Method and composition for calibrating a flow cytometer

Abstract

This disclosure provides improved and useful techniques for the mutual standardization of flow cytometry instruments, particularly spectral flow cytometry instruments. [Solution] An aspect of the present disclosure includes a method for calibrating a flow cytometer having multiple fluorescence channels. The method of interest uses a calibration set of bead ensembles, each bead ensemble of the calibration set having a different fluorophore attached to its surface, and the calibration set includes a number of bead ensembles less than the number of fluorescence channels in the flow cytometer. Further provided are a flow cytometer, a non-temporary computer-readable storage medium, and a kit comprising a calibration set of bead ensembles for carrying out, for example, the method of the subject.

Description

[Background technology]

[0001] The characterization of analytes in biological fluids is a crucial part of biological research, medical diagnosis, and the assessment of a patient's overall health and well-being. By detecting analytes in biological fluids such as human blood or blood-derived products, results can be obtained that may be relevant in determining treatment protocols for patients with various medical conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biomaterials, such as cells in blood samples or particles of interest in other types of biological or chemical samples. A flow cytometer typically comprises a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transports particles (including cells) from the fluid sample as a cellular flow into a flow cell, while further guiding the sheath fluid into the flow cell. Light is shone into the flow stream to characterize its components. Changes in biomaterials within the flow stream, such as morphology or the presence of fluorescent labels, can alter the observed light, and these changes enable characterization and separation. Light must be shone into the flow stream and collected to characterize its components. The light source of a flow cytometer can be a variety of light sources, including one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the irradiated particles is collected and quantified.

[0003] The separation of biological particles is achieved by adding a sorting or collection function to a flow cytometer. Particles present in the separated flow and detected to possess one or more desired characteristics are individually separated from the sample flow by mechanical or electrical removal. Common flow sorting techniques utilize droplet sorting, which divides a fluid stream containing linearly separated particles into droplets. Droplets containing the particles of interest are electrically charged and deflected into a collection tube by passing through an electric field. Typically, linearly separated particles in the flow are characterized as they pass through an observation point directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, the time it takes for that particle to reach the droplet separation point and separate from the flow into a droplet can be predicted. Ideally, the fluid stream is briefly charged just before the droplet containing the selected particles separates from the fluid stream, and then grounded immediately after the droplet separates. The droplet being sorted retains its charge when separating from the fluid stream, while all other droplets remain uncharged. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] In flow cytometry, standardization is essential to ensure that the changes in light collected and quantified by the flow cytometer are specific to the sample and not, for example, a result of the instrument's configuration. Standardization is particularly important in clinical cytometry, for example, when comparing data from various flow cytometers and laboratories to monitor the frequency of immune cell populations or the fluorescence intensity of cell markers used for disease diagnosis and monitoring. One of the main sources of variability that needs to be addressed through standardization throughout the flow cytometry experiment is the instrument's hardware.

[0005] To compensate for variations in instrument hardware, flow cytometers are typically calibrated using rigid dye beads to set a target mean fluorescence intensity (MFI) for each detector or channel of the flow cytometer. These beads encapsulate fluorescent polymers that emit fluorescence across a broad spectrum when excited at a wide range of wavelengths. These beads are widely used to set instruments to identical conditions, facilitating comparisons between experiments. However, the standardization process is time-consuming and requires technical expertise in error control and variability, for example, because it must account for a wide variety of subtle differences in instrument hardware. Furthermore, with the development of spectral flow cytometers that utilize multiple detectors to quantify light across the entire fluorescence spectrum emitted from a fluorescent particle of interest (e.g., a fluorophore), the standardization process has become even more difficult and complex.

[0006] Therefore, there is a need for improved and useful techniques for the mutual standardization of flow cytometry instruments, particularly spectral cytometry instruments. The inventors recognize that hard dye beads exhibit similar reactions to various minute differences in instrument hardware (e.g., laser wavelength, laser power, etc.), making it often difficult to account for specific variations in a particular instrument or experiment using such beads. In contrast, fluorophores exhibit a far more pronounced reaction to these minute differences, but due to the nature of spectral flow cytometers, it is extremely difficult to introduce fluorophores into the calibration of spectral instruments using conventional methods. Therefore, the inventors recognize that a process for efficiently and effectively utilizing fluorophores in the calibration of spectral instruments could enable the development of such improved and useful mutual standardization techniques. Accordingly, embodiments of the present disclosure provide methods and compositions for calibrating spectral instruments using fluorophores, thereby satisfying the aforementioned need. [Means for solving the problem]

[0007] Aspects of the present disclosure include a method for calibrating a flow cytometer characterized by a plurality of fluorescence channels. In the method of interest, a control composition comprising a calibration set of bead ensembles is irradiated with one or more light sources of the flow cytometer (each bead ensemble in the calibration set has a different fluorophore attached to its surface, and the calibration set comprises a number of bead ensembles less than the number of fluorescence channels in the flow cytometer), data signals generated by the plurality of fluorescence channels of the flow cytometer with respect to the calibration set of bead ensembles are measured, a quantitative index of fluorescence intensity is calculated for each of at least a portion of the plurality of fluorescence channels based on the data signal generated for the bead ensemble of the calibration set having the highest fluorescence intensity among the bead ensembles of the calibration set with respect to the fluorescence channel, and one or more fluorescence channels are adjusted based on the quantitative index calculated for each of the plurality of fluorescence channels. In some embodiments, the flow cytometer is a spectral flow cytometer.

[0008] In some embodiments, the quantitative index of fluorescence intensity is the average fluorescence intensity. In these cases, the average fluorescence intensity calculated for each fluorescence channel may be 500 or greater, for example, 1,000 or greater, or 2,000 or greater, or 5,000 or greater. In some embodiments, the average fluorescence intensity includes the arithmetic mean, geometric mean, and / or median of multiple fluorescence intensity values ​​measured for a given bead population and fluorescence channel.

[0009] In one embodiment, one or more fluorescence channels are adjusted such that the subsequent quantitative fluorescence index calculated for each of the one or more fluorescence channels falls within a predetermined threshold range of a standardized application setting generated for the fluorescence channels and the highest fluorescence intensity bead population of the calibration set corresponding to the fluorescence channels. In some embodiments, the voltage and / or electron gain settings of one or more fluorescence channels are changed during the adjustment.

[0010] In some embodiments, the bead collection of a calibration set includes beads having a core, a fluorophore, and a linker configured to adhere to the fluorophore and bind to the core. In some embodiments, the linker binds to the core, for example, so that the fluorophore is covalently bonded to the core via the linker. In some cases, the core contains an antigen and the linker contains an antibody. In some embodiments, the core contains polystyrene.

[0011] In one embodiment, each bead group in the calibration set has the highest fluorescence intensity among the bead groups in the calibration set with respect to at least one fluorescence channel of the flow cytometer. In some embodiments, the number of bead groups in the calibration set is less than half the number of fluorescence channels, for example, less than one-quarter or less the number of fluorescence channels, or less than one-eighth the number of fluorescence channels. In some embodiments, the plurality of fluorescence channels includes 10 or more fluorescence channels, for example, 40 or more fluorescence channels, or 60 or more fluorescence channels.

[0012] In some embodiments, the multiple fluorescence channels include at least one fluorescence channel for each of two or more emission spectra. In some embodiments, one or more light sources of the flow cytometer include multiple lasers, each emitting a different wavelength. For example, the multiple lasers may include ultraviolet (UV) spectra, violet spectra, blue spectra, green / yellow spectra, and red spectra. In some embodiments, the multiple fluorescence channels include at least one fluorescence channel for each of two or more excitation spectra. In some cases, each fluorescence channel of the multiple fluorescence channels differs by 10 nm or more in its respective excitation or emission spectrum. In some embodiments, for each laser of the flow cytometer, the multiple fluorescence channels are configured to detect a composite emission spectrum in the range of wavelengths from 20 nm or less longer than the laser wavelength to at least 810 nm. In some cases, for each laser of the flow cytometer, the calibration set of the bead ensemble includes one or more fluorophores having an excitation spectrum including the laser wavelength and a composite emission spectrum extending from a wavelength 20 nm or less longer than the laser wavelength to at least 810 nm in full fluorescence spectrum.

[0013] In some embodiments, the method further generates a set of standardized application settings for at least a portion of multiple fluorescence channels by calculating a quantitative fluorescence index for each fluorescence channel and for each bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel. In these cases, a set of standardized application settings may be generated using two or more flow cytometers of the same model. In some embodiments, the method further compares data generated by different flow cytometers using the generated set of standardized application settings. In some embodiments, the method further reduces variability between different flow cytometers using the generated set of standardized application settings.

[0014] In another embodiment, for example, a flow cytometer is provided which is configured to perform a calibration method as described above and herein. The flow cytometer of interest comprises one or more light sources configured to irradiate beads in the flow stream of the flow cytometer, a plurality of fluorescence channels configured to generate data signals from light received from the irradiated beads, and a processor to which memory is operablely connected, the memory storing instructions that, when executed by the processor, cause the processor to measure the data signals generated by the plurality of fluorescence channels with respect to a control composition including a calibration set of bead ensembles (each bead ensemble in the calibration set has a different fluorophore attached to its surface, and the calibration set includes a number of bead ensembles less than the number of fluorescence channels), calculate a quantitative index of fluorescence intensity for each of at least a portion of the plurality of fluorescence channels based on the data signal generated for the bead ensemble in the calibration set having the highest fluorescence intensity with respect to the fluorescence channel, and adjust one or more fluorescence channels based on the quantitative fluorescence index calculated for each of the plurality of fluorescence channels.

[0015] In another embodiment, a non-temporary computer-readable storage medium is provided which stores, for example, instructions for performing a calibration method as described above and herein. Embodiments of this disclosure further include a kit comprising, for example, the non-temporary computer-readable storage medium and / or a control composition including a calibration set of bead groups, as described above and herein. [Brief explanation of the drawing]

[0016] This disclosure can be best understood from the following detailed description when read in conjunction with the attached drawings. The drawings include the following figures.

[0017] [Figure 1A] This is a flowchart for implementing a method for calibrating a flow cytometer according to a certain embodiment. [Figure 1B]A flowchart for implementing a method of generating a set of standardized application settings for use in calibrating a flow cytometer according to an embodiment. [Figure 1C] A chart showing the emission profiles of a calibration set of bead populations overlaid on multiple fluorescence channels of a flow cytometer according to an embodiment. [Figure 2] A diagram showing a flow cytometry system according to an embodiment. [Figure 3-1] A diagram showing an image - corresponding particle sorter according to an embodiment. [Figure 3-2] A diagram showing an image - corresponding particle sorter according to an embodiment. [Figure 4] A functional block diagram showing a particle analysis system according to an embodiment. [Figure 5] A functional block diagram showing an example of a control system according to an embodiment. [Figure 6A] A schematic diagram showing a particle sorting system according to an embodiment. [Figure 6B] A schematic diagram showing a particle sorting system according to an embodiment. [Figure 7] A diagram showing an aspect of a computer control system according to an embodiment. **MODE FOR CARRYING OUT THE INVENTION**

[0018] This disclosure provides a method for calibrating a flow cytometer having multiple fluorescence channels. In the method of interest, a control composition containing a calibration set of bead ensembles is irradiated with one or more light sources of the flow cytometer (each bead ensemble in the calibration set has a different fluorophore attached to its surface, and the calibration set contains fewer bead ensembles than the number of fluorescence channels in the flow cytometer), data signals generated by the multiple fluorescence channels of the flow cytometer with respect to the calibration set of bead ensembles are measured, a quantitative index of fluorescence intensity is calculated for each of at least a portion of the multiple fluorescence channels based on the data signal generated for the bead ensemble of the calibration set having the highest fluorescence intensity among the bead ensembles of the calibration set with respect to the fluorescence channel, and one or more fluorescence channels are adjusted based on the quantitative fluorescence index calculated for each of the one or more fluorescence channels. A flow cytometer, a non-temporary computer-readable storage medium, and a kit for carrying out the method of the subject are further provided.

[0019] Before this disclosure is described in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, as it is, needless to say, subject to change. Since the scope of this disclosure is limited only by the appended claims, it should be further understood that the terms used herein are intended to describe, and not to limit, specific embodiments.

[0020] If a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless the context otherwise expresses, and all other stated or intervening values ​​within that stated range are included in this disclosure. These smaller upper and lower limits may be independently included within the smaller range and are further included in this disclosure, subject to any specifically excluded limits within the stated range. If the stated range includes one or both limits, the range excluding one or both of those included limits is also included in this disclosure.

[0021] In this specification, a range is presented preceded by the term “approximately.” The term “approximately” is used here to literally support the exact number preceding it, and any number that is close to or approximates it. When determining whether a number is close to or approximates a specifically stated number, the close or approximate unstated number may be a number that, in the context in which the specifically stated number is presented, gives a substantial equivalent of the specifically stated number.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar to or equivalent to those described herein may be further used in the practice or testing of this disclosure, but representative and exemplary methods and materials are described herein.

[0023] All publications and patents referenced herein are incorporated herein by reference as if each individual publication or patent were specifically and individually incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the references of the publications. Any reference to a publication is for its disclosure prior to the filing date, and this disclosure should not be construed as acknowledging that such publication has no prior rights on the grounds of prior invention. Furthermore, the dates of the publications provided may differ from the actual publication dates and may need to be verified individually.

[0024] It should be noted that, as used in this specification and the attached claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context otherwise indicates. It should also be noted that claims may be drafted to exclude any particular element. Therefore, this statement is intended to serve as a prior art for the use of exclusive terms such as "only," "only," or "negative" limitations relating to the description of elements of a claim.

[0025] As will be apparent to those skilled in the art upon reading this disclosure, each of the embodiments described and illustrated herein has separate components and features, which may be readily separated from or readily combined with any of the features of any of the other embodiments without departing from the scope or spirit of this disclosure. All described methods may be performed in the order of the described events or in any other logically possible order.

[0026] While systems and methods are described or stipulated for grammatical fluidity with respect to functional descriptions, claims should not necessarily be construed as being limited in any way by a limitation of the composition of “means” or “steps” unless expressly described under Section 112 of the United States Patent Act, and should be granted the meaning of the definitions and the full scope of equivalents given by claims under the judicial theory of equivalents, and should be clearly understood that if claims are expressly described under Section 112 of the United States Patent Act, they should be granted the full statutory equivalents under Section 112 of the United States Patent Act.

[0027] As summarized above, a method is provided for calibrating a flow cytometer (characterized by multiple different fluorescence channels, for example) using a control composition (characterized by multiple different fluorophores, for example). In further describing various embodiments of the present invention, the subject method, which includes, for example, a method for calibrating a flow cytometer and a method for generating a control composition and / or standardized application settings used to calibrate a flow cytometer, will be described in more detail first. Next, a kit including a flow cytometer, a non-temporary computer-readable storage medium, and, in either case, a calibration set of, for example, a bead collection, will be described for carrying out the subject method.

