EP4673722A1 - Qc particles and methods of use - Google Patents

Abstract

The present disclosure provides systems, methods, and kits to improve the characterization, including the quality control, of a flow cytometer. In an embodiment, a system for characterization of a flow cytometer comprises a first standard particle reagent, wherein the first standard particle reagent comprises a first particle mixture and a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye.

Description

QC PARTICLES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is being filed on March 1, 2024, as a PCT International patent application and claims the benefit of and priority to U.S. Provisional patent application Serial No. 63/488,067, filed March 2, 2023, the entire disclosure of which is incorporated by reference herein in its entirety.

INTRODUCTION

[0002] Biological laboratory instruments automatically measure analytes and compositions. Using automated instruments to perform blood chemistry' and cell analysis increases laboratory efficiency and the test accuracy. As laboratories became more dependent on these instruments, and as the instruments became more sensitive at measuring samples at lower and lower concentrations, it became necessary to calibrate the instruments frequently to assure that the data produced was both accurate and precise.

[0003] One such instrument is a flow cytometer. In flow cytometry, single cells in solution flow through a beam of laser light resulting in light scattered in the forward and the side directions. The scattered light is then collected by photodetectors, analyzed, and the cells are counted according to the analyzed characteristics. In addition to measuring scattered light, flow cytometers can also measure fluorescence, e.g., fluorescence labeled antibodies or markers. Flow cytometers have become more sensitive and capable of measuring more parameters as the technology has matured. With this technological maturity has come complexity such that a flow cytometer is now able to measure the intensity of multiple fluorescent stains, particle size, and structure as measured by scattering angle at substantially the same time.

[0004] To ensure the quality of results, flow cytometers require frequent, multiple calibrations and quality' control prior to analyzing and reporting results. Such calibrations can be time consuming, adding cost and reducing the useful daily working hours of the instrument in the lab.

[0005] Reliable evaluation and measurement of nanoparticles, e.g., extracellular vesicles (“EV”), using a flow cytometer presents numerous challenges. EVs have a size range of 30-2000 nm from the smallest exosomes to the larger apoptotic bodies. The detection of nanoparticles, e.g., EV, requires a flow cytometer be calibrated and sensitive enough to accurately measure 30 nm or even smaller particles. Furthermore, when detecting the number of epitopes for fluorophore-conj ugated antibodies to target, the difference in number of epitopes is several magnitudes lower on EVs than conventional cells. Accordingly, small changes can drastically alter the day-to-day performance of flow cytometer when studying nanoparticles, like EV.

[0006] Embodiments of the present invention include a characterization system, a quality control process, and a quality’ control kit for day-to-day monitoring and calibration of a flow cytometer instrument, e.g.. monitoring and calibration of the instrument scatter (nanoscale) and fluorescence sensitivity to ensure consistent and reliable detection of nanoparticles like extracellular vesicles.

[0007] Currently available quality' control fluorospheres, e g., CytoFLEX QC Fluorospheres (3 pm), are larger than extracellular vesicles. These beads do not reflect the size of extracellular vesicles, nor do they demonstrate the instrument sensitivity for fluorescently labeled extracellular vesicles because they are much brighter. The brightness of the amount of dye internalized in 3 pm beads are magnitudes greater than the brightness of a few fluorescently conjugated antibodies (as few as 10) binding epitopes on the surface of extracellular vesicles.

[0008] Accordingly, the present invention addresses this problem by providing a two-step daily quality’ control process that includes a system that comprises a first standard particle reagent, wherein the first standard particle reagent comprises a first particle mixture, and a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. This process and system allow a user to calibrate and standardize the day-to-day performance of a flow cytometer for measuring nanoparticles like EV. An embodiment of the process and system determines laser alignment, laser delay, flow rate, size scatter sensitivity, and performs calibration of a flow cytometer instrument in order to bring the cytometer into performance range.

[0009] An embodiment of the present invention includes a first standard particle reagent including a non-fluorescent polystyrene bead (e.g., 144 nm to 148 nm) as a quality control size standard optionally detectable by side scatter on one or more of the following: 405 nm (VSSC1 and VSSC2), 488 nm (BSSC), 561 nm (YSSC), and 638 nm (RSSC) lasers. The present invention allows an optimal gain range (1-3000) for scatter at which the non-fluorescent polystyrene bead shows linearity in Fisher distance. In this range of linearity, a flow cy tometer is sensitive enough to detect nanoparticles, e.g., extracellular vesicles. For example, using embodiments of the quality control process and system allows determination that a flow cytometer falls within the linearity' range, and therefore the flow cytometer is optimal for detection of exosomes and small EVs. Embodiments of the quality control process and system also allow the characterization of side scatter laser alignment and flow rate.

[0010] Embodiments of the present invention include a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. For example, in an embodiment, a second standard particle reagent includes a fluorescent 450-550 nm diameter quality control fluorosphere bead mixture detectable by fluorescence on one or more of the 405 nm, 488 nm, 561 nm, and 638 nm laser channels. In an embodiment, more than one dye is incorporated into the quality control fluorosphere beads with broad emission and excitability⁷ for detection on PB450, FITC, PE, APC, APC-A700, and APC-A750. In an embodiment, the second particle reagent including quality control fluorosphere beads help determine laser delay and fluorescent alignment of a flow cytometer.

[0011] In an optional embodiment, a process and system include a second standard particle reagent comprising 8 fluorospheres with different intensities of fluorescence for each channel containing set concentrations of dyes in each bead. The second standard particle reagent includes beads with 8 peaks of fluorescent intensity⁷ that range from blank to dim to intermediate to bright. A blank fluorosphere containing no dye may optionally be used to determine the lowest background level of autofluorescence. Using this embodiment of the second standard reagent allows the determination of an optimal MFI range for fluorescence at which the quality⁷ control fluorosphere mixture will show linearity in Fisher distance between the set peaks to the blank peak. Using this range of linearity, the flow cytometer is sensitive enough to detect the dim fluorescence on labeled extracellular vesicles.

