QC particles and their use

Abstract

The present disclosure provides systems, methods, and kits for improving the characterization (including quality control) of flow cytometers. In one embodiment, a system for characterizing a flow cytometer includes a first standard particle reagent (the first standard particle reagent includes a first particle mixture) and a second standard particle reagent (the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye). The systems, methods, and kits of the present invention offer significant advantages over currently available fluorospheres used for quality control of flow cytometers prior to evaluating nanoparticles (e.g., EVs).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT International patent application filed on March 1, 2024, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 488,067, filed on March 2, 2023, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Introduction Biological laboratory instruments automatically measure analytes and compositions. Using automated instruments to perform blood chemistry and cellular analyses increases laboratory efficiency and test accuracy. As laboratories become more reliant on these instruments, and as the instruments become more sensitive in measuring samples with lower concentrations, the instruments need to be calibrated more frequently to ensure the data generated is accurate and precise.

[0003] One such device is a flow cytometer. In flow cytometry, single cells in solution pass through a beam of laser light, resulting in light scattering in the forward and side directions. The scattered light is then collected and analyzed by a photodetector, and the cells are counted according to the analyzed properties. In addition to measuring scattered light, flow cytometers can also measure fluorescence (e.g., fluorescently labeled antibodies or markers). As flow cytometers have matured, they have become more sensitive and capable of measuring more parameters. With this technological maturity has come complexity, such that flow cytometers can now measure the intensity of multiple fluorescent stains, particle size, and structure as measured by scattering angle virtually simultaneously.

[0004] To ensure the quality of results, flow cytometers require frequent, multiple calibrations and quality controls before the results can be analyzed and reported. Such calibrations are time-consuming, increase costs, and can reduce the instrument's useful daily uptime in a laboratory.

[0005] Reliable evaluation and measurement of nanoparticles (e.g., extracellular vesicles ("EVs")) using a flow cytometer presents many challenges. EVs range in size from 30 to 2,000 nm, from the smallest exosomes to larger apoptotic bodies. Detecting nanoparticles (e.g., EVs) requires that the flow cytometer is calibrated and sensitive enough to accurately measure particles 30 nm or even smaller. Furthermore, when detecting the number of epitopes targeted by fluorophore-conjugated antibodies, the difference in the number of epitopes is several orders of magnitude lower in EVs than in conventional cells. Therefore, when studying nanoparticles like EVs, small changes can dramatically alter the day-to-day performance of the flow cytometer.

[0006] Embodiments of the present invention include characterization systems, quality control processes, and quality control kits for daily monitoring and calibration of flow cytometer instruments (e.g., monitoring and calibration of instrument scatter (nanoscale) and fluorescence sensitivity) to ensure consistent and reliable detection of nanoparticles such as extracellular vesicles.

[0007] Currently available quality control fluorospheres (e.g., CytoFLEX QC fluorospheres (3 µm)) are larger than extracellular vesicles. These beads do not reflect the size of extracellular vesicles and, because they are much brighter, do not demonstrate instrument sensitivity to fluorescently labeled extracellular vesicles. The brightness of the amount of dye internalized in a 3 µm bead is orders of magnitude greater than the brightness of a small number of fluorescently conjugated antibodies (as few as 10) that bind to epitopes on the surface of extracellular vesicles.

[0008] Therefore, the present invention addresses this issue by providing a two-step daily quality control process that includes a system containing a first standard particle reagent (the first standard particle reagent includes a first particle mixture) and a second standard particle reagent (the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye). This process and system allows users to calibrate and standardize the daily performance of a flow cytometer for measuring nanoparticles such as EVs. One embodiment of the process and system performs a calibration of the flow cytometer device to determine laser alignment, laser delay, flow velocity, size scatter sensitivity, and bring the flow cytometer into a performance range.

[0009] One embodiment of the present invention includes a first standard particle reagent comprising non-fluorescent polystyrene beads (e.g., 144 nm to 148 nm) as a quality control size standard, optionally detectable by side scatter at one or more of the following lasers: 405 nm (VSSC1 and VSSC2), 488 nm (BSSC), 561 nm (YSSC), and 638 nm (RSSC). The present invention allows for an optimal gain range (1-3000) for scatter, where non-fluorescent polystyrene beads exhibit linearity at the Fischer distance. In this range of linearity, the flow cytometer is sensitive enough to detect nanoparticles (e.g., extracellular vesicles). For example, by using embodiments of the quality control process and system, it can be determined that the flow cytometer falls within the linearity range and is therefore optimal for detecting exosomes and small EVs. Embodiments of the quality control process and system also allow for characterization of side scatter, laser alignment, and flow velocity.

[0010] An embodiment of the present invention includes a second standard particle reagent, the second standard particle reagent comprising a second particle mixture, the particles having a fluorescent dye. For example, in one embodiment, the second standard particle reagent comprises a mixture of fluorescent quality control fluorosphere beads with a diameter of 450-550 nm that are detectable by fluorescence in one or more of the 405 nm, 488 nm, 561 nm, and 638 nm laser channels. In one embodiment, more than one dye is incorporated into the quality control fluorosphere beads, which have a wide range of emission and excitation for detection on PB450, FITC, PE, APC, APC-A700, and APC-A750. In one embodiment, the second particle reagent containing the quality control fluorosphere beads is useful for determining laser delay and fluorescence alignment of a flow cytometer.

[0011] In an optional embodiment, the process and system include a second standard particle reagent containing eight fluorospheres with different fluorescence intensities for each channel, each containing a set concentration of dye in each bead. The second standard particle reagent contains beads with eight peaks of fluorescence intensity ranging from blank to dim, medium, and bright. Blank fluorospheres containing no dye can optionally be used to determine the minimum background level of autofluorescence. Using this embodiment of the second standard reagent, the quality control fluorosphere mixture allows for the determination of the optimal MFI range for fluorescence that exhibits linearity in the Fischer distance between the set peak and the blank peak. Using this linear range, the flow cytometer is sensitive enough to detect dim fluorescence on labeled extracellular vesicles.

[0012] One embodiment of the present invention has specifications of less than a 5 μs difference in delay settings, less than a 20% percent difference in target gain settings, and less than a 5% percent difference in target median fluorescence intensity. In one embodiment of the present invention, the robust coefficient of variation ("rCV") must be less than 6% in the target detector channel. Summary of the Invention [Means for solving the problem]

[0013] Brief Summary of the Invention The systems, methods, and kits of the present invention offer significant advantages over currently available fluorospheres, which are used for quality control of flow cytometers prior to evaluation of nanoparticles (e.g., EVs). The systems, methods, and kits of the present invention enable flow cytometer characterization that allows for accurate, reliable, and reproducible measurements of nanoparticles. Thus, nanoparticles can be reliably and accurately studied using a flow cytometer after flow cytometer characterization using the systems, methods, and kits disclosed herein.

