR Vδ1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, FcεR1α, IL-22, Lag-3, MIP-1β, perforin, TCR Vγ9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Igκ light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC class II (IA / IE), MHC II, TCRα / β, TCRβ, TCR Vα24, Akt(pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cd16 / 32, cd41a, CD3ε, 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, eFluor780Fix 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-α, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1RAcP, IL-6, IL-10, IL-12, IL-17, Integrin α4β7, Isotype control, 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, cleaved, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCRδ, TCRδ / γ, TCR Vα24Jα18, TCR Vβ11, TCRVγ1.1, TCR Vγ2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1. .

[0243] Embodiment 29. The composition of any one of embodiments 27 to 28, wherein the hydrogel comprises polyacrylamide.

[0244] Embodiment 30. A composition according to any one of Embodiments 21 to 29, comprising (iii) a third bead population, wherein the third bead population comprises a third biomarker, (iv) a fourth bead population, wherein the fourth bead population comprises a fourth biomarker, (v) a fifth bead population, wherein the fifth bead population comprises a fifth biomarker, and (vi) a sixth bead population, wherein the sixth bead population comprises a sixth biomarker.

[0245] Embodiment 31. A composition according to embodiment 30, wherein the first biomarker, the second biomarker, the third biomarker, the fourth biomarker, the fifth biomarker and the sixth biomarker are independently selected from the group consisting of: CD3, CD16, CD56, CD45, CD4, CD19 and CD8.

[0246] Embodiment 32. The composition of embodiment 30 or 31, wherein each population of polymer beads comprises a different fluorophore.

[0247] Embodiment 33. The composition of embodiment 27, wherein the hydrogel comprises a matrix comprising monomers.

[0248] Embodiment 34. A composition according to embodiment 33, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methacrylic acid Methoxy poly(ethylene glycol) ester, methacrylic acid, sodium methacrylate, glyceryl 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, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide , N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine or a combination thereof.

[0249] Embodiment 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.

[0250] Embodiment 35.1. The composition of any of Embodiments 21 to 34, at least one population of polymer beads exhibiting at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

[0251] Embodiment 36. The composition of Embodiment 35 or 35.1, wherein the at least one optical property is side scatter.

[0252] Embodiment 37. The composition of Embodiment 35 or 35.1, wherein the at least one optical property is forward scattering.

[0253] Embodiment 38. The composition of Embodiment 35 or 35.1, wherein the at least one optical property comprises side scatter and forward scatter.

[0254] Embodiment 39. The composition of embodiment 35, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

[0255] Embodiment 40. A method for calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (i) using the cytometry device to measure the fluorescence signal of a composition according to any one of embodiments 21 to 39, (ii) deconvolving the fluorescence signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix, and (iii) using the compensation or spectral unmixing matrix to calibrate the cytometry device.

[0256] Example 40.1. A method for calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (A) using the cytometry device to measure a fluorescence signal of a composition, the composition comprising (i) a first polymer bead population, the first polymer bead population comprising a first biomarker, and (ii) a second polymer bead population, the second polymer bead population comprising a second biomarker, (B) deconvolving the fluorescence signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix, and (C) using the compensation or spectral unmixing matrix to calibrate the cytometry device.

[0257] Embodiment 40.2. The method of Embodiment 40.1, wherein each population of polymer beads comprises a different fluorophore.

[0258] Example 41. A method for producing multicolor compensation control for a cytometry device, the method comprising the following steps: (A) contacting a composition with a plurality of fluorescent dyes in a single reaction, the composition comprising (i) a first polymer bead population, the first polymer bead population comprising a first biomarker, and (ii) a second polymer bead population, the second polymer bead population comprising a second biomarker, wherein each of the plurality of fluorescent dyes comprises a fluorophore having an excitation or emission spectrum that is different from the excitation or emission spectrum of the fluorophore in each other of the plurality of fluorescent dyes, and wherein each dye binds to the biomarker of only a single polymer bead population in the composition, such that each polymer bead population is bound to no more than one fluorescent dye, thereby producing multicolor compensation control.

[0259] Embodiment 41.1. The method of any one of Embodiments 40.1 to 41, wherein each biomarker of the population of polymer beads comprises an antigen configured to selectively bind to a fluorescent dye.

[0260] Embodiment 41.2. A method according to any one of Embodiments 41 to 41.1, wherein the plurality of fluorescent dyes are secondary antibody-fluorophore conjugates, and wherein each biomarker of the polymer bead population comprises an antigen that is configured to selectively bind to the secondary antibody-fluorophore conjugate.