[0012] An embodiment of the present invention will have specifications of less than 5ps difference in delay settings, less than 20% percent difference in target gain settings, and less than 5% percent difference in target median fluorescence intensity. In an embodiment of the present invention, the robust coefficient of variation (“rCV”) must be less than 6% in target detector channels.

BRIEF SUMMARY OF THE INVENTION

[0013] The systems, methods, and kits of the present invention offer significant advantages over the currently available fluorospheres used for quality⁷ control of a flow cytometer prior to evaluating nanoparticles, e.g., EV. The systems, methods, and kits of the present invention allow characterization of a flow cytometer that permits accurate, reliable, and repeatable measurements of nanoparticles. Accordingly, nanoparticles may be reliably and accurately studied using a flow cytometer following characterization of a flow cytometer using the systems, methods, and kits presently disclosed.

Systems for characterization of a flow cytometer.

[0014] An embodiment of the present invention is a system for characterization of a flow cytometer comprising a first standard particle reagent, wherein the first standard particle reagent comprises a first particle mixture; and a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. In an embodiment of the system, the first and second standard particle mixtures comprise beads or microparticles. In an embodiment, the standard particle mixtures comprise 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.

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

[0016] In an embodiment, the first standard particle mixture comprises a plurality of subpopulations of particles, each subpopulation having a different average diameter. In an embodiment, the first standard particle mixture has particles with a diameter between 140 nm and 148 nm. In an embodiment, the standard particle mixtures scatter light when excited by a laser between 325 to 808 nm.

[0017] In an embodiment, the standard particle mixtures comprise beads where at least one fluorosphere with no dye and at least one bead with a fluorescent dye. In an embodiment, the standard particle mixtures comprise beads with at least one peak of fluorescent intensity. In an embodiment, the standard particle mixtures comprise beads with at least two peaks of fluorescent intensity’, with at least 4 peaks of fluorescent intensity, with at least 8 peaks of fluorescent intensity⁷. [0018] In an embodiment, the second standard particle mixture comprises a dye, the dye comprising a small organic dye. a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof. In an embodiment, the second standard particles contain dyes excitable by lasers between 325 to 808 nm, in an embodiment 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).

Methods for characterization of a flow cytometer.

[0019] An embodiment of the present invention includes a method for quality controlling a flow cytometer comprising: (a) loading the first standard particle reagent and the second standard particle reagent of any one of clauses 1-15 into a flow cytometer; (b) evaluating at least one of the following: (i) evaluating side scatter sensitivity of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis; (ii) evaluating gain of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis; (iii) evaluating rCV of at least one laser in the flow cytometer based on a single peak analysis; (iv) evaluating fluorescence sensitivity⁷ of at least one fluorescent channel of at least one laser in the flow cytometer based on at least two peak fluorescence intensity analysis; (v) evaluating flow rate in the flow cytometer based on a single peak analysis; and (vi) comparing performance (day-to-day or across time of the same flow cytometer using the same first standard particle reagent and second standard particle reagent); and (c) based on the evaluations in step (b), determining at least one of the following: (i) whether the flow cytometer passes or fails quality control; (ii) details regarding current instrument component performance status.

[0020] In an embodiment, the evaluating in step (b) includes evaluating each of steps (b)(i) through (b)(vi). In an embodiment, the evaluation in step (b) is performed on an infrared laser and at least one laser with a wavelength of less than 808 nm. In an embodiment, the evaluation in step (b) is performed with a plurality of 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 an embodiment, methods of the present invention further comprise generating a quality control report following step (c). In an embodiment, methods for quality controlling a flow cytometer are performed at least once per day. In certain embodiments, methods for quality controlling a flow cytometer are performed before using the flow cytometer.

[0022] In an embodiment, methods of the present invention further comprise (d) unloading the quality control solution from the flow cytometer. In an embodiment, methods of the present invention further comprise the step of (e) performing an enhanced quality control analysis based on 1, 2. 4, 8, or 12 peaks of fluorescent intensity.

[0023] In an embodiment, the bead mixtures of the present invention are run to determine a distance between noise and a highest number of peaks that can be resolved by each fluorescence channel.

[0024] In an embodiment, the methods further comprise the step of (f) generating a quality control report to determine the sensitivity of detecting cellular structures between 30-2000 nm. In certain embodiments, the methods further comprise (g) loading a test a sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm.

Kits for characterization of a flow cytometer.

[0025] An embodiment of the present invention includes a for performing the methods of the present invention, the kit comprising: a system of the present invention; at least one vial to hold the first standard particle reagent; and instructions for using the kit.

[0026] In an embodiment, kits may further comprise a second vial to hold the second standard particle reagent solution.

BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. FIG. 1A-1C show the dynamic range of a 144 nm quality control polystyrene bead using a polystyrene particle mix (40-144 nm) standard. FIG. 1 A shows the VSSC-1 intensity by gain (linear). FIG. IB shows the VSSC SI V4/V1 versus VSSC-1 gain. FIG 1C shows the range of linearity in scatter detection for VSCC-1, VSCC-2, BSSC, and RSSC.

[0028] Figure 2 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. FIG. 2A-2C show a correlation of 144 nm quality control polystyrene bead by testing 40 nm LOD and E+O 5/6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain for target MFI with a 130,000 threshold followed by the collection of the median and standard deviation at 144 nm. The 144 nm quality control polystyrene beads were also run at a gain for target MFL with a 10 threshold followed by collecting the median and standard deviation of E+O noise peak. FIG. 2A shows a 40 nm versus 144 nm at E+O 5 sigma threshold at VSSC1-H. FIG. 2B shows a 144 nm 10 threshold versus E+O 5 sigma threshold at VSSC1-H. FIG. 2C shows a 40 nm 10 threshold versus E+O 5 sigma threshold at VSSC1-H.