[0014] A system for the characterization of flow cytometers. One embodiment of the present invention is a system for characterizing a flow cytometer, the system including a first standard particle reagent, the first standard particle reagent comprising a first particle mixture, and a second standard particle reagent, the second standard particle reagent comprising a second particle mixture, the particles having a fluorescent dye. In one embodiment of the system, the first and second standard particle mixtures comprise beads or microparticles. In one embodiment, the standard particle mixture comprises synthetic materials, metal materials, hollow spheres, latex beads, gold nanoparticles, lipid nanoparticles (LNPs), polystyrene beads, hydrogel particles, silica particles, poly(methyl methacrylate) (PMMA) particles, or a combination thereof.

[0015] In one embodiment, the first standard particle mixture comprises a plurality of particle subpopulations, each subpopulation having a different average diameter. In one embodiment, the second standard particle mixture comprises a plurality of particle subpopulations, each subpopulation having a different average diameter. In one embodiment, the standard particle mixture comprises particles having a diameter between 100 nm and 1000 nm.

[0016] In one embodiment, the first standard particle mixture includes a plurality of particle subpopulations, each subpopulation having a different average diameter. In one embodiment, the first standard particle mixture includes particles having diameters between 140 nm and 148 nm. In one embodiment, the standard particle mixture scatters light when excited by a laser between 325 and 808 nm.

[0017] In one embodiment, the standard particle mixture includes beads in which at least one fluorosphere has no dye and at least one bead has a fluorescent dye. In one embodiment, the standard particle mixture includes beads with at least one peak of fluorescent intensity. In one embodiment, the standard particle mixture includes beads with at least two peaks of fluorescent intensity, at least four peaks of fluorescent intensity, or at least eight peaks of fluorescent intensity.

[0018] In one embodiment, the second standard particle mixture includes a dye, the dye comprising a small organic dye, a phycobiliprotein, a quantum dot, a polymeric dye, a fluorescent protein, a tandem dye, or a combination thereof. In one embodiment, the second standard particle includes a dye excitable by a laser having one or more of the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). In one embodiment, the second standard particle includes a dye excitable by a laser between 325 and 808 nm.

[0019] Methods for flow cytometer characterization. One embodiment of the present invention is a method for quality control of a flow cytometer, comprising: (a) loading a first standard particle reagent and a second standard particle reagent according to any one of items 1 to 15 into a flow cytometer; (b) performing the following: (i) evaluating the side scatter sensitivity of at least one side scatter channel of at least one laser in the flow cytometer based on single peak analysis; (ii) evaluating the gain of at least one side scatter channel of at least one laser in the flow cytometer based on single peak analysis; (iii) evaluating the rCV of at least one laser in the flow cytometer based on single peak analysis; (iv) evaluating the rCV of at least one laser in the flow cytometer based on single peak analysis; (v) evaluating the fluorescence sensitivity of at least one fluorescence channel of at least one laser in the flow cytometer based on two-peak fluorescence intensity analysis; (v) evaluating the flow velocity in the flow cytometer based on single-peak analysis; and (vi) comparing the performance (day by day or over time of the same flow cytometer using the same first and second standard particle reagents); and (c) determining, based on the evaluation in step (b), at least one of the following: (i) whether the flow cytometer passes or fails quality control; and (ii) details regarding the performance status of current instrument components.

[0020] In one embodiment, evaluating in step (b) includes evaluating each of steps (b)(i) through (b)(vi). In one embodiment, evaluating in step (b) is performed with an infrared laser and at least one laser having a wavelength less than 808 nm. In one embodiment, evaluating in step (b) is performed using multiple lasers having one or more of the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared).

[0021] In one embodiment, the method of the present invention further comprises generating a quality control report after step (c). In one embodiment, the method for quality control of a flow cytometer is performed at least once a day. In certain embodiments, the method for quality control of a flow cytometer is performed before use of the flow cytometer.

[0022] In one embodiment, the method further comprises (d) removing the quality control solution from the flow cytometer. In one embodiment, the method further comprises (e) performing an enhanced quality control analysis based on the fluorescence intensity of 1, 2, 4, 8, or 12 peaks.

[0023] In one embodiment, a mixture of beads of the present invention are run to determine the distance between noise and the maximum number of peaks that can be separated by each fluorescence channel.

[0024] In one embodiment, the method further comprises (f) generating a quality control report to determine the sensitivity of detecting cellular structures between 30 and 2000 nm. In certain embodiments, the method further comprises (g) loading a test sample to detect fluorescently labeled cellular structures having diameters between 30 and 2000 nm.

[0025] Kit for flow cytometer characterization. One embodiment of the present invention includes a kit for carrying out a method of the present invention, the kit including a system of the present invention, at least one vial for holding a first standard particle reagent, and instructions for using the kit.

[0026] In one embodiment, the kit may further include a second vial for holding a second standard particle reagent solution. [Brief explanation of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1-1]Figure 1 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 1A-1C show the dynamic range of the 144 nm quality control polystyrene beads using the polystyrene particle mix (40-144 nm) standard. Figure 1A shows VSSC-1 intensity by gain (linear). Figure 1B shows VSSC S1 V4 / V1 vs. VSSC-1 gain. Figure 1C shows the range of linearity in scatter detection for VSCC-1, VSCC-2, BSSC, and RSSC. [Figure 1-2] Same as above.

[0028] [Figure 2] Figure 2 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 2A-2C show the correlation of the 144 nm quality control polystyrene beads by testing the 40 nm LOD and E+O 5 / 6 sigma thresholds. More specifically, the 144 nm quality control polystyrene beads were run at a gain against a target MFL using a threshold of 130,000, and then the median and standard deviation at 144 nm were collected. The 144 nm quality control polystyrene beads were also run at a gain against a target MFL using a threshold of 10, and then the median and standard deviation of the E+O noise peak were collected. Figure 2A shows the 40 nm vs. 144 nm E+O 5 sigma threshold for the VSSC1-H. Figure 2B shows the 10 threshold vs. E+O 5 sigma threshold for the VSSC1-H. Figure 2C shows the 10 threshold at 40 nm versus the E+O 5 sigma threshold in VSSC1-H.

[0029] [Figure 3]Figure 3 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 3A-3E show the correlation of the 144 nm quality control polystyrene beads by testing a 40 nm LOD and an E+O 5 / 6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain of 130,000 relative to the target MFL, and then the median and standard deviation at 144 nm were collected. The 144 nm quality control polystyrene beads were also run at a gain of 10 relative to the target MFL, and then the median and standard deviation of the E+O noise peak were collected. Figures 3A-3E separately show the median and standard deviation of the signal at the same gain but at different thresholds, using a high threshold and then allowing more noise by lowering the threshold to 10. Figure 3A shows the 10 threshold at 144 nm for VSSC1-H. Figure 3B shows the E+O 5 sigma threshold of 144 nm for VSSC1-H. Figure 3C shows the 10 threshold of 40 nm for VSSC1-H. Figure 3D shows the E+O 5 sigma threshold of 40 nm for VSSC1-H. Figure 3E shows the data (including median and noise) for the differential measurement.