[0261] Embodiment 42. The method of any one of Embodiments 41 to 41.2, wherein each of the plurality of fluorescent dyes comprises an antibody-fluorophore conjugate.

[0262] Embodiment 42.1. The method of any one of Embodiments 41 to 42, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

[0263] Embodiment 43. A method according to any one of embodiments 40.1 to 42.1, wherein each fluorophore is independently selected from any one of the following: Peridinium 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TMCarboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas -X STP ester or sodium salt; Texas -X succinimidyl ester; Texas -X succinimidyl ester; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-Diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7 -Dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl 4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone- 3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid Succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0264] Embodiment 44. A method according to any one of Embodiments 40.1 to 43, wherein the composition comprises at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each population of beads comprises a biomarker, and wherein the biomarker of each population of beads is different.

[0265] Embodiment 45. The method of any one of Embodiments 40.1 to 44, wherein each population of polymer beads comprises a different biomarker.

[0266] Embodiment 46. The method of any one of Embodiments 40.1 to 45, wherein each population of polymer beads comprises only a single biomarker.

[0267] Embodiment 47. The method of any one of Embodiments 40.1 to 46, comprising a population of beads that do not comprise a fluorophore or lack an epitope for association with an antibody-fluorophore conjugate.

[0268] Embodiment 48. A method according to any one of Embodiments 40.1 to 47, comprising a population of beads that do not contain a biomarker.

[0269] Embodiment 49. The method of any one of Embodiments 40.1 to 48, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by hydrated volume.

[0270] Embodiment 50. The method of any one of Embodiments 40.1 to 48, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by dehydrated volume.

[0271] Embodiment 51. The method of any one of Embodiments 40.1 to 50, wherein the polymer beads are hydrogel beads.

[0272] Embodiment 52. The method of any one of embodiments 40.1 to 51, wherein the biomarker is a polypeptide.

[0273] Embodiment 53. A method according to any one of embodiments 40.1 to 52, wherein the biomarker is an epitope of a fluorescent dye.

[0274] Embodiment 54. The method of any one of Embodiments 40.1 to 53, wherein each of the first population of polymer beads and the second population of polymer beads each comprises a different fluorophore.

[0275] Embodiment 55. A method according to any one of embodiments 40.1 to 54, wherein the biomarker is an epitope of an antibody.

[0276] Embodiment 56. The method of any one of Embodiments 40.1 to 55, wherein the antibody is configured to be conjugated to a fluorescent dye or is configured to be conjugated to a secondary antibody-fluorophore conjugate.

[0277] Embodiment 57. The method of 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.

[0278] Embodiment 58. A method according to Embodiment 57, wherein the epitope is the biomarker contained in the polymer bead.

[0279] Embodiment 59. The method of any one of embodiments 40.1 to 58, wherein the fluorophore is a commercially available antibody-label conjugate.

[0280] Embodiment 60. A method according to any one of embodiments 40.1 to 59, wherein the biomarker is selected from the biomarkers listed in Tables 1-3 of the present specification.

[0281] Embodiment 61. A method according to any one of embodiments 40.1 to 59, wherein each biomarker is independently selected from any one of the following: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-γ, CD20, TCRγ / δ, TNFα, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR Vα7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR Vδ2, 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, Igλ light chain, IgA, IL-17A, streptavidin, TCR Vδ1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, FcεR1α, IL-22, Lag-3, MIP-1β, perforin, TCR Vγ9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Igκ light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC class II (IA / IE), MHC II, TCRα / β, TCRβ, TCR Vα24, Akt(pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cd16 / 32, cd41a, CD3ε, 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, eFluor780Fix 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-α, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1RAcP, IL-6, IL-10, IL-12, IL-17, Integrin α4β7, Isotype control, 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, cleaved, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCRδ, TCRδ / γ, TCR Vα24Jα18, TCR Vβ11, TCRVγ1.1, TCR Vγ2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1. .

[0282] Embodiment 62. The method of any one of embodiments 51 to 61, wherein the hydrogel comprises polyacrylamide.

[0283] Embodiment 63. A method according to any one of Embodiments 40.1 to 62, comprising (iii) a third bead population, wherein the third bead population comprises a third biomarker, (iv) a fourth bead population, wherein the fourth bead population comprises a fourth biomarker, (v) a fifth bead population, wherein the fifth bead population comprises a fifth biomarker, and (vi) a sixth bead population, wherein the sixth bead population comprises a sixth biomarker.