[0028] How to calibrate a flow cytometer As summarized above, a method is provided for calibrating a flow cytometer having multiple different fluorescence channels. In an embodiment of the method, a control composition containing a calibration set of bead ensembles is irradiated with one or more light sources of the flow cytometer (each bead ensemble in the calibration set has a different fluorophore attached to its surface, and the calibration set contains fewer bead ensembles than the number of fluorescence channels in the flow cytometer), data signals generated by the multiple fluorescence channels of the flow cytometer with respect to the calibration set of bead ensembles are measured, a quantitative index of fluorescence intensity is calculated for each of at least a portion of the multiple fluorescence channels based on the data signal generated for the bead ensemble of the calibration set having the highest fluorescence intensity with respect to the fluorescence channel, and one or more fluorescence channels are adjusted based on the quantitative fluorescence index calculated for each of the one or more fluorescence channels. In some embodiments, one or more fluorescence channels are adjusted such that the subsequent quantitative fluorescence index calculated for each of the one or more fluorescence channels is within a predetermined threshold range of a standardization application setting generated for the fluorescence channel and the highest fluorescence intensity bead ensemble of the calibration set corresponding to the fluorescence channel.

[0029] As described above, embodiments of the method calibrate a flow cytometer having a plurality of different fluorescence channels. “Fluorescence channel” means a detector configured to measure fluorescence emission in a particular wavelength range, and a corresponding optical element (e.g., an optical filter) that selectively propagates, for example, a particular range of light to the detector. The fluorescence channels of the flow cytometer may be configured to measure fluorescence emission over any number or a variety of narrow or broad wavelength ranges. In some embodiments, one or more fluorescence channels of the flow cytometer may be configured to detect the maximum emission value of one or more fluorophores of interest. In some embodiments, the plurality of fluorescence channels include at least one fluorescence channel for each of two or more emission spectra (corresponding to the emission spectra of two or more fluorophores), e.g., five or more emission spectra, or ten or more emission spectra, or twenty or more emission spectra, or fifty or more emission spectra.

[0030] In some embodiments, one or more fluorescence channels in a flow cytometer are configured to effectively measure both the full emission spectrum and the total emission spectrum of a fluorophore of interest. For example, a flow cytometer may include multiple fluorescence channels configured to measure adjacent (or, for example, substantially adjacent) wavelength ranges, such that all fluorescence emitted by, for example, fluorescein isothiocyanate (FITC) is measured when irradiated with a 488 nm laser. In some embodiments, the flow cytometer is a spectral flow cytometer. A “spectral flow cytometer” means a flow cytometer that quantifies or measures light over the entire fluorescence spectrum emitted by a fluorescent particle (e.g., the full emission spectrum of each fluorescent dye or fluorophore used to stain cells or particles in an experiment), rather than simply measuring one emission wavelength per fluorescent particle. In this way, each fluorophore in a given experiment may be identified by its unique spectral characteristics, i.e., its unique shape, which is generated by measuring the emitted fluorescence at multiple wavelengths. For example, the flow cytometer may be a spectral flow cytometer configured to measure the full spectrum of synchrotron radiation in the range of 150 nm to 1200 nm, for example, 200 nm to 1000 nm, or 200 nm to 900 nm, or 300 nm to 900 nm, or 350 nm to 900 nm, or 350 nm to 850 nm, or 375 nm to 850 nm, or 400 nm to 850 nm, or 375 nm to 825 nm, or 375 nm to 810 nm, or 375 nm to 800 nm, or 400 nm to 800 nm.

[0031] In embodiments of the method, to measure the fluorescence emitted by a control composition of the present disclosure using each of several fluorescence channels (for example, as described in more detail below), the control composition is irradiated using one or more light sources of a flow cytometer. In some embodiments, one or more light sources of the flow cytometer irradiate the control composition as it passes through one or more investigation points of the flow stream of the flow cytometer. In these cases, the control composition may pass through different investigation points for each light source of the flow cytometer. In some embodiments, one or more light sources of the flow cytometer include multiple lasers, each emitting a different wavelength. For example, the multiple lasers may include ultraviolet (UV) spectra, violet spectra, blue spectra, green / yellow spectra, and red spectra.

[0032] In some embodiments, the multiple fluorescence channels include at least one fluorescence channel for each of two or more excitation spectra. In these cases, the two or more excitation spectra may correspond to the emission spectra of two or more lasers of the flow cytometer. For example, the flow cytometer may include a first fluorescence channel configured to measure the fluorescence emission in the range of 515 nm to 545 nm of a particle irradiated by a 349 nm excitation laser, and a second fluorescence channel configured to measure the fluorescence emission in the range of 515 nm to 545 nm of a particle irradiated by a 405 nm excitation laser. In these cases, the 349 nm and 405 nm excitation lasers may irradiate the particle at different non-overlapping (or, for example, minimally overlapping) positions along the flow stream. In other words, the investigation areas of each laser and the positions of each corresponding fluorescence channel along the flow stream may be different. In some embodiments, each fluorescence channel of the multiple fluorescence channels differs by 10 nm or more in its respective excitation or emission spectrum. For example, two or more fluorescence channels may be configured to measure substantially identical emission spectra, but they may also be arranged to measure light from different survey points corresponding to excitation light wavelengths that differ by 10 nm or more.

[0033] In some embodiments, for each laser in the flow cytometer, multiple fluorescence channels are configured to detect a composite emission spectrum in the range of wavelengths from 20 nm or less longer than the laser wavelength (e.g., the longest wavelength of the laser if the laser emits broad-spectrum light) to 810 nm or more. In some embodiments, the flow cytometer may be a spectral flow cytometer having two or more different excitation lasers. In other words, the flow cytometer may quantify or measure light across the entire fluorescence spectrum emitted by the fluorescent particles for each of the two or more different excitation wavelengths. In some embodiments, the flow cytometer may be a spectral flow cytometer including two or more different excitation lasers, e.g., five or more different excitation lasers, or eight or more, or ten or more, or fifteen or more different excitation lasers. In some embodiments, the flow cytometer may be a spectral flow cytometer including ten or more different fluorescence channels, e.g., twenty or more different fluorescence channels, or forty or more, or fifty or more, or 100 or more, or 500 or more different fluorescence channels.

[0034] In some cases, the flow cytometer may be a spectral flow cytometer that includes at least one laser emitting in the ultraviolet (UV) spectrum, violet spectrum, blue spectrum, green / yellow spectrum, and red spectrum. In these cases, the spectral flow cytometer may be configured to detect a composite emission spectrum for each laser ranging from a wavelength 20 nm or less (e.g., 10 nm or less, or 5 nm or less, or 2 nm or less) longer than the laser wavelength to 810 nm or more (e.g., 850 nm or more, 900 nm or more, or 1000 nm or more).

[0035] As described above, in embodiments of the method, a control composition is irradiated using one or more light sources of a flow cytometer, and the fluorescence emitted by the control composition is measured by each of the fluorescence channels of the flow cytometer. The control composition of this disclosure comprises a calibration set of bead ensembles, each bead ensemble of the calibration set having a different fluorophore attached to its surface. While hard dye beads, which are typically used to calibrate flow cytometry instruments, respond similarly to a variety of subtle changes in the instrument's hardware and environmental conditions, fluorophores respond far more distinctly to these subtle differences. For example, a slight change in either the wavelength or intensity of the excitation light may result in a similar change in the fluorescence emitted by hard dye beads, but a slight change in the wavelength of the excitation light may result in a significantly different change in the fluorescence emitted by a fluorophore compared to a slight change in the intensity of the excitation light. In this way, by including beads whose surfaces are stained with fluorophores (i.e., beads with fluorophores attached to their outer surfaces), the control compositions of the present disclosure can accurately and effectively account for minute or slight differences between instruments of separate flow cytometers or minute or slight differences between the same instrument at different points in time when used for calibration.

[0036] The reference compositions of this disclosure may be used to account for any number of distinct hardware or environmental differences when used for calibrating a flow cytometer. In some embodiments, the reference compositions of this disclosure may be used to account for any differences in instrument hardware that affect the wavelength or power (e.g., intensity) of the excitation light emitted from one or more light sources of the flow cytometer and irradiating the flow stream. For example, the reference compositions of this disclosure may be used to account for one or more minor differences among laser power, laser wavelength, laser alignment, and optical components (e.g., optical filters) that interact with the irradiated light. In some embodiments, the reference compositions of this disclosure may be used to account for any differences in instrument hardware that affect the collection or detection of fluorescence emitted by particles in the flow stream. For example, the reference compositions of this disclosure may be used to account for one or more minor differences among detector sensitivity, detector alignment, and optical components (e.g., optical filters) that interact with the synchrotron radiation.

[0037] In some embodiments of the method, the calibration set of the control composition includes a number of bead ensembles that is less than (i.e., smaller than) the number of fluorescence channels in the flow cytometer. In some embodiments, each bead ensemble of the calibration set includes one type or species of fluorophore. In other words, since different fluorophores are attached to the surface of each bead ensemble of the calibration set, the calibration set of the bead ensembles includes a number of different fluorophores that is less than the number of fluorescence channels in the flow cytometer. In some embodiments, the calibration sets of the control composition and the bead ensembles include the same number of different fluorophores. In some embodiments, the calibration set of the bead ensembles (including the fluorophores in the calibration set) is specifically configured or adapted for the hardware of the flow cytometer instrument. For example, the fluorophores in the calibration set of the bead ensembles may be selected based on the light source and fluorescence channels of the flow cytometer. In some cases, the calibration set of the bead ensembles is irrelevant to or unaffected by the flow cytometer experiment. In other words, the same control composition (containing the same bead population and its fluorophores) may be used to calibrate a flow cytometer regardless of the fluorophores used in experiments subsequently performed on the flow cytometer after calibration.

[0038] In some embodiments, the fluorophores of the calibration set of bead ensembles emit fluorescence for each fluorescence channel of the flow cytometer. In other words, for each fluorescence channel and the excitation / irradiation light source corresponding to the fluorescence channel, at least one bead ensemble of the calibration set emits light of detectable intensity. For example, when a control composition (and its calibration set) is run in a flow cytometer, each fluorescence channel of the flow cytometer may measure an average fluorescence intensity (MFI) of 500 or more for at least one bead ensemble of the calibration set (i.e., it may generate a data signal corresponding to the average fluorescence intensity). In some cases, each fluorescence channel of the flow cytometer may measure an MFI of 1,000 or more, or 2,000 or more, or 5,000 or more for at least one bead ensemble of the calibration set. In some embodiments, each bead ensemble of the calibration set has the highest fluorescence intensity (e.g., MFI) among the bead ensembles of the calibration set for at least one fluorescence channel of the flow cytometer.

[0039] In some embodiments, for each light source (e.g., laser) of the flow cytometer, the fluorophores of the bead ensemble calibration set emit fluorescence that extends to or includes the full fluorescence spectrum from wavelengths slightly longer than one or more wavelengths emitted from the light source to wavelengths greater than or equal to wavelengths detectable by the light source's fluorescence channel configured to detect the longest wavelength. In some embodiments, for each laser of the flow cytometer, the bead ensemble calibration set includes one or more fluorophores having an excitation spectrum that includes the wavelength of the laser and a composite emission spectrum that extends from wavelengths 20 nm or less (e.g., 10 nm or less, or 5 nm or less, or 2 nm or less) longer than the wavelength of the laser to a full fluorescence spectrum in the range of 810 nm or more (e.g., 850 nm or more, 900 nm or more, or 1000 nm or more).

[0040] As described above, beads stained with fluorophores can accurately and effectively account for minute or slight differences in instrument hardware, for example. However, beads stained with fluorophores may have a relatively narrow emission spectrum compared to hard dye beads. Consequently, introducing fluorophores into the calibration of flow cytometers with a large number of fluorescence channels (e.g., spectral flow cytometers) using conventional methods is time-consuming and costly, for example, because a different fluorophore is used to calibrate each fluorescence channel, or because a different set of fluorophore-stained beads is used for each experiment (e.g., depending on the fluorophore of the experiment). In contrast, the control composition of this disclosure is effective regardless of subsequent experiments performed on the flow cytometer and contains fewer beads (e.g., its fluorophores) than the number of fluorescence channels in the flow cytometer, so fluorophore-stained beads can be used for the calibration of, for example, spectral flow cytometers with higher efficiency and lower cost compared to conventional flow cytometer calibration techniques.

[0041] In some embodiments, the number of bead groups in the calibration set is less than half the number of fluorescent channels, for example, less than one-quarter, less than one-eighth, less than one-tenth, or less than one-twentieth of the number of fluorescent channels. In some embodiments, the multiple fluorescent channels include 10 or more fluorescent channels, for example, 40 or more fluorescent channels, or 60 or more fluorescent channels, or 100 or more fluorescent channels, or 200 or more fluorescent channels. For example, a flow cytometer may include 48 fluorescent channels, and the calibration set of bead groups may include 8 groups.

[0042] In some embodiments, the bead collection of the calibration set includes beads having a core, a fluorophore, and a linker configured to bind the fluorophore to the core. In some embodiments, the linker is attached to the fluorophore and configured to bind to the core. In other embodiments, the linker is attached to the core and configured to bind to the fluorophore. In some embodiments, the linker covalently binds the fluorophore to the core. For example, the beads may be modified to introduce a functional group such as a carboxyl group or an amino group, which may be used to covalently bind the fluorophore to the core by one or more chemical reactions. In some embodiments, the linker noncovalently binds the fluorophore to the core. In these cases, the core may contain an antigen, and the linker may contain an antibody attached to the fluorophore. In some embodiments, the core contains polystyrene. In other embodiments, the core may contain fixed cells.

[0043] In some embodiments, the control composition is provided to the user with the fluorophores already bound or attached to the core. In these cases, the bead population of the calibration set may each have a predetermined fluorophor-to-core ratio to ensure that the control composition can be effectively used, for example, for the cross-standardization of different instruments in different laboratories. In some embodiments, the method further binds fluorophores to the core. In these cases, the method may further examine and consider the lot number of each reagent used when performing the binding. In some embodiments, the control composition is lyophilized, for example, to extend the shelf life of the control composition. In these cases, the method may further reconstitute the lyophilized composition.

[0044] In some embodiments, the fluorophores of the bead ensemble in the calibration set may be polymer dyes. In some examples of the method, the polymer dyes include conjugated polymers. Conjugated polymers (CPs) are characterized by a delocalized electronic structure in which unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds) alternate in a backbone, and π electrons can move from one bond to the other. Thus, the conjugated backbone may confer an extended linear structure to the polymer dye, with the bond angles between the repeating units of the polymer restricted. For example, proteins and nucleic acids are similarly macromolecules, but in some cases they do not form an extended rod structure and rather fold into a higher-order three-dimensional shape. In addition, CPs may form a “rigid rod” polymer backbone, exhibiting restricted twist (e.g., torsion) angles between monomer repeating units along the polymer backbone chain. In some cases, the polymer dyes include CPs having a rigid rod structure. The structural properties of the polymer dye can affect the fluorescence properties of the molecule. In some embodiments, the polymer dye of the bead cluster may be, for example, BD Horizon Brilliant® Dye such as BD Horizon Brilliant® Violet Dye (e.g., BV421, BV480, BV510, BV570, BV605, BV650, BV711, BV750, BV786); BD Horizon Brilliant® Ultraviolet Dye (e.g., BUV395, BUV496, BUV563, BUV615, BUV661, BUV737, BUV805); and BD Horizon Brilliant® Blue Dye (e.g., BB515, BB630-P2, BB660-P2, BB700, BB755-P, BB-790P) (BD Biosciences, San Jose, CA).