[0030] [Figure 4]Figure 4 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 4A-4C show the correlation of the 144 nm quality control polystyrene beads by testing a 40 nm LOD and an E+O 5 / 6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain of 130,000 relative to the target MFL, and then the median and standard deviation at 144 nm were collected. Also, the 144 nm quality control polystyrene beads were run at a gain of 10 relative to the target MFL, and then the median and standard deviation of the E+O noise peak were collected. Figures 4A-4C show the median and standard deviation of the signal at the same gain and threshold. Figure 4A shows the 10 threshold at 144 nm for the VSSC1-H. Figure 4B shows the 40 nm E+O 5 sigma threshold for the VSSC1-H. FIG. 4C shows the data (including median and noise) for the differential measurement.

[0031] [Figure 5-1] Figure 5 illustrates an embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. More specifically, Figures 5A-5E show the relationship between 40 nm and VSSC1 (40 nm), BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm) for an embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads, with data for MFl and standard deviation shown in Figure 5E. Figures 5F-5J show the relationship between 40 nm and VSSC1 (40 nm), BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm) for an embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads, with data for MFl and standard deviation shown in Figure 5J. [Figure 5-2] Same as above.

[0032] [Figure 6]Figure 6 shows an embodiment of the first standard particle reagent containing 142 nm, 144 nm, or 141 nm quality control polystyrene beads. More specifically, rCV data for each embodiment in VSCC-1, VSCC-2, BSCC, and RSCC are shown.

[0033] [Figure 7A] Figure 7 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 7A shows measurements of 40 nm polystyrene beads (FP1 600 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 7B shows measurements of 80 nm polystyrene beads (FP1 600 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 7C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP1 600 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. [Figure 7B] Same as above. [Figure 7C] Same as above.

[0034] [Figure 8A] Figure 8 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 8A shows measurements of 40 nm polystyrene beads (FP1 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 8B shows measurements of 80 nm polystyrene beads (FP1 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 8C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP1 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. [Figure 8B] Same as above. [Figure 8C] Same as above.

[0035] [Figure 9A] Figure 9 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 9A shows measurements of 40 nm polystyrene beads (FP5 600 threshold) for VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 9B shows measurements of 80 nm polystyrene beads (FP5 600 threshold) for VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 9C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP5 600 threshold) for VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. [Figure 9B] Same as above. [Figure 9C] Same as above.

[0036] [Figure 10A] Figure 10 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 10A shows measurements of 40 nm polystyrene beads (FP5 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 10B shows measurements of 80 nm polystyrene beads (FP5 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 10C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP5 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. [Figure 10B] Same as above. [Figure 10C] Same as above.

[0037] [Figure 11A-1]Figure 11 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 11A shows the BV421 fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. The seven peaks are completely separated from noise at gains between 250 and 2000. Figure 11B shows the median gain titration of BV421 for both EP10003 and EP10004 for each of the eight peaks. Both EP10003 and EP10004 demonstrate a linear increase in BV421 median with increasing gain for the separated peaks. Figure 11C shows the staining index gain titration of BV421 for both EP10003 and EP10004 for each of the eight peaks. Figure 11D shows the BV421 MFl ratio (P# / P4) grain titration for both EP1003 and EP1004 for each of the eight peaks. Both EP10003 and EP10004 are linear from 250 to 3000 gain. Figure 11E shows the BV421 MFl ratio (P# / P6) gain titration for both EP10003 and EP10004. [Figure 11A-2] Same as above. [Figure 11A-3] Same as above. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above.

[0038] [Figure 12A-1]Figure 12 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 12A shows the FITC fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. Five peaks are separable from noise at gains between 250 and 2000. Figure 12B shows the median FITC gain titration for both EP10003 and EP10004 for each of P4 through P8. Both EP10003 and EP10004 demonstrate a linear increase in FITC median value with increasing gain for the isolated peaks. Figure 12C shows the FITC staining index titration for both EP10003 and EP10004 for each of peaks P4-P8. Figure 12D shows the FITC MFl ratio (P# / P4) gain titration for both EP10003 and EP10004 for each of P5-P8. Both EP10003 and EP10004 are linear from 500-3000 gain for P5-P8. Figure 12E shows the FITC MFl ratio (P# / P6) gain titration for both EP10003 and EP10004. Both EP10003 and EP10004 are linear from 250-3000 gain for P4-P5 and P7-P8. [Figure 12A-2] Same as above. [Figure 12A-3] Same as above. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above.

[0039] [Figure 13A-1]Figure 13 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 13A shows the PE fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. Five peaks are completely resolved from noise between 250 and 2000 gains, and the sixth peak is separable from 1000 and 3000 gains. Figure 13B shows the median PE gain titration for both EP10003 and EP10004 for each of the six peaks. Both EP10003 and EP10004 demonstrate a linear increase in median PE with increasing gain for the isolated peaks. Figure 13C shows the PE staining index gain titration for both EP10003 and EP10004 for each of the six peaks. Figure 13D shows the PE MFl ratio (P# / P4) grain titration for both EP10003 and EP10004 for each of P1-P8. Both EP10003 and EP10004 are linear from 500 to 3000 gain. Figure 13E shows the PE MFl ratio (P# / P6) gain titration for both EP10003 and EP10004. Both EP10003 and EP10004 are linear from 500 to 3000 gain for P4-P5 and P7-P8. [Figure 13A-2] Same as above. [Figure 13A-3] Same as above. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above.

[0040] [Figure 14A-1]Figure 14 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight-peak (light yellow, yellow, Nile red, purple, blue, sky blue) 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 14A shows the APC fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. P5-P8 are fully separable over a gain range of 250-1000 for EP10003, and P5-P8 are fully separable over a gain range of 250-500 for EP10004. Figure 14B shows the median APC gain titrations for both EP10003 and EP10004 for each of the eight peaks. EP10003 demonstrates a linear increase in median APC with increasing gain for the isolated peaks. Figure 14C shows the staining index gain titrations for APC for both EP10003 and EP10004 for each of P5-P8. Figure 14D shows the APC MFl ratio (P# / P6) grain titrations for both EP10003 and EP10004 for each of P5, P7, and P8. [Figure 14A-2] Same as above. [Figure 14A-3] Same as above. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above.

