Fluid retarding unit and flow cytometer and method involving same

Abstract

A fluid retarding unit is provided. A fluid blocker unit of interest includes a plurality of valves and a plurality of blockers, wherein each blocker of the plurality of blockers is fluidly coupled to and co-positioned with a different valve of the plurality of valves. In one embodiment, a fluid retarding unit includes an inlet for receiving a fluid, a valve line fluidly coupled to the inlet, a retarder line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the retarder line, and an outlet for discharging the fluid. The valve line of interest includes a series of fluidly coupled valves, and the blocker line of interest includes a series of fluidly coupled blockers. Methods and flow cytometers involving the subject fluid retardation units are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC § 119(e) to the filing date of U.S. Provisional Patent Application Serial No. 63 / 349,367, filed on June 6, 2022, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Flow cytometry is a technique used to characterize and often sort particles of interest in biological material such as cells of a blood sample or another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample such as a blood sample, and a sheath reservoir containing a sheath fluid. A flow cytometer delivers particles (including cells) in a fluid sample to a flow cell as a cell stream while also directing a sheath fluid to the flow cell. To characterize the components of the flow stream, the flow stream is irradiated with light. Changes in the material in the flow stream, such as morphology or the presence of fluorescent markers, can result in changes in the light observed, and these changes allow for characterization and separation. To characterize the components in the flow stream, light must be shone on the flow stream and collected. The light source in a flow cytometer can vary and can include one or more broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flow stream, and the optical response from the irradiated particles is collected and quantified.

[0004] Some flow cytometer systems have been implemented using pressure-driven fluidics, in which sample and sheath fluid are provided to a flow cell containing a detection area at a pressure greater than ambient pressure. Changes in the flow rate through the flow cell of a pressure-driven fluidics system are achieved by changing the pressure in the sample tube and / or sheath fluid reservoir that is supplied to the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is controlled by the pressure levels in the sample tube and sheath fluid reservoir and the ratio of the blockages of the sample fluid and sheath fluid paths.

[0005] Alternatively, flow cytometer systems have been implemented using vacuum-driven fluidics, where a vacuum pump draws a vacuum downstream of the flow cell, and the sample and sheath fluids are maintained at ambient pressure. The flow rate through the flow cell of the vacuum-driven fluidics system is changed by varying the vacuum drawn by the vacuum pump, and the ratio of sample fluid to sheath fluid flowing through the flow cell is controlled by the ratio of the retardation of the sample fluid and sheath fluid paths. Prior systems, such as the system disclosed in U.S. Pat. No. 8,528,427 to Vrane et al., have employed fluid retarders and valves to provide a limited number of sheath fluid retardation states. Summary of the invention

[0006] The inventors have recognized that in order to further improve sample flow rate control in vacuum driven fluidics systems, it is necessary to be able to change the resistance of the fluid as it is drawn through the fluidics system into the flow cell. Therefore, there is a need for systems and methods that involve increasing the resistance state. The embodiments of the fluid resistance unit, flow cytometer, and method described herein meet this need.

[0007] Aspects of the present invention include a fluid retardation unit. The fluid retardation unit includes a plurality of valves and a plurality of retarders, wherein each of the plurality of retarders is fluidically coupled to a different valve of the plurality of valves and co-located with a different valve of the plurality of valves. In an embodiment, the subject fluid retardation unit includes an inlet for receiving a fluid, a valve line fluidically coupled to the inlet, a retarder line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the retarder line, and an outlet for discharging the fluid. The valve line of interest includes a series of fluidically coupled valves, and the retarder line of interest includes a series of fluidically coupled retarders. In some instances, the valve line includes 2 to 6 valves (e.g., 4 valves). In some cases, the retarder line may include 2 to 6 retarders (e.g., 4 retarders). In some embodiments, the fluid retardation unit includes an equal number of retarders and valves. In such embodiments, the fluid retardation unit may include x 2 Blocking states, where x is the number of valves. In a selected version, each valve of the valve line can be switched individually between an open position and a closed position, in which the fluid is not blocked when passing through the valve, and in which the fluid is blocked when passing through the valve. In the closed position, the fluid is partially or completely blocked when passing through the valve. In some cases, each blocker of the blocker line includes an inner diameter ranging from 0.25 cm to 1.25 cm. In some instances, the inner diameter of the blocker of the blocker line is either increased in sequence or constant. In a selected embodiment, each blocker of the blocker line has a length ranging from 1.5 cm to 90 cm. In some cases, the length of the blocker in the blocker line can be increased in sequence. The number of connectors in a plurality of connectors can be in the range of, for example, 2 to 5 (for example, 3). In a selected instance, each connector in a plurality of connectors couples a portion of the valve line between two valves in a series of valves to a portion of the blocker line between two blockers in a series of blockers. The fluid blocking unit can include, for example, plastic or metal pipes.