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

[0030] Figure 4 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. FIG. 4A-4C show a correlation of 144 nm quality control polystyrene bead by testing 40 nm LOD and E+O 5/6 sigma threshold. More specifically, the 144 nm quality’ control polystyrene beads were run at a gain for target MFI with a 130,000 threshold followed by the collection of the median and standard deviation at 144 nm. The 144 nm quality' control polystyrene beads were also run at a gain for target MFL with a 10 threshold followed by collecting the median and standard deviation of E+O noise peak. FIG.4A-4C show the signal median and standard deviation together at the same gain and threshold. FIG. 4 A shows a 144 nm 10 threshold at VSSC1-H. FIG. 4B shows a 40 nm E+O 5 sigma threshold at VSSC1-H. FIG. 4C shows the data, including median and noise, for difference measurements. [0031] Figure 5 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. More specifically, FIG. 5A-5E show the relationship of 40 nm to an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads at VSSC1 (40 nm). BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm), with the data shown for MFI and standard deviation shown in FIG. 5E. FIG. 5F-5J show' the relationship of 40 nm to an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads at VSSC1 (40 nm), BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm), with the data shown for MFI and standard deviation shown in FIG. 5J. [0032] Figure 6 show s embodiments of a first standard particle reagent comprising 142 nm, 144 nm, or 141 nm quality control polystyrene beads. More specifically, shows the rCV data for each embodiment at VSCC-1, VSCC-2, BSCC, and RSCC.

[0033] Figure 7 show s embodiments of a first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 7A shows the measurement of 40 nm polystyrene beads (threshold of FP1 600) at VSSC1- H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 7B shows the measurement of 80 nm polystyrene beads at (threshold of FP1 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 7C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP1 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H. and RSSC-H, respectively.

[0034] Figure 8 show s embodiments of a first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 8A shows the measurement of 40 nm polystyrene beads (threshold of FP1 10) at VSSC1-H, VSSC2-H, BSSC-H. YSSC-H, and RSSC-H, respectively. FIG. 8B shows the measurement of 80 nm polystyrene beads at (threshold of FP1 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 8C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP1 10) at VSSC1-H, VSSC2-H. BSSC-H, YSSC-H, and RSSC-H, respectively.

[0035] Figure 9 show s embodiments of a first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 9A shows the measurement of 40 nm polystyrene beads (threshold of FP5 600) at VSSC1- H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 9B shows the measurement of 80 nm polystyrene beads at (threshold of FP5 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 9C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP5 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0036] Figure 10 shows embodiments of a first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 10A shows the measurement of 40 nm polystyrene beads (threshold of FP5 10) at VSSC1-H. VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 10B shows the measurement of 80 nm polystyrene beads at (threshold of FP5 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively . FIG. 10C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP5 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0037] Figure 11 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow. Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP10004, using a Cytoflex spectrometer. More specifically, the FIG. 11A shows the BV421 fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500. 2000 and 3000. 7 peaks are fully resolved from the noise at 250-2000 gain. FIG. 1 I B shows the BV421 median gain titration for both EP 10003 and EP10004 for each of the 8 peaks. Both EP10003 and EP10004 demonstrate linear increase in BV421 median with increasing gains for resolved peaks. FIG. 11C shows BV421 stain index gain titration for both EP 10003 and EP 10004 for each of the 8 peaks. FIG. 1 ID shows the BV421 MFI ratio (P#/P4) grain titration for both EP 1003 and EP 1004 for each of the 8 peaks. Both EP10003 and EP10004 are linear at 250-3000 gain. FIG. 1 IE shows BV421 MFI ratio (P#/P6) gain titration for both EP10003 and EP10004.

[0038] Figure 12 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 12A shows the FITC fluorescence gains for both EP 10003 and EP 10004 at 10, 100, 250. 500, 750, 1000, 1500, 2000 and 3000. 5 peaks are resolvable from the noise at 250-2000 gain. FIG. 12B shows the FITC median gain titration for both EP 10003 and EP 10004 for each of P4-P8. Both EP10003 and EP10004 demonstrate linear increase in FITC median with increasing gains for resolved peaks. FIG. 12C shows the FITC stain index titration for both EP10003 and EP10004 for each of peaks P4-P8. FIG. 12D shows the FITC MFI ratio (P#/P4) grain titration for both EP 10003 and EP 10004 for each of the P5-P8. Both EP10003 and EP10004 are linear at 500-3000 gain for P5-P8. FIG. 12E shows the FITC MFI ratio (P#/P6) gain titration for both EP 10003 and EP 10004. Both EP10003 and EP10004 are linear at 250-3000 gain for P4-P5 and P7-P8.

[0039] Figure 13 show s embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow. Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP10004, using a Cytoflex spectrometer. More specifically, the FIG. 13A shows the PE fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. 5 peaks are fully resolved from the noise at 250-2000 gain and a sixth peak is resolvable from 1000-3000 gain. FIG. 13B shows the PE median gain titration for both EP 10003 and EP 10004 for each of the 6 peaks. Both EP 10003 and EP 10004 demonstrate linear increase in PE median with increasing gains for resolved peaks. FIG. 13C shows PE stain index gain titration for both EP 10003 and EP 10004 for each of the 6 peaks. FIG. 13D shows the PE MFI ratio (P#/P4) grain titration for both EPl 0003 and EP 10004 for each of Pl -P8. Both EPl 0003 and EP10004 are linear at 500-3000 gain. FIG. 13E shows the PE MFI ratio (P#/P6) gain titration for both EP10003 and EP10004. Both EP10003 and EP10004 are linear at 500- 3000 gain for P4-P5 and P7-P8.