[0041] [Figure 15A-1]Figure 15 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 15A shows the APC-A700 fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. Peaks 5 through 8 are completely separated from noise at gains of 250 through 1000. Figure 15B shows the median gain titration of the APC-A700 for both EP10003 and EP10004 for each of P5 through P8. Both EP10003 and EP10004 demonstrate a linear increase in APC-A700 median with increasing gain for P6-P8. Figure 15C shows the staining index gain titration of APC-A700 for both EP10003 and EP10004 for each of P5-P8. The gain range for linearity is between 500-1000. Figure 15D shows the APC-A700 MFl ratio (P# / P6) grain titration for both EP1003 and EP1004 for P5, P7, and P8. Both EP10003 and EP10004 are linear from 250-1500 gain. [Figure 15A-2] Same as above. [Figure 15A-3] Same as above. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above.

[0042] [Figure 16A-1]Figure 16 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 16A shows the APC-A750 fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. The two peaks are completely separated from the noise at gains between 100 and 3000. Figure 16B shows the median gain titration of APC-A750 for both EP10003 and EP10004 for each of P6 through P8. FIG. 16C shows the staining index gain titration of APC-A750 against both EP10003 and EP10004 for each of P6-P8. [Figure 16A-2] Same as above. [Figure 16A-3] Same as above. [Figure 16B] Same as above. [Figure 16C] Same as above.

[0043] [Figure 17] FIG. 17 shows an overview of the EP1 fluorescence dynamic range of FIGS.

[0044] [Figure 18-1] Figure 18 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 18 shows the rCV comparison of 40 nm and 150 nm beads in VSSC1-H at gains of 25, 100, 150, 200, 500, and 1500. [Figure 18-2] Same as above.

[0045] [Figure 19-1]Figure 19 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 19 shows the gain at which other scattering channels saturate for 150 nm polystyrene beads in VSSC1 (10 gain and 200 gain), VSSC2 (10 gain and 3000 gain), BSSC (10 gain and 800 gain), YSSC (10 gain and 1700 gain), and RSSC (10 gain and 1300 gain). [Figure 19-2] Same as above.

[0046] [Figure 20-1] Figure 20 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye. Four-peak 500 nm polystyrene beads were tested. More specifically, the gain at which other scattering channels saturate for the 500 nm four-peak beads was analyzed using VSSC1 (10 gain and 22 gain), VSSC2 (10 gain and 2000 gain), BSSC (10 gain and 50 gain), YSSC (10 gain and 75 gain), and RSSC (10 gain and 35 gain). [Figure 20-2] Same as above.

[0047] [Figure 21] FIG. 21 shows a summary of data collected for both a first standard particle reagent embodiment (the first standard particle reagent includes 150 nm quality control polystyrene beads) and a second standard particle reagent embodiment (the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye (500 nm bead(s))).

[0048] [Figure 22-1] Figure 22 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 22 shows the rCV comparison of 40 nm and 150 nm beads in VSSC1-H at gains of 25, 100, 150, 200, 500, and 1500. [Figure 22-2]Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description While the concepts of the present disclosure will be illustrated and described in detail in the drawings and description herein, the results in the drawings and the description thereof are to be considered illustrative and not restrictive in nature, it being understood that only exemplary embodiments have been shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0050] Unless otherwise defined, scientific and technical nomenclature has the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0051] Those skilled in the art will appreciate that other suitable modifications and adaptations to the systems, methods, and kits described herein will be readily apparent from the description of the disclosure contained herein, in view of the information known to those skilled in the art, and may be made without departing from the scope of the disclosure or any embodiment thereof.

[0052] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0053] Definition. As used herein, "g" stands for gram, "L" stands for liter, "mg" stands for milligram (10-3 gram), and "mL" or "cc" stands for milliliter (10-3 liter). One "μL" is equal to one microliter (10-6 liter). The unit of temperature used herein is degrees Celsius (°C).

[0054] The term "about," when used in conjunction with a numerical value, includes normal variations in measurement as would be expected by one of ordinary skill in the art, and is understood to have the same meaning as "approximately," encompassing typical error ranges such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. Whether modified by the term "about," the claims include equivalents of the quantities.

[0055] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, a reference to a "method" includes having two or more methods that are either the same or different from each other. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise. As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as referring to and including no combination ("or") when otherwise construed.

[0056] For the sake of brevity and simplicity, any range of values ​​set forth herein should be construed as contemplating all values ​​within the range and as supporting a claim reciting any subrange having endpoints that are real values ​​within the specified range in question. As a hypothetical example, the disclosure of a range of 1 to 5 herein shall be construed as supporting a claim to any of the following ranges: 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, and 4 to 5.

[0057] The terms "substantially" or "about" are used herein to express the inherent degree of uncertainty that may result from any quantitative comparison, value, measurement, or other representation. The terms "substantially" or "about" are also used herein to express the degree to which a quantitative representation may vary from the stated reference without resulting in a change in the basic functionality of the subject matter in question.

[0058] As used herein, the terms "comprise," "comprises," and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0059] As used herein, the term "quality control" or "quality controlling" identifies the use of the disclosed compositions, methods, and kits to standardize a flow cytometer to ensure the reliability and accuracy of data collected by the flow cytometer.

[0060] As used herein, the term "characterization" identifies the use of the disclosed compositions, methods, and kits to optimize and / or quality control a flow cytometer to ensure the reliability and accuracy of data collected by the flow cytometer.

[0061] General description. A system for the characterization of flow cytometers.

[0062] One embodiment of the present invention is a system for characterizing a flow cytometer, comprising a first standard particle reagent, the first standard particle reagent comprising a first particle mixture, and a second standard particle reagent, the second standard particle reagent comprising a second particle mixture, the particles having a fluorescent dye. In one embodiment, the standard particle mixture comprises a synthetic material, a metal material, a hollow sphere, a latex bead, a gold nanoparticle, a lipid nanoparticle (LNP), a polystyrene bead, a hydrogel particle, a silica particle, a poly(methyl methacrylate) (PMMA) particle, or a combination thereof. In one embodiment of the system, the first and second standard particle mixtures comprise beads or microparticles. In certain embodiments, the first and second standard particle mixtures comprise the same beads or microparticles (e.g., polystyrene beads), but the beads or microparticles are of different sizes. The present invention is not limited to any particular particles and contemplates the use of particles made of any material suitable for use in a flow cytometer.

[0063] In one embodiment, the first standard particle mixture comprises a plurality of particle subpopulations, each subpopulation having a different average diameter. In one embodiment, the second standard particle mixture comprises a plurality of particle subpopulations, each subpopulation having a different average diameter. In one embodiment, the standard particle mixture comprises particles having a diameter between 100 nm and 1000 nm. In certain embodiments, the standard particle mixture comprises particles having a diameter greater than about 100 nm, greater than about 120 nm, greater than about 140 nm, greater than about 150 nm, greater than about 160 nm, greater than about 400 nm, greater than about 500 nm, or greater than about 600 nm. In certain embodiments, the standard particle mixture comprises particles having a diameter less than about 1000 nm, less than about 550 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, or less than about 100 nm.