[0284] Embodiment 64. A method according to embodiment 63, wherein the first biomarker, the second biomarker, the third biomarker, the fourth biomarker, the fifth biomarker and the sixth biomarker are independently selected from the group consisting of: CD3, CD16, CD56, CD45, CD4, CD19 and CD8.

[0285] Embodiment 65. The method of any one of Embodiments 40.1 to 64, wherein each population of polymer beads comprises a different fluorophore.

[0286] Embodiment 66. The method of any one of embodiments 51 to 65, wherein the hydrogel comprises a matrix comprising monomers.

[0287] Embodiment 67. The method of embodiment 66, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methyl methacrylate Oxyethylene glycol ester, methacrylic acid, sodium methacrylate, glyceryl 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, propylene glycol 2-Naphthyl acrylate, 2-naphthyl methacrylate, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide , N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine or a combination thereof.

[0288] Embodiment 68. The method of any one of Embodiments 40.1 to 67, wherein the polymer beads exhibit at least one optical property substantially similar to an optical property of a target cell.

[0289] Embodiment 69. The method of any one of Embodiments 40.1 to 68, wherein at least one population of polymer beads exhibits at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

[0290] Embodiment 70. A method according to Embodiment 68 or 69, wherein the at least one optical property is side scattering.

[0291] Embodiment 71. A method according to any one of Embodiments 68 to 69, wherein at least one of the optical properties is forward scattering.

[0292] Embodiment 72. A method according to any one of Embodiments 68 to 69, wherein the at least one optical property comprises side scatter and forward scatter.

[0293] Embodiment 73. The method of any one of Embodiments 40.1 to 72, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

[0294] Embodiment 74. A method for calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (i) providing a composition comprising (a) a first polymer bead population, the first polymer bead population comprising a first polymer bead population of a first biomarker, and (b) a second polymer bead population, the second polymer bead population comprising a second polymer bead population of a second biomarker, each of the first polymer bead population and the second polymer bead population comprising a different fluorophore, (ii) contacting the composition with at least two antibody populations or fragments thereof, each antibody population being capable of binding to only one of the biomarkers in the composition, wherein the antibodies or fragments thereof are conjugated to fluorophores capable of generating a fluorescent signal, (iii) measuring the fluorescent signal of the composition using the cytometry device, (iv) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix, and (v) using the compensation or spectral unmixing matrix to calibrate the cytometry device.

[0295] Embodiment 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.

[0296] Embodiment 76. The method of any one of embodiments 40 to 74, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the optical properties of each polymer bead population.

[0297] ********

[0298] All documents, patents, patent applications, publications, product descriptions, and schemes cited throughout this application are incorporated herein 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; US 2021 / 0341469; US2021 / 0231552; US2020 / 0400546; PCT / US2023 / 06668; and PCT / US2023 / 067893.

[0299] The embodiments shown and discussed in this specification are intended only to teach those skilled in the art to make and use the invention disclosed herein in the best manner known to the inventors. Modifications and variations of the above-described embodiments of the present disclosure are possible without departing from the spirit of the present invention, as understood by those skilled in the art based on the above teachings. Therefore, it should be understood that within the scope of the claims and their equivalents, the invention disclosed herein may be practiced in a manner different from that specifically described.

Claims

1. A composition comprising: (i) a first population of polymer beads, the first population of polymer beads comprising a first fluorophore; and (ii) a second population of polymer beads, the second population of polymer beads comprising a second fluorophore.

2. The composition of claim 1, comprising at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each population of beads comprises a fluorophore, and wherein the fluorophore of each population of beads is different.

3. The composition of claim 1, wherein the first fluorophore and the second fluorophore are different fluorophores.

4. The composition of any one of claims 1 to 3, wherein each population of polymer beads comprises only a single type of fluorophore.

5. The composition of any one of claims 1 to 4, further comprising a final polymer bead population, the final polymer bead population not comprising any fluorophore.