[0040] Figure 14 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple. Blue. Sky’ Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 14A shows the APC fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. P5-P8 are fully resolvable for EP10003 in the gain range of 250-1000 and P5-P8 are fully resolvable for EP10004 in the gain range of 250-500. FIG. 14B shows the APC median gain titration for both EP 10003 and EP 10004 for each of the 8 peaks. EP 10003 demonstrates linear increase in APC median with increasing gains for resolved peaks. FIG. 14C shows APC stain index gain titration for both EP10003 and EP10004 for each of P5-P8. FIG. 14D shows the APC MFI ratio (P#/P6) grain titration for both EP10003 and EP10004 for each of P5, P7, and P8. [0041] Figure 15 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 15A shows the APC-A700 fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. Peaks 5-8 are fully resolved from the noise at 250- 1000 gain. FIG. 15B shows the APC-A700 median gain titration for both EP 10003 and EP10004 for each of P5-P8. Both EP10003 and EP10004 demonstrate linear increase in APC-A700 median with increasing gains for P6-P8. FIG. 15C shows the APC-A700 stain index gain titration for both EP 10003 and EP 10004 for each of P5-P8. The gain range for linearity is between 500-1000. FIG. 15D shows the APC-A700 MFI ratio (P#/P6) grain titration for both EP 1003 and EP 1004 for P5, P7, and P8. Both EP 10003 and EP 10004 are linear at 250 to 1500 gain.

[0042] Figure 16 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EPl 0003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 16A shows the APC-A750 fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. 2 peaks are fully resolved from the noise at 100-3000 gain. FIG. 16B shows the APC-A750 median gain titration for both EP 10003 and EP10004 for each of P6-P8. FIG. 16C shows APC-A750 stain index gain titration for both EP 10003 and EP 10004 for each of P6-P8.

[0043] Figure 17 shows the EPl fluorescence dynamic range summary of FIGI 1- FIG16.

[0044] Figure 18 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 18 shows the 40 nm and 150 nm rCV comparison on VSSC1-H at a gain of 25, 100, 150, 200, 500, and 1500. [0045] Figure 19 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 19 shows gain at which other scatter channels saturate for 150 nm polystyrene beads at VSSC 1 (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).

[0046] Figure 20 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 4 peak 500 nm polystyrene beads were tested. More specifically, the gain at which other scatter channels saturate for 500 nm 4 peaks beads were analyzed VSSC 1 (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).

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

[0048] Figure 22 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 22 shows the 40 nm and 150 nm rCV comparison on VSSC1-H at a gain of 25, 100, 150, 200, 500, and 1500.

DETAILED DESCRIPTION

[0049] While the concepts of the present disclosure are illustrated and described in detail in the figures and descriptions herein, results in the figures and their description are to be considered as examples and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0050] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

[0051] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the systems, methods, and kits described herein are readily apparent from the description of the disclosure contained herein in view of information known to the ordinarily skilled artisan, 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 the present disclosure, preferred methods and materials are now described.

Definitions.

[0053] As used herein, “g” represents gram; “L” represents liter; “mg” represents “milligram (10-3 gram);” “mL” or “cc” represents milliliter (10-3 liter). One “pL” equals to one microliter (10-6 liter). The unit of temperature used herein is degree Celsius (°C).

[0054] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0055] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, 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 employed in its sense including “and/or” unless the content clearly dictates otherwise. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0056] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By way of a hypothetical illustrative example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[0057] The term “substantially” or “about” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” or “about” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0058] The term “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0059] The term “quality control” or “quality controlling” as used herein, specify the use of disclosed compositions, methods, and kits to standardize a flow cytometer in order to ensure the reliability and accuracy of data collected by the flow cytometer.

[0060] The term “characterization” as used herein, specify the use of the disclosed composition, methods, and kits to optimize and/or quality control a flow cytometer in order to ensure reliability and accuracy of data collected by the flow cytometer.

General Description.

[0061] Systems for characterization of a flow cytometer.

[0062] An embodiment of the present invention is a system for characterization of a flow cytometer comprising a first standard particle reagent, wherein the first standard particle reagent comprises a first particle mixture; and a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. In an embodiment, the standard particle mixtures comprise 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 an 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 difference sizes. The present invention is not limited to any specific particle and contemplates the use of particles made of any material that is suitable for use with a flow cytometer.

[0063] In an embodiment, the first standard particle mixture comprises a plurality of subpopulations of particles, each subpopulation having a different average diameter. In an embodiment, the second standard particle mixture comprises a plurality of subpopulations of particles, each subpopulation having a different average diameter. In an embodiment, the standard particle mixtures have particles with a diameter between 100 nm and 1000 nm. In certain embodiments, the standard particle mixtures have particles with a diameter of 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, greater than about 600 nm. In certain embodiments, the standard particle mixtures have particles with a diameter of 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, less than about 100 mu.

[0064] In an embodiment, the first standard particle mixture has particles with a diameter between 100 nm and 300 nm, between 120 nm and 200 nm, between 140 mu and I48nm. In an embodiment, the standard particle mixtures are excited by a laser channel between 325 to 808 mu. In certain embodiments, the standard particle mixtures are excited by at least one laser channel, at least 2 laser channels, at least 3 laser channels, at least 4 laser channels, or at least 5 laser channels. In certain embodiments, only the second standard particle mixture has particles designed to be excitable by a laser channel.