[0064] In one embodiment, the first standard particle mixture has particles with diameters between 100 nm and 300 nm, between 120 nm and 200 nm, or between 140 nm and 148 nm. In one embodiment, the standard particle mixture is excited by a laser channel between 325 and 808 nm. In certain embodiments, the standard particle mixture is excited by at least one laser channel, at least two laser channels, at least three laser channels, at least four laser channels, or at least five laser channels. In certain embodiments, only the second standard particle mixture has particles designed to be excitable by the laser channels.

[0065] In one embodiment, the standard particle mixture includes beads in which at least one fluorosphere does not have a dye and at least one bead has a fluorescent dye. In one embodiment, the standard particle mixture includes beads with at least one peak of fluorescent intensity. In one embodiment, the standard particle mixture includes beads with at least two peaks of fluorescent intensity, at least four peaks of fluorescent intensity, or at least eight peaks of fluorescent intensity. In certain embodiments, only the second standard particle mixture includes beads with at least one fluorescent dye.

[0066] In one embodiment, the second standard particle mixture includes a dye, the dye comprising a small organic dye, a phycobiliprotein, a quantum dot, a polymeric dye, a fluorescent protein, a tandem dye, or a combination thereof. In one embodiment, the second standard particles are coated with a dye that can be excited by a laser having one or more of the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). In certain embodiments, the second standard particle mixture includes between 2 and 15 dyes, between 2 and 13 dyes, between 2 and 11 dyes, between 2 and 9 dyes, between 2 and 8 dyes, or between 2 and 7 dyes. In certain embodiments, the second standard particle mixture includes more than 2 dyes, more than 3 dyes, more than 4 dyes, more than 5 dyes, more than 6 dyes, more than 7 dyes, or more than 8 dyes. In certain embodiments, the second standard particle mixture includes fewer than 15 dyes, fewer than 13 dyes, fewer than 11 dyes, fewer than 9 dyes, fewer than 7 dyes, fewer than 5 dyes, or fewer than 3 dyes.

[0067] One embodiment of the present invention is a method for quality control of a flow cytometer, comprising: (a) loading a first standard particle reagent and a second standard particle reagent of the present invention; (b) performing the following: (i) evaluating the side scatter sensitivity of at least one side scatter channel of at least one laser in the flow cytometer based on single peak analysis; (ii) evaluating the gain of at least one side scatter channel of at least one laser in the flow cytometer based on single peak analysis; (iii) evaluating the rCV of at least one laser in the flow cytometer based on single peak analysis; and (iv) analyzing the fluorescence intensity of at least two peaks. (v) evaluating the fluorescence sensitivity of at least one fluorescence channel of at least one laser in the flow cytometer based on single peak analysis; (v) evaluating the flow velocity in the flow cytometer based on single peak analysis; (vi) comparing the performance (day by day or over time of the same flow cytometer using the same first and second standard particle reagents); and (c) determining at least one of the following based on the evaluation in step (b): (i) whether the flow cytometer passes or fails quality control; and (ii) details regarding the performance status of current instrument components.

[0068] In one embodiment, evaluating in step (b) includes evaluating each of steps (b)(i) through (b)(vi). In one embodiment, evaluating in step (b) is performed with an infrared laser and at least one laser having a wavelength less than 808 nm. In one embodiment, evaluating in step (b) is performed using multiple lasers having one or more of the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared).

[0069] In one embodiment, the method of the present invention further comprises generating a quality control report after step (c). In one embodiment, the method for quality control of the flow cytometer is performed at least once a day. In certain embodiments, the method for quality control of the flow cytometer is performed before use of the flow cytometer.

[0070] In one embodiment, the method further comprises (d) removing the quality control solution from the flow cytometer. In one embodiment, the method further comprises (e) performing an enhanced quality control analysis based on the fluorescence intensity of 1, 2, 4, 8, or 12 peaks.

[0071] In one embodiment, a mixture of beads of the present invention is run to determine the distance between noise and the maximum number of peaks that can be separated by each fluorescence channel.

[0072] In one embodiment, the method further includes (f) generating a quality control report to determine the sensitivity for detecting cellular structures between 30 and 2000 nm. In certain embodiments, the method further includes (g) loading the test sample and detecting fluorescently labeled cellular structures having diameters between 30 and 2000 nm. In certain embodiments, (f) generating a quality control report determines the sensitivity for detecting cellular structures between 20 nm and 3000 nm, between 30 nm and 2000 nm, or between 40 nm and 1000 nm. In certain embodiments, (f) generating a quality control report determines the sensitivity for detecting cellular structures less than about 3000 nm, less than about 2000 nm, less than about 1000 nm, or less than about 500 nm, or less than about 250 nm.

[0073] One embodiment of the present invention includes a kit for carrying out a method of the present invention, the kit including a system of the present invention, at least one vial for holding a first standard particle reagent, and instructions for using the kit.

[0074] In one embodiment, the kit may further include a second vial for holding a second standard particle reagent solution. In certain embodiments, the vials are each 10 ml. In embodiments, the kit may include more than one vial, more than two vials, more than three vials, more than four vials, or more than five vials. In embodiments, the kit may include less than five vials, less than four vials, less than three vials, or less than two vials. The vials may be of any suitable size, and the vials within the kit may be of different sizes.

[0075] Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the disclosure. [Example]

[0076] Example The following examples are presented to fully disclose and explain to those skilled in the art how to practice and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0077] Example 1 To test embodiments of the present disclosure, first standard particle reagent embodiments having different polystyrene bead sizes were prepared and tested for flow cytometer characterization / quality control in the range starting at 40 nm.

[0078] Figure 1 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 1A-1C show the dynamic range of the 144 nm quality control polystyrene beads using the polystyrene particle mix (40-144 nm) standard. Figure 1A shows VSSC-1 intensity by gain (linear). Figure 1B shows VSSC S1 V4 / V1 vs. VSSC-1 gain. Figure 1C shows the range of linearity in scatter detection for VSCC-1, VSCC-2, BSSC, and RSSC.

[0079] Figure 2 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 2A-2C show the correlation of the 144 nm quality control polystyrene beads by testing the 40 nm LOD and E+O 5 / 6 sigma thresholds. More specifically, the 144 nm quality control polystyrene beads were run at a gain against a target MFL using a threshold of 130,000, and then the median and standard deviation at 144 nm were collected. The 144 nm quality control polystyrene beads were also run at a gain against a target MFL using a threshold of 10, and then the median and standard deviation of the E+O noise peak were collected. Figure 2A shows the 40 nm vs. 144 nm E+O 5 sigma threshold for the VSSC1-H. Figure 2B shows the 10 threshold vs. E+O 5 sigma threshold for the VSSC1-H. Figure 2C shows the 10 threshold at 40 nm versus the E+O 5 sigma threshold in VSSC1-H.