[0045] In some embodiments, the fluorophores of the bead ensemble in the calibration set may be nonpolymer dyes. For example, the fluorophores of the bead ensemble may be, for example, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine, and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1- Naphthyl maleimide; anthranilamide; brilliant yellow; coumarin, and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanine, and derivatives such as cyanosine, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5', 5''-Dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; Diethylaminocoumarin; Diethylenetriaminepentaacetate; 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dancyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-Dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); Eosin, and derivatives such as eosin and eosin isothiocyanate; Erythrosine, and derivatives such as erythrosine B and erythrosine isothiocyanate; Ethidium;Fluorescein, and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazine-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, QFITC ​​(XRITC); fluoresamine; IR144; IR1446; green fluorescent protein (GF) P); Coral reef fluorescent protein (RCFP); Lissamine (trademark); Lissamine rhodamine, Lucifer Yellow; Malachite green isothiocyanate; 4-Methylumbelliferone; Orthocresolphthalein; Nitrotyrosine; Pararoseaniline; Nile Red; Oregon Green; Phenol Red; β-Phycoerythrin (PE); o-Phthaldaldehyde; Pyrene, and Pyrene, Pyrene butyrate, Succinimidyl 1-Pyrenebutyrate Derivatives such as rate; Reactive Red 4 (Cibacron (trademark) Brilliant Red 3B-A); Rhodamine, and 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lysamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulforhodamine 101 Derivatives such as sulfonyl chloride derivatives (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthenes; carotenoid-protein complexes such as peridinine-chlorophyll protein (PerCP); allophycocyanin (APC); or combinations thereof.

[0046] As described above, in embodiments of the method, data signals generated by multiple fluorescence channels of a flow cytometer are measured with respect to a calibration set of beads, and for each of at least a portion of the multiple fluorescence channels, a quantitative index of fluorescence intensity is calculated based on the data signal generated for the calibration set of beads that has the highest fluorescence intensity among the calibration set of beads with respect to the fluorescence channel. In other words, a quantitative index of fluorescence intensity is calculated with respect to the fluorescence channel based on the data signal generated for the calibration set of beads that emits the highest intensity light within the wavelength range that the fluorescence channel is configured to detect when irradiated by the light source corresponding to the fluorescence channel. In some embodiments, the quantitative index of fluorescence intensity is the mean fluorescence intensity. In these cases, the mean fluorescence intensity calculated for each fluorescence channel may be 500 or greater, for example, 1,000 or greater, or 2,000 or greater, or 5,000 or greater. In some embodiments, the mean fluorescence intensity is calculated using the arithmetic mean, geometric mean, and / or median. In some embodiments, the quantitative index of fluorescence intensity is calculated for more than half of the fluorescence channels. In some embodiments, the quantitative index of fluorescence intensity is calculated for all of the fluorescence channels.

[0047] As described above, in embodiments of the method, one or more fluorescence channels are adjusted based on a quantitative fluorescence index calculated for each of the one or more fluorescence channels. In some embodiments, one or more fluorescence channels are adjusted so that the subsequent quantitative fluorescence index calculated for each of the one or more fluorescence channels falls within a predetermined threshold range of a standardized application setting generated for the fluorescence channels and the highest fluorescence intensity bead population of the calibration set corresponding to the fluorescence channels. In some cases, the standardized application setting is MFI, and one or more fluorescence channels are adjusted so that the subsequent MFI calculated for each fluorescence channel from the light emitted from each highest fluorescence intensity bead population of the calibration set falls within a predetermined threshold range of the MFI in the standardized application setting. In some embodiments, all fluorescence channels of the flow cytometer are adjusted so that the subsequent quantitative fluorescence index calculated for each of the fluorescence channels falls within a predetermined threshold range of a standardized application setting generated for the fluorescence channels.

[0048] When adjusting one or more fluorescence channels, any number of parameters of the flow cytometer hardware associated with one or more fluorescence channels may be changed. In some embodiments, when adjusting one or more fluorescence channels, the voltage and / or electron gain settings of one or more fluorescence channels are changed. In some embodiments, the adjustment is performed automatically, for example, using a processor that operationally communicates with one or more fluorescence channels.

[0049] In some embodiments, the method further generates a set of standardized application settings for at least some of the multiple fluorescence channels by calculating a quantitative fluorescence index for each fluorescence channel and for each bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel. In some cases, a set of standardized application settings is generated for all fluorescence channels of the flow cytometer. In some embodiments, a set of standardized application settings may be generated using two or more flow cytometers of the same model. In some cases, the method further compares data generated by different flow cytometers using the generated set of standardized application settings. In some embodiments, the method further reduces variability between different flow cytometers using the generated set of standardized application settings.

[0050] In some embodiments, the fluorophores of the bead ensemble calibration set are further determined by the method. In these cases, the fluorophores may be determined based on the instrument hardware of a particular flow cytometer model in which a control composition is used for calibration. For example, the bead ensemble calibration set may be determined based on the light source and fluorescence channels of the flow cytometer. In some cases, computer software may be used to determine a possible set of fluorophores for the bead ensemble calibration set. For example, BD® SpectrumViewer or FlowJo® may be used to perform a preliminary screening of fluorophores that emit fluorescence together across all fluorescence channels of the flow cytometer. In some embodiments, to generate the bead ensemble calibration set, a possible set of fluorophores having a minimum number of fluorophores is selected while satisfying the limitations of the control composition of this disclosure (as described above and herein, for example).

[0051] In some cases, the sample analyzed by this method is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungus, or subset of tissue, cells, or components of an animal, which may, in some cases, be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, or semen. Thus, “biological sample” refers to both a natural organism or a subset of its tissue, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissue, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, airways, gastrointestinal tract, cardiovascular system, urogenital tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be tissue from any type of organism, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, or semen, and in some cases, the sample is a blood sample containing whole blood, such as blood obtained by venipuncture or fingertip puncture (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0052] In some embodiments, the sample source is “mammal” or “mammal,” and these terms are used broadly to describe organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs and rats), and Primates (e.g., humans, chimpanzees and monkeys). In some cases, the subject is human. The method may be applied to samples obtained from human subjects of both sexes and at any developmental stage (i.e., neonates, infants, young children, adolescents, and adults), and in some embodiments, the human subject is young children, adolescents, or adults. While this disclosure may be applied to samples from human subjects, it should be understood that the method may be further carried out on samples from other animal subjects (i.e., “non-human subjects”), including but not limited to birds, mice, rats, dogs, cats, livestock, and horses.

[0053] The cells of interest may be characterized according to various parameters, such as phenotypic characteristics identified by attaching a specific fluorescent label to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. Various cells may be characterized using the method of the subject. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythrocytes. Target cells of interest include cells having a convenient cell surface marker or antigen that may be taken up by a convenient affinity agent or its conjugate, or to which a label may be affixed. For example, target cells may contain cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0054] When performing the method described in the subject, a certain amount of the initial fluid sample is injected into the flow cytometer. The amount of sample injected into the flow cytometer may vary, for example, within the range of 0.001 mL to 1000 mL, for example 0.005 mL to 900 mL, for example 0.01 mL to 800 mL, for example 0.05 mL to 700 mL, for example 0.1 mL to 600 mL, for example 0.5 mL to 500 mL, for example 1 mL to 400 mL, for example 2 mL to 300 mL, for example 5 mL to 100 mL.

[0055] In the methods according to the embodiments of this disclosure, labeled particles (e.g., target cells) in a sample are counted and optionally sorted. When carrying out the method of the subject, first, a fluid sample containing particles is introduced into the flow nozzle of the system. As the particles exit the flow nozzle, each particle passes through a sample investigation area irradiated with a light source substantially one at a time, and the results of light scattering parameter measurements, and optionally fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements), are recorded separately for each particle as desired. Depending on the characteristics of the flow stream being investigated, the light may be irradiated to a flow stream of 0.001 mm or more, for example, 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more. In one embodiment, the method irradiates a planar cross section of the flow stream within the sample investigation area with, for example, a laser (as described above). In other embodiments, the method involves irradiating a flow stream of a predetermined length within the sample investigation area to correspond to the irradiation profile of a diffuse laser beam or lamp.

[0056] In one embodiment, the flowstream is irradiated at or near the nozzle orifice of the flow cell by the method. For example, the flowstream may be irradiated at a position of about 0.001 mm or more from the nozzle orifice, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more from the nozzle orifice by the method. In one embodiment, the flowstream is irradiated right next to the nozzle orifice of the flow cell by the method.

[0057] In embodiments of the method, a detector such as a photomultiplier tube (PMT) is used to record the light passing through each particle (sometimes referred to as forward scattered light), the light reflected perpendicular to the direction of the particle flow passing through the detection area (sometimes referred to as orthogonal scattered light or side scattered light), and, if the particles are labeled with one or more fluorescent markers, the fluorescence emitted from the particles when they pass through the detection area and are irradiated by an energy source is recorded. Each of the forward scattered light (FSC), side scattered light (SSC), and fluorescence emission has separate parameters for each particle (or "event"). Thus, for example, two, three, or four parameters may be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle can be analyzed in real time as desired or stored in data storage and analysis means such as a computer.

[0058] In one embodiment, particles are detected and specifically identified by exposing them to excitation light as desired and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted by the detection channels used to identify the particles and their associated binding complexes may be measured after excitation by a single light source, or individually after excitation by different light sources. When particle labels are excited using separate excitation light sources, the particle labels may be selected so that all particle labels can be excited by each of the excitation light sources used.

[0059] One embodiment of the method further includes, for example, computer-aided data acquisition, analysis, and recording, where multiple data channels record data from each detector regarding the light scattering and fluorescence emitted by each particle as it passes through the sample examination area of ​​the particle sorting module. In these embodiments, during analysis, particles are classified and counted such that each particle exists as a set of digitized parameter values. The system of the subject may be configured to trigger on selected parameters to distinguish the particle of interest from background and noise. "Trigger" refers to a preset threshold for parameter detection, which may be used as a means to detect when a particle has passed through a light source. When an event exceeding the threshold of the selected parameter is detected, the collection of light scattering and fluorescence data for the particle is triggered. Data for particles or other components in the analytical medium that cause a reaction below the threshold is not acquired. The trigger parameter may be the detection of forward scattered light produced when a particle passes through a light beam. In this way, the flow cytometer detects and collects light scattering and fluorescence data for the particle.

[0060] Subsequently, a specific subpopulation of interest is further analyzed by "gating" based on data collected for the entire population. The data is plotted to separate the subpopulation as best as possible in order to select the appropriate gate. This procedure may be performed by plotting forward scattered light (FSC) versus side-scattered (i.e., orthogonal) scattered light (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles (i.e., cells within the gate) is selected, and particles not within the gate are excluded. If desired, the gate may be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Then, only those particles within the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to calculate the number of particles of interest in the sample.

[0061] In the methods of interest, particles may also be used in research, clinical trials, or treatment. In some embodiments, the methods of the subject matter obtain individual cells prepared from biological samples of a target fluid or tissue. For example, the methods of the subject matter obtain cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods of the subject matter obtain cells from fluid or tissue samples used for therapeutic purposes. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissues, is prepared and introduced into a subject as a therapeutic procedure. Conditions that can be treated by administration of flow cytometry-selected samples include, but are not limited to, blood disorders, immune system disorders, and organ damage.

[0062] A typical cell therapy protocol may include steps such as sample collection, cell isolation, genetic modification, culture, in vitro growth, cell harvesting, sample volume reduction, sample washing, in vivo preservation, storage, and introduction of cells into the subject. The protocol may be initiated by collecting viable cells and tissues from the subject's tissue source to produce a cell and / or tissue sample. Samples may be collected by any appropriate procedure, such as administering a cell recruiter to the subject, drawing blood from the subject, or removing bone marrow from the subject. After sample collection, cell enrichment may be performed by several methods, such as centrifugation-based methods, filter-based methods, elutriation, magnetic separation, or fluorescence-activated cell sorting (FACS). In some cases, the enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and grown in vitro. In some cases, the cells may be preserved, for example, cryopreserved, and stored for future use. When ready for use, the cells may be thawed and then administered to the patient, for example, by injecting the cells into the patient.

[0063] Figure 1A is a flowchart for carrying out a method for calibrating a flow cytometer according to one embodiment. Step 101 involves irradiating a reference composition of the Disclosure (e.g., as described above and herein) with one or more light sources of the flow cytometer. Step 102 involves measuring the data signals generated by the multiple fluorescence channels of the flow cytometer for a calibration set of beads. Step 103 involves calculating a quantitative index of fluorescence intensity for each of at least a portion of the multiple fluorescence channels, based on the data signal generated for the bead set of the calibration set that has the highest fluorescence intensity among the bead set of the calibration set with respect to the fluorescence channel. Step 104 involves adjusting one or more fluorescence channels based on the quantitative fluorescence index calculated for each of the one or more fluorescence channels.

[0064] Figure 1B is a flowchart for carrying out a method for generating a set of standardized application settings used to calibrate a flow cytometer according to one embodiment. Step 111 involves irradiating a reference composition of the Disclosure (e.g., as described above and herein) with one or more light sources of the flow cytometer. Step 112 involves measuring the data signals generated by the fluorescence channels of the flow cytometer for a calibration set of bead ensembles. Step 113 involves calculating a quantitative index of fluorescence intensity for each of at least a portion of the fluorescence channels, based on the data signal generated for the bead ensemble of the calibration set having the highest fluorescence intensity among the bead ensembles of the calibration set with respect to the fluorescence channel. Step 114 involves generating a set of standardized application settings by calculating a quantitative index for at least a portion of the fluorescence channels, for each fluorescence channel and for the bead ensemble with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel.

[0065] Flow cytometer devices and flow cytometer systems Aspects of the present disclosure further include flow cytometers and flow cytometer systems, including a computer-controlled system, for carrying out embodiments of the above-described methods. Aspects of flow cytometers and flow cytometer systems include one or more light sources configured to irradiate beads in a flow stream of a flow cytometer, a plurality of fluorescence channels configured to generate data signals from light received from the irradiated beads, and a processor to which memory is operably coupled, the memory storing instructions that, when executed by the processor, cause the processor to measure the data signals generated by the plurality of fluorescence channels with respect to a control composition including a calibration set of bead ensembles (each bead ensemble in the calibration set has a different fluorophore attached to its surface, and the calibration set includes a number of bead ensembles less than the number of fluorescence channels), calculate a quantitative index of fluorescence intensity for each of at least a portion of the plurality of fluorescence channels based on the data signal generated for the bead ensemble in the calibration set having the highest fluorescence intensity with respect to the fluorescence channel, and adjust one or more fluorescence channels based on the quantitative index calculated for each of the plurality of fluorescence channels.