[0065] In an embodiment, the standard particle mixtures comprise beads where at least one fluorosphere with no dye and at least one bead with a fluorescent dye. In an embodiment, the standard particle mixtures comprise beads with at least one peak of fluorescent intensity. Tn an embodiment, the standard particle mixtures comprise beads with at least two peaks of fluorescent intensity, with at least 4 peaks of fluorescent intensity, with at least 8 peaks of fluorescent intensity. In certain embodiments, only the second standard particle mixture comprises beads with at least one fluorescent dye.

[0066] In an embodiment, the second standard particle mixture comprises a dye, the dye comprising a small organic dye, a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof. In an embodiment, the second standard particles are coated with dyes capable of being excited by 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). In certain embodiments, the second standard particle mixture comprises 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 comprises greater than 2 dyes, greater than 3 dyes, greater than 4 dyes, greater than 5 dyes, greater than 6 dyes, greater than 7 dyes, or greater than 8 dyes. In certain embodiments, the second standard particle mixture comprises less than 15 dyes, less than 13 dyes, less than 11 dyes, less than 9 dyes, less than 7 dyes, less than 5 dyes, or less than 3 dyes.

[0067] An embodiment of the present invention includes a method for quality controlling a flow cytometer comprising: (a) loading the first standard particle reagent and the second standard particle reagent of the present invention; (b) evaluating at least one of the following: (i) evaluating side scatter sensitivity of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis; (ii) evaluating gain of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis; (iii) evaluating rCV of at least one laser in the flow cytometer based on a single peak analysis; (iv) evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow cytometer based on at least two peak fluorescence intensity analysis; (v) evaluating flow rate in the flow cytometer based on a single peak analysis; (vi) comparing performance (day- to-day or across time of the same flow cytometer using the same first standard particle reagent and second standard particle reagent); and (c) based on the evaluations in step (b), determining at least one of the following: (i) whether the flow cytometer passes or fails quality control; (ii) details regarding current instrument component performance status.

[0068] In an embodiment, the evaluating in step (b) includes evaluating each of steps (b)(i) through (b)(vi). In an embodiment, the evaluation in step (b) is performed on an infrared laser and at least one laser with a wavelength of less than 808 nm. In an embodiment, the evaluation in step (b) is performed with a plurality of 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 an embodiment, methods of the present invention further comprise generating a quality control report following step (c). In an embodiment, methods for quality controlling a flow cytometer are performed at least once per day. In certain embodiments, methods for quality controlling a flow cytometer are performed before using the flow cytometer.

[0070] In an embodiment, methods of the present invention further comprise (d) unloading the quality control solution from the flow cytometer. In an embodiment, methods of the present invention further comprise the step of (e) performing an enhanced quality control analysis based on 1, 2, 4, 8, or 12 peaks of fluorescent intensity.

[0071] In an embodiment, the bead mixtures of the present invention are run to determine a distance between noise and a highest number of peaks that can be resolved by each fluorescence channel.

[0072] In an embodiment, the methods further comprise the step of (f) generating a quality control report to determine the sensitivity of detecting cellular structures between 30-2000 nm. In certain embodiments, the methods further comprise (g) loading a test a sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 mu. In certain embodiments, the step of (f) generating a quality control report is to determine the sensitivity of detecting cellular structures between 20 nm and 3000 nm, between 30nm and 2000 nm, between 40 nm and 1000 nm. In certain embodiments, the step of (f) generating a quality control report is to determine the sensitivity of detecting cellular structures of less than about 3000 nm, of less than about 2000 nm, of less than about 1000 nm, or less than about 500 nm, or of less than about 250 nm.

[0073] An embodiment of the present invention includes a for performing the methods of the present invention, the kit comprising: a system of the present invention; at least one vial to hold the first standard particle reagent; and instructions for using the kit.

[0074] In an embodiment, kits may further comprise a second vial to hold the second standard particle reagent solution. In certain embodiments, the vials are each lOmls. In embodiments, a kit may include more than 1 vial, more than 2 vials, more than 3 vials, more than 4 vials, or more than 5 vials. In embodiments, a kit may include less than 5 vials, less than 4 vials, less than 3 vials, or less than 2 vials. The vials may be of appropriate 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 herewith for purposes of illustration only and are not intended to be limiting of the disclosure.

EXAMPLES

[0076] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the disclosure, 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, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1

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

[0078] Figure 1 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. FIG. 1A-1C show the dynamic range of a 144 nm quality control polystyrene bead using a polystyrene particle mix (40-144 nm) Standard. FIG. 1A shows the VSSC-1 intensity by gain (linear). FIG. IB shows the VSSC SI V4/V1 versus VSSC-1 gain. FIG. 1C shows the range of linearity in scatter detection for VSCC-1, VSCC-2, BSSC, and RSSC.

[0079] Figure 2 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. FIG. 2A-2C show a con-elation of 144 nm quality control polystyrene bead by testing 40 nm LOD and E+O 5/6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain for target MFI with a 130,000 threshold followed by the collection of the median and standard deviation at 144 nm. The 144 nm quality control polystyrene beads were also ran at a gain for target MFL with a 10 threshold followed by collecting the median and standard deviation of E+O noise peak. FIG. 2A shows a 40 nm versus 144 mu at E+O 5 sigma threshold at VSSC1-H. FIG. 2B shows a 144 nm 10 threshold versus E+O 5 sigma threshold at VSSC1 -H. FIG. 2C shows a 40 nm 10 threshold versus E+O 5 sigma threshold at VSSC1-H.

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

[0081] Figure 4 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. FIG. 4A-4C show a correlation of 144 nm quality control polystyrene bead by testing 40 nm LOD and E+O 5/6 sigma threshold. More specifically, the 144 nm quality control polystyrene beads were run at a gain for target MFI with a 130,000 threshold followed by the collection of the median and standard deviation at 144 nm. The 144 nm quality control polystyrene beads were also run at a gain for target MFL with a 10 threshold followed by collecting the median and standard deviation of E+O noise peak. FIG.4A-4C show the signal median and standard deviation together at the same gain and threshold. FIG. 4A shows a 144 nm 10 threshold at VSSC1-H. FIG. 4B shows a 40 nm E+O 5 sigma threshold at VSSC1-H. FIG. 4C shows the data, including median and noise, for difference measurements.