[0080] Figure 3 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 3A-3E show the correlation of the 144 nm quality control polystyrene beads by testing a 40 nm LOD and an E+O 5 / 6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain of 130,000 relative to the target MFL, and then the median and standard deviation at 144 nm were collected. The 144 nm quality control polystyrene beads were also run at a gain of 10 relative to the target MFL, and then the median and standard deviation of the E+O noise peak were collected. Figures 3A-3E separately show the median and standard deviation of the signal at the same gain but at different thresholds, using a high threshold and then allowing more noise by lowering the threshold to 10. Figure 3A shows the 10 threshold at 144 nm for VSSC1-H. Figure 3B shows the E+O 5 sigma threshold of 144 nm for VSSC1-H. Figure 3C shows the 10 threshold of 40 nm for VSSC1-H. Figure 3D shows the E+O 5 sigma threshold of 40 nm for VSSC1-H. Figure 3E shows the data (including median and noise) for the differential measurement.

[0081] Figure 4 shows one embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. Figures 4A-4C show the correlation of the 144 nm quality control polystyrene beads by testing a 40 nm LOD and an E+O 5 / 6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain of 130,000 relative to the target MFL, and then the median and standard deviation at 144 nm were collected. Also, the 144 nm quality control polystyrene beads were run at a gain of 10 relative to the target MFL, and then the median and standard deviation of the E+O noise peak were collected. Figures 4A-4C show the median and standard deviation of the signal at the same gain and threshold. Figure 4A shows the 10 threshold at 144 nm for the VSSC1-H. Figure 4B shows the 40 nm E+O 5 sigma threshold for the VSSC1-H. FIG. 4C shows the data (including median and noise) for the differential measurement.

[0082] Figure 5 illustrates an embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads. More specifically, Figures 5A-5E show the relationship between 40 nm and VSSC1 (40 nm), BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm) for an embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads, with data for MFl and standard deviation shown in Figure 5E. Figures 5F-5J show the relationship between 40 nm and VSSC1 (40 nm), BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm) for an embodiment of a first standard particle reagent containing 144 nm quality control polystyrene beads, with data for MFl and standard deviation shown in Figure 5J.

[0083] Figure 6 shows an embodiment of the first standard particle reagent containing 142 nm, 144 nm, or 141 nm quality control polystyrene beads. More specifically, rCV data for each embodiment in VSCC-1, VSCC-2, BSCC, and RSCC are shown.

[0084] Example 2 To test embodiments of the present disclosure, first standard particle reagent embodiments having different polystyrene bead sizes were prepared and tested for flow cytometer characterization / quality control in the range starting at 40 nm.

[0085] Figure 7 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 7A shows measurements of 40 nm polystyrene beads (FP1 600 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 7B shows measurements of 80 nm polystyrene beads (FP1 600 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 7C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP1 600 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0086] Figure 8 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 8A shows measurements of 40 nm polystyrene beads (FP1 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 8B shows measurements of 80 nm polystyrene beads (FP1 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 8C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP1 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0087] Figure 9 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 9A shows measurements of 40 nm polystyrene beads (FP5 600 threshold) for VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 9B shows measurements of 80 nm polystyrene beads (FP5 600 threshold) for VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 9C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP5 600 threshold) for VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0088] Figure 10 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 10A shows measurements of 40 nm polystyrene beads (FP5 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 10B shows measurements of 80 nm polystyrene beads (FP5 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. Figure 10C shows measurements of a first standard particle reagent containing 150 nm quality control polystyrene beads (FP5 10 threshold) in VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0089] Example 3 To test embodiments of the present disclosure, an embodiment of a second standard particle reagent (the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye) was tested for characterization / quality control of a flow cytometer for measuring nanoparticles such as EVs.

[0090] Eight-peak (light yellow, yellow, Nile red, purple, blue, sky blue) 500 nm polystyrene beads were tested in duplicate using a Cytoflex spectrometer, EP10003 and EP10004. The purpose of the test was to determine the dynamic range of fluorescence gain using the 500 nm 8-peak beads.

[0091] The method involved titrating the gain against fluorescence, setting the gain to the target MFl, allowing it to stabilize for 2 minutes, and recording the sample for 1 minute. MFl, SD, and rCV were collected. The MFl ratio (MFl ration) was calculated as (MedFl ピークX ) / (MedFl 参照ピーク The staining index was determined by (MedFl ピークx -MedFl ノイズ ) / (2*SD ノイズ ) was determined.

[0092] Figure 11 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 11A shows the BV421 fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. The seven peaks are completely separated from noise at gains between 250 and 2000. Figure 11B shows the median gain titration of BV421 for both EP10003 and EP10004 for each of the eight peaks. Both EP10003 and EP10004 demonstrate a linear increase in BV421 median with increasing gain for the separated peaks. Figure 11C shows the staining index gain titration of BV421 for both EP10003 and EP10004 for each of the eight peaks. Figure 11D shows the BV421 MFl ratio (P# / P4) grain titration for both EP1003 and EP1004 for each of the eight peaks. Both EP10003 and EP10004 are linear from 250 to 3000 gain. Figure 11E shows the BV421 MFl ratio (P# / P6) gain titration for both EP10003 and EP10004.

[0093] Figure 12 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 12A shows the FITC fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. Five peaks are separable from noise at gains between 250 and 2000. Figure 12B shows the median FITC gain titration for both EP10003 and EP10004 for each of P4 through P8. Both EP10003 and EP10004 demonstrate a linear increase in FITC median value with increasing gain for the isolated peaks. Figure 12C shows the FITC staining index titration for both EP10003 and EP10004 for each of peaks P4-P8. Figure 12D shows the FITC MFl ratio (P# / P4) gain titration for both EP10003 and EP10004 for each of P5-P8. Both EP10003 and EP10004 are linear from 500-3000 gain for P5-P8. Figure 12E shows the FITC MFl ratio (P# / P6) gain titration for both EP10003 and EP10004. Both EP10003 and EP10004 are linear from 250-3000 gain for P4-P5 and P7-P8.