[0066] The embodiments of flow cytometers and flow cytometer systems covered include flow cytometers having one or more light sources and multiple fluorescence channels, as described above and herein. In some embodiments, the processor is fully integrated with the flow cytometer (i.e., the flow cytometer comprises one or more light sources, multiple fluorescence channels, and a processor, as described above and herein). In other embodiments, the processor may be located outside the flow cytometer. In these cases, the processor is configured to receive data signals from the multiple fluorescence channels and to adjust one or more fluorescence channels, for example, by communicating with the flow cytometer.

[0067] In some embodiments, the processor may be a collection of processors that may or may not operate in parallel. In these cases, some of the processors may be integrated with the flow cytometer, while other processors are operably coupled to the flow cytometer but are located outside the flow cytometer. For example, the processor may have a first processor integrated with the flow cytometer and a second processor located outside the flow cytometer and operably coupled to the first processor, where the first processor performs measurements, the second processor performs calculations, and the first processor performs adjustments (for example, based on adjustment results determined by the second processor). In this regard, the terms “flow cytometer” and “flow cytometer system” may be used interchangeably herein, and embodiments of the subject flow cytometer system may be directly integrated with the subject flow cytometer, while embodiments of the subject flow cytometer may be included in the subject flow cytometer system located outside the flow cytometer of the flow cytometer system, for example, based on user needs and preferences. For example, the integration or externalization may be carried out based on, for instance, constraints on processing or computing power, a desire to incorporate user feedback, compatibility with existing equipment, or labor constraints.

[0068] In some embodiments, one or more fluorescence channels are adjusted such that the subsequent quantitative fluorescence index calculated for each of the one or more fluorescence channels falls within a predetermined threshold range of a standardized application setting generated for the fluorescence channels and the highest fluorescence intensity bead population of the calibration set corresponding to the fluorescence channels. In some embodiments, the voltage and / or electron gain settings of one or more fluorescence channels are changed during adjustment. In some embodiments, the quantitative index of fluorescence intensity is the mean fluorescence intensity (MFI). In some embodiments, the flow cytometer is a spectral flow cytometer.

[0069] In some embodiments, the processor stores instructions that, when executed by the processor, cause the processor to generate a set of standardized application settings for at least some of the multiple fluorescence channels by calculating a quantitative fluorescence index for each fluorescence channel and for each bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel. In some embodiments, the processor stores instructions that, when executed by the processor, cause the processor to compare data generated by different flow cytometers using the generated set of standardized application settings. In some embodiments, the processor stores instructions that, when executed by the processor, cause the processor to reduce variability between different flow cytometers using the generated set of standardized application settings.

[0070] In some embodiments, the processor may be configured to automatically perform the flow cytometer calibration method described above. In some cases, the flow cytometer system further comprises one or more computers for full or partial automation of the method described herein. In some embodiments, the system comprises a computer having a computer-readable storage medium in which computer programs are stored.

[0071] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard or mouse. The processing module has a processor that can access memory in which instructions for performing steps of the method of the subject are stored. The processing module may also have an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or will be available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs, which may be written in various programming languages ​​such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system generally works with the processor to coordinate and execute the functions of other components of the computer. The operating system further provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all according to known techniques. In some embodiments, the processor has analog electronic equipment that provides feedback control, such as negative feedback control.

[0072] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or diskette drives. Such types of memory storage devices generally read from and / or write to program storage media such as compact disks (not shown). Any of these program storage media, or any other program storage media currently in use or to be developed in the future, may be considered computer program products. As is understood, these program storage media generally store computer software programs and / or data. Computer software programs, also called computer control logic, are generally stored in system memory and / or in program storage devices used in conjunction with memory storage devices.

[0073] In some embodiments, a computer program product is described that includes a computer-usable medium in which control logic (a computer software program including program code) is stored. When executed by a processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of hardware state machines for performing the functions described herein will be apparent to those skilled in the art.

[0074] Memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic device, optical device, or solid-state storage device (including magnetic disks or optical disks or tapes or RAM or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium that stores the necessary program code. The program may be provided remotely to the processor via a communication channel, or may be pre-recorded in a computer program product such as memory, or on other portable or fixed computer-readable storage media using any of those devices connected to memory. For example, a magnetic disk or optical disk may store a program and may be read by a disk writer / reader. The system of this disclosure further includes, for example, a program in the form of a computer program product, and algorithms for use in carrying out the methods described above. The program of this disclosure may be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as magnetic disks, hard disk storage media and magnetic tapes; optical storage media such as CD-ROMs; electromechanical storage media such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0075] The processor may also access communication channels to communicate with the user at a remote location. A remote location means that the user does not have direct contact with the system, but rather relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0076] In some embodiments, the system relating to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, and wireless communication includes, but is not limited to, radio frequency (RF) communication, such as radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0077] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, e.g., USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, enabling data communication between the system of the subject and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0078] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the system of the subject to communicate with other devices, such as computer terminals and / or networks, communicative mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with it.

[0079] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet Protocol (IP) via a mobile phone network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0080] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11, Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer; or a larger device, such as a desktop computer or electrical equipment. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as buttons, a keyboard, a mouse, or a touchscreen.

[0081] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device using one or more of the communication protocols and / or communication mechanisms described above, for example, data stored in the subject system, such as any data storage unit.

[0082] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may generally be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be implemented using a network or other type of remote communication in alternative embodiments. The output manager may further provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone or satellite network, according to known techniques. The presentation of data by the output manager may be carried out according to various known techniques. As some examples, the data may include SQL, HTML or XML documents, email or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve additional SQL, HTML, XML or other documents or data from a remote source. One or more platforms present within the subject system are typically a class of computers commonly referred to as servers, but may be any type of known or future computer platform. However, the platforms may also be mainframe computers, workstations, or other computer types. The platforms may be connected via any type of known or future cabling, wireless or other communication systems, whether networked or not. The platforms may be located in the same location or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be used on any of the computer platforms. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android®, SGI IRIX®, Oracle Solaris®, and others.

[0083] In some embodiments, the flow cytometer of the subject matter of this disclosure comprises a flow cell. The flow cell of interest has a cuvette configured to carry particles in a flow stream. As used herein, “flow cell” is used in the conventional sense to refer to an element having a channel for a liquid flow stream to carry particles in a sheath fluid. The cuvette of interest has a passage (i.e., a channel) through the cuvette. The flow stream in which the channel is formed may contain a liquid sample injected from a sample tube. In some cases, the flow cell has a light-transmitting channel. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed of silica, such as fused silica. In some cases, the flow cell is configured to be irradiated with light from a light source at one or more investigation points. As used herein, “investigation point” refers to an area within the flow cell where particles are irradiated with light from a light source for analysis, for example. The size of the investigation points may vary as desired. For example, if 0 μm represents the axis of light emitted by the light source, the investigation points may be within the range of -50 μm to 50 μm, for example, -25 μm to 40 μm, or for example, -15 μm to 30 μm. Depending on certain considerations (e.g., the number and arrangement of lasers), there may be multiple irradiation points within the flow cell.

[0084] In some embodiments, the flow cell has a sample injection port configured to supply a sample to the flow cell, or is configured to be used in conjunction with such a sample injection port. In embodiments, the sample injection system is configured to supply a suitable flow of the sample to the internal chamber (i.e., the flow path) of the flow cell. Depending on the desired characteristics of the flowstream, the flow rate of the sample delivered to the flow cell chamber by the sample injection port may be 1 μL / min or more, for example 2 μL / min or more, for example 3 μL / min or more, for example 5 μL / min or more, for example 10 μL / min or more, for example 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, for example 100 μL / min or more, and in some cases, the flow rate of the sample delivered to the flow cell chamber by the sample injection port may be 1 μL / second or more, for example 2 μL / second or more, for example 3 μL / second or more, for example 5 μL / second or more, for example 10 μL / second or more, for example 15 μL / second or more, for example 25 μL / second or more, for example 50 μL / second or more, for example 100 μL / second or more.

[0085] The sample injection port may be an orifice provided in the wall of the internal chamber, or a tube provided at the proximal end of the internal chamber. If the sample injection port is an orifice provided in the wall of the internal chamber, the sample injection port orifice may have any suitable shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal shapes, curved cross-sectional shapes such as circular and oval shapes, and irregular shapes such as a parabolic bottom joined to a planar top. In one embodiment, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and may have an opening in the range of 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, for example 1.25 mm to 1.75 mm, for example 1.5 mm.

[0086] In some cases, the sample injection port is a tube located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a tube positioned such that its orifice is aligned with the orifice of the flow cell. If the sample injection port is a tube positioned so as to be aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube may have any suitable shape, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curvilinear cross-sectional shapes such as circles and ovals, and irregular shapes such as a parabolic bottom joined to a planar top. The orifice of the tube may vary depending on its shape and may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may have a bevel angle in the range of 1° to 10°, for example 2° to 9°, for example 3° to 8°, for example 4° to 7°, or a bevel angle of 5°.

[0087] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, for example, together with the sample, into the internal chamber of the flow cell, thereby creating a layered flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of the sheath fluid delivered to the chamber of the flow cell may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more.

[0088] In some embodiments, the sheath fluid injection port is an orifice provided in the wall of the internal chamber. The orifice of the sheath fluid injection port may have any suitable shape, and the cross-sectional shapes of interest include, but are not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ovals, and irregular shapes such as a parabolic bottom joined to a planar top. The size of the orifice of the sheath fluid injection port may vary depending on the shape, and may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm.

[0089] As described above, the flow cytometer of this disclosure comprises a light source configured to irradiate particles in the flow stream at an investigation point in the flow cell. The number of light sources in the flow cytometer may vary. In some embodiments, the flow cytometer comprises one light source. Alternatively, the flow cytometer may optionally comprise multiple light sources. In some such cases, the number of light sources is in the range of 2 to 10, e.g., 2 to 5, e.g., 2 to 4. Any convenient light source may be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, e.g., an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer of the subject is equipped with a dye laser, such as a stilbene laser, coumarin laser, or rhodamine laser. In yet other cases, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser, copper laser, or gold laser, and combinations thereof. In other cases, the flow cytometer of the subject comprises solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0090] A laser light source according to one embodiment may further include one or more optical adjustment components. In one embodiment, the optical adjustment components are positioned between the light source and the flow cell and may include any device that can change the spatial width of the irradiation from the light source or other characteristics of the irradiation, such as the direction of irradiation, wavelength, beam width, beam intensity and focus. The optical adjustment protocol may include, but is not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols and combinations thereof, any convenient device for adjusting one or more characteristics of the light source. In one embodiment, the flow cytometer of interest includes one or more focusing lenses. The focusing lenses may, in one example, be reduction lenses. In yet another embodiment, the flow cytometer of interest includes optical fibers.

[0091] The light source may be placed at any appropriate distance from the flow cell, for example, the light source and the flow cell may be separated by 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, for example 25 mm or more, or separated by a gap of 100 mm or more. In addition, the light source may be placed at any appropriate angle with respect to the flow cell, for example, within the range of 10 to 90 degrees, for example 15 to 85 degrees, for example 20 to 80 degrees, for example 25 to 75 degrees, for example 30 to 60 degrees, for example 90 degrees.

[0092] In some embodiments, the light source of interest has multiple lasers configured to provide laser light for separate irradiation of a flowstream, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, e.g., fifteen or more lasers configured to provide laser light for separate irradiation of a flowstream. Depending on the desired wavelength of light for irradiating the flowstream, each laser may have various specific wavelengths within the range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. In some embodiments, the laser of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0093] In one embodiment, the light source is a light beam generator configured to generate two or more beams of frequency-shifted light. In some cases, the light beam generator has a laser, a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optical device to generate two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. For example, the laser of the light beam generator of interest includes the lasers listed above.

[0094] The acousto-optic device may be any simple acousto-optic protocol configured to frequency-shift laser light using applied sound waves. In one embodiment, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), arbitrary waveform generator (AWG), or electrical pulse generator.

[0095] In this embodiment, the controller is configured to apply high-frequency drive signals to an acoustic-optical device to generate a desired number of angularly deflected laser beams of the output laser beam, and is configured to apply, for example, 3 or more high-frequency drive signals, 4 or more high-frequency drive signals, 5 or more high-frequency drive signals, 6 or more high-frequency drive signals, 7 or more high-frequency drive signals, 8 or more high-frequency drive signals, 9 or more high-frequency drive signals, 10 or more high-frequency drive signals, 15 or more high-frequency drive signals, 25 or more high-frequency drive signals, 50 or more high-frequency drive signals, and is configured to apply, for example, 100 or more high-frequency drive signals.

[0096] In some cases, to generate an intensity profile of the angularly deflected laser beam of the output laser beam, the controller is configured to apply a high-frequency drive signal having various amplitudes within the following ranges: for example, approximately 0.001 V to approximately 500 V, approximately 0.005 V to approximately 400 V, for example, approximately 0.01 V to approximately 300 V, for example, approximately 0.05 V to approximately 200 V, for example, approximately 0.1 V to approximately 100 V, for example, approximately 0.5 V to approximately 75 V, for example, approximately 1 V to approximately 50 V, for example, approximately 2 V to approximately 40 V, for example, approximately 3 V to approximately 30 V, for example, approximately 5 V to approximately 25 V. In some embodiments, the applied high-frequency drive signal has frequencies within the ranges of approximately 0.001 MHz to approximately 500 MHz, for example, approximately 0.005 MHz to approximately 400 MHz, for example, approximately 0.01 MHz to approximately 300 MHz, for example, approximately 0.05 MHz to approximately 200 MHz, for example, approximately 0.1 MHz to approximately 100 MHz, for example, approximately 0.5 MHz to approximately 90 MHz, for example, approximately 1 MHz to approximately 75 MHz, for example, approximately 2 MHz to approximately 70 MHz, for example, approximately 3 MHz to approximately 65 MHz, for example, approximately 4 MHz to approximately 60 MHz, for example, approximately 5 MHz to approximately 50 MHz.

[0097] In one embodiment, the controller has a processor to which the memory is operablely connected, such that the memory stores instructions, when executed by the processor, for generating an output laser beam including an angle-deflected laser beam having a desired intensity profile. For example, the memory may have instructions for generating two or more angle-deflected laser beams of the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, e.g., different intensities, e.g., the memory may have instructions for generating 100 or more angle-deflected laser beams of different intensities.