[0008] Aspects of the present invention also include a flow cytometer. The flow cytometer of interest includes a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidly coupling to a sheath fluid reservoir, and a fluid retardation unit located between the sheath fluid line and the flow cell. The fluid retardation unit for the subject flow cytometer is as described above and includes an inlet fluidically coupled to the sheath fluid line, a valve line fluidically coupled to the inlet, a retardation line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the retardation line, and an outlet fluidically coupling the fluid retardation unit to the flow cell. As discussed above, the valve line of interest includes a series of fluidically coupled valves, and the retardation line of interest includes a series of fluidically coupled retarders. In some instances, the flow cytometer includes a processor operably connected to each valve of the valve line. In such instances, the processor can be configured to change the retardation state of the fluid retardation unit by initiating switching of the valve in the valve line from an open state to a closed state or vice versa. In select versions, the flow cytometer includes a light source configured to irradiate the flow cell at an interrogation point, a detector configured to collect particle modulated light from the flow cell, and a sheath fluid reservoir.

[0009] Additional aspects of the invention include methods for analyzing samples. The methods of interest include: (a) introducing a particle sample into a flow cytometer having a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidically coupling to a sheath fluid reservoir, and a fluidic retardation unit located between the sheath fluid line and the flow cell, and (b) performing a flow cytometric analysis on the sample. The fluidic retardation unit for use in the subject method includes an inlet fluidically coupled to the sheath fluid line, a valve line fluidically coupled to the inlet, a retardation line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the retardation line, and a fluidic retardation unit fluidically coupled to an outlet of the flow cell. As discussed above, the valve line of interest includes a series of fluidically coupled valves, and the retardation line of interest includes a series of fluidically coupled retarders. An embodiment of the method includes changing the retardation state of the fluidic retardation unit by switching a valve of the valve line from an open state to a closed state or vice versa. In some cases, the particle sample of interest includes a biological sample, such as a cell.

[0010] Elements of the present invention also include methods of assembling a flow cytometer. The method of interest includes fluidly coupling a fluid retardation unit to a flow cell for transporting particles in a flow stream and a sheath fluid line for fluidly coupling to a sheath fluid reservoir. The fluid retardation unit for the subject method includes an inlet fluidically coupled to the sheath fluid line, a valve line fluidically coupled to the inlet, a retarder line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the retarder line, and a fluidly coupling the fluid retardation unit to an outlet of the flow cell. As discussed above, the valve line of interest includes a series of fluidically coupled valves, and the retarder line of interest includes a series of fluidically coupled retarders. Methods according to some embodiments include operably connecting a processor to each valve of the valve line. As discussed above, the processor of interest can be configured to change the retarding state of the fluid retardation unit by initiating switching of a valve in the valve line from an open state to a closed state or vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which the following figures are included:

[0012] Figures 1A-1C A fluid retardation unit according to certain embodiments of the present invention is depicted.

[0013] Figure 2 A fluid system having a fluid retardation unit according to certain embodiments of the present invention is depicted.

[0014] Figure 3 A fluid system configured to vary the retardation of a fluid retardation unit is depicted.

[0015] Figure 4 Depicted is a functional block diagram of a flow cytometry system according to certain embodiments.

[0016] Figure 5 Systems for analyzing and displaying biological events according to certain embodiments of the present invention are depicted.

[0017] Figure 6 A block diagram of a computing system is depicted in accordance with certain embodiments.

[0018] Figure 7A-7B Depicted is the effect of varying the sheath fluid retardation on sample flow rate using the subject fluid retardation unit. DETAILED DESCRIPTION

[0019] A fluid block unit is provided. The fluid block unit of interest includes a plurality of valves and a plurality of blocks, wherein each of the plurality of blocks is fluidically coupled to a different valve of the plurality of valves and co-located with a different valve of the plurality of valves. In one embodiment, the fluid block unit includes an inlet for receiving a fluid, a valve line fluidically coupled to the inlet, a blocker line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the blocker line, and an outlet for discharging the fluid. The valve line of interest includes a series of fluidically coupled valves, and the blocker line of interest includes a series of fluidically coupled blocks. Methods and flow cytometers relating to the subject fluid block unit are also provided.

