Ready-to-use routine QC fluorospheres

Abstract

The present disclosure provides compositions, methods, and kits for improving quality control of flow cytometers by using fluorospheres encapsulating at least one dye having an infrared fluorescent emission and at least one dye having at least one fluorescent emission between 355 nm and 800 nm. In one embodiment, the composition is a suspension for quality control of a flow cytometer, the suspension of fluorospheres comprising individual fluorospheres that have an infrared fluorescent emission above 800 nm when excited with an infrared laser and at least one fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm, at least one surfactant, and at least one stabilizer or preservative.

Description

[Technical Field]

[0001] This application is a PCT International patent application filed on January 10, 2024, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 479,305, filed on January 10, 2023, the entire disclosure of which is incorporated by reference in its entirety. [Background technology]

[0002] Introduction Biological laboratory instruments automatically measure analytes and compositions. The use of 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 increasingly lower concentration samples, it has become necessary to frequently calibrate the instruments to ensure the data generated is accurate and precise.

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

[0004] To ensure the quality of patient results, flow cytometers require frequent, multiple calibrations and quality control before analyzing and reporting patient results. Such calibrations are time-consuming, increase costs, and can reduce the effective daily uptime of the instrument within a laboratory.

[0005] Currently, flow cytometers equipped with both an infrared ("IR") laser (e.g., 808 nm) and an additional laser (e.g., below 800 nm) require the use of two different quality control kits or processes (e.g., two different sets of fluorospheres) before the performance of all lasers in the flow cytometer can be verified. For example, a Beckman Coulter CytoFLEX Platform flow cytometer equipped with an IR (808 nm) laser requires the use of two different single-peak quality control ("QC") fluorospheres to perform quality control on the instrument. CytoFLEX Ready to Use Daily QC Fluorospheres (PNC65719) are used to evaluate the performance of the five non-IR lasers (UV, violet, blue, yellow, and red), while CytoFLEX Daily IR QC Fluorospheres (PNC06147) are used to evaluate the performance of the IR laser on the instrument.

[0006] Quality control fluorospheres designed for non-IR lasers, such as CytoFLEX Ready to Use QC Fluorospheres, are not excited by IR lasers and therefore are not suitable for qualifying the IR channel on flow cytometers with IR lasers. On the other hand, quality control fluorospheres designed for IR lasers, such as CytoFLEX IR QC Fluorospheres, contain dyes that are excited by IR lasers and cannot be used to qualify UV, violet, blue, yellow-green, or red lasers on flow cytometers.

[0007] Therefore, users of flow cytometers with IR lasers must currently use two different quality control fluorospheres to ensure that the flow cytometer is calibrated and sample ready. The need to use two different quality control fluorospheres, one for non-IR lasers and one for IR lasers, is time-consuming and burdensome for the user.

[0008] The present invention eliminates the need for two different sets of quality control fluorospheres when performing quality control on a flow cytometer equipped with an IR laser by providing one set of QC fluorospheres that can be used for quality control. Briefly, the present invention provides a single set of fluorospheres, rather than two different sets of fluorospheres, for quality control of all lasers in a flow cytometer, thereby eliminating the need to quality control the flow cytometer twice with two different sets of fluorospheres.

[0009] Embodiments of the compositions, methods, and kits of the present invention include fluorospheres composed of at least two dyes incorporated into polystyrene beads that can be excited by all lasers in a flow cytometer (UV, violet, blue, yellow, red, and IR). The present invention includes a complete routine quality control fluorosphere solution for flow cytometers. The fluorosphere suspensions, methods of use, and kits of the present invention can be used to assess the alignment of all lasers, calculate laser delays, and evaluate flow stability in a flow cytometer.

[0010] One embodiment of the present invention has specifications of less than a 5 μsec 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 channels. In one embodiment, the target detector channels are either detector 3 or detector 4 for the UV, violet, blue, yellow-green, and red channels, and detector 1 or detector 2 for the IR channel. These channels are chosen because the dyes are brightest in these regions with low rCV.

[0011] Currently, non-IR fluorospheres are sold in 1-peak, 3-peak, 4-peak, 6-peak, 8-peak, and 9-peak formats for equivalent soluble fluorophore ("MESF") calibration to determine detector sensitivity; however, these fluorospheres do not contain IR dyes and are not suitable for quality control of IR lasers in flow cytometers. To meet the needs of users seeking to evaluate flow cytometry sensitivity as part of their QC process, an alternative embodiment of the present invention includes QC fluorospheres in a multi-peak format (peak 7, peak 4, peak 2) and assigned equivalent soluble fluorophore ("MESF") values. This embodiment is used to calculate the MESF sensitivity of an instrument. This embodiment is an optional quality control composition, method, and kit for determining sensitivity and can be used in conjunction with the single-peak embodiment. Summary of the Invention [Means for solving the problem]

[0012] Brief summary of the invention The compositions, methods, and kits of the present invention offer significant advantages over currently available fluorospheres used for quality control of flow cytometers that have both IR and non-IR lasers. The compositions, methods, and kits of the present invention eliminate the need to use two different quality control fluorospheres and perform two different quality controls by combining dyes that are excited by both non-IR and IR lasers into a single fluorosphere. Thus, a single suspension of the present invention has fluorophores that contain both IR and non-IR excited dyes, thus eliminating the need to quality control the IR laser with one set of quality control fluorospheres and the non-IR laser with a second set of quality control fluorospheres.

[0013] Composition for quality control of flow cytometers. One embodiment of the present invention is a suspension for quality control of a flow cytometer, the suspension comprising fluorospheres, the fluorospheres including individual fluorospheres having an infrared fluorescence emission greater than 800 nm when excited with an infrared laser and at least one fluorescence emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm, at least one surfactant, and at least one stabilizer or preservative. The individual fluorospheres encapsulate at least one dye having an infrared fluorescence emission greater than 800 nm when excited with an infrared laser and at least one dye having a fluorescence emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm.

[0014] In one embodiment, the at least one dye having an infrared fluorescence emission greater than 800 nm when excited with an infrared laser is selected from the group consisting of aqua green, jade green, Cy green, indocyanine green (ICG), Cy7 or Cy7.5, IR dye 800CW, or any combination thereof. In one embodiment, the at least one dye having an infrared fluorescence emission greater than 800 nm is excited with an infrared laser having a wavelength of 808 nm.

[0015] In one embodiment, the at least one dye having at least one fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm is selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile Red, purple, blue, sky blue, or any combination thereof. In certain embodiments, individual fluorospheres encapsulate at least seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited at wavelengths less than 800 nm, selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile Red, purple, blue, sky blue, or any combination thereof. In certain embodiments, the individual fluorospheres encapsulate seven dyes that have seven fluorescent emissions between 355 nm and 800 nm when excited by five or six lasers with wavelengths of 355 nm, 375 nm, 405 nm, 488 nm, 561 nm, and 638 nm. In certain embodiments, the individual fluorospheres encapsulate eight dyes that have eight fluorescent emissions when excited by six or seven lasers with wavelengths of 355 nm (UV), 375 nm, 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared).

[0016] The fluorospheres of the present invention can be polystyrene beads. In one embodiment, the fluorospheres have a diameter of 2.5 μm to 6.5 μm, 2.8 μm to 3.4 μm, or about 3.0 μm.

[0017] In certain embodiments, the fluorospheres in the suspension comprise 0.4 x 10 fluorospheres. 6 1.5 x 10 fluorospheres / mL 6 Fluorospheres 0.9 x 10 particles / mL 6 1.1 x 10 fluorospheres / mL 6 1.0 x 10 fluorospheres / mL6 It has a concentration of 10 ...

[0018] In one embodiment of the present invention, the at least one surfactant is selected from the group consisting of an ionic surfactant, a non-ionic surfactant, sodium dodecyl sulfate (SDS), NP-40, Ecosurf EH-9, Ecosurf SA-9, Ecosurf Tween® 20, Triton® X-11, or any combination thereof. In certain embodiments, the at least one surfactant is at a concentration of 0.01% to 1% based on the total volume of the suspension. In certain embodiments, the at least one surfactant is EcoSurf EH-9 at a concentration of 0.05% based on the total volume of the suspension.

[0019] In one embodiment of the present invention, the at least one stabilizer is selected from the group consisting of a stabilizing reducing agent, a stabilizing thiol-containing compound, (S)-2-aminobutane-1,4-dithiol hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof. In one embodiment of the present invention, the at least one preservative is selected from the group consisting of sodium azide, thimerosal, or any combination thereof.

[0020] Methods for quality control of flow cytometers. One embodiment of the present invention includes a method for quality control of a flow cytometer using a single peak, the method comprising: (a) loading a quality control suspension of the present invention; (b) evaluating at least one of the following steps: (i) evaluating the output power of at least one laser in the flow cytometer based on single peak analysis; (ii) evaluating the EPS of at least one laser in the flow cytometer based on single peak analysis; (iii) evaluating the laser delay of at least one laser in the flow cytometer based on single peak analysis; (iv) evaluating the gain of at least one laser in the flow cytometer based on single peak analysis; (v) evaluating the rCV of at least one laser in the flow cytometer based on single peak analysis; and (c) determining whether the flow cytometer passes or fails quality control based on the evaluation in step (b).

[0021] In one embodiment, the evaluating step in step (b) includes evaluating each of steps (b)(i) through (b)(v). In one embodiment, the evaluating step in step (b) is performed with an infrared laser and at least one laser having a wavelength less than 800 nm. In a specific embodiment, the evaluating step in step (b) is performed with seven lasers, including 355 nm (UV), 375 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). In a specific embodiment, the 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), and 638 nm (red) lasers are evaluated on selected detector channel 3 or 4 of the flow cytometer, and the 808 nm (infrared) laser is evaluated on selected detector channel 1 or 2 of the flow cytometer.