6. The composition of any one of claims 1 to 5, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

7. The composition of any one of claims 1 to 6, wherein each fluorophore is independently selected from any one of the following: Peridinium 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 Amber imide ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; Eosin-5-isothiocyanate; Erythrosine 5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; Fluorescein-S-EX succinimidyl ester; Fluorescein-5-isothiocyanate; Fluorescein-6-isothiocyanate; 488 Carboxylic acid or succinimide ester; Oregon 488 isothiocyanate; 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas STP ester or sodium salt; Texas Succinimide ester; Texas Succinimidyl esters; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-XSTP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4 -borax-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-borax-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-borax-3a,4a-diaza-s-dicyclopentaphenazone-3-propanoic acid; 4,4- Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-dicyclopenta-benzo-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -dicyclopentaphenazone-3-propionyl)sulfoalanine, 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-s-dicyclopentaphenazone-3-propionic acid; 4,4-difluoro-5,7 -Diphenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

8. The composition of any one of claims 1 to 7, wherein each fluorophore emits fluorescence at one, two, three, four, five, six, seven, eight or nine wavelengths.

9. The composition of any one of claims 1 to 8, wherein each fluorophore has a diameter between about 500 nm and about 10 μm.

10. The composition of any one of claims 1 to 9, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by hydrated volume.

11. The composition of any one of claims 1 to 9, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by dehydrated volume.

12. The composition of any one of claims 1 to 11, wherein the polymer beads are hydrogel beads.

13. The composition of claim 12, wherein the hydrogel comprises monomers.

14. The composition of claim 13, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxymethacrylate, Poly(ethylene glycol) ester, methacrylic acid, sodium methacrylate, glyceryl 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, propylene glycol 2-Naphthyl acrylate, 2-naphthyl methacrylate, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide , N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine or a combination thereof.

15. The composition of any one of claims 1 to 14, wherein the polymer beads exhibit at least one optical property that is substantially similar to an optical property of a target cell.

16. The composition of any one of claims 1 to 14, at least one population of polymer beads exhibiting at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

17. The composition of claim 15 or 16, wherein the at least one optical property is side scattering.

18. The composition of claim 15 or 16, wherein the at least one optical property is forward scattering.

19. The composition of claim 15 or 16, wherein the at least one optical property comprises side scatter and forward scatter.

20. The composition of any one of claims 15 to 19, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

21. The composition according to any one of claims 1 to 20, further comprising one or more of the following: (iii) a third population of polymer beads, the third population of polymer beads comprising a third fluorophore; (iv) a fourth population of polymer beads, the fourth population of polymer beads comprising a fourth fluorophore; (v) a fifth population of polymer beads, the fifth population of polymer beads comprising a fifth fluorophore; and / or (vi) a sixth population of polymer beads, the sixth population of polymer beads comprising a sixth fluorophore.

22. The composition of claim 21, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

23. The composition of claim 21, wherein the first fluorophore, the second fluorophore, the third fluorophore, the fourth fluorophore, the fifth fluorophore, and the sixth fluorophore are independently selected from the group consisting of: Peridinium chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5); Phycoerythrin-cyanine 7 (PE-Cy5.5); 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas STP ester or sodium salt; Texas Succinimide ester; Texas Succinimidyl esters; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-Diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7 -Dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionyl)sulfoalanine, succinimide 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-s-dicyclopenta-3-propionyl 4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone- 3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid Succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3NHS ester; Cy 5NHS ester; Cy5.5NHS ester; and Cy7 NHS ester.

24. The composition of any one of claims 1 to 23, wherein the fluorophore is conjugated to an antibody or fragment thereof that binds to an epitope within the polymer bead.

25. The composition of claim 24, wherein the epitope is a biomarker contained in the polymer bead.

26. The composition of claim 24 or 25, wherein the fluorophore is a commercially available antibody-label conjugate.

27. The composition according to claim 25 or 26, wherein the biomarker is selected from the biomarkers listed in Tables 1-3 of the present specification.

28. A method of calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (i) measuring the fluorescent signal of the composition according to any one of claims 1 to 26 using the cytometry device; (ii) deconvolving the fluorescent signal from each population of polymer beads of the composition to calculate a compensation or spectral unmixing matrix; and (iii) calibrating the cytometry device using the compensation or spectral unmixing matrix.

29. A method of calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (A) using the cytometry device to measure a fluorescent signal of a composition, the composition comprising (i) a first population of polymer beads, the first population of polymer beads comprising a first fluorophore, and (ii) a second population of polymer beads, the second population of polymer beads comprising a second fluorophore; (B) deconvolving the fluorescent signal from each population of polymer beads of the composition to calculate a compensation or spectral unmixing matrix; and (C) Calibration of the cytometry device using the compensation or spectral unmixing matrix.