[0020] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, but can certainly be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention is limited only by the appended claims.

[0021] Where a numerical range is provided, it is understood that unless the context clearly indicates otherwise, each intermediate value to the tenth lower limit unit between the upper and lower limits of the range and any other stated value or intermediate value in the stated range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, but are subject to any explicitly excluded limitations in the stated ranges. Where the stated range includes one or both of the limitations, ranges excluding either or both of those included limitations are also encompassed within the present invention.

[0022] Certain ranges are presented herein with a numerical value preceded by the term "about." The term "about" is used herein to provide literal support for the exact numerical value that follows it, as well as numbers that are close to or approximately the numerical value that follows the enumerated term. In determining whether a number is close to or approximately a specifically enumerated number, a close or approximate unenumerated number may be a number that, in the context in which the number is given, provides a substantial equivalence to the specifically enumerated number.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0024] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publication. The citation of any publication is due to its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0025] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. As such, this statement is intended to serve as antecedent basis for use of special terminology, such as "solely," "only," etc., in connection with the recitation of claim elements, or use of a "negative" limitation.

[0026] It will be apparent to those skilled in the art after reading this disclosure that each of the various embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any enumerated method can be performed in the order of the enumerated events or in any other order that is logically possible.

[0027] Although the system and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be clearly understood that unless expressly recited pursuant to 35 USC §112, the claims should not be interpreted as necessarily being limited in any way to "means" or "step" limitations, but should be given the full scope of meaning and equivalents provided by the claims pursuant to the judicial doctrine of equivalents, and to the extent a claim is expressly recited pursuant to 35 USC §112, it will be given full legal equivalents pursuant to 35 USC §112.

[0028] Fluid Retardation Unit

[0029] As discussed above, aspects of the present invention include a fluid retardation unit. "Fluid retardation unit" means a device configured to provide a certain amount of retardation (e.g., using pressure or vacuum) to a fluid being transported therethrough. For example, where the subject fluid retardation unit is employed in a flow cytometer (although other applications are apparent to one of ordinary skill in the art), the relative proportions of sheath fluid and sample fluid drawn through a flow cell may be modulated by employing a certain retardation. The modulation of the relative proportions of sheath fluid and sample fluid drawn through the flow cell also controls the sample fluid flow rate through the flow cell while the total flow rate through the flow cell remains constant. In some cases, for example, the subject fluid retardation unit increases the number of retardation states that can be applied to a fluid flow relative to conventional devices for providing fluid retardation. "Retardation state" means a specific level of retardation associated with a fluid retardation unit that is applied at a given time during the flow of a fluid. In some cases, the fluid retardation unit of the present disclosure increases the number of retardation states by two or more times, three or more times, four or more times, and including five or more times. The increase in the number of retardation states increases the precision with which the fluid flow rate can be controlled. The blocking state can be discrete or continuous. In some embodiments, the blocking state is discrete. In other embodiments, the blocking state is continuous.

[0030] The fluid retardation unit can include any convenient material. In some cases, the fluid retardation unit includes a conduit (e.g., a pipe). In such cases, the conduit can include, for example, glass, plastic, or steel, and combinations thereof. The conduit can have any convenient inner diameter, where the range of diameters of interest is 0.025 cm to 1.25 cm, such as 0.05 cm to 0.50 cm and including 0.05 to 0.50 cm. The inner diameter of the conduit can be constant throughout the fluid retardation unit, or can vary as desired. In an embodiment, the fluid retardation unit includes an inlet for receiving a liquid and an outlet for discharging the liquid. Any convenient inlet can be used. For example, the inlet can be a portal in a conduit through which a fluid can enter. In some versions, the inlet is or includes a fluid fitting that is configured to couple to, for example, a sheath fluid line. Similarly, a convenient outlet can be used. In some embodiments, the outlet is configured to couple to the flow cell fluid, for example, via a fitting.