[0098] In one embodiment, the controller has a processor to which the memory is operablely connected, such that the memory stores instructions for the processor to generate an output laser beam whose intensity increases from the center to the edge of the output laser beam along the horizontal axis when executed by the processor. In these cases, the intensity of the angular deflection laser beam at the center of the output beam may be in the range of 0.1% to about 99% of the intensity of the angular deflection laser beam at the edge of the output laser beam along the horizontal axis, for example, in the range of 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%, or, for example, in the range of about 10% to about 50% of the intensity of the angular deflection laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor to which the memory is operablely connected, such that the memory stores instructions for the processor to generate an output laser beam whose intensity increases from the edge to the center of the output laser beam along the horizontal axis when executed by the processor. In these cases, the intensity of the angular deflection laser beam at the edge of the output beam may be in the range of 0.1% to about 99% of the intensity of the angular deflection laser beam at the center of the output laser beam along the horizontal axis, for example, in the range of 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%, or, for example, in the range of about 10% to about 50% of the intensity of the angular deflection laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller has a processor to which the memory is operablely coupled, such that the memory stores instructions for the processor to generate an output laser beam having a Gaussian intensity profile along the horizontal axis when executed by the processor.In yet another embodiment, the controller has a processor to which the memory is operablely coupled, such that the memory stores instructions for the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis when executed by the processor.

[0099] In some embodiments, the light beam generator of interest may be configured to generate spatially separated angular deflection laser beams of the output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angular deflection laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, e.g., 1000 μm or more, e.g., 5000 μm or more, e.g., 5000 μm or more. In some embodiments, the system is configured to generate an angular deflection laser beam of the output laser beam that overlaps, for example, adjacent angular deflection laser beams along the horizontal axis of the output laser beam. The overlap of adjacent angle-deflected laser beams (e.g., beam spot overlap) may be 0.001 μm or more, for example 0.005 μm or more, for example 0.01 μm or more, for example 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm or more, for example 1 μm or more, for example 5 μm or more, for example 10 μm or more, for example 100 μm or more.

[0100] In some cases, a light beam generator configured to generate two or more beams of frequency-shifted light is included herein by reference in U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 1,000,6852, 1,003,6699, 1,007,8045, 1,022,2316, 1,028,8546, 1,032,4019, 1,040,8758, 1,045,1538, 1,062,0111, 1,068,4211, 1,084,5295, 1,093,5482, and U.S. Patent Nos. 1,093,5482, which are incorporated herein by reference. It includes a laser excitation module as described in U.S. Patent No. 10935485, U.S. Patent No. 11105728, U.S. Patent No. 11280718, U.S. Patent No. 11327016, U.S. Patent No. 11366052, U.S. Patent No. 11371937, U.S. Patent No. 11692926, U.S. Patent No. 11630053, U.S. Patent No. 11774343, U.S. Patent No. 11940369, and U.S. Patent No. 11946851.

[0101] As described above, the flow cytometer includes a photodetector configured to collect light emitted from an irradiated particle. The photodetector is configured to detect particle-modulated light transmitted by an optical fiber focusing element and generate a signal based on the characteristics of the light (e.g., intensity). For example, one or more particle-modulated photodetectors may have one or more side-scatter photodetectors for detecting light of side-scatter wavelengths (i.e., light refracted and reflected by the surface and internal structure of the particle). In some embodiments, the flow cytometer includes one side-scatter photodetector. In other embodiments, the flow cytometer includes multiple side-scatter photodetectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more side-scatter photodetectors.

[0102] Any convenient detector for detecting the light to be collected may be used in the side scatter light detector described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors within the detector, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoreistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the light to be collected is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, for example 0.01 cm 2 ~10 cm 2 、 for example 0.05 cm 2 ~9 cm 2 、 for example 0.1 cm 2 ~8 cm 2 、 for example 0.5 cm 2 ~7 cm 2 、 for example 1 cm 2 ~5 cm 2 and having an active detection surface area in each region within the range of.

[0103] As described above, the subject flow cytometer may include a fluorescence detector or fluorescence channel configured to detect light at one or more fluorescence wavelengths. In other embodiments, the flow cytometer includes a plurality of fluorescence detectors (i.e., fluorescence channels), such as two or more, such as three or more, such as four or more, such as five or more, such as ten or more, such as fifteen or more, such as twenty or more fluorescence detectors (i.e., fluorescence channels).

[0104] Any convenient detector for detecting the collected light may be used with the fluorescence detector described herein. Detectors of interest may include, but are not limited to, light sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), ICCDs (intensified charge-coupled devices), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In some embodiments, the collected light is measured by a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, for example, 0.01 cm². 2 ~10 cm 2 For example, 0.05 cm 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 For example, 1cm 2 ~5cm 2 This is a photomultiplier tube having an active detection surface area in each region within the specified range.

[0105] When the flow cytometer in question is equipped with multiple fluorescence detectors, each fluorescence detector may be identical, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, when the flow cytometer in question is equipped with two fluorescence detectors, in some embodiments the first fluorescence detector is a CCD type device and the second fluorescence detector (or image sensor) is a CMOS type device. In other embodiments both the first and second fluorescence detectors are CCD type devices. In yet another embodiment both the first and second fluorescence detectors are CMOS type devices. In yet another embodiment the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In yet another embodiment the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment both the first and second fluorescence detectors are photomultiplier tubes.

[0106] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure collected light at one or more wavelengths, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, 25 or more different wavelengths, for example, 50 or more different wavelengths, for example, 100 or more different wavelengths, for example, 200 or more different wavelengths, for example, 300 or more different wavelengths, and is configured to measure light emitted from a sample in a flow stream at, for example, 400 or more different wavelengths. In some embodiments, two or more detectors of a module as described herein are configured to measure collected light at the same or overlapping wavelengths.

[0107] In some embodiments, the fluorescence detector of interest (i.e., the fluorescence channel) is configured to measure the collected light over a range of wavelengths (e.g., 200 nm to 1000 nm). In some embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, a flow cytometer may have one or more detectors configured to collect the spectrum of light over one or more wavelengths within the 200 nm to 1000 nm range. In yet another embodiment, the detector of interest is configured to measure the light emitted from a sample in the flow stream at one or more specific wavelengths. For example, the module may have one or more detectors configured to measure light at one or more of the following wavelengths: 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, and 617nm, and any combination thereof. In one embodiment, one or more detectors may be configured to pair with a specific fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0108] The flow cytometer may have any suitable one or more mechanisms for supplying the sheath fluid and sample fluid to the sheath fluid input coupler and the sample fluid input coupler. For example, the sample fluid input coupler may be fluidically connected to a sample fluid line (e.g., tubing) that is fluidically connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidically connected to a sheath fluid line that is fluidically connected to a sheath fluid reservoir. Similarly, the flow cytometer may have any suitable one or more mechanisms for managing waste from the flow stream. The fluid output coupler may be fluidically connected to a waste line that is fluidically connected to a waste reservoir. A fluid management system that may be adapted for use in the subject flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the entire disclosure of which is incorporated herein by reference.

[0109] Appropriate flow cytometry systems are disclosed herein by reference in the following publications: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), FlowCytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1):17-28; Linden, et al., Semin Throm Hemost. 2004 Oct;30(5):502-11; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. Flow cytometry systems described in 24(3):203-255 may be included, but are not limited to, those described.In some cases, the flow cytometry systems of interest include BD Biosciences FACSCanto(trademark) flow cytometer, BD Biosciences FACSCanto(trademark) II flow cytometer, BD Accuri(trademark) flow cytometer, BD Accuri(trademark) C6 Plus flow cytometer, BD Biosciences FACSCelesta(trademark) flow cytometer, BD Biosciences FACSLyric(trademark) flow cytometer, BD Biosciences FACSVerse(trademark) flow cytometer, BD Biosciences FACSymphony(trademark) flow cytometer, BD Biosciences LSRFortessa(trademark) flow cytometer, BD Biosciences LSRFortessa(trademark) X-20 flow cytometer, BD Biosciences FACSPresto(trademark) flow cytometer, BD Biosciences FACSVia(trademark) flow cytometer, and BD Biosciences FACSCalibur(trademark) cell sorter, BD Biosciences FACSCount(trademark) cell sorter, BD Biosciences FACSLyric(trademark) cell sorter, and BD This includes Biosciences Via (trademark) cell sorters, BD Biosciences Influx (trademark) cell sorters, BD Biosciences Jazz (trademark) cell sorters, BD Biosciences Aria (trademark) cell sorters, BD Biosciences FACSAria (trademark) II cell sorters, BD Biosciences FACSAria (trademark) III cell sorters, BD Biosciences FACSAria (trademark) Fusion cell sorters, BD Biosciences FACSMelody (trademark) cell sorters, BD Biosciences FACSymphony (trademark) S6 cell sorters, BD Biosciences FACSDiscover (trademark) cell sorters, and others.

[0110] In some embodiments, the subject system is incorporated herein by reference in its entirety by reference to U.S. Patent Nos. 10663476, 10620111, 10613017, 10605713, 10585031, 10578542, 10578469, 10481074, 10302545, 10145793, 10113967, 10006852, 9952076, 9933341, 9726527, 9453789, 9200334, 9097640, and 9095494. These are flow cytometry systems such as those described in U.S. Patent No. 9092034, U.S. Patent No. 8975595, U.S. Patent No. 8753573, U.S. Patent No. 8233146, U.S. Patent No. 8140300, U.S. Patent No. 7544326, U.S. Patent No. 7201875, U.S. Patent No. 7129505, U.S. Patent No. 6821740, U.S. Patent No. 6813017, U.S. Patent No. 6809804, U.S. Patent No. 6372506, U.S. Patent No. 5700692, U.S. Patent No. 5643796, U.S. Patent No. 5627040, U.S. Patent No. 5620842, U.S. Patent No. 5602039, U.S. Patent No. 4987086, and U.S. Patent No. 4498766.

[0111] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the system described in the subject is as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013), and U.S. Patent No. 9423353, U.S. Patent No. 9784661, and U.S. Patent No. 9983132. U.S. Patent No. 10006852, U.S. Patent No. 10036699, U.S. Patent No. 10078045, U.S. Patent No. 10222316, U.S. Patent No. 10288546, U.S. Patent No. 10324019, U.S. Patent No. 10408758, U.S. Patent No. 10451538, U.S. Patent No. 10620111, U.S. Patent No. 10684211, U.S. Patent No. 10845295, U.S. Patent No. 10935482, U.S. Patent No. 10935485, U.S. Patent No. 11105728, U.S. Patent No. 11280 A flow cytometry system configured to image particles in a flow stream by fluorescence imaging with radio frequency tag emission (FIRE), as described in U.S. Patent No. 718, U.S. Patent No. 11327016, U.S. Patent No. 11366052, U.S. Patent No. 11371937, U.S. Patent No. 11692926, U.S. Patent No. 11630053, U.S. Patent No. 11774343, U.S. Patent No. 11940369, and U.S. Patent No. 11946851 (their disclosures are incorporated herein by reference). In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-responsive particle sorters are described in U.S. Patent No. 1,0324019, U.S. Patent No. 1,0620111, U.S. Patent No. 1,1105728, and U.S. Patent No. 1,1774343, and U.S. Patent Application No. 18 / 537103, U.S. Patent Application No. 18 / 657618, U.S. Patent Application No. 18 / 657623, and U.S. Patent Application No. 18 / 657633, the entirety of which is incorporated herein by reference.

[0112] Figure 2 shows a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 comprises a laser 201 configured to irradiate particles 211 in a flow stream 214 at an investigation point 215 within a flow cell 210. Although one laser is shown in the example in Figure 2, it is understood that multiple lasers may be used. The laser beam from laser 201 is guided to a focusing lens 202, which focuses the laser beam onto the portion of the fluid stream where the particles 211 of the sample in the flow cell 210 are present. The flow cell 210 is part of a fluid system that guides particles in the stream to the focused laser beam, usually one at a time, for investigation. Alternatively, if the flow cytometer is a stream-in-air cytometer, a nozzle top may be used.

[0113] As shown in Figure 2, the flow cell 210 is fluidically connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. The sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a tube (i.e., sheath fluid line) 207. In addition, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a tube (i.e., sample fluid line) 205. The sample injection port 206 is fluidically connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce particles 211 into the flow cell 210. The particles 211 are hydrodynamically focused via the sheath fluid flowing in from the sheath fluid injection port 208, so that a flowstream 214 is formed downstream of the tapered portion 212 of the flow cell 210. Particles emitted from the distal end of the flow cell 210 may be discarded and / or collected via any appropriate protocol. For example, depending on the type of flow cytometry performed, the particles may be collected at the distal end of the flow cell 210, for example, via a waste line. Alternatively, the particles may be sorted.

[0114] Light from one or more laser beams interacts with particles 211 in the sample by diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various different wavelengths depending on the characteristics of the particles, such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles. The fluorescent emission, as well as the diffracted, refracted, reflected, and scattered light, may be sent to one or more detectors. In particular, forward scatter (FSC) is sent to a forward scatter detector 223. The forward scatter detector 223 is positioned slightly axially offset from the direct beam passing through the flow cell 210 and is configured to detect diffracted light, i.e., excitation light that travels mainly forward through or around the particles. The intensity of the light detected by the forward scatter detector 223 depends on the overall size of the particles. The forward scatter detector may have, for example, a photodiode. A scattering bar 222 is positioned between the forward scatter detector 223 and the optical filter 221a. The optical filter 221a may be configured to remove non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scatter light detector 223.

[0115] In addition, side-scattered light (SSC) is detected by a side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the complexity of the particle structure increases. In the example in Figure 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 while allowing non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scattered light detector 224. Further fluorescence detectors 225a to 225c, each configured to detect fluorescence at different wavelengths, are shown. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while allowing light of other wavelengths to pass through. The optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to the first wavelength (or wavelength range) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to the second wavelength (or wavelength range) to the fluorescence detector 225b, while allowing light of the third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. The optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to the second wavelength (or wavelength range) from being detected by the fluorescence detector 225b. In addition, the optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to the third wavelength (or wavelength range) from being detected by the fluorescence detector 225c.

[0116] Those skilled in the art will recognize that the flow cytometer according to the embodiments of this disclosure is not limited to the flow cytometer shown in Figure 2, but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations. For example, although three fluorescence detectors are shown for illustrative purposes in the embodiment of Figure 2, it will be understood that any suitable number of fluorescence detectors may be used.

[0117] During operation, the flow cytometer's operation is controlled by the controller / processor 290, and measurement data from the detector is stored in memory 295 and can be processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is connected to the detector to receive output signals from the detector and may be further connected to the flow cytometer's electrical and electromechanical components to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function unit 297 may be further provided in the system. The memory 295, controller / processor 290, and I / O function unit 297 may be provided entirely as an integrated part of the flow cytometer. In such embodiments, a display may further form part of the I / O function unit 297 to present experimental data to the user of the flow cytometer 200. Alternatively, part or all of the memory 295, controller / processor 290, and I / O function unit 297 may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and the controller / processor 290 can communicate with the flow cytometer wirelessly or via a wired connection. Together with the memory 295 and the I / O function unit 297, the controller / processor 290 may be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0118] The various fluorescent molecules in the fluorescent dye panel used in flow cytometry experiments emit light in their respective characteristic wavelength bands. The specific fluorescent labels used in the experiment, and the associated fluorescence emission bands of the fluorescent labels, may be selected to roughly match the filter window of the detector. The I / O function unit 297 may be configured to receive data relating to a flow cytometry experiment having a panel of fluorescent labels and multiple cell populations having multiple markers (each cell population having a subset of multiple markers). The I / O function unit 297 may be further configured to receive biodata assigning one or more markers to one or more cell populations, marker concentration data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, may be further stored in memory 295. The controller / processor 290 may be configured to evaluate one or more assignments of labels to markers.