[0022] In certain embodiments, the method includes generating a quality control report after step (c). In additional embodiments, the method for quality controlling a flow cytometer is performed at least once daily. In certain embodiments, the method for quality controlling a flow cytometer is performed prior to analyzing a sample using the flow cytometer.

[0023] An alternative embodiment of the present invention includes a method for quality control of a flow cytometer using multiple peaks, comprising the steps of: (a) loading a quality control suspension of the present invention into a flow cytometer and performing a quality control analysis based on two or more peaks; (b) adjusting the brightest peak to a target median fluorescence intensity; (c) reading the median fluorescence intensity and rCV of each of the peaks; (d) removing the quality control suspension from the flow cytometer after step (c); (e) loading polystyrene beads without encapsulated fluorescence into the flow cytometer; (f) reading the median fluorescence intensity of the polystyrene beads without encapsulated fluorescence; and (g) calculating sensitivity and background. In certain embodiments, the multiple peaks include three peaks: a bright peak, a medium peak, and a dim peak.

[0024] In one embodiment, the target median fluorescence intensity in step (f) is between 500,000 and 4×10 6 In certain embodiments, the sensitivity and background include MESF sensitivity, quantum efficiency, or background.

[0025] In certain embodiments, step (a) of loading a quality control suspension of the present invention into a flow cytometer and performing a quality control analysis based on two or more peaks is simultaneous with step (e) of loading non-fluorescent polystyrene beads into the flow cytometer. In certain embodiments, the method further includes, after step (k), generating a quality control report.

[0026] A kit for quality control of flow cytometers. One embodiment of the present invention includes a kit for carrying out a method of the present invention, comprising a suspension of the present invention, at least one vial for holding the suspension, and instructions for using the kit. In certain embodiments, the kit may further comprise a second vial for holding the suspension of the present invention. In certain embodiments, the vials are each 10 ml.

[0027] In certain embodiments, the kit includes a second vial containing a suspension of dye-free polystyrene beads. In certain embodiments, the diameter of the dye-free polystyrene beads in the second vial is about 1 μm. In certain embodiments, the concentration of the polystyrene beads in the second vial is 0.4×10 fluorospheres. 6 1.5 x 10 fluorospheres / mL 6 pieces / mL. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows an embodiment of the method disclosed herein for quality control of a flow cytometer with a suspension of the present invention using a single peak. [Figure 2] FIG. 2 shows an alternative embodiment of the method disclosed herein for quality control of a flow cytometer with a suspension of the present invention using multiple peaks. [Figure 3-1]Figure 3 shows the analysis of dye leakage over time from fluorospheres with a single dye to understand dye leakage and its effect on rCV and singlet percentage. Specifically, Figure 3 shows the gating strategy for each dye. Figure 3A shows the gating strategy for fluorospheres with a UV dye. Figure 3B shows the gating strategy for fluorophores with a light yellow dye. Figure 3C shows the gating strategy for fluorophores with a yellow dye. Figure 3D shows the gating strategy for fluorospheres with a Nile Red dye. Figure 3E shows the gating strategy for fluorospheres with a purple dye. Figure 3F shows the gating strategy for fluorospheres with a blue dye. Figure 3G shows the gating strategy for fluorospheres with a sky blue dye. Figure 3H shows the gating strategy for fluorospheres with an aqua green dye (IR excitation dye). Figure 3I shows the gating strategy for fluorospheres with a Cy green dye (IR excitation dye). Figure 3J shows the average diameter (um) of each of the single dyes. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1]Figure 4 shows an analysis of dye leakage over time (days 0, 3, 16, 22, and 24) from fluorospheres with a single dye. Specifically, Figure 4 shows the median fluorescence intensity ("MdF1") % difference in the emission channel on days 0, 3, 16, 22, and 24 (Figures 4A, 4C, 4E), as well as the MdF1 for all spectra on days 0, 3, 16, 22, and 24 (Figures 4B, 4D, 4F). Figure 4A shows the MdF1 % difference for fluorospheres with UV on the emission channel on days 0, 3, 16, 22, and 24, while Figure 4B shows the MdF1 for the same fluorospheres across the spectrum. Figure 4C shows the MdF1 % difference for fluorospheres with a light yellow dye on the emission channel on days 0, 3, 16, 22, and 24, while Figure 4D shows the MdF1 for the same fluorospheres across the spectrum. Figure 4E shows the MdF1% difference for fluorospheres with a yellow dye across emission channels on days 0, 3, 16, 22, and 24, and Figure 4F shows the MdF1 for the same fluorospheres across the spectrum. Figure 4G shows the MdF1% difference for fluorospheres with a Nile Red dye across emission channels on days 0, 3, 16, 22, and 24, and Figure 4H shows the MdF1 for the same fluorospheres across the spectrum. Figure 4I shows the MdF1% difference for fluorospheres with a purple dye across emission channels on days 0, 3, 16, 22, and 24, and Figure 4J shows the MdF1 for the same fluorospheres across the spectrum. Figure 4K shows the MdF1% difference for fluorospheres with a blue dye across emission channels on days 0, 3, 16, 22, and 24, and Figure 4L shows the MdF1 for the same fluorospheres across the spectrum. Figure 4M shows the MdFl % difference for fluorospheres with sky blue dye in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4N shows the MdFl for the same fluorospheres across the spectrum.Figure 4O shows the MdFl % difference for fluorospheres with Jade Green dye (IR excitation) across emission channels on days 0, 3, 16, 22, and 24, while Figure 4P shows the MdFl for the same fluorospheres across the spectrum. Figure 4Q shows the MdFl % difference for fluorospheres with Cy Green dye (IR excitation) across emission channels on days 0, 3, 16, 22, and 24, while Figure 4R shows the MdFl for the same fluorospheres across the spectrum. Figure 4S shows the MdFl % difference for fluorospheres with Aqua Green dye (IR excitation) across emission channels on days 0, 3, 16, 22, and 24, while Figure 4T shows the MdFl for the same fluorospheres across the spectrum. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Figure 4-7] Same as above. [Figure 5-1]Figure 5 shows an analysis of dye leakage from fluorospheres of the present invention based on dilution in three different volumes (0.5 ml, 1.0 ml, and 2.0 ml) to a concentration of 1 x 10 beads / sample using two different buffers: Ecosurf EH-9 (CAS: 64366-70-7) and Ecosurf SA-9 (CAS: 68937-66-6). Figures 5A-5C are based on Table 10 herein and disclose gated analyses of fluorospheres of the present invention (Set 7.2) diluted to 0.5 ml in Ecosurf EH-9 four days after dilution (Figure 5A), diluted to 1.0 ml in Ecosurf EH-9 four days after dilution (Figure 5B), and diluted to 2.5 ml in Ecosurf EH-9 four days after dilution (Figure 5C). Figures 5D-5F show the MdF1% difference for the samples analyzed in Figures 5A-5C, respectively. Figures 5G-5I show the MdF1% difference for fluorospheres of the present invention diluted to 0.5 ml in Ecosurf SA-9 (Figure 5G), 1.0 ml in Ecosurf SA-9 (Figure 5H), and 2.5 ml in Ecosurf SA-9 (Figure 5I). Figures 5J-5K show the MdF1 over time (days 0, 4, 7, 18, 20, 25, and 27) for fluorospheres of the present invention diluted to 0.5 ml in Ecosurf EH-9 (Figure 5J) and 2.5 ml in Ecosurf EH-9 (Figure 5K). Figures 5L-5M show the MdF1 over time (days 0, 4, 7, 18, 20, 25, and 27 (for Ecosurf EH-9 only)) of fluorospheres of the present invention diluted to 2.5 ml in Ecosurf EH-9 (Figure 5L) and diluted to 2.5 ml in Ecosurf SA-9 (Figure 5M). [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 6]Figure 6 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 6A-6D show rCV analysis of fluorospheres of the present invention in channel U3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 6A shows the rCV analysis of Set 7.2. Figure 6B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in sodium dodecyl sulfate ("SDS"). Figure 6C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 6D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. [Figure 7] Figure 7 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on analysis of rCV compared to day 0. Figures 7A-7D show rCV analysis of fluorospheres of the present invention in channel U3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 7A shows the rCV analysis of Set 7.2. Figure 7B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 7C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 7D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. [Figure 8]Figure 8 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on analysis of rCV compared to day 0. Figures 8A-8D show rCV analysis of fluorospheres of the present invention in channel B3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 8A shows the rCV analysis of Set 7.2. Figure 8B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 8C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 8D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. [Figure 9] Figure 9 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 9A-9D show rCV analysis of fluorospheres of the present invention in channel Y3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 9A shows the rCV analysis of Set 7.2. Figure 9B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 9C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 9D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. [Figure 10]Figure 10 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 10A-10D show rCV analysis of fluorospheres of the present invention in channel R3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 10A shows the rCV analysis of Set 7.2. Figure 10B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 10C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 10D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. [Figure 11] Figure 11 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 11A-11D show rCV analysis of fluorospheres of the present invention in channel IR1A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 11A shows the rCV analysis of Set 7.2. Figure 11B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 11C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 11D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. [Figure 12]Figure 12 shows a comparison of MdF1 for channels IR1A (Figures 12A-12C) and IR2A (Figures 12D-12F) of the same samples evaluated in Figures 11 and 13. Specifically, Figure 12A shows an MdF1 analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 12B shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12C shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. Figure 12D shows an MdF1 analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 12E shows the MdF1 analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12F shows the MdF1 analysis of Set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide. [Figure 13] Figure 13 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 13A-13D show rCV analysis of fluorospheres of the present invention in channel IR2A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 13A shows the rCV analysis of Set 7.2. Figure 13B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 13C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 13D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide. [Figure 14]Figure 14 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 14A-14D show rCV analysis of fluorospheres of the present invention in channel IR3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 14A shows the rCV analysis of Set 7.2. Figure 14B shows the rCV analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 14C shows the rCV analysis of Set 10.2 diluted and resuspended in SDS containing 0.05% Ecosurf EH-9 and 0.02% sodium azide. FIG. 14D shows the rCV analysis of set 10.2 diluted and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide. [Figure 15-1]Figure 15 shows MdF1 analysis of Set 10.1 and Set 10.2 (channels IR1A and IR2A) based on exposure to 1500 lux or 3500 lux at 0, 15, 30, 1, 2, 4, and 6 hours. Figure 15A shows MdF1 analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS with exposure to 3500 lux (channel IR1A). Figure 15B shows MdF1 analysis of Set 10.1 washed in SDS and resuspended in SDS with 0.05% Ecosurf EH-9 and 0.02% sodium azide with exposure to 3500 lux (channel IR1A). Figure 15C shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.05% Ecosurf EH-9 and 0.02% sodium azide (channel IR1A). Figure 15D shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide (channel IR1A) with an exposure of 1500 lux. Figure 15E shows an MdF1 analysis of Set 10.2 washed with SDS and resuspended in 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide with an exposure of 1500 lux (channel IR1A). Figure 15F shows an MdF1 analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS with an exposure of 1500 lux (channel IR2A). Figure 15G shows an MdF1 analysis of Set 10.1 washed in SDS and resuspended in SDS with 0.05% Ecosurf EH-9 and 0.02% sodium azide with an exposure of 1500 lux (channel IR2A). Figure 15H shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.05% Ecosurf EH-9 and 0.02% sodium azide with an exposure of 1500 lux (channel IR2A).Figure 15I shows an MdF1 analysis of set 10.2 diluted in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide (channel IR2A) with an exposure of 1500 lux. Figure 15J shows an MdF1 analysis of set 10.2 washed in SDS and resuspended in SDS containing 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide (channel IR2A) with an exposure of 1500 lux. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. [Figure 15-5] Same as above. [Figure 15-6] Same as above. [Figure 16] Figure 16 illustrates the fluorosphere preparation process of an embodiment of the present invention, specifically Sets 11 and 12. Following fluorosphere polymerization (300), a non-IR dye is added (302), followed by the addition of an IR dye (304), followed by dialysis (306), washing (308), and resuspension (310). [Figure 17-1]Figure 17 shows the evaluation of fluorospheres of the present invention containing aqua green dye or jade green dye as the IR-excitable dye. Both sets of fluorospheres were prepared as shown in Figure 16. MdF1 analyses are for peak 2 (Figures 17A-17F), peak 4 (Figures 17G-17L), and peak 7 (Figures 17M-17R). Figure 17A shows the MdF1 analyses (peak 2) of fluorospheres with aqua green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17B shows the MdF1 analyses (peak 2) of fluorospheres with jade green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17C shows the MdF1 analyses (peak 2) of fluorospheres with aqua green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17D shows the MdF1 analysis (peak 2) of fluorospheres with Jade Green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17E shows the MdF1 analysis (peak 2) of fluorospheres with Aqua Green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17F shows the MdF1 analysis (peak 2) of fluorospheres with Jade Green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17G shows the MdF1 analysis (peak 4) of fluorospheres with Aqua Green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17H ​​shows the MdF1 analysis (peak 4) of fluorospheres with Jade Green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 171 shows the MdF1 analysis (peak 4) of fluorospheres with Aqua Green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18.Figure 17J shows the MdF1 analysis (peak 4) of fluorospheres with Jade Green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17K shows the MdF1 analysis (peak 4) of fluorospheres with Aqua Green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17L shows the MdF1 analysis (peak 4) of fluorospheres with Jade Green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17M shows the MdF1 analysis (peak 7) of fluorospheres with Aqua Green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17N shows the MdF1 analysis (peak 7) of fluorospheres with Jade Green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17O shows the MdF1 analysis (peak 7) of fluorospheres with Aqua Green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17P shows the MdF1 analysis (peak 7) of fluorospheres with Jade Green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17Q shows the MdF1 analysis (peak 7) of fluorospheres with Aqua Green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17R shows the MdF1 analysis (peak 7) of fluorospheres with Jade Green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figures 17S and 17T show the differences in peaks 2, 4, and 7 in channel IR1A between fluorospheres with Aqua Green dye (Figure 17S) and fluorospheres with Jade Green dye (Figure 17T).Figures 17U and 17V show the differences in peak 2, peak 4, and peak 7 in channels U3A, V3A, B3A, Y3A, and R3A between fluorospheres with aqua green dye (Figure 17S) and fluorospheres with jade green dye (Figure 17T). [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 17-5] Same as above. [Figure 17-6] Same as above. [Figure 17-7] Same as above. [Figure 17-8] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description While the concepts of the present disclosure have been illustrated and described in detail in the figures and description herein, it is understood that the results in the figures and description are to be considered as examples and not as limiting in character, merely that exemplary embodiments have been shown and described, and that all changes and modifications that come within the spirit of the present disclosure are desired to be protected.