[0094] Figure 13 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 13A shows the PE fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. Five peaks are completely resolved from noise between 250 and 2000 gains, and the sixth peak is separable from 1000 and 3000 gains. Figure 13B shows the median PE gain titration for both EP10003 and EP10004 for each of the six peaks. Both EP10003 and EP10004 demonstrate a linear increase in median PE with increasing gain for the isolated peaks. Figure 13C shows the PE staining index gain titration for both EP10003 and EP10004 for each of the six peaks. Figure 13D shows the PE MFl ratio (P# / P4) grain titration for both EP10003 and EP10004 for each of P1-P8. Both EP10003 and EP10004 are linear from 500 to 3000 gain. Figure 13E shows the PE MFl ratio (P# / P6) gain titration for both EP10003 and EP10004. Both EP10003 and EP10004 are linear from 500 to 3000 gain for P4-P5 and P7-P8.

[0095] Figure 14 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight-peak (light yellow, yellow, Nile red, purple, blue, sky blue) 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 14A shows the APC fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. P5-P8 are fully separable over a gain range of 250-1000 for EP10003, and P5-P8 are fully separable over a gain range of 250-500 for EP10004. Figure 14B shows the median APC gain titrations for both EP10003 and EP10004 for each of the eight peaks. EP10003 demonstrates a linear increase in median APC with increasing gain for the isolated peaks. Figure 14C shows the staining index gain titrations for APC for both EP10003 and EP10004 for each of P5-P8. Figure 14D shows the APC MFl ratio (P# / P6) grain titrations for both EP10003 and EP10004 for each of P5, P7, and P8.

[0096] Figure 15 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 15A shows the APC-A700 fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. Peaks 5 through 8 are completely separated from noise at gains of 250 through 1000. Figure 15B shows the median gain titration of the APC-A700 for both EP10003 and EP10004 for each of P5 through P8. Both EP10003 and EP10004 demonstrate a linear increase in APC-A700 median with increasing gain for P6-P8. Figure 15C shows the staining index gain titration of APC-A700 for both EP10003 and EP10004 for each of P5-P8. The gain range for linearity is between 500-1000. Figure 15D shows the APC-A700 MFl ratio (P# / P6) grain titration for both EP1003 and EP1004 for P5, P7, and P8. Both EP10003 and EP10004 are linear from 250-1500 gain.

[0097] Figure 16 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having fluorescent dyes. Eight peaks (light yellow, yellow, Nile red, purple, blue, and sky blue) of 500 nm polystyrene beads were tested in duplicate with EP10003 and EP10004 using a Cytoflex spectrometer. More specifically, Figure 16A shows the APC-A750 fluorescence gain for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000, and 3000. The two peaks are completely separated from the noise at gains between 100 and 3000. Figure 16B shows the median gain titration of APC-A750 for both EP10003 and EP10004 for each of P6 through P8. FIG. 16C shows the staining index gain titration of APC-A750 against both EP10003 and EP10004 for each of P6-P8.

[0098] FIG. 17 shows an overview of the EP1 fluorescence dynamic range of FIGS.

[0099] Example 4 To test embodiments of the present disclosure, first standard particle reagent embodiments having different polystyrene bead sizes were prepared and tested for flow cytometer characterization / quality control in the range starting at 40 nm.

[0100] Figure 18 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 18 shows the rCV comparison of 40 nm and 150 nm beads in VSSC1-H at gains of 25, 100, 150, 200, 500, and 1500.

[0101] Figure 19 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 19 shows the gain at which other scattering channels saturate for 150 nm polystyrene beads in VSSC1 (10 gain and 200 gain), VSSC2 (10 gain and 3000 gain), BSSC (10 gain and 800 gain), YSSC (10 gain and 1700 gain), and RSSC (10 gain and 1300 gain).

[0102] Example 5 To test embodiments of the present disclosure, an embodiment of a second standard particle reagent (the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye) was tested for characterization / quality control of a flow cytometer for measuring nanoparticles such as EVs.

[0103] Four peaks of 500 nm polystyrene beads were tested. The beads were diluted to 1 x 10 beads / mL for each of peaks 1, 4, 5, and 7 for a total volume of 500 μl (10 μl peak 1, 10 μl peak 4, 10 μl peak 5, 10 μl peak 7, and 460 μl water).

[0104] Figure 20 shows an embodiment of a second standard particle reagent, where the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye. Four-peak 500 nm polystyrene beads were tested. More specifically, the gain at which other scattering channels saturate for the 500 nm four-peak beads was analyzed using VSSC1 (10 gain and 22 gain), VSSC2 (10 gain and 2000 gain), BSSC (10 gain and 50 gain), YSSC (10 gain and 75 gain), and RSSC (10 gain and 35 gain).

[0105] Example 6 FIG. 21 shows a summary of data collected for both a first standard particle reagent embodiment (the first standard particle reagent includes 150 nm quality control polystyrene beads) and a second standard particle reagent embodiment (the second standard particle reagent includes a second particle mixture, the particles having a fluorescent dye (500 nm bead(s))).

[0106] Figure 22 shows an embodiment of a first standard particle reagent containing 150 nm quality control polystyrene beads. More specifically, Figure 22 shows the rCV comparison of 40 nm and 150 nm beads in VSSC1-H at gains of 25, 100, 150, 200, 500, and 1500.