[0082] Figure 5 shows an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads. More specifically, FIG. 5A-5E show the relationship of 40 nm to an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads at VSSC1 (40 nm), BSSC1 (50 nm), YSSC1 (50 nm), and RSSC1 (50 nm), with the data shown for MFI and standard deviation shown in FIG. 5E. FIG. 5F-5J show the relationship of 40 nm to an embodiment of a first standard particle reagent comprising 144 nm quality control polystyrene beads at VSSC1 (40 nm), BSSC1 (50 mu), YSSC1 (50 mu), and RSSC1 (50 nm), with the data shown for MFI and standard deviation shown in FIG. 5J.

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

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

[0085] Figure 7 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 7A shows the measurement of 40 nm polystyrene beads (threshold of FP1 600) at VSSC1- H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 7B shows the measurement of 80 mn polystyrene beads at (threshold of FP1 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 7C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP1 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0086] Figure 8 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 8A shows the measurement of 40 nm polystyrene beads (threshold of FP1 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 8B shows the measurement of 80 mn polystyrene beads at (threshold of FP1 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 8C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP1 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0087] Figure 9 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 9A shows the measurement of 40 nm polystyrene beads (threshold of FP5 600) at VSSC1- H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 9B shows the measurement of 80 nm polystyrene beads at (threshold of FP5 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 9C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP5 600) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively.

[0088] Figure 10 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 10A shows the measurement of 40 nm polystyrene beads (threshold of FP5 10) at VSSC1-H, VSSC2-H, BSSC-H. YSSC-H, and RSSC-H, respectively. FIG. 10B shows the measurement of 80 nm polystyrene beads at (threshold of FP5 10) at VSSC1-H, VSSC2-H, BSSC-H, YSSC-H, and RSSC-H, respectively. FIG. 10C shows the measurement of a first standard particle reagent comprising 150 nm quality control polystyrene beads (threshold of FP5 10) at VSSC1-H, VSSC2-H. BSSC-H, YSSC-H, and RSSC-H, respectively.

Example 3

[0089] To test embodiments of the present disclosure, embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye were tested for characterization/quality control of a flow cytometer for measuring nanoparticles, like EVs.

[0090] 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004. using a Cytoflex spectrometer. The purpose of the tests was to determine the dynamic fluorescence gain range using 500 nm 8 peak beads.

[0091] Methods included performing titration of gains on fluorescence and setting gain to target MFI, letting it stabilize for 2 minutes, and recording a sample for 1 minute. MFI. SD, and rCV were collected. MFI ration was determined by (MedFl Peak x)/(MedFI Reference peak). Stain index was determined by (MedFl p_e k x - MedFl noise)/(2* SD noise).

[0092] Figure 11 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP10004, using a Cytoflex spectrometer. More specifically, the FIG. 11A shows the BV421 fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000. 1500. 2000 and 3000. 7 peaks are fully resolved from the noise at 250-2000 gain. FIG. 11B shows the BV421 median gain titration for both EP 10003 and EP 10004 for each of the 8 peaks. Both EP10003 and EP10004 demonstrate linear increase in BV421 median with increasing gains for resolved peaks. FIG. 11C shows BV421 stain index gain titration for both EP 10003 and EP 10004 for each of the 8 peaks. FIG. 11D shows the BV421 MFI ratio (P#/P4) grain titration for both EP 1003 and EP 1004 for each of the 8 peaks. Both EP10003 and EP10004 are linear at 250-3000 gain. FIG. HE shows BV421 MFI ratio (P#/P6) gain titration for both EP 10003 and EP 10004.

[0093] Figure 12 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple. Blue. Sky’ Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 12A shows the FITC fluorescence gains for both EP 10003 and EP 10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. 5 peaks are resolvable from the noise at 250-2000 gain. FIG. 12B shows the FITC median gain titration for both EP 10003 and EP 10004 for each of P4-P8. Both EP10003 and EP10004 demonstrate linear increase in FITC median with increasing gains for resolved peaks. FIG. 12C shows the FITC stain index titration for both EP10003 and EP10004 for each of peaks P4-P8. FIG. 12D shows the FITC MFI ratio (P#/P4) grain titration for both EP 10003 and EP 10004 for each of the P5-P8. Both EP10003 and EP 10004 are linear at 500-3000 gain for P5-P8. FIG. 12E shows the FITC MFI ratio (P#/P6) gain titration for both EP 10003 and EP 10004. Both EP10003 and EP10004 are linear at 250-3000 gain for P4-P5 and P7-P8.

[0094] Figure 13 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow. Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EPl 0003 and EP10004, using a Cytoflex spectrometer. More specifically, the FIG. 13A shows the PE fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. 5 peaks are fully resolved from the noise at 250-2000 gain and a sixth peak is resolvable from 1000-3000 gain. FIG. 13B shows the PE median gain titration for both EP 10003 and EP 10004 for each of the 6 peaks. Both EP 10003 and EP 10004 demonstrate linear increase in PE median with increasing gains for resolved peaks. FIG. 13C shows PE stain index gain titration for both EP 10003 and EP 10004 for each of the 6 peaks. FIG. 13D shows the PE MFI ratio (P#/P4) grain titration for both EP10003 and EP10004 for each of P1-P8. Both EP10003 and EP10004 are linear at 500-3000 gain. FIG. 13E shows the PE MFI ratio (P#/P6) gain titration for both EP10003 and EP10004. Both EP10003 and EP10004 are linear at 500- 3000 gain for P4-P5 and P7-P8. [0095] Figure 14 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 14A shows the APC fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500, 2000 and 3000. P5-P8 are fully resolvable for EP10003 in the gain range of 250-1000 and P5-P8 are fully resolvable for EP10004 in the gain range of 250-500. FIG. 14B shows the APC median gain titration for both EP 10003 and EP 10004 for each of the 8 peaks. EP10003 demonstrates linear increase in APC median with increasing gains for resolved peaks. FIG. 14C shows APC stain index gain titration for both EP10003 and EP10004 for each of P5-P8. FIG. 14D shows the APC MFI ratio (P#/P6) grain titration for both EP10003 and EP10004 for each of P5, P7, and P8.