[0030] Unless otherwise defined, scientific and technical terminology has the same meaning as commonly understood by one of ordinary skill in the art relevant to this disclosure.

[0031] Those skilled in the relevant art will appreciate that, in view of information known to those skilled in the art, other suitable modifications and adaptations to the compositions, methods and kits described herein will be readily apparent from the description of the disclosure contained herein and can be made without departing from the scope of the disclosure or any embodiment thereof.

[0032] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0033] 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).

[0034] The term "about," when used in conjunction with a numerical value, includes normal variations in measurement as would be estimated by one of ordinary skill in the art, and is understood to have the same meaning as "approximately," encompassing typical tolerances 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 to the quantities.

[0035] 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 context clearly dictates otherwise. For example, a reference to "a method" includes having two or more methods that are the same or different from each other. It should also be noted that the term "or" is generally used in the sense of including "and / or" unless the context 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, and the lack of combination when interpreted as alternatives ("or").

[0036] For simplicity and brevity, any range of values ​​set forth herein should be construed as supporting a claim that contemplates all values ​​within the range and recites any subranges with endpoints that are real values ​​within the stated range in question. As a hypothetical example, the disclosure herein of a range of 1 to 5 shall be deemed to support a claim to any of the 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.

[0037] The terms "substantially" or "about" are used herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The terms "substantially" or "about" are also used herein to represent the degree to which a quantitative representation may vary from a stated reference value without resulting in a change in the basic function of the subject matter in question.

[0038] The terms "comprise", "comprises" and "comprising" as used herein 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.

[0039] The terms "quality control" or "quality controlling," as used herein, designate the use of the disclosed compositions, methods, and kits to standardize a flow cytometer for the purpose of ensuring the reliability and accuracy of data collected by the flow cytometer.

[0040] Brief explanation. The present invention provides a suspension for quality control in a flow cytometer, comprising fluorospheres, the suspension of fluorospheres comprising individual fluorospheres having an infrared fluorescence emission above 800 nm when excited with an infrared laser and at least one fluorescence emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm, at least one surfactant, and at least one stabilizer or preservative.

[0041] The individual fluorospheres encapsulate at least one dye having an infrared fluorescence emission above 800 nm when excited with an infrared laser and at least one dye having a fluorescence emission between 355 nm and 800 nm when excited with wavelengths below 800 nm.

[0042] Embodiments of the present invention have at least two dyes (one IR-excitable dye and one non-IR-excitable dye), but may have three or more dyes. Fluorospheres of the present invention may have 2 to 15 dyes, 2 to 13 dyes, 2 to 11 dyes, 2 to 9 dyes, 2 to 8 dyes, or 2 to 7 dyes. Fluorospheres of the present invention may have three or more dyes, four or more dyes, five or more dyes, six or more dyes, seven or more dyes, eight or more dyes, or nine or more dyes. Fluorospheres of the present invention may have 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.

[0043] In one embodiment, the at least one dye having an infrared fluorescence emission greater than 800 nm when excited by an infrared laser is selected from the group consisting of aqua green, jade green, Cy green, indocyanine green (ICG), Cy7 or Cy7.5, IR dye 800CW, or any combination thereof. Embodiments may include any commercially available dye excitable by an IR laser. In certain embodiments, the fluorosphere contains two or more IR-excitable dyes, three or more IR-excitable dyes, or four or more IR-excitable dyes. In one embodiment, the at least one dye having an infrared fluorescence emission greater than 800 nm is excited by an infrared laser with a wavelength of 808 nm. In certain embodiments, the at least one dye having an infrared fluorescence emission is excited by an IR laser with any suitable wavelength.

[0044] In one embodiment, the at least one dye having at least one fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm is selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof. Embodiments may include any commercially available dye excitable at wavelengths less than 800 nm. In certain embodiments, the fluorospheres have 1-12 non-IR-excitable dyes (e.g., dyes excitable at less than 800 nm with fluorescent emissions between 355 nm and 800 nm), 1-10 non-IR-excitable dyes, 1-8 non-IR-excitable dyes, 1-6 non-IR-excitable dyes, 1-4 non-IR-excitable dyes, or 1-3 non-IR-excitable dyes. In certain embodiments, the fluorospheres have 3 or more non-IR-excitable dyes, 5 or more non-IR-excitable dyes, 7 or more non-IR-excitable dyes, 9 or more non-IR-excitable dyes, or 11 or more non-IR-excitable dyes, hi certain embodiments, the fluorospheres have fewer than 12 non-IR-excitable dyes, fewer than 10 non-IR-excitable dyes, fewer than 8 non-IR-excitable dyes, fewer than 6 non-IR-excitable dyes, fewer than 4 non-IR-excitable dyes, or fewer than 2 non-IR-excitable dyes.