[0107] The following numbered items define further exemplary aspects and features of the present disclosure: 1. A system for characterization of a flow cytometer, comprising: a first standard particle reagent, the first standard particle reagent comprising a first particle mixture; and a second standard particle reagent, the second standard particle reagent comprising a second particle mixture, the particles having a fluorescent dye; Including, the system. 2. The system of item 1, wherein the first and second standard particle mixtures comprise beads or microparticles. 3. The system of item 1, wherein the first standard particle mixture comprises a plurality of particle subpopulations, each subpopulation having a different average diameter. 4. The system of item 1, wherein the second standard particle mixture comprises multiple particle subpopulations, each subpopulation having a different average diameter. 5. The system of any one of items 1 to 4, wherein the standard particle mixture comprises synthetic materials, metallic materials, hollow spheres, latex beads, gold nanoparticles, lipid nanoparticles (LNPs), polystyrene beads, hydrogel particles, silica particles, poly(methyl methacrylate) (PMMA) particles, or a combination thereof. 6. The system according to any one of items 1 to 5, wherein the standard particle mixture has particles with diameters between 100 nm and 1000 nm. 7. The system according to any one of items 1 to 3, wherein the first standard particle mixture has particles having a diameter between 140 nm and 148 nm. 8. The system according to any one of items 1 to 4, wherein the standard particle mixture is excited by a laser channel between 325 and 808 nm. 9. The system of any one of items 1 to 7, wherein the standard particle mixture comprises beads, wherein at least one fluorosphere has no dye and at least one bead has a fluorescent dye. 10. The system according to any one of items 1 to 9, wherein the standard particle mixture contains beads having at least one peak of fluorescent intensity. 11. The system according to any one of items 1 to 9, wherein the standard particle mixture contains beads having at least two peaks of fluorescent intensity. 12. The system according to any one of items 1 to 9, wherein the standard particle mixture contains beads having at least four peaks of fluorescence intensity. 13. The system according to any one of items 1 to 9, wherein the standard particle mixture contains beads having at least 8 peaks of fluorescence intensity. 14. The system of any one of items 1 to 13, wherein the second standard particle mixture comprises a dye, and the dye comprises a small organic dye, a phycobiliprotein, a quantum dot, a polymeric dye, a fluorescent protein, a tandem dye, or a combination thereof. 15. The system according to any one of items 1 to 14, wherein the second standard particles are coated with a dye that can be excited by a laser having the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). 16. A method for performing quality control on a flow cytometer, comprising: (a) loading the first standard particle reagent and the second standard particle reagent according to any one of items 1 to 15 into a flow cytometer; (b) Below: (i) assessing side scatter resolution of at least one side scatter channel of at least one laser in said flow cytometer based on single peak analysis; (ii) assessing the gain of at least one side scatter channel of at least one laser in said flow cytometer based on single peak analysis; (iii) assessing the rCV of at least one laser in said flow cytometer based on single peak analysis; (iv) assessing the fluorescence sensitivity of at least one fluorescence channel of at least one laser in the flow cytometer based on the fluorescence intensity analysis of at least two peaks; (v) assessing the flow rate in the flow cytometer based on single peak analysis; and (vi) comparing the performance (day to day or over time) of the same flow cytometer using the same first and second standard particle reagents; and (c) determining, based on the evaluation in step (b), at least one of the following: (i) whether the flow cytometer passes or fails quality control; and (ii) details regarding the performance status of the current instrument components. A method comprising: 17. The method according to item 16, wherein the evaluating in step (b) includes evaluating each of steps (b)(i) to (b)(vi). 18. The method according to any one of items 16 to 17, wherein the evaluating in step (b) is carried out with an infrared laser and at least one laser having a wavelength of less than 808 nm. 19. The method according to any one of items 16 to 17, wherein the evaluation in step (b) is performed using multiple lasers having the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). 20. The method according to any one of items 16 to 19, further comprising the step of generating a quality control report after step (c). 21. The method according to any one of items 16 to 20, wherein the method for quality control of a flow cytometer is performed at least once a day. 22. The method according to any one of items 16 to 21, wherein the method for quality control of a flow cytometer is performed before using the flow cytometer. 23. The method according to any one of items 16 to 22, further comprising the step of (d) removing the quality control solution from the flow cytometer. 24. The method according to any one of items 16 to 23, further comprising the step of (e) performing an enhanced quality control analysis based on the fluorescence intensity of 1, 2, 4, 8, or 12 peaks. 25. The method according to any one of items 16 to 24, wherein the bead mixture according to items 1 to 15 is run and the distance between noise and the maximum number of peaks that can be separated by each fluorescence channel is determined. 26. The method according to any one of items 16 to 25, further comprising the step of (f) generating a quality control report to determine the sensitivity for detecting cellular structures between 30 and 2000 nm. 27. The method according to item 26, further comprising the step of (g) loading a test sample to detect fluorescently labeled cellular structures having a diameter between 30 and 2000 nm. 28. A kit for carrying out the method according to any one of items 16 to 27, The system according to any one of items 1 to 15, at least one vial for holding the first standard particle reagent; and Instructions for using the kit Includes a kit. 29. The kit of item 28, further comprising a second vial for holding the second standard particle reagent solution.

Claims

1. 1. A system for characterization of a flow cytometer, comprising: a first standard particle reagent, the first standard particle reagent comprising a first particle mixture; and a second standard particle reagent, the second standard particle reagent comprising a second particle mixture, the particles having a fluorescent dye; Including, the system.

2. 2. The system of claim 1, wherein the first and second standard particle mixtures comprise beads or microparticles, and the first and second standard particle mixtures comprise a plurality of particle subpopulations, each subpopulation having a different average diameter.

3. 3. The system of claim 1, wherein the standard particle mixture comprises synthetic materials, metallic materials, hollow spheres, latex beads, gold nanoparticles, lipid nanoparticles (LNPs), polystyrene beads, hydrogel particles, silica particles, poly(methyl methacrylate) (PMMA) particles, or combinations thereof.

4. The system of any one of claims 1 to 3, wherein the standard particle mixture comprises particles having diameters between 100 nm and 1000 nm.

5. The system of any one of claims 1 to 4, wherein the standard particle mixture is excited by a laser channel between 325 and 808 nm.

6. The system of any one of claims 1 to 5, wherein the standard particle mixture comprises beads in which at least one fluorosphere has no dye and at least one bead has a fluorescent dye.

7. The system according to any one of claims 1 to 6, wherein the standard particle mixture comprises beads having at least eight peaks of fluorescent intensity.

8. 8. The system of claim 1, wherein the second standard particles are coated with a dye that can be excited by lasers having the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared).

9. 1. A method for performing quality control of a flow cytometer, comprising: (a) loading the first standard particle reagent and the second standard particle reagent according to any one of claims 1 to 8 into a flow cytometer; (b) Below: (i) assessing the side scatter resolution of at least one side scatter channel of at least one laser in said flow cytometer based on single peak analysis; (ii) assessing the gain of at least one side scatter channel of at least one laser in said flow cytometer based on single peak analysis; (iii) assessing the rCV of at least one laser in the flow cytometer based on single peak analysis; (iv) evaluating the fluorescence sensitivity of at least one fluorescence channel of at least one laser in the flow cytometer based on the fluorescence intensity analysis of at least two peaks; (v) assessing the flow rate in the flow cytometer based on single peak analysis; and (vi) comparing performance (day to day or over time of the same flow cytometer using the same first and second standard particle reagents); and (c) determining, based on the evaluation in step (b), at least one of the following: (i) whether the flow cytometer passes or fails quality control; and (ii) details regarding the performance status of current instrument components. A method comprising:

10. 10. The method of claim 9, wherein the evaluating in step (b) is performed using multiple lasers having the following wavelengths: 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared).

11. The method of any one of claims 9 to 10, further comprising the step of generating a quality control report after step (c).

12. 12. The method of any one of claims 9 to 11, further comprising the step of: (d) performing an enhanced quality control analysis based on the fluorescence intensity of 1, 2, 4, 8, or 12 peaks.

13. 13. The method of any one of claims 9 to 12, wherein a bead mixture according to claims 1 to 8 is run to determine the distance between noise and the maximum number of peaks that can be separated by each fluorescence channel.

14. 14. The method of any one of claims 9 to 13, further comprising the step of: (e) generating a quality control report to determine the sensitivity for detecting cellular structures between 30 and 2000 nm.

15. 15. The method of claim 14, further comprising the step of: (f) loading a test sample to detect fluorescently labeled cellular structures having a diameter between 30 and 2000 nm.

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