[0119] In some embodiments, the subject system is a particle sorting system configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed on 28 March 2017 (the disclosure of which is incorporated herein by reference). In some embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed on 23 December 2019 (the disclosure of which is incorporated herein by reference). In some embodiments, the system for sorting components of a sample comprises a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed on 28 March 2017 (the disclosure of which is incorporated herein by reference).

[0120] In one embodiment, the system is fluorescence imaging using a high-frequency tag emission image-compatible particle sorter, as shown in Figure 3. The particle sorter 300 includes a light irradiation section 300a containing a light source 301 (e.g., a 488 nm laser), the light source 301 generating an output beam of light 301a, which is split into beams 302a and 302b by a beam splitter 302. The light beam 302a is propagated through an acousto-optical device (e.g., an acousto-optic deflector (AOD)) 303 to generate an output beam 303a having one or more angularly deflected beams of light. In some cases, the output beam 303a generated from the acousto-optical device 303 includes a local oscillation beam and multiple high-frequency comb beams. The light beam 302b is propagated through an acousto-optical device (e.g., an acousto-optic deflector (AOD)) 304 to generate an output beam 304a having one or more angularly deflected beams of light. In some cases, the output beam 304a generated from the acousto-optical device 304 includes a local oscillation beam and multiple high-frequency comb beams. The output beams 303a and 304a generated from the acousto-optical devices 303 and 304, respectively, are combined in a beam combiner 305 to generate an output beam 305a, which is transmitted through an optical component 306 (e.g., an objective lens) to irradiate particles in the flow cell 307. In one embodiment, the acousto-optical device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in the beam combiner 305. In some embodiments, a light irradiation system having a light source and an acousto-optical device may further include those described in Schraivogel, et al. (“High-speed fluorescence image-enabled cell sorting” Science (2022), 375 (6578): 315-320) and U.S. Patent Application Publication No. 2021 / 0404943 (the disclosure of which is incorporated herein by reference).

[0121] The output beam 305a irradiates the sample particles 308 propagating through the flow cell 307 (for example, together with the sheath fluid 309) in the irradiation area 310. As shown in the irradiation area 310, multiple beams (for example, angle-bending high-frequency shift beams of light shown as dots across the irradiation area 310) overlap with the reference local oscillation beam (shown as a shaded line across the irradiation area 310). The overlapping beams exhibit beat behavior because they have different optical frequencies, with each beamlet having a different frequency f 1-n This transmits sine wave modulation.

[0122] Light from the irradiated sample is transmitted to a photodetector system 300b having multiple photodetectors. The photodetector system 300b includes a forward scatter photodetector 311 for generating a forward scatter image 311a and a side scatter photodetector 312 for generating a side scatter image 312a. The photodetector system 300b further includes a bright-field photodetector 313 for generating an optical loss image 313a. In some embodiments, the forward scatter photodetector 311 and the side scatter photodetector 312 are photodiodes (e.g., avalanche photodiodes (APDs)). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the irradiated sample is further detected by fluorescence detectors 314-317. In some cases, the photodetectors 314-317 are photomultiplier tubes. Light from the irradiated sample is directed via the beam splitter 320 to the side-scatter detection channel 312 and the fluorescence detection channels 314-317. The photodetection system 300b includes bandpass optical components 321-324 (e.g., dichroic mirrors) for propagating light of predetermined wavelengths to the photodetectors 314-317, respectively. In some cases, optical component 321 is a 534 nm / 40 nm bandpass. In some cases, optical component 322 is a 586 nm / 42 nm bandpass. In some cases, optical component 323 is a 700 nm / 54 nm bandpass. In some cases, optical component 324 is a 783 nm / 56 nm bandpass. The first digit represents the center of the spectral band. The second digit represents the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on both sides from the center of the spectral band, i.e., from 500 nm to 520 nm.

[0123] Data signals generated in response to light detected by the forward scatter detection channel 311, the side scatter detection channel 312, the bright-field detection channel 313, and the fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Based on the data signals generated by processors 350 and 351, images 311a-317a may be generated in each light detection channel. Image-based sorting is performed in response to a sorting signal generated by the sorting trigger 352. The sorting unit 300c has deflection plates 331 for deflecting particles towards the sample container 332 or the waste stream 333. In some cases, the sorting unit 300c is configured to sort particles using a sealed particle sorting module, as described in U.S. Patent Application Publication No. 2017 / 0299493, filed on March 28, 2017 (the disclosure of which is incorporated herein by reference). In one embodiment, the sorting unit 300c has a sorting decision module having a plurality of sorting decision units, as described in U.S. Patent Application Publication No. 2020 / 0256781 (the disclosure of which is incorporated herein by reference).

[0124] In some embodiments, the system is a particle analyzer and can analyze and characterize particles using the particle analysis system 401 (Figure 4), with or without physically sorting the particles into a collection container. Figure 4 is a functional block diagram of the particle analysis system for computational sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid system 402. The fluid system 402 has a sample tube 405 and a moving fluid column within the sample tube through which sample particles 403 (e.g., cells) move along a common sample path 409, or can be connected to the sample tube 405 and the moving fluid column.

[0125] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. During detection, in some embodiments, it is possible to detect light or one or more other properties of a particle 403 as it passes through the monitoring area 407. Figure 4 shows one detection station 408 with one monitoring area 407. In some embodiments of the particle analysis system 401, multiple detection stations may be provided. Furthermore, some detection stations may monitor two or more areas.

[0126] Each signal is assigned a signal value, generating a data point for each particle. As described above, this data may be referred to as event data. The data points may be multidimensional data points containing the values ​​of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0127] The particle analysis system 401 may further comprise a control system 406. The control system 406 may have one or more processors, amplitude control circuits, and / or frequency control circuits. The illustrated control system may be operably associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first time interval, based on the Poisson distribution and number of data points collected by the detection system 404 during a first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may further compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0128] Figure 5 is a functional block diagram of an example of a particle analysis control system, including an analysis controller (i.e., processor) 500 for analyzing and displaying biological events. The analysis controller 500 may be configured to perform various processes for controlling the graphical display of biological events.

[0129] The particle analyzer or particle sorting system 502 may be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or particle sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0130] The analysis controller 500 may be configured to receive bioevent data from a particle analyzer or particle sorting system 502. The bioevent data received from the particle analyzer or particle sorting system 502 may include flow cytometry event data. The analysis controller 500 may be configured to provide a display device 506 with a graphical display including a first plot of bioevent data. The analysis controller 500 may be further configured to render regions of interest as gates around the collection of bioevent data displayed by the display device 506, for example, overlaid on the first plot. In some embodiments, the gates may be logical combinations of one or more illustrated regions of interest drawn on a histogram or bivariate plot of one parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0131] The analysis controller 500 may be further configured to display bio-event data on the display device 506 within the gate differently from other events in the bio-event data outside the gate. For example, the analysis controller 500 may be configured to render the colors of the bio-event data contained within the gate differently from the colors of the bio-event data outside the gate. The display device 506 may be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0132] The analysis controller 500 may be configured to receive gate selection signals from a first input device that identify gates. For example, the first input device may be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 that identify gates to be displayed or manipulated via the display device 506 (for example, by clicking on or inside a desired gate when the cursor is at the desired gate). In some embodiments, the first device may be implemented as a keyboard 508, or as other means for providing input signals to the analysis controller 500, such as a touchscreen, input pen, photodetector, or voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, as shown in Figure 5, the mouse 810 may include a right mouse button and a left mouse button, and the right mouse button and the left mouse button may each generate trigger events.

[0133] In response to a trigger event, the analysis controller 500 can provide input for further processing, such as changing how the data is displayed, which portion of the data is actually displayed on the display device 506, and / or selecting a population of interest for particle sorting.

[0134] In some embodiments, the analysis controller 500 may be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 may be further configured to automatically modify the plot visualization to facilitate gate processing. This modification can be made based on a specific distribution of biological event data received by the analysis controller 500.

[0135] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may be further configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may be further configured by the analysis controller 500 to enable retrieval of biological event data, such as flow cytometry event data.

[0136] The display device 506 may be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that show an overview of the divisions of the plots. The display device 506 may be further configured to change the information displayed in response to input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0137] In some embodiments, the analysis controller 500 can generate a user interface to receive exemplary events for sorting. For example, the user interface may include controls for receiving exemplary events or exemplary images. The exemplary events or images, or exemplary gates, may be provided before the collection of event data for the sample, or based on a first set of events for a portion of the sample.

[0138] Figure 6A is a schematic diagram showing a particle sorting system 600 (e.g., a particle analyzer or particle sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is connected to a fluid tube 601, which may be connected to a nozzle 603, may include a nozzle 603, or may be a nozzle 603. Within the fluid tube 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a line and irradiated by an irradiation source 612 (e.g., a laser) across a monitoring area 611 (e.g., where the laser and stream intersect). The vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to split into multiple droplets 610, some of which contain particles 609.

[0139] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring area 611. The detection station 614 supplies input to a timing circuit 628, which then supplies input to a flash charge circuit 630. At droplet splitting points indicated by a timed droplet delay (Δt), a flash charge may be applied to the moving fluid column 608 so that the droplet of interest carries charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect them into a collection tube or a container such as a multi-well or microwell sample plate, and the wells or microwells may be associated with the droplet of interest in particular. As shown in Figure 6A, the droplets may be collected in a drain container 638.

[0140] A detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet driving signal as the particle of interest passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679039, the entirety of which is incorporated herein by reference. The detection system 616 enables the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 can supply an amplitude signal 620 and / or a phase signal 618, which are then supplied (via amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet-forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be located within a control system.

[0141] In some embodiments, the sorting electronic equipment (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and sorting decision results based on the detected events. The sorting decision results may be included in the event data for the particles. In some embodiments, the detection system 616 and detection station 614 may be implemented as a single detection unit, or they may be communicatively coupled so that event measurements can be collected by either the detection system 616 or the detection station 614 and provided to non-collecting elements.

[0142] Figure 6B is a schematic diagram showing a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in Figure 6B comprises deflection plates 652 and 654. Charge can be applied via stream-charging wires in barbs. This generates a stream of droplets 610 containing particles 609 for analysis. The particles can be irradiated using one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronic equipment (not shown in Figure 6B) or other detection systems. The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets and guide the droplets toward a target collection container (e.g., one of 672, 674, 676, or 678). As shown in Figure 6B, deflection plates 652 and 654 can be controlled to guide particles toward container 674 along a first path 662 or toward container 678 along a second path 668. If the particles are not of interest (e.g., do not show scattering or irradiation information within a specified sorting range), the deflection plates may allow the particles to continue along the flow path 664. Such uncharged droplets may flow into the waste container via a suction device 670 or the like.

[0143] Sorting electronics may be included to initiate the collection of measurement data, receive fluorescence signals related to particles, and determine how to adjust the deflection plates to sort the particles. An exemplary embodiment of the embodiment shown in Figure 6B is a BD FACSAria® flow cytometer, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0144] Figure 7 shows a general configuration of an exemplary computing device 700 according to one embodiment. The general configuration of the computing device 700 shown in Figure 7 includes the arrangement of computer hardware and software components. However, it is not necessary to show all of these common conventional elements in order to provide a valid disclosure. As shown, the computing device 700 comprises a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may further communicate with a memory 770 and may further provide output information for any display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or program, e.g., FlowJo®) stored as execution instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 740 may further accept input from any input device 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, speech recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0145] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that are executed by the processing unit 710 to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Data may be stored in a data storage device 790. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0146] Non-temporary computer-readable storage medium Aspects of this disclosure further include a non-temporary computer-readable storage medium having instructions for carrying out the subject method. The computer-readable storage medium may be used in one or more computers for the full or partial automation of a system for carrying out the method described herein. In some embodiments, the instructions relating to the method described herein may be coded on a computer-readable medium in the form of a “program,” and the term “computer-readable medium” as used herein refers to any non-temporary storage medium involved in providing instructions and data to a computer for execution and processing. Suitable examples of non-temporary storage mediums include magnetic disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray® discs, solid-state disks, flash drives, and network-attached storage (NAS), whether such devices are located inside or outside a computer. A file containing information may be “stored” on a computer-readable medium, and “stored” means recording the information so that it is later accessible and retrievable by a computer. The computer implementations described herein may be executed using programs that can be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Java, Python, Visual Basic, and C++, as well as many other languages.

[0147] In some embodiments, a computer program is stored on a computer-readable storage medium of interest, and the computer program, when loaded into a computer, has instructions for calibrating the flow cytometer (for example, automatically) by irradiating a control composition containing a calibration set of bead ensembles with one or more light sources of a flow cytometer (each bead ensemble in the calibration set has a different fluorophore attached to its surface, and the calibration set contains a number of bead ensembles less than the number of fluorescence channels in the flow cytometer), measuring data signals generated by the fluorescence channels of the flow cytometer with respect to the calibration set of bead ensembles, calculating a quantitative index of fluorescence intensity for each of at least a portion of the fluorescence channels based on the data signal generated for the bead ensemble in the calibration set that has the highest fluorescence intensity among the bead ensembles in the calibration set with respect to the fluorescence channel, and adjusting one or more fluorescence channels based on the quantitative fluorescence index calculated for each of the fluorescence channels.

[0148] kit Aspects of the present disclosure further include a kit, which comprises a storage medium, such as a magneto-optical disk, CD-ROM, CD-R, magnetic tape, non-volatile memory card, ROM, DVD-ROM, Blu-ray® disk, solid-state disk, and network-attached storage (NAS). Any of these programmable storage media, or other programmable storage media currently in use or to be developed in the future, may be included in the subject kit. In some embodiments, the programmable storage medium has instructions for calibrating a flow cytometer, for example, as described above and herein. In some embodiments, the programmable storage medium has instructions for generating a set of standardized application settings for the fluorescence channels of a flow cytometer, for example, as described above and herein. In some embodiments, instructions or a portion thereof contained in a computer-readable medium provided in the subject kit may be implemented as software components of software for analyzing data. In these embodiments, the computer control system according to the present disclosure may function as a software "plug-in" to an existing software package (e.g., FlowJo®).