[0031] Aspects of the fluid retardation unit include multiple valves. Any valve suitable for fluid applications can be used, including but not limited to needle valves, stop valves, gate valves, plug valves, ball valves, butterfly valves, pinch valves, angle valves, rotary valves (e.g., plug valves), etc. Each valve in the multiple valves can be a valve of the same type or a different type. In some cases, each valve in the multiple valves is a valve of the same type. In other cases, one or more valves in the multiple valves are valves of a different type relative to another valve in the multiple valves. The number of valves in the multiple valves can vary as needed. In some embodiments, the number of valves in the multiple valves ranges from 2 to 10, such as 2 to 8, such as 2 to 6, and includes 2 to 4. In some embodiments, the multiple valves include 4 valves.

[0032] Aspects of the fluid retardation unit also include a plurality of retarders. The retarders described herein are configured to restrict fluid flow and can take any convenient form. In some embodiments of the present invention, the fluid retardation unit is configured to provide a plurality of discrete retardation levels. This is achieved by providing alternative selectable fluid paths, each of which has different fluid retardations. In such embodiments, each discrete path can constitute or include different retarders in a plurality of retarders. The fluid path can be a conduit (e.g., a pipe segment) with different lengths or inner diameters. A valve can be located in the fluid path to allow selection of a desired fluid path from a plurality of alternative fluid paths.

[0033] In some cases, the fluid retardation unit includes a viscosity-dominated restrictor. In an embodiment, the viscosity-dominated restrictor is implemented by using a section of pipe or similar conduit with a substantially constant inner diameter. Such a retarder can be characterized as having discrete levels of retardation. For a fluid of a given viscosity, the retardation force depends on the length and internal cross-sectional area of ​​the conduit, and the length is selected to provide the desired retardation. In contrast, simply clamping the conduit at a single point, or using a valve (such as a needle valve) to provide flow restriction, will result in convection acceleration. Due to the effect of temperature on viscosity, the temperature dependence of the retardation of the sheath fluid line incorporating a viscosity-dominated fluid retarder is the same as the temperature dependence of the retardation of the sample fluid line. Therefore, when the temperature changes, the retardation ratio of the sample fluid line and the sheath fluid line remains unchanged. In contrast, since clamping is convection-dominated and the retardation does not change with temperature, the use of a clamping-type retardation will cause the ratio of path retardation to change with temperature.

[0034] In an alternative embodiment of the present invention, the fluid retardation unit is continuously variable. In some such embodiments, at least one retarder includes a conduit made of at least partially compressible or deformable material, such as a section of plastic pipe, and an adjustable pressure is applied to the outside of the pipe. Modulation of the pressure applied to the outside of the pipe changes the diameter and / or shape of the pipe, thereby changing the cross-sectional area of ​​the fluid path and, subsequently, the fluid retardation of the pipe. The continuously variable retarder can be composed of a section of mechanically compressed pipe. For example, the pipe can be wrapped around a cylindrical column and compressed between plates, at least one plate being movable and having a surface perpendicular to the column. Alternatively, the continuously variable retarder can be composed of a section of pipe passing through the inside of a pressure chamber with an adjustable internal pressure. The internal pressure of the pressure chamber can be modulated by connecting the chamber to an adjustable pressure source, such as a compressed air source, or by changing the size and / or shape of the pressure chamber, such as by mechanical means.

[0035] Each of the multiple blockers can be a blocker of the same type or different types. In some cases, each of the multiple valves is a blocker of the same type. In other cases, one or more of the multiple blockers are different types of blockers relative to another blocker in the multiple blockers. In some such cases, one or more of the multiple blockers are continuously variable (i.e., a certain range of blockage can be provided), while one or more other blockers are discrete. The number of blockers in the multiple blockers can vary as needed. In some embodiments, the number of blockers in the multiple blockers ranges from 2 to 10, such as 2 to 8, such as 2 to 6, and includes 2 to 4. In some embodiments, the multiple blockers include 4 blockers. Each blocker can have any convenient length. In some embodiments, each blocker has a length ranging from 1.5 cm to 90 cm, such as 1.5 cm to 50 cm, and includes 1.5 cm to 45 cm. In some cases, at least one blocker has a length ranging from 35 cm to 45 cm, such as 40.64 cm. In some cases, at least one blocker has a length ranging from 15 cm to 25 cm, such as 20.32 cm. In some cases, at least one blocker has a length ranging from 5 cm to 15 cm, such as 10.16 cm. In some cases, at least one blocker has a length ranging from 1 cm to 10 cm, such as 5.08 cm. In the case where the blocker includes multiple sections of conduit (e.g., tubing), the tubing can be arranged in any convenient manner. In some cases, the tubing can be wound around a cylindrical post, such as into a coil. In some such cases, the tubing can be compressed between movable plates having a surface perpendicular to the post. However, any other suitable arrangement (e.g., an arrangement that does not produce unintentional kinks in the tubing) can also be adopted.