30. The method of claim 28 or 29, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the fluorescence emission maximum.

31. The method of claim 28 or 29, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the optical properties of each polymer bead population.

32. The method of any one of claims 28 to 31, wherein the composition measured comprises at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each bead population comprises a fluorophore, and wherein the fluorophore of each bead population is different.

33. The method of any one of claims 28 to 32, wherein the first fluorophore and the second fluorophore are different fluorophores.

34. The method of any one of claims 28 to 32, wherein each population of polymer beads comprises only a single fluorophore.

35. The method of any one of claims 28 to 34, wherein the measured composition comprises a final polymer bead population that does not comprise any fluorophore.

36. The method of any one of claims 28 to 34, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

37. The method of any one of claims 28 to 35, wherein each fluorophore is independently selected from any one of the following: Peridinium 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas STP ester or sodium salt; Texas Succinimide ester; Texas Succinimidyl esters; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-XSTP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4 -borax-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-borax-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-borax-3a,4a-diaza-s-dicyclopentaphenazone-3-propanoic acid; 4,4- Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-dicyclopenta-benzo-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -dicyclopentaphenazone-3-propionyl)sulfoalanine, 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-s-dicyclopentaphenazone-3-propionic acid; 4,4-difluoro-5,7 -Diphenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

38. The method of any one of claims 28 to 37, wherein each fluorophore emits fluorescence at one, two, three, four, five, six, seven, eight or nine wavelengths.

39. The method of any one of claims 28 to 38, wherein each fluorophore has a diameter between about 500 nm and about 10 μm.

40. A method according to any one of claims 28 to 39 wherein the polymer beads comprise less than 10%, 20%, 30%, 40% polystyrene by hydrated volume.

41. The method of any one of claims 28 to 39, wherein the polymer beads comprise less than 10%, 20%, 30%, 40% polystyrene by dehydrated volume.

42. The method of any one of claims 28 to 41, wherein the polymer beads are hydrogel beads.

43. The method of claim 42, wherein the hydrogel comprises monomers.

44. The method of claim 43, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxymethacrylate Poly(ethylene glycol) ester, methacrylic acid, sodium methacrylate, glyceryl 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, propylene 2-Naphthyl acrylate, 2-naphthyl methacrylate, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

45. The method of any one of claims 28 to 35, wherein the polymer beads exhibit at least one optical property that is substantially similar to an optical property of a target cell.

46. ​​The method of any one of claims 28 to 45, wherein at least one population of polymer beads exhibits at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

47. The method of claim 45 or 46, wherein the at least one optical property is side scatter.

48. The method of claim 45 or 46, wherein the at least one optical property is forward scattering.

49. The method of claim 45 or 46, wherein the at least one optical property comprises side scatter and forward scatter.

50. The method of any one of claims 45 and 47 to 49, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

51. The method of any one of claims 29 to 50, wherein the measured composition comprises one or more of: (iii) a third population of polymer beads, the third population of polymer beads comprising a third fluorophore; (iv) a fourth population of polymer beads, the fourth population of polymer beads comprising a fourth fluorophore; (v) a fifth population of polymer beads, the fifth population of polymer beads comprising a fifth fluorophore; and / or (vi) a sixth population of polymer beads, the sixth population of polymer beads comprising a sixth fluorophore.

52. The method of claim 51, wherein the first fluorophore, the second fluorophore, the third fluorophore, the fourth fluorophore, the fifth fluorophore, and the sixth fluorophore are independently selected from the group consisting of PerCP-Cy5.5, PE Cy7, APC-Cy7, FITC, PE, and APC.

53. A method of calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (A) providing a composition comprising (i) a first population of polymer beads, the first population of polymer beads comprising a first fluorophore, and (ii) a second population of polymer beads, the second population of polymer beads comprising a second fluorophore; (B) measuring a fluorescent signal of the composition using the cytometry device; (C) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix; as well as (D) Calibration of the cytometry device using the compensation or spectral unmixing matrix.

54. The method of any one of claims 28 to 53, wherein each population of polymer beads contains a sufficiently high fluorophore content to produce a fluorescent signal at least as strong as the fluorescent signal of the cell population to be analyzed by the cytometry device.

55. A composition comprising: (i) a first population of polymer beads comprising a first biomarker; and (ii) a second population of polymer beads, the second population of polymer beads comprising a second biomarker.

56. A composition according to claim 55, comprising at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each population of beads comprises a biomarker, and wherein the biomarker of each population of beads is different.