[0149] In some embodiments, the subject kit may comprise a control composition including a calibration set of bead ensembles, for example, as described above and herein. In some cases, the calibration set of bead ensembles may comprise 12 or fewer ensembles, e.g., 10 or fewer ensembles, or 6 or fewer ensembles, or 4 or fewer ensembles. In some embodiments, the bead ensembles of the calibration set comprise beads having a core, a fluorophore, and a linker configured to adhere to the fluorophore and bind to the core. In some embodiments, the linker binds to the core such that, for example, the fluorophore is covalently bonded to the core via the linker. In some embodiments, the core may comprise an antigen and the linker may comprise an antibody. In some embodiments, the core may comprise polystyrene. In some embodiments, the control composition is contained in a liquid medium comprising, for example, a solvent, a buffer, and / or a stabilizer. In some embodiments, the control composition is lyophilized. In some embodiments, the control composition is stored in a liquid container, for example, a tube or vial. In these cases, the liquid container may be sealed. In some embodiments, the subject kit may comprise, for example, a control composition and a program storage medium containing instructions, as described above and herein.

[0150] In addition to the components described above, the subject kit may further include (in some embodiments) instructions for use. These instructions may be present in the subject kit in various forms, and one or more of these instructions may be present in the kit. One form in which these instructions may be provided is information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, the kit packaging, or accompanying documents. Yet another form of these instructions may be a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), or portable flash drive. Yet another form of these instructions may be a website address, which may be used via the Internet to access the information remotely.

[0151] usefulness The method and system are used for various applications where it is desirable to analyze and optionally sort particulate components in a sample in a fluid medium, such as a biological sample, and then store the sorted product for subsequent applications, such as therapeutic applications. The disclosure is particularly useful when it is desirable to calibrate a flow cytometer having multiple fluorescence channels, for example, by adjusting the voltage and / or electron gain settings of one or more of the fluorescence channels.

[0152] The subject method and system are further used in applications where it is desirable to reduce variability between different flow cytometers and / or to compare data generated by different flow cytometers (or, for example, to compare the same flow cytometer at different time points). Embodiments of the present disclosure are further used when it is desirable to provide a flow cytometer with improved cell sorting accuracy, improved particle collection, improved performance, and reduced setup time.

[0153] Embodiments of this disclosure are used in applications where cells prepared from biological samples may be desirable for use in research, clinical trials, or therapy. In some embodiments, the methods and devices of the subject may facilitate obtaining and / or analyzing individual cells prepared from biological samples of a target fluid or tissue. For example, the methods and systems of the subject may facilitate obtaining cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods and systems of the subject may facilitate obtaining cells from fluid or tissue samples used for therapeutic purposes.

[0154] The following are examples and are not limiting.

[0155] experiment The following examples are provided to give a complete disclosure and explanation of how the present invention is made and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or only those that have been performed. Efforts have been made to ensure accuracy with respect to the figures used (e.g., mean fluorescence intensity (MFI), wavelength, etc.), but some degree of experimental error and deviation should be taken into consideration.

[0156] Experiment 1 Production of control composition An initial screening was performed using the Spectral Populations plugin of FlowJo® (v1 0.1 0.0) to find combinations of fluorescent dyes that would produce emission signals from all photomultiplier tubes (PMTs) of the FACS Symphony A5 SE spectral instrument. The initial screening revealed that using a limited number of unique fluorescent dyes covering the full spectrum from 375 to 810 nm across all laser lines of the spectral instrument enables sufficient signal detection for inter-instrumental standardization of spectral cytometry across all detectors of the spectral instrument. Examples of fluorescent dye combinations sufficient for inter-instrumental standardization of spectral cytometry in the FACS Symphony A5 SE spectral instrument were selected for further analysis. Selected combinations include BUV395, BUV661, BV421, BV480, BV650, FITC, PE, and PE-Cy5 (see, e.g., Figure 1C). After selecting a combination of fluorescent dyes, BD CompBeads were stained with an antibody against human CD4 conjugated to the selected fluorophore.

[0157] Generating standardized application settings Individually stained BD CompBeads were examined in two different FACS Symphony A5 SE spectral instruments configured with the same application settings (3*r-SD), and the MFI of each fluorophore within each PMT was recorded. The average MFI of each detector in both instruments was calculated for each fluorophore to set a target MFI and generate a standardized application setting. The fluorophore giving the highest signal in each channel was identified, and its MFI was assigned as the inter-instrument fluorophore for setting the corresponding channel on the instrument. Fluorophores with a minimum MFI of 1000 were determined to be sufficient for inter-instrument standardization.

[0158] To generate standardized application settings, the tubes of each monochromatic stained bead were examined, and the PMT voltage was adjusted until the target MFI for each detector was reached. The monochromatic stained beads were then examined again, and the resulting MFI was compared to the target MFI (see, for example, Table 1).

[0159] [Table 1-1] [Table 1-2]

[0160] Notwithstanding the attached claims, this disclosure is further defined by the following annotations:

[0161] Note 1. A method for calibrating a flow cytometer equipped with multiple fluorescence channels, (a) A control composition including a calibration set of bead clusters is irradiated using one or more light sources of a flow cytometer, wherein different fluorophores are attached to the surface of each bead cluster in the calibration set, and the calibration set includes a number of bead clusters less than the number of fluorescence channels of the flow cytometer. (b) Measure the data signals generated by multiple fluorescence channels of the flow cytometer with respect to the calibration set of bead populations. (c) For each of at least a portion of the multiple fluorescence channels, a quantitative index of fluorescence intensity is calculated based on the data signal generated for the calibration set bead population that has the highest fluorescence intensity among the calibration set bead population with respect to the fluorescence channel. (d) A method for adjusting one or more fluorescent channels based on a quantitative index calculated for each of the one or more fluorescent channels.

[0162] Appendix 2. The method according to Appendix 1, wherein one or more fluorescence channels are adjusted such that the subsequent quantitative index calculated for each of the one or more fluorescence channels is within a predetermined threshold range of a standardized application setting generated for the fluorescence channel and the bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel.

[0163] Note 3. The method according to Note 1 or 2, wherein the voltage and / or electron gain settings of one or more fluorescence channels are changed during adjustment.

[0164] Appendix 4. The method according to any one of Appendix 1 to 3, wherein each bead cluster in the calibration set has the highest fluorescence intensity among the bead clusters in the calibration set for at least one fluorescence channel of the flow cytometer.

[0165] Note 5. The quantitative indicator of fluorescence intensity is mean fluorescence intensity (MFI), as described in any one of the methods in Notes 1 to 4.

[0166] Note 6. The MFI calculated for each fluorescence channel is 1,000 or more, as described in Note 5.

[0167] Note 7. The flow cytometer is a spectral flow cytometer, as described in any one of the methods in Notes 1 to 6.

[0168] Note 8. The method according to any one of Notes 1 to 7, wherein the number of beads in the calibration set is less than or equal to half the number of fluorescent channels.

[0169] Note 9. The method according to Note 8, wherein the number of beads in the calibration set is less than or equal to one-quarter of the number of fluorescent channels.

[0170] Note 10. The method according to Note 9, wherein the number of beads in the calibration set is less than or equal to one-eighth of the number of fluorescent channels.

[0171] Note 11. The method according to any one of Notes 1 to 10, wherein the multiple fluorescent channels include 10 or more fluorescent channels.

[0172] Note 12. The method according to Note 11, wherein the multiple fluorescence channels include 40 or more fluorescence channels.

[0173] Note 13. The method according to any one of Notes 1 to 12, wherein the multiple fluorescence channels include at least one fluorescence channel for each of two or more emission spectra.

[0174] Appendix 14. The method according to Appendix 13, wherein one or more light sources of the flow cytometer include multiple lasers, each emitting a different wavelength.

[0175] Note 15. The method according to Note 14, wherein the plurality of lasers include at least one laser that emits in the ultraviolet (UV) spectrum, violet spectrum, blue spectrum, green / yellow spectrum, and red spectrum.

[0176] Appendix 16. The method according to Appendix 14 or 15, wherein the multiple fluorescence channels include at least one fluorescence channel for each of two or more excitation spectra.

[0177] Note 17. The method according to Note 16, wherein each of the multiple fluorescence channels differs by 10 nm or more in its respective excitation spectrum or emission spectrum.

[0178] Appendix 18. The method according to Appendix 16 or 17, wherein for each laser of the flow cytometer, multiple fluorescence channels are configured to detect a composite emission spectrum in the range of wavelengths from 20 nm or less longer than the laser wavelength to at least 810 nm.

[0179] Note 19. The method according to any one of Notes 16 to 18, wherein for each laser of the flow cytometer, the calibration set of the bead ensemble includes one or more fluorophores having an excitation spectrum that includes the wavelength of the laser and a composite emission spectrum that extends from a wavelength 20 nm or less longer than the wavelength of the laser to a full fluorescence spectrum at least 810 nm.

[0180] Appendix 20. The flow cytometer is provided with 48 fluorescence channels and 5 lasers, according to any one of the methods described in Appendix 14 to 19.

[0181] Appendix 21. The method according to Appendix 20, wherein the five lasers include lasers emitting at 349 nm, 405 nm, 488 nm, 561 nm, and 637 nm.

[0182] Note 22. The flow cytometer is equipped with a BD FACSymphony® A5 SE cell analyzer, as described in Note 20 or 21.

[0183] Note 23. The calibration set for the bead group includes groups of 8 or fewer, as described in Note 22.

[0184] Note 24. The fluorophores of the bead group calibration set are as described in Note 23, including BUV395, BUV661, BV421, BV480, BV650, FITC, PE, and PE-Cy5.

[0185] Note 25. The bead collection in the calibration set is (i) Core, (ii) Fluorophores, and (iii) Linkers configured to adhere to the fluorophores and bond to the core The method according to any one of appendices 1 to 24, comprising beads having the following characteristics.

[0186] Note 26. The linker is bonded to the core as described in Note 25.

[0187] Note 27. The method described in Note 26, wherein the fluorophore is covalently bonded to the core via a linker.

[0188] Note 28. The method according to Note 25 or 26, wherein the core contains an antigen and the linker contains an antibody.

[0189] Note 29. The core is polystyrene, as described in any one of Notes 25-28.

[0190] Appendix 30. The method according to any one of the appendices 1 to 29, wherein a set of standardized application settings is generated by calculating a quantitative fluorescence index for each fluorescence channel and for each bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel, for at least a portion of the multiple fluorescence channels.

[0191] Appendix 31. The method according to Appendix 30, which generates a set of standardized application settings using two or more flow cytometers of the same model.

[0192] Appendix 32. The method according to Appendix 30 or 31, which involves comparing data generated by different flow cytometers using a set of standardized application settings that have been generated.

[0193] Appendix 33. The method according to any one of the appendices 30-32, which reduces variability between different flow cytometers using a set of standardized application settings that have been generated.

[0194] Appendix 34. A kit for calibrating a flow cytometer, comprising a calibration set of bead ensembles described in any one of Appendices 1 to 33.

[0195] Appendix 35. The kit described in Appendix 34, further comprising instructions for calibrating the flow cytometer in accordance with the method described in any one of Appendix 1 to 33.

[0196] Note 36. The instructions are stored in a non-temporary computer-readable storage medium, as described in Note 35 of the kit.

[0197] Appendix 37. The kit described in any one of the appendices 34-36, further comprising a set of standardized application settings generated in accordance with Appendix 30 or 31.

[0198] Note 38. A set of standardized application settings is stored in a non-temporary computer-readable storage medium, as described in the kit in Note 37.

[0199] Note 39. A flow cytometer, One or more light sources configured to irradiate beads in the flow stream of a flow cytometer, Multiple fluorescence channels configured to generate data signals from light received from irradiated beads, A processor with memory that is operable and It is equipped with, When memory is executed by the processor, the processor, (a) With respect to a control composition containing a calibration set of bead ensembles, the data signals generated by multiple fluorescence channels are measured, wherein different fluorophores are attached to the surface of each bead ensemble in the calibration set, and the calibration set contains a number of bead ensembles less than the number of fluorescence channels. (b) For each of at least a portion of the multiple fluorescence channels, calculate a quantitative index of fluorescence intensity based on the data signal generated for the calibration set bead population that has the highest fluorescence intensity among the calibration set bead population with respect to the fluorescence channel. (c) Adjust one or more fluorescence channels based on quantitative indicators calculated for each of the one or more fluorescence channels. A flow cytometer that stores commands.

[0200] Appendix 40. A flow cytometer as described in Appendix 39, wherein one or more fluorescence channels are adjusted such that the subsequent quantitative index calculated for each of the one or more fluorescence channels is within a predetermined threshold range of a standardized application setting generated for the fluorescence channels and the bead population with the highest fluorescence intensity of the calibration set corresponding to the fluorescence channels.

[0201] Note 41. A flow cytometer as described in Note 39 or 40, wherein the voltage and / or electron gain settings of one or more fluorescence channels are changed during adjustment.

[0202] Appendix 42. A flow cytometer according to any one of the appendices 39 to 41, wherein each bead cluster in the calibration set has the highest fluorescence intensity among the bead clusters in the calibration set for at least one fluorescence channel of the flow cytometer.

[0203] Note 43. The quantitative index of fluorescence intensity is mean fluorescence intensity (MFI), as specified in any one of Notes 39-42 for the flow cytometer.

[0204] Note 44. The flow cytometer described in Note 43 has an MFI of 1,000 or more calculated for each fluorescence channel.

[0205] Note 45. The flow cytometer is a spectral flow cytometer, as described in any one of the notes 39-44.

[0206] Note 46. A flow cytometer as described in any one of Notes 39-45, wherein the number of beads in the calibration set is less than half the number of fluorescent channels.

[0207] Note 47. The flow cytometer described in Note 46, wherein the number of beads in the calibration set is less than or equal to one-quarter of the number of fluorescent channels.

[0208] Note 48. The flow cytometer described in Note 47, wherein the number of beads in the calibration set is less than or equal to one-eighth of the number of fluorescent channels.

[0209] Note 49. A flow cytometer described in any one of Notes 39 to 48, wherein the multiple fluorescence channels include 10 or more fluorescence channels.

[0210] Note 50. The flow cytometer described in Note 49, wherein the multiple fluorescence channels include 40 or more fluorescence channels.

[0211] Note 51. A flow cytometer according to any one of Notes 39 to 50, wherein the multiple fluorescence channels include at least one fluorescence channel for each of two or more emission spectra.

[0212] Appendix 52. The flow cytometer described in Appendix 51, wherein one or more light sources of the flow cytometer include multiple lasers, each laser emitting a different wavelength.

[0213] Note 53. The flow cytometer according to Note 52, wherein the plurality of lasers include at least one laser configured to emit in the ultraviolet (UV) spectrum, violet spectrum, blue spectrum, green / yellow spectrum, and red spectrum.

[0214] Appendix 54. The flow cytometer according to Appendix 52 or 53, wherein the multiple fluorescence channels include at least one fluorescence channel for each of two or more excitation spectra.

[0215] Note 55. The flow cytometer described in Note 54, wherein each of the multiple fluorescence channels differs by 10 nm or more in its respective excitation spectrum or emission spectrum.

[0216] Appendix 56. A flow cytometer according to Appendix 54 or 55, wherein for each laser of the flow cytometer, multiple fluorescence channels are configured to detect a composite emission spectrum in the range of wavelengths from 20 nm or less longer than the laser wavelength to at least 810 nm.