[0036] Each of the multiple blockers can apply the same level of retardation or apply different levels of retardation. In some instances, all blockers apply the same level of retardation relative to each other. In some such instances, each blocker can be characterized by the same inner diameter and length, that is, the inner diameter and length remain constant. In other embodiments, each of the multiple blockers applies different levels of retardation relative to each other. In some cases, the retardation of multiple blockers increases in sequence. In other words, multiple blockers include a blocker characterized by a minimum retardation, a blocker characterized by a higher retardation than the minimum retardation, a blocker characterized by a higher retardation than the previous retardation, and the like. In some such embodiments, the inner diameter and / or length of the blocker may be different. In certain embodiments, the inner diameter of the blocker increases in sequence. In additional embodiments, the length of the blocker increases in sequence. In other embodiments, the inner diameter of the blocker is constant, and the length of the blocker increases in sequence.

[0037] In some cases, each of the multiple retarder fluid is coupled to a different valve in a plurality of valves and is co-located with a different valve in a plurality of valves. As discussed herein, if two entities have a conduit between them that is configured to transfer liquid from one entity to another entity, they can be described as "fluid coupling". Similarly, if two entities are positioned adjacent to each other and / or very closely in the fluid retardation unit, they can be described as "co-located". For example, if entity A and entity B are fluidically coupled to each other via one or more pipes, and there is no intermediate entity in or along these pipes, entities A and B can be described as co-located. Therefore, in some instances, each retarder can be coupled to the valve fluid and co-located with the valve, so that the open or closed state of the valve affects whether and / or how the fluid passes through the co-located retarder. In a selected version, the fluid retardation unit does not include a valve that is not co-located and / or fluidically connected with a specific retarder. In these embodiments, the fluid retardation unit includes an equal number of retarders and valves.

[0038] In selected cases, each valve in the plurality of valves is capable of being individually switched between an open position in which fluid is unobstructed through the valve and a closed position in which fluid is obstructed through the valve. In the closed position, fluid may be partially or completely obstructed through each valve. In some cases in which the valve is completely obstructed in the closed position, the fluid retardation unit is configured to provide discrete levels of retardation. In these cases, the valve may select a retarder through which the fluid will pass, thereby achieving different retardation states. In some other cases in which the valve is partially obstructed in the closed position, the fluid retardation unit is configured to provide a continuous level of retardation. The valves may include any convenient mechanism for switching between open and closed positions. In some cases, the valves are under automatic control, such as using a servo mechanism.

[0039] The components of the subject fluid retardation unit can be arranged in any suitable configuration. Exemplary configurations include series configurations, parallel configurations, and configurations including series and parallel elements. In some instances, the fluid retardation unit includes a series configuration. In some such embodiments, the fluid retardation unit includes an inlet for receiving a fluid, a valve line coupled to the inlet, and a retarder line coupled to the inlet. Any convenient inlet can be used. As discussed herein, a "valve line" refers to a plurality of (i.e., a series of) valves that are fluidically coupled to each other, for example, via a conduit. Similarly, a "retarder line" refers to a plurality of (i.e., a series of) retarders that are fluidically coupled to each other, for example, via a conduit. Embodiments of the fluid retardation unit also include a plurality of connectors that fluidly couple the valve line to the retarder line. The "connector" described herein is a conduit that connects a portion of the valve line to a portion of the retarder line. In some instances, each of the plurality of connectors couples a portion of the valve line between two valves in a series of valves to a portion of the retarder line between two retarders in a series of retarders. Any convenient number of connectors can be used. The number of connectors in the plurality of connectors can vary as desired. In some embodiments, the number of connectors in the plurality of connectors ranges from 2 to 10, such as 2 to 8, such as 2 to 5, and includes 2 to 4. In some embodiments, the plurality of connectors includes 3 connectors. In some cases, the plurality of connectors includes n-1 connectors, where n is the number of retarders and / or valves.