[0096] Figure 15 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow. Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP10003 and EP10004, using a Cytoflex spectrometer. More specifically, the FIG. 15A shows the APC-A700 fluorescence gains for both EP10003 and EP10004 at 10, 100, 250, 500, 750, 1000, 1500. 2000 and 3000. Peaks 5-8 are fully resolved from the noise at 250- 1000 gain. FIG. 15B shows the APC-A700 median gain titration for both EP 10003 and EP10004 for each of P5-P8. Both EP10003 and EP10004 demonstrate linear increase in APC-A700 median with increasing gains for P6-P8. FIG. 15C shows the APC-A700 stain index gain titration for both EP10003 and EP10004 for each of P5-P8. The gain range for linearity is between 500-1000. FIG. 15D shows the APC-A700 MFI ratio (P#/P6) grain titration for both EP1003 and EP1004 for P5, P7, and P8. Both EP10003 and EP 10004 are linear at 250 to 1500 gain.

[0097] Figure 16 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 8 peak (Light Yellow, Yellow, Nile Red, Purple, Blue, Sky Blue) 500 nm polystyrene beads were tested in duplicate, EP 10003 and EP 10004, using a Cytoflex spectrometer. More specifically, the FIG. 16A shows the APC-A750 fluorescence gains for both EP 10003 and EP 10004 at 10, 100, 250. 500, 750, 1000, 1500, 2000 and 3000. 2 peaks are fully resolved from the noise at 100-3000 gain. FIG. 16B shows the APC-A750 median gain titration for both EP10003 and EP10004 for each of P6-P8. FIG. 16C shows APC-A750 stain index gain titration for both EP10003 and EP10004 for each of P6-P8.

[0098] Figure 17 shows the EPl fluorescence dynamic range summary of FIGI 1- FIG16.

Example 4

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

[0100] Figure 18 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 18 shows the 40 nm and 150 nm rCV comparison on VSSC1-H at a gain of 25, 100, 150, 200, 500, and 1500.

[0101] Figure 19 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically. FIG. 19 shows gain at which other scatter channels saturate for 150 nm polystyrene beads at VSSC 1 (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).

Example 5

[0102] To test embodiments of the present disclosure, embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye were tested for characterization/quality control of a flow cytometer for measuring nanoparticles, like EVs.

[0103] 4 peak 500 nm polystyrene beads were tested. The beads were diluted to

1x107 beads/mL for each of peaks 1, 4, 5, and 7 for a total volume of 500 pl (10 pl Peak 1, 10 pl Peak 4, 10 pl Peak 5, 10 pl peak 7, and 460 pl water).

[0104] Figure 20 shows embodiments of a second standard particle reagent, wherein the second standard particle reagent comprises a second particle mixture wherein the particles have a fluorescent dye. 4 peak 500 nm polystyrene beads were tested. More specifically, the gain at which other scatter channels saturate for 500 nm 4 peaks beads were analyzed VSSC 1 (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).

Example 6

[0105] Figure 21 shows a summan- of data collected for both embodiments of a first standard particle reagent, the first standard particle reagent comprising 150 nm quality control polystyrene beads and embodiments of a second standard particle reagent, wherein the second standard particle reagent compnses a second particle mixture wherein the particles have a fluorescent dye (500 nm bead(s)).

[0106] Figure 22 shows embodiments of the first standard particle reagent comprising 150 nm quality control polystyrene beads. More specifically, FIG. 22 shows the 40 nm and 150 nm rCV comparison on VSSC1-H at a gain of 25. 100, 150, 200, 500, and 1500.

[0107] The following numbered clauses define further example aspects and features of the present disclosure:

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

2. The system of clause 1, wherein the first and second standard particle mixtures comprise beads or microparticles.

3. The system of clause 1, wherein the first standard particle mixture comprises a plurality of subpopulations of particles, each subpopulation having a different average diameter.

4. The system of clause 1, wherein the second standard particle mixture comprises a plurality of subpopulations of particles, each subpopulation having a different average diameter. 5. The system of any one of clauses 1-4, wherein the standard particle mixtures comprise 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.

6. The system of any one of clauses 1-5, wherein the standard particle mixtures have particles with a diameter between 100 nm and 1000 nm.

7. The system of any one of clauses 1-3, wherein the first standard particle mixture has particles with a diameter between 140 nm and 148 nm.

8. The system of any one of clauses 1-4, wherein the standard particle mixtures are excited by a laser channel between 325 to 808 nm.

9. The system of any one of clauses 1-7, wherein the standard particle mixtures comprise beads where at least one fluorosphere with no dye and at least one bead with a fluorescent dye.

10. The system of any one of clauses 1-9, wherein the standard particle mixtures comprise beads with at least one peak of fluorescent intensity.

11. The system of any one of clauses 1 -9, wherein the standard particle mixtures comprise beads with at least two peaks of fluorescent intensity.

12. The system of any one of clauses 1-9, wherein the standard particle mixtures comprise beads with at least 4 peaks of fluorescent intensity.

13. The system of any one of clauses 1-9, wherein the standard particle mixtures comprise beads with at least 8 peaks of fluorescent intensity.