[0045] In certain embodiments, the individual fluorospheres encapsulate at least seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited at wavelengths less than 800 nm, selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof. In certain embodiments, the individual fluorospheres encapsulate seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited by six lasers having wavelengths of 355 nm, 405 nm, 488 nm, 561 nm, and 638 nm. While specific laser wavelengths are mentioned, any commercially available laser having a non-IR wavelength is contemplated by the present invention. In certain embodiments, individual fluorospheres encapsulate eight dyes that have eight fluorescent emissions when excited by seven lasers with wavelengths of 355 nm (UV), 375 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). In certain embodiments, the fluorospheres can be excited by at least two lasers (one IR and one non-IR). In embodiments, the fluorospheres are excited by at least three lasers, at least four lasers, at least five lasers, at least six lasers, at least seven lasers, or at least eight lasers. In embodiments, the fluorospheres are excited by 2-12 lasers, 2-10 lasers, 2-8 lasers, 2-6 lasers, or 2-4 lasers.

[0046] The fluorospheres of the present invention can be polystyrene beads. The present invention is not limited to polystyrene beads and contemplates the use of fluorospheres made of any material suitable for application of two or more dyes and use with a flow cytometer. In one embodiment, the fluorospheres have a diameter of 2.5 μm to 6.5 μm, 2.8 μm to 3.4 μm, or about 3.0 μm. In embodiments, the fluorospheres have a diameter of less than 10 μm, less than 8.0 μm, less than 6.5 μm, less than 5.0 μm, less than 4.0 μm, less than 3.5 μm, or less than 3.0 μm. In embodiments, the fluorospheres have a diameter greater than 1.0 μm, greater than 2.0 μm, greater than 2.5 μm, greater than 2.8 μm, greater than 3.0 μm, greater than 4.0 μm, or greater than 4.0 μm. In embodiments, the fluorospheres in the suspension have different diameters and are not all the same diameter. In one embodiment, the fluorophores in the suspension are substantially the same diameter.

[0047] In certain embodiments, the fluorospheres in the suspension comprise 0.4 x 10 fluorospheres. 6 1.5 x 10 fluorospheres / mL 6 Fluorospheres 0.9 x 10 particles / mL 6 1.1 x 10 fluorospheres / mL 6 1.0 x 10 fluorospheres / mL 6 In embodiments, the fluorospheres in the suspension have a concentration of 2.0 x 10 fluorospheres / mL. 6 <1.5 x 10 fluorospheres / mL 6 Less than 1.0 x 10 fluorospheres / mL 6 In embodiments, the fluorospheres in the suspension have a concentration of less than 0.3 x 10 fluorospheres / mL. 6 >0.8 x 10 fluorospheres / mL 6 >1.0 x 10 fluorospheres / mL 6 >1.1 x 10 fluorospheres / mL 6 >1.5 x 10 fluorospheres / mL 6The above concentrations of fluorospheres refer to the concentrations of fluorospheres when used for quality control of a flow cytometer. Higher or lower concentrations are contemplated for storage and transportation of fluorospheres.

[0048] In one embodiment of the present invention, the at least one surfactant is selected from the group consisting of an ionic surfactant, a non-ionic surfactant, sodium dodecyl sulfate (SDS), NP-40, Ecosurf EH-9, Ecosurf SA-9, Ecosurf Tween 20, Triton X-11, or any combination thereof. The present invention contemplates the use of any commercially available surfactant. In certain embodiments, the at least one surfactant is present at a concentration of 0.01% to 1% by total volume of the suspension, 0.05% to 0.9% by total volume of the suspension, 0.1% to 0.8% by total volume of the suspension, or 0.3% to 0.6% by total volume of the suspension. In embodiments, the at least one surfactant is present at a concentration of less than 1.0% by total volume of the suspension, less than 0.8% by total volume of the suspension, or less than 0.6% by total volume of the suspension. In certain embodiments, the concentration of at least one surfactant is greater than 0.01% based on the total volume of the suspension, greater than 0.1% based on the total volume of the suspension, greater than 0.3% based on the total volume of the suspension, or greater than 0.5% based on the total volume of the suspension. In certain embodiments, the concentration of at least one surfactant is EcoSurf EH-9 at a concentration of 0.05% based on the total volume of the suspension. The above concentrations of surfactant refer to the concentration of surfactant when the fluorosphere suspension is used for quality control of a flow cytometer. Higher or lower concentrations are contemplated for storage and transportation.

[0049] In embodiments, at least two surfactants, at least three surfactants, at least four surfactants, or at least five surfactants are present in the suspension, while in embodiments, fewer than five surfactants, fewer than four surfactants, fewer than three surfactants, or fewer than two surfactants are present in the suspension.

[0050] In one embodiment of the present invention, the at least one stabilizer is selected from the group consisting of a stabilizing reducing agent, a stabilizing thiol-containing compound, (S)-2-aminobutane-1,4-dithiol hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof. The present invention contemplates the use of any commercially available stabilizer.

[0051] In embodiments, at least two stabilizers, at least three stabilizers, at least four stabilizers, or at least five stabilizers are present in the suspension, hi embodiments, fewer than five stabilizers, fewer than four stabilizers, fewer than three stabilizers, or fewer than two stabilizers are present in the suspension.

[0052] In one embodiment of the present invention, the at least one preservative is selected from the group consisting of sodium azide, thimerosal, or any combination thereof. The present invention contemplates the use of any commercially available preservative.

[0053] In embodiments, at least two preservatives, at least three preservatives, at least four preservatives, or at least five preservatives are present in the suspension, hi embodiments, fewer than five preservatives, fewer than four preservatives, fewer than three preservatives, or fewer than two preservatives are present in the suspension.

[0054] One embodiment of the present invention includes a method for quality control of a flow cytometer using a single peak, the method comprising: (a) loading a quality control suspension of the present invention; (b) evaluating at least one of the following: (i) evaluating the output power of at least one laser in the flow cytometer based on single-peak analysis; (ii) evaluating the EPS of at least one laser in the flow cytometer based on single-peak analysis; (iii) evaluating the laser delay of at least one laser in the flow cytometer based on single-peak analysis; (iv) evaluating the gain of at least one laser in the flow cytometer based on single-peak analysis; and (v) evaluating the rCV of at least one laser in the flow cytometer based on single-peak analysis; and (c) determining whether the flow cytometer passes or fails quality control based on the at least one evaluation in step (b). In one embodiment, the evaluating step in step (b) includes evaluating each of steps (b)(i) through (b)(v), or any combination thereof.

[0055] In one embodiment, the evaluation in step (b) is performed with an infrared laser and at least one laser having a wavelength less than 800 nm. In embodiments, the evaluation in step (b) is performed with fewer than all lasers in the flow cytometer, and may include evaluation of any combination of lasers in the flow cytometer. In certain embodiments, the evaluation in step (b) is performed with seven lasers having wavelengths of 355 nm (UV), 375 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). In certain embodiments, the 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), and 638 nm (red) lasers are evaluated on detector channel 3 or detector channel 4 of the flow cytometer, and the 808 nm (infrared) laser is evaluated on detector channel 1 or detector channel 2 of the flow cytometer. In other embodiments, the laser evaluated in step (b) is evaluated in an additional detector channel.

[0056] In certain embodiments, the method includes generating a quality control report after step (c). The quality control report may include a pass or fail analysis of each evaluated laser in the flow cytometer. It may also include specific measurements of each evaluated laser in the flow cytometer. In additional embodiments, the method for quality controlling the flow cytometer is performed at least once a day. In certain embodiments, the method for quality controlling the flow cytometer is performed prior to using the flow cytometer.

[0057] An alternative embodiment of the present invention includes a method for quality control of a flow cytometer using multiple peaks rather than a single peak, comprising the steps of: (a) loading a quality control suspension of the present invention into a flow cytometer and performing a quality control analysis based on two or more peaks; (b) adjusting the brightest peak to a target median fluorescence intensity; (c) reading the median fluorescence intensity and rCV of each of the peaks; (d) removing the quality control suspension from the flow cytometer after step (c); (e) loading non-fluorescent (e.g., dye-free) polystyrene beads into the flow cytometer (or loading a mixture of fluorescent and non-fluorescent polystyrene beads); (f) reading the median fluorescence intensity of the non-fluorescent (e.g., dye-free) polystyrene beads; and (g) calculating sensitivity and background. In certain embodiments, the multiple peaks include three peaks: a bright peak, a medium peak, and a dim peak. In one embodiment, the multiple peaks include two or more peaks, three or more peaks, four or more peaks, five or more peaks, six or more peaks, or seven or more peaks. In one embodiment, the multiple peaks include two or more but less than seven peaks, less than six peaks, less than five peaks, or less than four peaks.

[0058] In one embodiment, the target median fluorescence intensity in step (f) is between 500,000 and 4×10 6 In one embodiment, the target median fluorescence intensity is greater than 500,000, 1 x 10 6 Super, 2×10 6 Super, or 4x10 6 In one embodiment, the target median fluorescence intensity is greater than 5×10 6 Less than 4 x 10 6 Less than 3 x 10 6 Less than or 2 x 10 6 In certain embodiments, the sensitivity and background include MESF sensitivity, quantum efficiency, or background.

[0059] In certain embodiments, step (a) of loading a quality control suspension of the present invention into a flow cytometer and performing a quality control analysis based on two or more peaks is simultaneous with step (e) of loading non-fluorescent polystyrene beads into the flow cytometer. In certain embodiments, the method further includes generating a quality control report after step (k). In embodiments, the quality control report may include a pass or fail analysis of each evaluated laser in the flow cytometer. It may also include specific measurements of each evaluated laser in the flow cytometer.

[0060] One embodiment of the present invention includes a kit for carrying out the method of the present invention, comprising a suspension of the present invention, at least one vial for holding the suspension, and instructions for using the kit. In certain embodiments, the kit may further comprise a second vial for holding the suspension of the present invention. In certain embodiments, the vials are 10 ml each. In embodiments, the kit may include two or more vials, three or more vials, four or more vials, five or more vials, or six or more vials. In embodiments, the kit may include fewer than five vials, fewer than four vials, fewer than three vials, or fewer than two vials. The vials may be of any suitable size, and the vials within the kit may be of different sizes.