[0040] In some embodiments involving a series configuration, each valve of the valve line can be switched individually (e.g., via a servo mechanism) between an open position and a closed position, in which the fluid is not blocked when passing through the valve, and in which the fluid is blocked when passing through the valve, for example, wherein the blockage can be complete or partial. In this way, the blocker through which the fluid passes can be adjusted. In some embodiments, the blockage of the blocker in the blocker line can be increased in sequence. In some cases, each blocker of the blocker line includes an inner diameter ranging from 0.025 to 1.25 cm, such as 0.050 to 0.50 cm and including 0.050 to 0.10 cm. In selected instances, the inner diameter of the blocker in the blocker line increases in sequence. In other instances, the inner diameter of the blocker in the blocker line is constant. In some versions, each blocker of the blocker line has a length ranging from 1.5 cm to 90 cm. In selected cases, the length of the blocker in the blocker line increases in sequence. In some embodiments involving a series configuration, the fluid retardation unit includes an equal number of retarders and valves. In some such embodiments, the fluid retardation unit includes x 2, where x is the number of valves. In an exemplary embodiment comprising 4 valves in series, the fluid blocking unit comprises 16 blocking states.

[0041] In one example involving a series configuration, a fluid may enter through an inlet and then branch off into a retarder line and a valve line (e.g., via a Y-connector). If each valve in the valve line is closed, the fluid will pass through each retarder in the retarder line before reaching the outlet. This scenario constitutes a highest retarder state. If the valve closest to the inlet in the valve line is open, a certain amount of fluid may bypass the first retarder. Since the next valve in the valve line is closed, the bypassed fluid will enter the retarder line via a connector and pass through the remaining retarders. This will result in an incrementally lower retarder state than the retarder state when each valve is closed. In this way, any combination of open and closed valves produces a different retarder state.

[0042] Figure 1A 100a is depicted as having a series configuration according to some embodiments of the present invention. The fluid retardation unit 100a includes an inlet A, a plurality of retarders R1-R4 in a retardation line 101, valves V1-V4 in a valve line 102, connectors 103a-103c, and an outlet B. Since the fluid retardation unit 100a includes four valves, it consists of four 2 = 16 blocking states are represented. If V1-V4 are all closed, the fluid entering at inlet A must pass through each of R1-R4, and therefore encounters a maximum level of blocking. If valve V1 is open and valves V2-V4 are closed, a certain amount of fluid that would otherwise pass through R1 will pass through V1 and connector 103a before passing through valves R2-R4. If valve V3 is open and valves V1-V2 and V4 are closed, a certain amount of fluid that would otherwise pass through R3 will pass through V3 and return to the blocker pipeline 103c. In this way, any of the 16 combinations of open and closed valves can be achieved, resulting in 16 blocking states.

[0043] In some cases, the fluid retardation unit is characterized by a parallel configuration. In such embodiments, the fluid retardation unit includes an inlet for receiving fluid, a plurality of fluid lines extending in parallel, and an outlet for discharging fluid. Each fluid line in the fluid lines contains a valve and a retarder. A portion of each fluid line closer to the inlet includes a valve, and a portion of each fluid line closer to the outlet includes a retarder. In some embodiments, there are no parallel fluid lines that include a valve but not a retarder or include a retarder but not a valve. In other words, each parallel fluid line includes both a valve and a retarder. In some cases, the parallel fluid retardation unit includes an equal number of retarders and valves.

[0044] Figure 1B A fluid retardation unit 100b having a parallel configuration is depicted according to certain embodiments of the present invention. The fluid retardation unit 100b includes an inlet A, a plurality of valves V1-V4, a plurality of retarders R1-R4, parallel fluid lines 104a-104d, and an outlet B. Valve V1 is fluidically coupled to and co-located with the retarder R1 on the fluid line 104a, valve V2 is fluidically coupled to and co-located with the retarder R2 on the fluid line 104b, valve V3 is fluidically coupled to and co-located with the retarder R3 on the fluid line 104c, and valve V4 is fluidically coupled to and co-located with the retarder R4 on the fluid line 104d. Figure 1B In an embodiment of the invention, each of the retarder R1-R4 is characterized by a different level of retarder (e.g., they have successively increasing lengths, although this is not shown). Thus, depending on which of the valves V1-V4 is open or closed at a given time, different retarder states can be achieved.

[0045] In some cases, the fluid retardation unit is characterized by a configuration involving parallel and series arrangements. In these cases, at least two sets of valves and retarders are located in parallel fluid lines (e.g., as described with respect to Figure 1B In addition, at least one of the resistor-valve pairs is located upstream and / or downstream of the parallel fluid lines, where "upstream" and "downstream" are defined relative to the direction of fluid flow. In other words, the resistor-valve pair is "in series" with the "parallel" resistor-valve pair.