57. The composition of claim 55 or 56, wherein each population of polymer beads comprises a different biomarker.

58. A composition according to any one of claims 55 to 57, wherein each population of polymer beads comprises only a single biomarker.

59. A composition according to any one of claims 55 to 58, comprising a population of beads which do not comprise a fluorophore.

60. The composition of any one of claims 55 to 59, comprising a population of beads that do not comprise a biomarker.

61. A composition according to any one of claims 55 to 60, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by hydrated volume.

62. The composition of any one of claims 55 to 60, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by dehydrated volume.

63. The composition of any one of claims 55 to 61, wherein the polymer beads are hydrogel beads.

64. The composition of any one of claims 55 to 63, wherein the biomarker is a polypeptide.

65. The composition of any one of claims 55 to 64, wherein the biomarker is an epitope of a fluorescent dye.

66. The composition of any one of claims 55 to 65, wherein each of the first population of polymer beads and the second population of polymer beads each comprises a different fluorophore.

67. The composition of any one of claims 55 to 66, wherein the biomarker is an epitope of an antibody.

68. The composition of claim 67, wherein the antibody is configured to be conjugated to a fluorescent dye or is configured to be conjugated to a secondary antibody-fluorophore conjugate.

69. The composition of any one of claims 55 to 68, wherein the fluorophore is conjugated to an antibody or fragment thereof that binds to an epitope within the polymer bead.

70. The composition of claim 69, wherein the epitope is the biomarker contained in the polymer bead.

71. The composition of claim 69 or 70, wherein the fluorophore is a commercially available antibody-label conjugate.

72. The composition of any one of claims 69 to 71, wherein the biomarker is selected from the biomarkers listed in Tables 1-3 of the present specification.

73. The composition of any one of claims 55 to 65, wherein each biomarker is independently selected from any one of the following: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-γ, CD20, TCRγ / δ, TNFα, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR Vα7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR Vδ2, 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, Igλ light chain, IgA, IL-17A, streptavidin, TCR Vδ1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, FcεR1α, IL-22, Lag-3, MIP-1β, perforin, TCR Vγ9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Igκ light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC Class II (IA / IE), MHCII, TCRα / β, TCRβ, TCR Vα24, Akt (pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, CD16 / 32, CD41a, CD3ε, 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 780Fix 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-α, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1RAcP, IL-6, IL-10, IL-12, IL-17, Integrin α4β7, Isotype control, 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, cleaved, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCRδ, TCRδ / γ, TCR Vα24Jα18, TCR Vβ11, TCR Vγ1.1, TCR Vγ2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1.

74. The composition of any one of claims 63 to 73, wherein the hydrogel comprises polyacrylamide.

75. A composition according to any one of claims 55 to 74, 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, the sixth population of beads comprising a sixth biomarker.

76. The composition of claim 75, wherein the first biomarker, the second biomarker, the third biomarker, the fourth biomarker, the fifth biomarker, and the sixth biomarker are independently selected from the group consisting of: CD3, CD16, CD56, CD45, CD4, CD19, and CD8.

77. A composition according to claim 75 or 76, wherein each population of polymer beads comprises a different fluorophore.

78. The composition of claim 63, wherein the hydrogel comprises a matrix comprising monomers.

79. The composition of claim 78, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxymethacrylate, Poly(ethylene glycol) ester, methacrylic acid, sodium methacrylate, glyceryl 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, propylene glycol 2-Naphthyl acrylate, 2-naphthyl methacrylate, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide , N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine or a combination thereof.

80. The composition of any one of claims 55 to 79, wherein the polymer beads exhibit at least one optical property substantially similar to an optical property of a target cell.

81. The composition of any one of claims 55 to 79, at least one population of polymer beads exhibiting at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

82. The composition of claim 80 or 81, wherein the at least one optical property is side scattering.

83. The composition of claim 80 or 81, wherein the at least one optical property is forward scattering.

84. The composition of claim 80 or 81, wherein the at least one optical property comprises side scattering and forward scattering.

85. The composition of claim 80, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

86. A method of calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (i) measuring the fluorescent signal of the composition according to any one of claims 55 to 85 using the 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.

87. A method of calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (A) using the cytometry device to measure a fluorescent signal of a composition, the composition comprising (i) a first population of polymer beads, the first population of polymer beads comprising a first biomarker, and (ii) a second population of polymer beads, the second population of polymer 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; as well as (C) Calibration of the cytometry device using the compensation or spectral unmixing matrix.