[0061] In certain embodiments, the kit includes at least a second vial (although additional vials may be included) containing a suspension of dye-free fluorospheres, e.g., dye-free polystyrene beads. In certain embodiments, the dye-free polystyrene beads in the second vial have a diameter of about 1 μm, but may have different or mixed diameters. In certain embodiments, the concentration of polystyrene beads in the second vial is 0.4×10 fluorospheres. 6 1.5 x 10 fluorospheres / mL 6 Fluorospheres 0.9 x 10 particles / mL 61.1 x 10 fluorospheres / mL 6 1.0 x 10 fluorospheres / mL 6 In an embodiment, the polystyrene beads in the second vial are 2.0 x 10 fluorospheres / mL. 6 <1.5 x 10 fluorospheres / mL 6 Less than 1.0 x 10 fluorospheres / mL 6 In an embodiment, the polystyrene beads in the second vial have a concentration of less than 0.3 x 10 fluorospheres / mL. 6 >0.8 x 10 fluorospheres / mL 6 >1.0 x 10 fluorospheres / mL 6 >1.1 x 10 fluorospheres / mL 6 >1.5 x 10 fluorospheres / mL 6 The concentrations of polystyrene beads without dyes refer to the concentrations of polystyrene beads used for quality control of flow cytometers. Higher or lower concentrations are contemplated for storage and transportation of fluorospheres.

[0062] Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are for illustrative purposes only and are not intended to limit the disclosure. [Example]

[0063] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make 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 specified, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric.

[0064] Example 1 One embodiment of the method disclosed herein for quality controlling a flow cytometer using a single peak is shown in FIG. 1. Referring to FIG. 1, which shows a flowchart of the steps for quality controlling a flow cytometer, a user of the flow cytometer selects a quality control bead target file for the flow cytometer (100). The user then loads a quality control suspension for the flow cytometer of the present invention, designed for single-peak analysis, into the flow cytometer (102). The user then initiates the quality control process (104) to allow the bead fluorescence to reach a target value. The quality control process includes one or more of: evaluating the laser power of one or more lasers in the flow cytometer (106); evaluating the EPS of one or more lasers in the flow cytometer (108); evaluating the laser delay of one or more lasers in the flow cytometer (110); evaluating the gain of one or more lasers in the flow cytometer (112); and / or evaluating the rCV of one or more lasers in the flow cytometer (114). If any evaluation step fails for any laser, the quality control fails (120) and the user is notified of the quality control report (118). If each evaluation step passes, the quality control passes (116) and the user is notified of the quality control report (118). After the quality control report (pass or fail) is generated (118), the quality control process ends (122) and the user removes the quality control suspension and cleans the flow cytometer (124).

[0065] Example 2 One embodiment of the method disclosed herein for quality control of a flow cytometer using a single peak and then multiple peaks is shown in FIG. 2. Referring to FIG. 2, steps 200-224 are the same as those shown in FIG. 1 and represent the steps for quality control of a flow cytometer using a single peak. After step 216, the user can perform additional quality control analyses using multiple peaks, which is initiated by the user selecting an enhanced quality control analysis (226). The enhanced quality control analysis may be performed daily, weekly, monthly, on a specific number of days, or never (226). If the timing to perform an enhanced quality control analysis has not yet occurred, for example, if only one day has passed since the last enhanced quality control analysis and the timing is set to every three days (228), the flow cytometer will not perform the enhanced quality control analysis and will generate a quality control report (218). If it is time to perform the enhanced quality control analysis (228), the user can decide whether or not to perform the enhanced quality control analysis (230). If the user chooses to proceed with the enhanced quality control analysis, the user retrieves the single-peak suspension (232), selects the enhanced quality control target file (234), and loads the enhanced multi-peak suspension of the present invention into the flow cytometer (236). The user then selects the enhanced quality control analysis (238), adjusts the brightest peak to the target MdF1 (240), and the flow cytometer, in embodiments using a three-peak analysis, reads the MdF1 and rCV for the bright, medium, and dim peaks (242). The user then retrieves the enhanced multi-peak suspension of the present invention (244), loads blank fluorospheres (beads without dye) (246), and then selects to continue the enhanced quality control analysis (248). The MdF1 is read against the blank fluorospheres (250), followed by calculating the MESF sensitivity for the specific channel 252, generating an enhanced quality control report (254), and the enhanced quality control analysis is completed (256).

[0066] Example 3 To better understand dye leakage from fluorospheres and thereby provide a better understanding of how best to use non-IR and IR dyes in combination as a single fluorosphere, single-dye fluorospheres were evaluated for dye leakage, rCV, and singlet percentage over time. Fluorospheres with single dyes of UV, light yellow, yellow, Nile red, purple, blue, sky blue, aqua green, and Cy green were evaluated. All fluorospheres were prepared and ordered by Spherotech, Inc. (https: / / www.spherotech.com / ) (27845 Irma Lee Circle, Unit 101, Lake Forest, IL 60045).

[0067] Each single dye fluorosphere suspension was prepared by dissolving 1 × 10 fluorospheres in 0.05% Ecosurf EH-9 available from Sigma Aldrich (CAS: 64366-70-7). 6 The samples were diluted to 10000 / mL. Once diluted, the samples were stored in FACS tubes, covered with foil to protect them from light, and stored at 4°C. The day of dilution is Day 0. Prior to measurement, the samples were vortexed for 5 seconds and acquired for 60 seconds at an FSC height threshold of 600,000. Each single dye suspension was acquired and evaluated for rCV, singlet percentage, and MdFl difference compared to Day 0. An rCV of less than 5% was considered a pass. Singlet events divided by total fluorosphere events (% singlets) greater than 85% was considered a pass. MdFl minus MdFl on Day 0 divided by MdFl on Day 0 equals % MdFl difference.

[0068] Table 1 presents the preparation of each single dye fluorosphere suspension evaluated. [Table 1]

[0069] For each of the single-dye fluorosphere suspensions, the gating strategy is disclosed in Figure 3. The percentage of singlets at day 0 and day 3 are disclosed in Tables 2 and 3, respectively. The day 3 control, representing the freshly diluted sample at day 3, is disclosed in Table 4. [Table 2] [Table 3] [Table 4]

[0070] rCV analysis of each single-dye fluorosphere suspension was performed in the U3-A, U4-A, V3-A, V4-A, B3-A, B4-A, Y3-A, Y4-A, R3-A, R4-A, IR1-A, IR2-A, and IR3-A channels on days 0 (Table 5), 3 (Table 6), 16 (Table 7), 22 (Table 8), and 24 (Table 9). A CytoFlex LX flow cytometer from Beckman Coulter was used for all readings. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9-1] [Table 9-2]

[0071] The rCV failed for Fluorospheres with Aqua Green on day 0. The rCV for Fluorospheres with Cy Green failed on day 24. The rCV for Fluorospheres with Jade Green failed on day 20.

[0072] The MdFl calculations and MdFl % differences were also evaluated and are disclosed in Figure 4. More specifically, Figure 4A shows the MdFl % differences of fluorospheres with UV in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4B shows the MdFl of the same fluorospheres across the spectrum.

[0073] FIG. 4C shows the MdFl % difference for fluorospheres with a light yellow dye in the emission channel on days 0, 3, 16, 22, and 24, and FIG. 4D shows the MdFl for the same fluorospheres across the spectrum.

[0074] FIG. 4E shows the MdFl % difference for fluorospheres with yellow dye in the emission channel on days 0, 3, 16, 22, and 24, and FIG. 4F shows the MdFl for the same fluorospheres across the spectrum.

[0075] Figure 4G shows the MdFl % difference of fluorospheres with Nile Red dye in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4H shows the MdFl of the same fluorospheres across the spectrum.

[0076] FIG. 4I shows the MdFl % difference of fluorospheres with purple dye in the emission channel on days 0, 3, 16, 22, and 24, and FIG. 4J shows the MdFl of the same fluorospheres across the spectrum.

[0077] FIG. 4K shows the MdFl % difference for fluorospheres with blue dye in the emission channel on days 0, 3, 16, 22, and 24, and FIG. 4L shows the MdFl for the same fluorospheres across the spectrum.

[0078] Figure 4M shows the MdFl % difference for fluorospheres with sky blue dye in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4N shows the MdFl for the same fluorospheres across the spectrum.

[0079] Figure 4O shows the MdFl % difference for fluorospheres with Jade Green dye (IR excitation) in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4P shows the MdFl for the same fluorospheres across the spectrum.

[0080] Figure 4Q shows the MdFl % difference for fluorospheres with Cy Green dye (IR excitation) in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4R shows the MdFl for the same fluorospheres across the spectrum.

[0081] Figure 4S shows the MdFl % difference for fluorospheres with aqua green dye (IR excitation) in the emission channel on days 0, 3, 16, 22, and 24, and Figure 4T shows the MdFl of the same fluorospheres across the spectrum.

[0082] The dyes became dimmer after 22 days. Cy Green was dimmer in the IR laser, followed by Cy Green. Jade Green provided the brightest MdF1. Jade Green fluorospheres had some dye leakage over time.

[0083] Example 4 The stability of the fluorospheres of the present invention was evaluated after dilution to different volumes using different buffers. Eight dye-containing fluorospheres of the present invention (Set 7.2) were prepared for evaluation. The fluorospheres contained UV dye, light yellow dye, yellow dye, Nile red dye, purple dye, blue dye, sky blue dye, and jade green. Jade green was added to the fluorospheres after the other dyes had been added. The fluorospheres were washed with 0.01% NP-40 and suspended in 0.01% NP-40 buffer.