[0046] Figure 1C A fluid retardation unit 100c is depicted that is characterized by a configuration involving parallel and series arrangements according to certain embodiments of the present invention. The fluid retardation unit 100c includes an inlet A, valves V1-V3, retarders R1-R3, parallel fluid lines 105a-105b, and an outlet B. Valve V2 is fluidically coupled to and co-located with the retarder R2 on the fluid line 105a, and valve V3 is fluidically coupled to and co-located with the retarder R3 on the fluid line 105b. Valve V1 and retarder R1 are co-located and fluidically coupled to each other and are located upstream of the fluid lines 105a-105b. Although in Figure 1C Not shown, but valve V1 may optionally be fluidly coupled to a conduit that bypasses resistor R1. If a lower blocking state is desired, valve V1 may be configured to divert fluid to this conduit.

[0047] Flow cytometry

[0048] Aspects of the present invention also include flow cytometers. The flow cytometer of interest includes a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidically coupling to a sheath fluid reservoir, and a fluidic retardation unit located between the sheath fluid line and the flow cell. As discussed above, the fluidic retardation unit of the present invention includes a plurality of valves and a plurality of retarders, wherein each of the plurality of retarders is fluidically coupled to a different valve of the plurality of valves and co-located with a different valve of the plurality of valves. In some cases, the fluidic retardation unit of the present invention includes an inlet fluidically coupled to the sheath fluid line, a valve line fluidly coupled to the inlet, a retardation line fluidly coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the retardation line, and an outlet fluidically coupling the fluidic retardation unit to the flow cell.

[0049] As discussed herein, "flow cell" is described in its conventional sense and refers to a component containing a flow channel having a liquid flow stream for transporting particles in a sheath fluid. In an embodiment, the subject flow cell comprises a cuvette. The cuvette of interest comprises a container having a passageway therethrough. The flow stream may include a liquid sample injected from a sample tube. The flow cell of interest comprises a light accessible flow channel. In some instances, the flow cell comprises a transparent material (e.g., quartz) that allows light to pass through. Any convenient flow cell that propagates a fluid sample to a sample interrogation area can be used as a flow cell described herein, wherein in some embodiments, the flow cell comprises a cylindrical flow cell, a frustoconical flow cell, or a flow cell comprising a proximal cylindrical portion and a distal frustoconical portion, the proximal cylindrical portion defining a longitudinal axis, the distal frustoconical portion terminating in a flat surface having an orifice transverse to the longitudinal axis.

[0050] In certain embodiments, the flow cytometer includes a sample fluid source. The sample fluid source can be any suitable reservoir or container (e.g., having rigid or flexible walls) for containing the sample fluid. The sample fluid container can have a volume ranging from 1 mL to 100 mL; for example, the volume of the container can be in the range of 1 mL to 90 mL, 1 mL to 80 mL, 1 mL to 70 mL, 1 mL to 60 mL, 1 mL to 50 mL, 1 mL to 40 mL, 1 mL to 30 mL, 1 mL to 20 mL, or 1 mL to 10 mL.

[0051] In some embodiments, the flow cytometer includes a sheath fluid reservoir. The sheath fluid reservoir can be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding a sheath fluid. In certain embodiments, the sheath fluid reservoir is fluidically coupled to an input of a flow cell. The sheath fluid container can have a volume ranging from 1 L to 100 L; for example, the volume of the container can be in the range of 1 L to 90 L, 1 L to 80 L, 1 L to 70 L, 1 L to 60 L, 1 L to 50 L, 1 L to 40 L, 1 L to 30 L, 1 L to 20 L, or 1 L to 10 L. The sheath fluid reservoir can be fluidically connected to a sheath fluid line configured to deliver the sheath fluid from the reservoir to the flow cell.

[0052] In some embodiments, the flow cell includes a sample injection port configured to provide a sample from a sample fluid source to the flow cell. The sample injection port may be an orifice located in the wall of the inner chamber, or may be a conduit located at the proximal end of the inner chamber. In the case where the sample injection port is an orifice located in the wall of the inner chamber, the sample injection port orifice may be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to, a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, an ellipse, etc.; and an irregular shape, such as a parabola bottom coupled to a plane top. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain instances, its opening is in the range of 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0053] In some instances, the sample injection port is a conduit located at the proximal end of the flow cell chamber. For example, the sample injection port can be a conduit that is positioned so that the orifice of the sample injection port is in line with the orifice of the flow cell. In the case where the sample injection port is a conduit positioned in line with the orifice of the flow cell, the cross-sectional shape of the sample injection tube can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to: a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, an ellipse; and an irregular shape, such as a parabola bottom coupled to a plane top. The orifice of the conduit can vary depending on the shape, and in some instances, its opening is in the range of 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm, for example 1.5 mm. The shape of the tip of the sample injection port can be the same or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip having a bevel angle ranging from 1 to 10 degrees, such as 2 to 9 degrees, such as 3 to 8 degrees, such as 4 to 7 degrees and including a bevel angle of 5 degrees.