88. The method of claim 87, wherein each population of polymer beads comprises a different fluorophore.

89. A method for generating multicolor compensation controls for a cytometry device, the method comprising the steps of: (A) contacting a composition with a plurality of fluorescent dyes in a single reaction, the composition comprising (i) a first population of polymer beads, the first population of polymer beads comprising a first biomarker, and (ii) a second population of polymer beads, said second population of polymer beads comprising a second biomarker, wherein each dye in the plurality of fluorescent dyes comprises a fluorophore having an excitation or emission spectrum that is different from the excitation or emission spectrum of the fluorophore in each other dye in the plurality of fluorescent dyes, and wherein each dye is bound to the biomarker of only a single polymer bead population in the composition such that each polymer bead population is bound to no more than one fluorescent dye, thereby generating a multicolor compensation control.

90. The method of any one of claims 87 to 89, wherein each biomarker of the population of polymer beads comprises an antigen configured to selectively bind to a fluorescent dye.

91. The method of any one of claims 89 to 90, wherein the plurality of fluorescent dyes are secondary antibody-fluorophore conjugates, and wherein each biomarker of the polymer bead population comprises an antigen configured to selectively bind to the secondary antibody-fluorophore conjugate.

92. The method of any one of claims 89 to 91, wherein each dye of the plurality of fluorescent dyes comprises an antibody-fluorophore conjugate.

93. The method of any one of claims 89 to 92, wherein each fluorophore is independently selected from the fluorophores listed in Table 4.

94. The method of any one of claims 87 to 93, wherein each fluorophore is independently selected from any one of the following: Peridinium 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 Amine ester; 6-carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(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 or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas STP ester or sodium salt; Texas Succinimide ester; Texas Succinimidyl esters; X-rhodamine-5-(and -6) isothiocyanate, FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-XSTP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4 -borax-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-borax-3a,4a-diaza-s-dicyclopentaphenazone-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-borax-3a,4a-diaza-s-dicyclopentaphenazone-3-propanoic acid; 4,4- Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-dicyclopenta-benzo-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -dicyclopentaphenazone-3-propionyl)sulfoalanine, 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-s-dicyclopentaphenazone-3-propionic acid; 4,4-difluoro-5,7 -Diphenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-dicyclopenta-3-propionic acid 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid succinimide ester 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopenta-benzo-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a- Diaza-s-dicyclopentaphenazone-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-dicyclopentaphenazone-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimidyl ester, Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 Carboxylic acid; Alexa 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

95. The method of any one of claims 87 to 94, wherein the composition comprises at most 5, at most 10, at most 12, at most 18, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 populations of polymer beads, wherein each population of beads comprises a biomarker, and wherein the biomarker of each population of beads is different.

96. The method of any one of claims 87 to 95, wherein each population of polymer beads comprises a different biomarker.

97. The method of any one of claims 87 to 96, wherein each population of polymer beads comprises only a single biomarker.

98. The method of any one of claims 87 to 97, comprising a population of beads that do not comprise a fluorophore or lack an epitope for association with an antibody-fluorophore conjugate.

99. The method of any one of claims 87 to 98, comprising a population of beads that do not comprise a biomarker.

100. The method of any one of claims 87 to 99, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by hydrated volume.

101. The method of any one of claims 87 to 99, wherein the polymer beads comprise less than 10%, 20%, 30% or 40% polystyrene by dehydrated volume.

102. The method of any one of claims 87 to 101, wherein the polymer beads are hydrogel beads.

103. The method of any one of claims 87 to 102, wherein the biomarker is a polypeptide.

104. The method of any one of claims 87 to 103, wherein the biomarker is an epitope of a fluorescent dye.

105. The method of any one of claims 87 to 104, wherein each of the first population of polymer beads and the second population of polymer beads each comprises a different fluorophore.

106. The method of any one of claims 87 to 105, wherein the biomarker is an epitope of an antibody.

107. The method of any one of claims 87 to 106, wherein the antibody is configured to be conjugated to a fluorescent dye or is configured to be conjugated to a secondary antibody-fluorophore conjugate.

108. The method of any one of claims 87 to 107, wherein the fluorophore is conjugated to an antibody or fragment thereof that binds to an epitope within the polymer bead.

109. The method of claim 108, wherein the epitope is the biomarker contained in the polymer bead.

110. The method of any one of claims 87 to 109, wherein the fluorophore is a commercially available antibody-label conjugate.