[0084] Using either Ecosurf EH-9 (CAS: 64366-70-7) or Ecosurf SA-9 (CAS: 68937-66-6), add 1 x 10 fluorospheres to a total volume of either 0.5 ml, 1.0 ml, or 2.5 ml. 6 The fluorosphere suspensions were diluted to a concentration of 10 ...

[0085] On day 0, Fluorospheres (Set 7.2) were diluted with Ecosurf EH-9 to final volumes of 0.5 ml, 1.0 ml, and 2.5 ml. Also on day 0, Fluorospheres (Set 7.2) were diluted with Ecosurf SA-9 to final volumes of 0.5 ml, 1.0 ml, and 2.5 ml. Percent singlet analysis was performed on days 0, 4, 7, 18, 20, 25, and 27, as disclosed in Tables 10 and 11. The gated analysis of the day 4 sample diluted with Ecosurf EH-9 is shown in Figure 5. [Table 10] [Table 11-1] [Table 11-2]

[0086] On day 0, Fluorospheres (Set 7.2) were diluted with Ecosurf EH-9 to final volumes of 0.5 ml, 1.0 ml, and 2.5 ml. Also on day 0, Fluorospheres (Set 7.2) were diluted with Ecosurf SA-9 to final volumes of 0.5 ml, 1.0 ml, and 2.5 ml. For each sample, rCV analysis was performed on days 0, 4, 7, 18, 20, 25, and 27 in each of the following channels: U3-A, U4-A, V3-A, V4-A, B3-A, B4-A, Y3-A, Y4-A, R3-A, R4-A, IR1-A, IR2-A, and IR3-A. [Table 12] [Table 13]

[0087] MdF1 calculations and MdF1 % differences were also evaluated and are shown in Figures 5D-5M. More specifically, Figures 5D-5F show the MdF1 % differences for fluorospheres of the present invention diluted to 0.5 ml with Ecosurf EH-9 (Figure 5D), 1.0 ml with Ecosurf EH-9 (Figure 5E), and 2.5 ml with Ecosurf EH-9 (Figure 5F). Figures 5G-5I show the MdF1 % differences for fluorospheres of the present invention diluted to 0.5 ml with Ecosurf SA-9 (Figure 5G), 1.0 ml with Ecosurf SA-9 (Figure 5H), and 2.5 ml with Ecosurf SA-9 (Figure 5I). Figures 5J-5K show the MdF1 over time (days 0, 4, 7, 18, 20, 25, 27) for fluorospheres of the present invention diluted to 0.5 ml in Ecosurf EH-9 (Figure 5J) and diluted to 2.5 ml in Ecosurf EH-9 (Figure 5K). Figures 5L-5M show the MdF1 over time (days 0, 4, 7, 18, 20, 25, 27 (for Ecosurf EH-9 only)) for fluorospheres of the present invention diluted to 2.5 ml in Ecosurf EH-9 (Figure 5L) and diluted to 2.5 ml in Ecosurf SA-9 (Figure 5M).

[0088] Example 5 The stability of the fluorospheres of the present invention was evaluated after exposure to light. Eight dye-containing fluorospheres of the present invention (Set 10) were prepared for evaluation. The fluorospheres contained UV dye, light yellow dye, yellow dye, Nile red dye, purple dye, blue dye, sky blue dye, and Jade Green. Jade Green was added to the fluorospheres at the same time as the other dyes. The fluorospheres were washed with 0.01% NP-40. One set of fluorospheres of the present invention (Set 10.1) was washed with 0.01% NP-40, and then 1 x 10 fluorospheres were dissolved in SDS. 6The fluorospheres were diluted to 1000pg / mL (Set 10.1 SDS Dilution). A second set of fluorospheres (Set 10.1) was washed with 0.05% Ecosurf EH-9 and 0.02% sodium azide and then resuspended in the same buffer (Set 10.1 Wash + Resuspended in Ecosurf + SA). A third set of fluorospheres (Set 10.2) was diluted and resuspended in a buffer of 0.05% Ecosurf EH-9 and 0.02% sodium azide (Set 10.2 SDS Dilution Ecosurf + SA). A fourth set of fluorospheres (Set 10.2) was diluted and resuspended in a buffer of 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide (Set 10.2 SDS Dilution ETA + Ecosurf + SA). All fluorosphere suspensions contain 1 x 10 fluorospheres 6 The cells were diluted or resuspended to a working concentration of 1000 cells / mL.

[0089] All samples were vortexed for 5 seconds before acquisition. Samples were exposed to 1500 lux for different periods: 0 min (control), 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h. Normal exposure in the laboratory is estimated to be 400-800 lux. The FSC height threshold was 100,000. Fluorospheres were acquired and evaluated for rCV, singlet percentage, and MdFl difference compared to day 0.

[0090] Figures 6 to 14 show the rCV analysis of each sample.

[0091] Figure 6 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 6A-6D show rCV analysis of fluorospheres of the present invention in channel U3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 6A shows the rCV analysis of Set 7.2. Figure 6B shows the rCV analysis of 1 x 10 fluorospheres per ml in sodium dodecyl sulfate ("SDS"). 6Figure 6C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and resuspended in SDS with 0.02% sodium azide. Figure 6D shows the rCV analysis of Set 10.2 diluted to 0.1% ethanolamine, 0.05% Ecosurf EH-9, and resuspended in SDS with 0.02% sodium azide.

[0092] Figure 7 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 7A-7D show rCV analysis of fluorospheres of the present invention in channel U3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 7A shows the rCV analysis of Set 7.2. Figure 7B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6 Figure 7C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and resuspended in SDS with 0.02% sodium azide. Figure 7D shows the rCV analysis of Set 10.2 diluted to 0.1% ethanolamine, 0.05% Ecosurf EH-9, and resuspended in SDS with 0.02% sodium azide.

[0093] Figure 8 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 8A-8D show rCV analysis of fluorospheres of the present invention in channel B3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Sets 10.1 and 10.2). Specifically, Figure 8A shows the rCV analysis of Set 7.2. Figure 8B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6Figure 8C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 8D shows the rCV analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide.

[0094] Figure 9 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 9A-9D show rCV analysis of fluorospheres of the present invention in channel Y3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Set 10.1 and Set 10.2). Specifically, Figure 9A shows the rCV analysis of Set 7.2. Figure 9B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6 Figure 9C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 9D shows the rCV analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide.

[0095] Figure 10 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 10A-10D show rCV analysis of fluorospheres of the present invention in channel R3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Sets 10.1 and 10.2). Specifically, Figure 10A shows the rCV analysis of Set 7.2. Figure 10B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6Figure 10C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 10D shows the rCV analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide.

[0096] Figure 11 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 11A-11D show rCV analysis of fluorospheres of the present invention in channel IR1A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Sets 10.1 and 10.2). Specifically, Figure 11A shows the rCV analysis of Set 7.2. Figure 11B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6 Figure 11C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 11D shows the rCV analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide.

[0097] Figure 12 shows a comparison of MdF1 in channels IR1A (Figures 12A-12C) and IR2A (Figures 12D-12F) of the same samples evaluated in Figures 11 and 13. Specifically, Figure 12A ... 6Figure 12B shows an MdF1 analysis of Set 10.1 diluted to 1 x 10 fluorospheres per ml in SDS. Figure 12B shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12C shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12D shows an MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9 and 0.02% sodium azide. 6 Figure 12B shows the MdF1 analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12C shows the MdF1 analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide. Figure 12D shows the MdF1 analysis of Set 10.3 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12E shows the MdF1 analysis of Set 10.4 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 12F shows the MdF1 analysis of Set 10.5 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide.

[0098] Figure 13 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 13A-13D show rCV analysis of fluorospheres of the present invention in channel IR2A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Sets 10.1 and 10.2). Specifically, Figure 13A shows the rCV analysis of Set 7.2. Figure 13B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6 Figure 13C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and resuspended in SDS with 0.02% sodium azide. Figure 13D shows the rCV analysis of Set 10.2 diluted to 0.1% ethanolamine, 0.05% Ecosurf EH-9, and resuspended in SDS with 0.02% sodium azide.

[0099] Figure 14 shows an analysis of light exposure (1500 lux at different time points) to fluorospheres of the present invention based on an analysis of rCV compared to day 0. Figures 14A-14D show rCV analysis of fluorospheres of the present invention in channel IR3A at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 9 hours (Set 7.2 compared to Sets 10.1 and 10.2). Specifically, Figure 14A shows the rCV analysis of Set 7.2. Figure 14B shows the rCV analysis of 1 x 10 fluorospheres per ml in SDS. 6 Figure 14C shows the rCV analysis of Set 10.1 diluted to 0.05% Ecosurf EH-9 and 0.02% sodium azide. Figure 14D shows the rCV analysis of Set 10.2 diluted and resuspended in SDS with 0.1% ethanolamine, 0.05% Ecosurf EH-9, and 0.02% sodium azide.

[0100] Table 14 shows the rCV analysis of each sample over time after exposure to 3500 lux. rCV analysis was performed on channels U3-A, V3-A, B3-A, Y3-A, IR1-A and IR2-A. [Table 14-1] [Table 14-2] [Table 14-3]

[0101] Table 15 shows the % singlet analysis of each sample over time after exposure to 3500 lux. [Table 15-1] [Table 15-2]

[0102] Example 6 To investigate the most potential IR dyes for use with the fluorospheres of the present invention, fluorospheres of the present invention containing either Jade Green, Aqua Green, or Cy Green were evaluated. Fluorospheres of the present invention containing eight dyes (Set 11) were prepared for evaluation. The fluorospheres contained a UV dye, a light yellow dye, a yellow dye, a Nile Red dye, a purple dye, a blue dye, a sky blue dye, and either Aqua Green or Cy Green. Additionally, fluorospheres of the present invention containing eight dyes (Set 12) were prepared for evaluation. The fluorospheres contained a UV dye, a light yellow dye, a yellow dye, a Nile Red dye, a purple dye, a blue dye, a sky blue dye, and Jade Green. All three sets (Set 11 with Aqua Green, Set 11 with Cy Green, and Set 12 with Jade Green) were prepared as shown in Figure 16.