[0217] Note 57. For each laser of the flow cytometer, the calibration set of the bead ensemble includes one or more fluorophores having an excitation spectrum that includes the wavelength of the laser and a composite emission spectrum that extends from a wavelength 20 nm or less longer than the wavelength of the laser to at least 810 nm in full fluorescence spectrum, as described in any one of Notes 54 to 56.

[0218] Note 58. The flow cytometer is a flow cytometer described in any one of Notes 52 to 57, comprising 48 fluorescence channels and 5 lasers.

[0219] Appendix 59.5 The flow cytometer according to Appendix 58, comprising lasers configured to emit light at 349 nm, 405 nm, 488 nm, 561 nm, and 637 nm.

[0220] Note 60. A flow cytometer as described in Note 58 or 59, equipped with a BD FACSymphony(trademark) A5 SE cell analyzer.

[0221] Note 61. The calibration set of the bead group includes a group of 8 or fewer, as described in Note 60 for the flow cytometer.

[0222] Note 62. The flow cytometer described in Note 61, wherein the fluorophores of the bead group calibration set include BUV395, BUV661, BV421, BV480, BV650, FITC, PE, and PE-Cy5.

[0223] Note 63. The bead collection in the calibration set is (i) Core, (ii) Fluorophores, and (iii) Linkers configured to adhere to the fluorophores and bond to the core A flow cytometer according to any one of appendices 39 to 62, comprising beads having the following properties.

[0224] Note 64. The linker is connected to the core, as described in Note 63 for the flow cytometer.

[0225] Note 65. The flow cytometer described in Note 64, wherein the fluorophores are covalently bonded to the core via a linker.

[0226] Note 66. A flow cytometer as described in Note 63 or 64, wherein the core contains an antigen and the linker contains an antibody.

[0227] Note 67. A flow cytometer as described in any one of Notes 63 to 66, wherein the core contains polystyrene.

[0228] Note 68. The flow cytometer according to any one of Notes 39 to 67, wherein the memory further stores instructions that, when executed by the processor, cause the processor to generate a set of standardized application settings by calculating a quantitative fluorescence index for each fluorescence channel and for each bead population with the highest fluorescence intensity of the calibration set corresponding to the fluorescence channel, for at least a portion of the multiple fluorescence channels.

[0229] Note 70. The flow cytometer described in Note 68, wherein the memory, when executed by the processor, further stores instructions causing the processor to compare data generated by different flow cytometers using a set of standardized application settings that have been generated.

[0230] Note 71. The flow cytometer as described in Note 69 or 70, wherein the memory, when executed by the processor, further stores instructions for the processor to reduce variability between different flow cytometers using a set of standardized application settings that have been generated.

[0231] Note 72.(a) A control composition including a calibration set of bead ensembles is irradiated using one or more light sources of a flow cytometer, wherein different fluorophores are attached to the surface of each bead ensemble in the calibration set, and the calibration set includes a number of bead ensembles less than the number of fluorescence channels of the flow cytometer. (b) Measure the data signals generated by multiple fluorescence channels of the flow cytometer with respect to the calibration set of bead populations. (c) For each of at least a portion of the multiple fluorescence channels, a quantitative index of fluorescence intensity is calculated based on the data signal generated for the calibration set bead population that has the highest fluorescence intensity among the calibration set bead population with respect to the fluorescence channel. (d) Adjust one or more fluorescence channels based on quantitative indicators calculated for each of the one or more fluorescence channels. A non-temporary, computer-readable storage medium that stores instructions for calibrating a flow cytometer, depending on the method.

[0232] Appendix 73. A non-temporary computer-readable storage medium as described in Appendix 72, which adjusts one or more fluorescence channels such that the subsequent quantitative fluorescence index calculated for each of the one or more fluorescence channels is within a predetermined threshold range of a standardization application setting generated for the fluorescence channels and the highest fluorescence intensity bead population of the calibration set corresponding to the fluorescence channels.

[0233] Appendix 74. A non-temporary computer-readable storage medium as described in Appendix 72 or 73, which, when adjusting, changes the voltage and / or electron gain settings of one or more fluorescence channels.

[0234] Appendix 75. A non-temporary computer-readable storage medium according to any one of Appendix 72 to 74, wherein each bead cluster in the calibration set has the highest fluorescence intensity among the bead clusters in the calibration set for at least one fluorescence channel of the flow cytometer.

[0235] Note 76. The quantitative indicator of fluorescence intensity is mean fluorescence intensity (MFI), as described in any one of Notes 72-75 for non-temporary computer-readable storage media.

[0236] Note 77. The MFI calculated for each fluorescence channel is 1,000 or more, for the non-temporary computer-readable storage medium described in Note 76.

[0237] Note 78. A flow cytometer is a spectral flow cytometer, a non-temporary computer-readable storage medium as described in any one of Notes 72 to 77.

[0238] Note 79. A non-temporary computer-readable storage medium as described in any one of Notes 72-78, wherein the number of beads in the calibration set is less than half the number of fluorescent channels.

[0239] Note 80. The non-temporary computer-readable storage medium described in Note 79, wherein the number of beads in the calibration set is less than or equal to one-quarter of the number of fluorescent channels.

[0240] Note 81. The non-temporary computer-readable storage medium described in Note 80, wherein the number of beads in the calibration set is less than or equal to one-eighth of the number of fluorescent channels.

[0241] Note 82. A non-temporary computer-readable storage medium as described in any one of Notes 72 to 81, wherein the multiple fluorescence channels include 10 or more fluorescence channels.

[0242] Note 83. The multiple fluorescence channels include 40 or more fluorescence channels in the non-temporary computer-readable storage medium as described in Note 82.

[0243] Note 84. A non-temporary computer-readable storage medium according to any one of Notes 72 to 83, wherein the multiple fluorescence channels include at least one fluorescence channel for each of two or more emission spectra.

[0244] Note 85. A non-temporary computer-readable storage medium as described in Note 84, wherein one or more light sources of the flow cytometer include multiple lasers, each emitting a different wavelength.

[0245] Appendix 86. The non-temporary computer-readable storage medium as described in Appendix 85, comprising multiple lasers, including at least one laser emitting in the ultraviolet (UV) spectrum, violet spectrum, blue spectrum, green / yellow spectrum, and red spectrum.

[0246] Note 87. A non-temporary computer-readable storage medium according to Note 85 or 86, wherein the multiple fluorescence channels include at least one fluorescence channel for each of two or more emission spectra.

[0247] Note 88. A non-temporary computer-readable storage medium as described in Note 87, wherein each of the multiple fluorescence channels differs by 10 nm or more in its respective excitation spectrum or emission spectrum.

[0248] Appendix 89. A non-temporary computer-readable storage medium according to Appendix 87 or 88, wherein for each laser of the flow cytometer, multiple fluorescence channels are configured to detect a composite emission spectrum in the range of wavelengths from 20 nm or less longer than the laser wavelength to at least 810 nm.

[0249] Appendix 90. For each laser of the flow cytometer, the calibration set of the bead ensemble comprises one or more fluorophores having an excitation spectrum including the wavelength of the laser and a composite emission spectrum extending from a wavelength 20 nm or less longer than the wavelength of the laser to at least 810 nm in full fluorescence spectrum, as described in any one of the appendices 87 to 89.

[0250] Note 91. A non-temporary computer-readable storage medium as described in any one of Notes 85-90, comprising 48 fluorescence channels and 5 lasers.

[0251] Appendix 92.5 The non-temporary computer-readable storage medium as described in Appendix 91, comprising lasers emitting at 349 nm, 405 nm, 488 nm, 561 nm, and 637 nm.

[0252] Note 93. The flow cytometer is equipped with a BD FACSymphony® A5 SE cell analyzer, and is a non-temporary computer-readable storage medium as described in Note 91 or 92.

[0253] Note 94. The calibration set of the bead collection is a non-temporary computer-readable storage medium as described in Note 93, containing a collection of 8 or fewer beads.

[0254] Note 95. The fluorophores of the bead group calibration set are non-temporary computer-readable storage media as described in Note 94, including BUV395, BUV661, BV421, BV480, BV650, FITC, PE, and PE-Cy5.

[0255] Note 96. The bead collection in the calibration set is (i) Core, (ii) Fluorophores, and (iii) Linkers configured to adhere to the fluorophores and bond to the core A non-temporary computer-readable storage medium according to any one of appendices 72 to 95, comprising beads having [a certain property].

[0256] Note 97. The linker is a non-temporary computer-readable storage medium as described in Note 96, which is linked to the core.

[0257] Note 98. A non-temporary computer-readable storage medium as described in Note 97, wherein the fluorophore is covalently bonded to the core via a linker.

[0258] Note 99. A non-temporary computer-readable storage medium as described in Note 96 or 97, wherein the core contains an antigen and the linker contains an antibody.

[0259] Note 100. The core is a non-temporary computer-readable storage medium as described in any one of Notes 96-99, containing polystyrene.

[0260] Appendix 101. A non-temporary computer-readable storage medium according to any one of Appendix 72 to 100, which generates a set of standardized application settings by calculating a quantitative fluorescence index for each fluorescence channel and for each bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel, for at least a portion of the multiple fluorescence channels.

[0261] Appendix 102. A non-temporary computer-readable storage medium as described in Appendix 101, which generates a set of standardized application settings using two or more flow cytometers of the same model.

[0262] Appendix 103. A non-temporary computer-readable storage medium as described in Appendix 101 or 102, which compares data generated by different flow cytometers using a set of standardized application settings that have been generated.

[0263] Appendix 104. A non-temporary computer-readable storage medium described in any one of Appendix 101 to 103, which reduces variability between different flow cytometers using a set of standardized application settings that have been generated.

[0264] While the above disclosure is described in some detail as an illustration and example for ease of understanding, it will be readily apparent to those skilled in the art that, in view of the teachings of this disclosure, several changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0265] Therefore, the foregoing is only illustrative of the essence of the present disclosure. It is clear that those skilled in the art can devise various configurations that embody the essence of the present disclosure and are within the spirit and scope of the present disclosure but are not explicitly described or shown in this specification. Furthermore, all the examples and conditional terms described in this specification are essentially intended to assist the reader in understanding the essence of the present disclosure and the concepts given by the inventors to advance the technical field, and should not be construed as being limited to the specifically described examples and conditions. Moreover, all the descriptions in this specification that describe the essence, aspects, and embodiments of the present disclosure as well as specific examples of the present disclosure are intended to include both structural and functional equivalents of the present disclosure. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, that is, all developed elements that perform the same function regardless of structure. Furthermore, regardless of whether such disclosure is explicitly recited in the claims, the disclosure in this specification is not generally intended to be publicly disclosed.

[0266] Therefore, it is intended that the scope of the present disclosure not be limited to the exemplary embodiments shown and described in this specification. Rather, the scope and spirit of the present disclosure are embodied by the appended claims. With respect to the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is specifically defined to be applied to limitations in a claim only when the exact phrase "means for" or "step for" begins such a limitation in the claim, and 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is not applied when such exact phrase is not used in the claim limitation.

[0267] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 687668, filed on 27 August 2024, pursuant to Section 119(e) of the U.S. Patent Act, and the entire disclosure of that application is incorporated herein by reference.

Claims

1. A method for calibrating a flow cytometer equipped with multiple fluorescence channels, (a) A control composition comprising a calibration set of bead ensembles is irradiated using one or more light sources of the flow cytometer, wherein different fluorophores are attached to the surface of each bead ensemble of the calibration set, and the calibration set comprises a number of bead ensembles less than the number of fluorescence channels of the flow cytometer. (b) With respect to the calibration set of the bead ensemble, measure the data signals generated by the multiple fluorescence channels of the flow cytometer, (c) For each of at least a portion of the plurality of fluorescence channels, a quantitative index of fluorescence intensity is calculated based on the data signal generated for the bead group of the calibration set having the highest fluorescence intensity among the bead group of the calibration set with respect to the fluorescence channel. (d) A method for adjusting one or more fluorescent channels based on the quantitative index calculated for each of the one or more fluorescent channels.

2. The method according to claim 1, wherein the one or more fluorescence channels are adjusted such that a subsequent quantitative index calculated for each of the one or more fluorescence channels is within a predetermined threshold range of a standardized application setting generated for the fluorescence channel and the bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel.

3. The method according to claim 1 or 2, wherein the voltage and / or electron gain settings of one or more fluorescence channels are changed during adjustment.

4. The method according to any one of claims 1 to 3, wherein each bead group of the calibration set has the highest fluorescence intensity among the bead groups of the calibration set with respect to at least one fluorescence channel of the flow cytometer.

5. The method according to any one of claims 1 to 4, wherein the quantitative index of the fluorescence intensity is the mean fluorescence intensity (MFI).

6. The method according to any one of claims 1 to 5, wherein the flow cytometer is a spectral flow cytometer.

7. The method according to any one of claims 1 to 6, wherein the number of beads in the calibration set is less than or equal to half the number of fluorescent channels.

8. The method according to any one of claims 1 to 7, wherein the plurality of fluorescence channels include at least one fluorescence channel for each of two or more emission spectra.

9. The bead collection of the calibration set is (i) Core, (ii) Fluorophores, and (iii) Linker configured to adhere to the fluorophore and bond to the core The method according to any one of claims 1 to 8, comprising beads having the following properties.

10. The method according to any one of claims 1 to 9, wherein a set of standardized application settings is generated by calculating a quantitative index for each fluorescence channel and for each bead population with the highest fluorescence intensity in the calibration set corresponding to the fluorescence channel, for at least a portion of the plurality of fluorescence channels.

11. The method according to claim 10, comprising generating the set of standardized application settings using two or more flow cytometers of the same model.

12. The method according to claim 10 or 11, wherein a set of generated standardized application settings is used to compare data generated by different flow cytometers.

13. The method according to any one of claims 10 to 12, wherein variations between different flow cytometers are reduced using a set of generated standardized application settings.

14. A kit for calibrating a flow cytometer, comprising a bead group calibration set according to any one of claims 1 to 13.

15. It is a flow cytometer, One or more light sources configured to irradiate beads in the flow stream of the flow cytometer, Multiple fluorescence channels configured to generate data signals from light received from irradiated beads, A processor with memory that is operable and It is equipped with, When the memory is executed by the processor, the processor will (a) A control composition comprising a calibration set of bead ensembles is subjected to the measurement of data signals generated by multiple fluorescence channels, wherein different fluorophores are attached to the surface of each bead ensemble in the calibration set, and the calibration set comprises a number of bead ensembles less than the number of fluorescence channels. (b) For each of at least a portion of the plurality of fluorescence channels, a quantitative index of fluorescence intensity is calculated based on the data signal generated for the bead group of the calibration set having the highest fluorescence intensity among the bead group of the calibration set with respect to the fluorescence channel. (c) Adjust the one or more fluorescence channels based on a quantitative index calculated for each of the one or more fluorescence channels. A flow cytometer that stores commands.

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