111. The method of any one of claims 87 to 110, wherein the biomarker is selected from the biomarkers listed in Tables 1-3 of the present specification.

112. The method of any one of claims 87 to 110, wherein each biomarker is independently selected from any one of the following: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-γ, CD20, TCRγ / δ, TNFα, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CD11c, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR Vα7.2, CD11b, CD21, CD24, IL-4, biotin, CCR10, CD31, CD44, CD138, CD294, NKp46, TCR Vδ2, 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, Igλ light chain, IgA, IL-17A, streptavidin, TCR Vδ1, CD1d, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD137, CD163, CD193, CTLA-4, CX3CR1, FcεR1α, IL-22, Lag-3, MIP-1β, perforin, TCR Vγ9, CD1a, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Igκ light chain, IgE, IgG1, IgG3, IL-5, IL-8, IL-21R, KIR3dl05, KLRC1 / 2, Ly-6C, Ly-6G, MHC Class II (IA / IE), MHCII, TCRα / β, TCRβ, TCR Vα24, Akt (pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, CD16 / 32, CD41a, CD3ε, 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 780Fix 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-α, IgA1, IgA1 / IgA2, IgA2, IgG2, IgG4, IL-1RAcP, IL-6, IL-10, IL-12, IL-17, Integrin α4β7, Isotype control, 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, cleaved, RT1B, S6(pS235 / pS236), STIM1, STIM2, TCRδ, TCRδ / γ, TCR Vα24Jα18, TCR Vβ11, TCR Vγ1.1, TCR Vγ2, TER-119, TIMP-3, TRAF3, TSLP receptor, VDAC1, vimentin, XCR1, and YAP1.

113. The method of any one of claims 101 to 112, wherein the hydrogel comprises polyacrylamide.

114. The method of any one of claims 87 to 113, 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, the sixth population of beads comprising a sixth biomarker.

115. The method of claim 114, wherein the first biomarker, the second biomarker, the third biomarker, the fourth biomarker, the fifth biomarker, and the sixth biomarker are independently selected from the group consisting of: CD3, CD16, CD56, CD45, CD4, CD19, and CD8.

116. The method of any one of claims 87 to 115, wherein each population of polymer beads comprises a different fluorophore.

117. The method of any one of claims 101 to 116, wherein the hydrogel comprises a matrix comprising monomers.

118. The method of claim 66, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxymethacrylate, Poly(ethylene glycol) ester, methacrylic acid, sodium methacrylate, glyceryl 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, propylene glycol 2-Naphthyl acrylate, 2-naphthyl methacrylate, 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,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide , N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylmethylacrylamide, N-1-naphthylmethylacrylamide, N-4-nitrophenylmethylacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethylacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine or a combination thereof.

119. The method of any one of claims 87 to 118, wherein the polymer beads exhibit at least one optical property substantially similar to an optical property of a target cell.

120. The method of any one of claims 87 to 119, wherein at least one population of polymer beads exhibits at least one optical property that is different from a corresponding optical property of another population of polymer beads within the composition.

121. The method of claim 119 or 120, wherein the at least one optical property is side scatter.

122. The method of any one of claims 119 to 120, wherein the at least one optical property is forward scattering.

123. The method of any one of claims 119 to 120, wherein the at least one optical property comprises side scatter and forward scatter.

124. The method of any one of claims 87 to 123, wherein each target cell is independently selected from any one of the following: a T cell, a B cell, and a natural killer cell.

125. A method of calibrating a cytometry device for compensation or spectral unmixing, the method comprising: (i) providing a composition comprising (a) a first population of polymer beads, the first population of polymer beads comprising a first biomarker, and (b) a second population of polymer beads, the second population of polymer beads comprising a second biomarker, each of the first population of polymer beads and the second population of polymer beads comprising a different fluorophore; (ii) contacting the composition with at least two antibody populations or fragments thereof, each antibody population being 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 a fluorescent signal of the composition using the cytometry device; (iv) deconvolving the fluorescent signal from each bead population of the composition to calculate a compensation or spectral unmixing matrix; as well as (v) calibrating the cytometry device using the compensation or spectral unmixing matrix.

126. The method of any one of claims 86 to 125, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the fluorescence emission maximum.

127. The method of any one of claims 86 to 125, wherein the fluorescent signal from each polymer bead population is deconvoluted based on the optical properties of each polymer bead population.

Images