[0103] Fluorospheres with Cy Green (Set 11 with Cy Green) did not perform as well as fluorospheres with aqua green (Set 11 with aqua green) or fluorospheres with jade green (Set 12 with jade green). Therefore, only analyses for fluorospheres with aqua green or jade green are included.

[0104] MdF1 analysis was performed on the samples. Figure 17 shows the evaluation of fluorospheres of the present invention containing aqua green dye or jade green dye as the IR-excitable dye. MdF1 analysis was performed on peak 2 (Figures 17A-17F), peak 4 (Figures 17G-17L), and peak 7 (Figures 17M-17R).

[0105] Figure 17A shows the MdF1 analysis (peak 2) of fluorospheres with aqua green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17B shows the MdF1 analysis (peak 2) of fluorospheres with jade green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17C shows the MdF1 analysis (peak 2) of fluorospheres with aqua green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17D shows the MdF1 analysis (peak 2) of fluorospheres with jade green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17E shows the MdF1 analysis (peak 2) of fluorospheres with aqua green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17F shows the MdF1 analysis (peak 2) of fluorospheres with jade green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18.

[0106] Figure 17G shows the MdF1 analysis (peak 4) of fluorospheres with aqua green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17H ​​shows the MdF1 analysis (peak 4) of fluorospheres with jade green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17I shows the MdF1 analysis (peak 4) of fluorospheres with aqua green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17J shows the MdF1 analysis (peak 4) of fluorospheres with jade green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17K shows the MdF1 analysis (peak 4) of fluorospheres with aqua green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17L shows the MdF1 analysis (peak 4) of fluorospheres with jade green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18.

[0107] Figure 17M shows the MdF1 analysis (peak 7) of fluorospheres with aqua green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17N shows the MdF1 analysis (peak 7) of fluorospheres with jade green dye incubated at 22°C on days 0, 5, 7, 11, 14, and 18. Figure 17O shows the MdF1 analysis (peak 7) of fluorospheres with aqua green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17P shows the MdF1 analysis (peak 7) of fluorospheres with jade green dye incubated at 32°C on days 0, 5, 7, 11, 14, and 18. Figure 17Q shows the MdF1 analysis (peak 7) of fluorospheres with aqua green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18. Figure 17R shows the MdF1 analysis (peak 7) of fluorospheres with jade green dye incubated at 50°C on days 0, 5, 7, 11, 14, and 18.

[0108] Figures 17S and 17T show the differences in peaks 2, 4, and 7 in channel IR1A between fluorospheres with aqua green dye (Figure 17S) and fluorospheres with jade green dye (Figure 17T). Figures 17U and 17V show the differences in peaks 2, 4, and 7 in channels U3A, V3A, B3A, Y3A, and R3A between fluorospheres with aqua green dye (Figure 17S) and fluorospheres with jade green dye (Figure 17T).

[0109] An evaluation of the MdFl% difference for each sample as a function of time (days 0, 5, 7, 11, 14 and 22) in combination with temperature (22°C, 32°C and 50°C) was also performed and is shown in Tables 16-18 below. [Table 16A] [Table 16B] [Table 16C] [Table 17A] [Table 17B] [Table 17C] [Table 18A] [Table 18B] [Table 18C]

[0110] Numbered clauses 1. A suspension for quality control in a flow cytometer, fluorospheres, wherein the suspension of fluorospheres comprises individual fluorospheres that have an infrared fluorescence emission above 800 nm when excited with an infrared laser and at least one fluorescence emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm; at least one surfactant; At least one stabilizer or preservative; A suspension comprising: 2. The suspension of clause 1, wherein the individual fluorospheres encapsulate at least one dye having an infrared fluorescence emission above 800 nm when excited with an infrared laser and at least one dye having a fluorescence emission between 355 nm and 800 nm when excited at wavelengths below 800 nm. 3. The suspension of clause 2, wherein the at least one dye having an infrared fluorescence emission greater than 800 nm when excited by an infrared laser is selected from the group consisting of aqua green, jade green, Cy green, indocyanine green (ICG), Cy7 or Cy7.5, IR dye 800CW, or any combination thereof. 4. The suspension according to clause 3, wherein the at least one dye having infrared fluorescence emission above 800 nm when excited by an infrared laser is aqua green. 5. The suspension according to any one of clauses 2 to 4, wherein at least one dye having an infrared fluorescence emission above 800 nm is excited with an infrared laser at a wavelength of 808 nm. 6. The suspension of any one of clauses 2 to 5, wherein the at least one dye having at least one fluorescent emission between 355 nm and 800 nm when excited at a wavelength less than 800 nm is selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof. 7. The suspension of any one of clauses 2-6, wherein individual fluorospheres encapsulate at least seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited at wavelengths less than 800 nm, selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof. 8. The suspension of any one of clauses 2 to 7, wherein individual fluorospheres encapsulate seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited by five lasers having wavelengths of 355 nm, 405 nm, 488 nm, 561 nm, and 638 nm. 9. The suspension of any one of clauses 2 to 8, wherein individual fluorospheres encapsulate eight dyes that have eight fluorescent emissions when excited by six lasers having wavelengths of 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). 10. The suspension of any one of clauses 1 to 9, wherein the fluorospheres are polystyrene beads. 11. The suspension of any one of clauses 1 to 10, wherein the fluorospheres have a diameter of between 2.5 μm and 6.5 μm. 12. A suspension according to any one of clauses 1 to 11, wherein the fluorospheres have a diameter of between 2.8 μm and 3.4 μm. 13. The suspension of any one of clauses 1-12, wherein the fluorospheres have a diameter of about 3.0 μm. 14. The fluorospheres in the suspension are 0.4 x 10 fluorospheres 6 1.5 x 10 fluorospheres / mL 6 14. The suspension of any one of clauses 1 to 13, having a concentration of 0.15% by weight / mL. 15. The fluorospheres in the suspension are 0.9 x 10 fluorospheres 6 1.1 x 10 fluorospheres / mL 6 15. The suspension of any one of clauses 1 to 14, having a concentration of 0.15% by weight / mL. 16. The fluorospheres in the suspension are approximately 1.0 x 10 fluorospheres. 6 16. The suspension of any one of clauses 1 to 15, having a concentration of 10 ... 17. The suspension of any one of clauses 1-16, wherein the at least one surfactant is selected from the group consisting of an ionic surfactant, a non-ionic surfactant, sodium dodecyl sulfate (SDS), NP-40, Ecosurf EH-9, Ecosurf SA-9, Ecosurf Tween 20, Triton X-11, or any combination thereof. 18. The suspension according to any one of clauses 1 to 17, wherein the at least one surfactant is at a concentration of 0.01% to 1%, based on the total volume of the suspension. 19. The suspension according to any one of clauses 1 to 18, wherein the at least one surfactant is EcoSurf EH-9 at a concentration of 0.05% based on the total volume of the suspension. 20. The suspension of any one of clauses 1-19, wherein the at least one stabilizer is selected from the group consisting of a stabilizing reducing agent, a stabilizing thiol-containing compound, (S)-2-aminobutane-1,4-dithiol hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof. 21. The suspension of any one of clauses 1-20, wherein at least one preservative is selected from the group consisting of sodium azide, thimerosal, or any combination thereof. 22. A method for quality control of a flow cytometer using a single peak, comprising: (a) loading the quality control suspension of any one of clauses 1-21 into a flow cytometer; (b) (i) evaluating the output of at least one laser in a flow cytometer based on single peak analysis; (ii) evaluating the EPS of at least one laser in the flow cytometer based on single peak analysis; (iii) evaluating the laser delay of at least one laser in the flow cytometer based on single peak analysis; (iv) evaluating the gain of at least one laser in the flow cytometer based on single peak analysis; (v) evaluating the rCV of at least one laser in the flow cytometer based on single peak analysis; evaluating at least one of the following: (c) determining whether the flow cytometer passes or fails quality control based on the evaluation in step (b); A method comprising: 23. The method of clause 22, wherein the evaluating step in step (b) includes evaluating each of steps (b)(i) through (b)(v). 24. A method according to any one of clauses 22-23, wherein the evaluation in step (b) is carried out with an infrared laser and at least one laser having a wavelength of less than 800 nm. 25. A method according to any one of clauses 22 to 24, wherein the evaluation in step (b) is carried out with seven lasers having wavelengths of 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). 26. The method described in clause 25, wherein the 355 (UV) laser, 405 nm (violet) laser, 488 nm (blue) laser, 561 nm (yellow-green) laser, and 638 nm (red) laser are evaluated on detector channel 3 or detector channel 4 of the flow cytometer, and the 808 nm (infrared) laser is evaluated on detector channel 1 or detector channel 2 of the flow cytometer. 27. The method of any one of clauses 22 to 26, further comprising, after step (c), generating a quality control report. 28. The method according to any one of clauses 22 to 27, wherein the method for quality control of the flow cytometer is carried out at least once a day. 29. The method according to any one of clauses 22 to 27, wherein the method for quality control of the flow cytometer is carried out before using the flow cytometer. 30. A method for quality control of a flow cytometer using multiple peaks, comprising: (a) loading the quality control suspension of any one of clauses 1-21 into a flow cytometer and performing a quality control analysis based on two or more peaks; (b) adjusting the brightest peak to a target median fluorescence intensity; (c) reading the median fluorescence intensity and rCV of each of the peaks; (d) after step (c), removing the quality control suspension from the flow cytometer; (e) loading non-fluorescent polystyrene beads into a flow cytometer; (f) reading the median fluorescence intensity of the non-fluorescent polystyrene beads; (g) calculating sensitivity and background; A method comprising: 31. The method of clause 30, wherein there are three peaks: a bright peak, a medium peak, and a dim peak. 32. The target median fluorescence intensity in step (f) is between 500,000 and 4 × 10 6 32. The method according to any one of clauses 30 to 31, wherein 33. The method of any one of clauses 30-32, wherein the sensitivity and background comprise MESF sensitivity, quantum efficiency, or background. 34. The method of any one of clauses 30 to 33, wherein step (a) is simultaneous with step (e). 35. The method of any one of clauses 30 to 34, further comprising, after step (k), generating a quality control report. 36. A kit for carrying out the method according to any one of clauses 22 to 35, comprising: a suspension according to any one of clauses 1 to 21; at least one vial for holding the suspension; Instructions for using the kit and A kit comprising: 37. The kit of clause 36, further comprising a second vial for holding the suspension. 38. The kit according to clause 37, wherein the two vials are each 10 ml. 39. The kit of clause 36, further comprising a second vial containing a suspension of dye-free polystyrene beads. 40. The kit of clause 39, wherein the diameter of the polystyrene beads in the second vial is about 1 μm. 41. The concentration of polystyrene beads in the second vial is 0.4 x 10 fluorospheres. 6 1.5 x 10 fluorospheres / mL6 41. The kit according to any one of clauses 39 to 40, wherein the concentration is 1000 mg / mL. 42. A suspension for quality control in a flow cytometer, comprising: fluorospheres encapsulating at least one dye having a fluorescent emission, wherein the suspension of fluorospheres comprises individual fluorospheres having an infrared fluorescent emission above 800 nm when excited with an infrared laser and at least one fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm; at least one surfactant; At least one stabilizer or preservative; A suspension comprising: 43. The suspension of clause 42, wherein the suspension of fluorospheres having a fluorescent emission above 800 nm when excited by an infrared laser has encapsulated therein at least one dye selected from the group consisting of aqua green, jade green, Cy green, indocyanine green (ICG), Cy7 or Cy7.5, IR dye 800CW, or any combination thereof. 44. The suspension of clause 42 or 43, wherein the suspension of fluorospheres having a fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm has encapsulated therein at least one dye selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof. 45. The suspension of any one of clauses 42 to 44, wherein individual fluorospheres encapsulate at least seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited at wavelengths less than 800 nm, selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof. 46. ​​A suspension according to any one of clauses 42 to 45, wherein individual fluorospheres encapsulate eight dyes having eight fluorescent emissions when excited by six lasers having wavelengths of 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared). 47. A suspension according to any one of clauses 42 to 46, wherein the fluorospheres have a diameter of between 2.5 μm and 6.5 μm. 48. The fluorospheres in suspension are 0.4 x 10 fluorospheres 6 1.5 x 10 fluorospheres / mL 6 48. The suspension of any one of clauses 42 to 47, having a concentration of 0.1g / mL. 49. The suspension of any one of clauses 42-48, wherein the at least one surfactant is selected from the group consisting of an ionic surfactant, a non-ionic surfactant, sodium dodecyl sulfate (SDS), NP-40, Ecosurf EH-9, Ecosurf SA-9, Ecosurf Tween 20, Triton X-11, or any combination thereof; the at least one stabilizer is selected from the group consisting of a stabilizing reducing agent, a stabilizing thiol-containing compound, (S)-2-aminobutane-1,4-dithiol hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof; and the at least one preservative is selected from the group consisting of sodium azide, thimerosal, or any combination thereof. 50. A suspension according to any one of clauses 42 to 49, wherein the at least one surfactant is at a concentration of 0.01% to 1%, based on the total volume of the suspension. 51. A method for quality control of a flow cytometer using a single peak, comprising: (a) loading the quality control suspension of any one of clauses 42-50 into a flow cytometer; (b) (i) evaluating the output of at least one laser in a flow cytometer based on single peak analysis; (ii) evaluating the EPS of at least one laser in the flow cytometer based on single peak analysis; (iii) evaluating the laser delay of at least one laser in the flow cytometer based on single peak analysis; (iv) evaluating the gain of at least one laser in the flow cytometer based on single peak analysis; (v) evaluating the rCV of at least one laser in the flow cytometer based on single peak analysis; evaluating at least one of the following: (c) determining whether the flow cytometer passes or fails quality control based on the evaluation in step (b); A method comprising: 52. The method of clause 51, wherein the evaluating step in step (b) includes evaluating each of steps (b)(i) through (b)(v). 53. A method for quality control of a flow cytometer using multiple peaks, comprising: (a) loading the quality control suspension of any one of clauses 42-49 into a flow cytometer and performing a quality control analysis based on two or more peaks; (b) adjusting the brightest peak to a target median fluorescence intensity; (c) reading the median fluorescence intensity and rCV of each of the peaks; (d) after step (c), removing the quality control suspension from the flow cytometer; (e) loading non-fluorescent polystyrene beads into a flow cytometer; (f) reading the median fluorescence intensity of the non-fluorescent polystyrene beads; (g) calculating sensitivity and background; A method comprising: 54. The method of clause 53, wherein there are three peaks: a bright peak, a medium peak, and a dim peak. 55. The target median fluorescence intensity in step (f) is between 500,000 and 4 × 10 6 55. The method of any one of clauses 53 to 54, wherein 56. The method of any one of clauses 53 to 55, wherein the sensitivity and background comprise MESF sensitivity, quantum efficiency, or background.