14. The system of any one of clauses 1-13, wherein the second standard particle mixture comprises a dye, the dye comprising a small organic dye, a phycobiliprotein, quantum dots, a polymer dye, a fluorescent protein, a tandem dye, or a combination thereof. 15. The system of any one of clauses 1-14, wherein the second standard particles are coated with dyes capable of being excited by lasers having the follow ing wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Y ellow-Green), 638 nm (Red), and 808 nm (Infrared).

16. A method for performing flow cytometer quality control comprising:

(a) loading the first standard particle reagent and the second standard particle reagent of any one of clauses 1-15 into a flow cytometer;

(b) evaluating at least one of the following:

(i) evaluating side scatter resolution of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis;

(ii) evaluating gain of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis;

(iii) evaluating rCV of at least one laser in the flow cytometer based on a single peak analysis

(iv) evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow cytometer based on at least tw o peak fluorescence intensity analysis;

(v) evaluating flow rate in the flow cytometer based on a single peak analysis; and

(vi) comparing performance (day-to-day or across time of the same flow cytometer using the same first standard particle reagent and second standard particle reagent); and

(c) based on the evaluations in step (b), determining at least one of the following: (i) whether the flow cytometer passes or fails quality control; (ii) details regarding current instrument component performance status.

17. The method of clause 16, wherein the evaluating in step (b) includes evaluating each of steps (b)(i) through (b)(vi).

18. The method of any one of clauses 16-17, wherein the evaluation in step (b) is performed on an infrared laser and at least one laser with a wavelength of less than 808 nm. 19. The method of any one of clauses 16-17, wherein the evaluation in step (b) is performed with a plurality of lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Y ellow-Green), 638 nm (Red), and 808 nm (Infrared).

20. The method of any one of clauses 16-19, further comprising: generating a quality control report following step (c).

21. The method of any one of clauses 16-20, wherein the method for qualitycontrolling a flow cytometer is performed at least once per day.

22. The method of any one of clauses 16-21, wherein the method for quality controlling a flow cytometer is performed before using the flow cytometer.

23. The method of any one of clauses 16-22, further comprising (d) unloading the quality control solution from the flow cytometer

24. The method of any one of clauses 16-23, further comprising the step of (e) performing an enhanced quality control analysis based on 1, 2, 4. 8, or 12 peaks of fluorescent intensity.

25. The method of any one of clauses 16-24, wherein the bead mixture of clauses 1- 15 is run to determine a distance between noise and a highest number of peaks that can be resolved by each fluorescence channel.

26. The method of any one of clauses 16-25, further comprising the step of (f) generating a quality control report to determine the sensi tivi ty of detecting cellular structures between 30-2000 nm.

27. A method of clause 26, further comprising, (g) loading a test a sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm. 28. A kit for performing the method according to any one of clauses 16-27, the kit comprising: a system of any one of clauses 1-15; at least one vial to hold the first standard particle reagent; and instructions for using the kit.

29. The kit of clause 28, further comprising a second vial to hold the second standard particle reagent solution.

Claims

CLAIMS What is claimed is:

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

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 mixture comprises a plurality⁷ of subpopulations of particles, each subpopulation having a different average diameter.

3. The system of any one of claims 1-2, wherein the standard particle mixtures comprise a synthetic material, a metal material, a hollow sphere, a latex bead, a gold nanoparticle, a lipid nanoparticle (LNP), a polysty rene bead, a hydrogel particle, a silica particle, a Poly(methyl methacrylate) (PMMA) particle, or a combination thereof.

4. The system of any one of claims 1-3, wherein the standard particle mixtures have particles with a diameter betw een 100 nm and 1000 nm.

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

6. The system of any one of claims 1-5, wherein the standard particle mixtures comprise beads where at least one fluorosphere with no dye and at least one bead with a fluorescent dye.

7. The system of any one of claims 1-6, wherein the standard particle mixtures comprise beads with at least 8 peaks of fluorescent intensity.

8. The system of any one of claims 1-7, wherein the second standard particles are coated with dyes capable of being excited by lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Y ellow-Green), 638 nm (Red), and 808 nm (Infrared).

9. A method for performing flow cytometer quality control comprising:

(a) loading the first standard particle reagent and the second standard particle reagent of any one of claims 1-8 into a flow⁷ cytometer;

(b) evaluating at least one of the follow ing:

(i) evaluating side scatter resolution of at least one side scatter channel of at least one laser in the flow cytometer based on a single peak analysis;

(ii) evaluating gain of at least one side scatter channel of at least one laser in the flow⁷ cytometer based on a single peak analysis;

(iii) evaluating rCV of at least one laser in the flow cytometer based on a single peak analysis

(iv) evaluating fluorescence sensitivity of at least one fluorescent channel of at least one laser in the flow⁷ cytometer based on at least tw o peak fluorescence intensity analysis;

(v) evaluating flow rate in the flow cytometer based on a single peak analysis; and

(vi) comparing performance (day-to-day or across time of the same flow cytometer using the same first standard particle reagent and second standard particle reagent); and

(c) based on the evaluations in step (b), determining at least one of the following: (i) whether the flow cytometer passes or fails quality control; (ii) details regarding current instrument component performance status.

10. The method of claim 9, wherein the evaluation in step (b) is performed with a plurality of lasers having the following wavelengths: 355 nm (UV), 405 nm (Violet), 488 nm (Blue), 561 nm (Y ellow-Green), 638 nm (Red), and 808 nm (Infrared).

11. The method of any one of claims 9-10, further comprising: generating a quality control report following step (c).

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

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

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

15. The method of claim 14, further comprising, (f) loading a test a sample to detect a fluorescently labeled cellular structure with a diameter between 30 - 2000 nm.