Claims

1. A suspension for quality control in a flow cytometer, comprising: fluorospheres encapsulating at least one dye having a fluorescent emission, said suspension of fluorospheres comprising individual fluorospheres having an infrared fluorescent emission above 800 nm when excited with an infrared laser and at least one fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm; at least one surfactant; at least one stabilizer or preservative; A suspension comprising:

2. 2. The suspension of claim 1, wherein the suspension of fluorospheres having a fluorescent emission above 800 nm when excited with an infrared laser has encapsulated therein at least one dye selected from the group consisting of aqua green, jade green, Cy green, indocyanine green (ICG), Cy7 or Cy7.5, IR dye 800CW, or any combination thereof.

3. 3. The suspension of claim 1 or 2, wherein the suspension of fluorospheres having a fluorescent emission between 355 nm and 800 nm when excited at wavelengths less than 800 nm has encapsulated therein at least one dye selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof.

4. 4. The suspension of any one of claims 1 to 3, wherein the individual fluorospheres encapsulate at least seven dyes having seven fluorescent emissions between 355 nm and 800 nm when excited at wavelengths less than 800 nm, selected from the group consisting of small molecule organic dyes, phycobiliproteins, quantum dots, polymeric dyes, fluorescent proteins, tandem dyes, UV, light yellow, yellow, Nile red, purple, blue, sky blue, or any combination thereof.

5. 5. The suspension of any one of claims 1 to 4, wherein the individual fluorospheres encapsulate eight dyes that have eight fluorescent emissions when excited by six lasers having wavelengths of 355 nm (UV), 405 nm (violet), 488 nm (blue), 561 nm (yellow-green), 638 nm (red), and 808 nm (infrared).

6. 6. The suspension of any one of claims 1 to 5, wherein the fluorospheres have a diameter of from 2.5 μm to 6.5 μm.

7. The fluorospheres in the suspension are 0.4 x 10 fluorospheres ６ 1.5 x 10 fluorospheres / mL ６ The suspension according to any one of claims 1 to 6, having a concentration of 1000 / mL.

8. 8. The suspension of any one of claims 1 to 7, wherein the at least one surfactant is selected from the group consisting of an ionic surfactant, a non-ionic surfactant, sodium dodecyl sulfate (SDS), NP-40, Ecosurf EH-9, Ecosurf SA-9, Ecosurf Tween 20, Triton X-11, or any combination thereof; the at least one stabilizer is selected from the group consisting of a stabilizing reducing agent, a stabilizing thiol-containing compound, (S)-2-aminobutane-1,4-dithiol hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof; and the at least one preservative is selected from the group consisting of sodium azide, thimerosal, or any combination thereof.

9. 9. The suspension according to any one of claims 1 to 8, wherein the at least one surfactant is at a concentration of 0.01% to 1% based on the total volume of the suspension.

10. 1. A method for quality control of a flow cytometer using a single peak, comprising: (a) loading a quality control suspension according to any one of claims 1 to 9 into a flow cytometer; (b) (i) evaluating the output of at least one laser in said flow cytometer based on single peak analysis; (ii) evaluating the EPS of at least one laser in said flow cytometer based on single peak analysis; (iii) estimating a laser delay of at least one laser in said flow cytometer based on single peak analysis; (iv) estimating the gain of at least one laser in said flow cytometer based on single peak analysis; (v) evaluating the rCV of at least one laser in said flow cytometer based on single peak analysis. evaluating at least one of the following: (c) determining whether the flow cytometer passes or fails quality control based on the evaluation in step (b); A method comprising:

11. 11. The method of claim 10, wherein the evaluating in step (b) comprises evaluating each of steps (b)(i) through (b)(v).

12. 1. A method for quality control of a flow cytometer using multiple peaks, comprising: (a) loading a quality control suspension according to any one of claims 1 to 9 into the flow cytometer and performing a quality control analysis based on two or more peaks; (b) adjusting the brightest peak to a target median fluorescence intensity; (c) reading the median fluorescence intensity and rCV of each of the peaks; (d) after step (c), removing the quality control suspension from the flow cytometer; (e) loading non-fluorescent polystyrene beads into the flow cytometer; (f) reading the median fluorescence intensity of the polystyrene beads that have no fluorescence; (g) calculating the sensitivity and background; A method comprising:

13. 13. The method of claim 12, wherein there are three peaks: a bright peak, a medium peak, and a dim peak.

14. The target median fluorescence intensity in step (f) is between 500,000 and 4×10 ６ The method according to any one of claims 12 to 13, wherein

15. The method of any one of claims 12 to 14, wherein the sensitivity and background comprise MESF sensitivity, quantum efficiency, or background.

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