[0054] In some embodiments, the flow cell further comprises a sheath injection port configured to provide sheath fluid from a sheath fluid source to the flow cell. In embodiments, the sheath injection system is configured to provide a flow of sheath fluid to the flow cell chamber, for example in combination with the sample to produce a layered sheath fluid flow stream surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the rate of sheath fluid delivered to the flow cell chamber can be 25 microliters / second to 2500 microliters / second, such as 50 microliters / second to 1000 microliters / second, and includes 75 microliters / second or greater to 750 microliters / second.

[0055] In some embodiments, the sheath injection port is an orifice located in the wall of the inner chamber. The sheath injection port orifice can be any suitable shape, wherein the cross-sectional shapes of interest include, but are not limited to, straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as a parabolic bottom coupled to a flat top. The size of the sample injection port orifice can vary depending on the shape, and in some instances, its opening is in the range of 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0056] The flow cytometer of the present invention includes a fluid retardation unit located between a sheath fluid reservoir and a flow cell (e.g., a sheath fluid injection port of the flow cell). For example, where the sheath fluid reservoir includes a sheath fluid line fluidically coupled thereto, the inlet of the fluid retardation unit can be fluidically coupled to the sheath fluid line (e.g., via a fitting). Similarly, the outlet of the fluid retardation unit can be coupled to the sheath fluid injection port of the flow cell (e.g., via a fitting). Any fluid retardation unit, such as those described above, can be employed in the flow cytometer. In some embodiments, the fluid retardation units are in series. In other embodiments, the fluid retardation units are in parallel. In yet other embodiments, the fluid retardation unit includes parallel and series elements.

[0057] In some embodiments, the system also includes a pump (e.g., a vacuum pump) that is in fluid communication with the flow cell to propagate the flow stream through the flow cell. Any convenient fluid pump scheme can be used to control the flow of the flow stream through the flow cell. In some instances, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the subject system is configured to transmit the fluid through the flow cell at a rate suitable for multi-photon counting of the light from the sample in the flow stream. For example, the system can include a pump that is configured to make the sample flow through the flow cell at a certain rate, and the rate is 1 nanoliter / minute to 500 nanoliters / minute, such as 1 nanoliter / minute to 250 nanoliters / minute, such as 1 nanoliter / minute to 100 nanoliters / minute, such as 2 nanoliters / minute to 90 nanoliters / minute, such as 3 nanoliters / minute to 80 nanoliters / minute, such as 4 nanoliters / minute to 70 nanoliters / minute, such as 5 nanoliters / minute to 60 nanoliters / minute and including 10 nanoliters / minute to 50 nanoliters / minute in the range. In certain embodiments, the flow rate of the flowing stream is between 5 nanoliters / minute and 6 nanoliters / minute.

[0058] Figure 2 A schematic diagram of the elements of the flow cytometric system of the present invention is depicted. The system vacuum is created by a vacuum pump 211, which draws sheath fluid from the sheath reservoir 202 and draws sample fluid containing particles to be analyzed from the sample tube 201 through the flow cell 200, where optical analysis is performed (optics not shown). The waste effluent, i.e., a mixture of sample and sheath fluid leaving the flow cell, is discharged into the waste reservoir 203.

[0059] The pulsations in the vacuum created by the vacuum pump 211, which is typically a diaphragm type pump, are dampened by the accumulator 255, which is also known as a pulsation dampener. The accumulator may be a sealed canister having an internal volume that is a multiple (e.g., 10 to 1000 times) of the stroke volume of the vacuum pump.

[0060] The transducer 231 measures the pressure drop relative to atmospheric pressure formed by the vacuum pump 211. This pressure drop is referred to herein as the "static pressure drop". The static pressure drop can be measured from the inside of the accumulator 255, thereby obtaining a stable measurement. The transducer 231 is usually connected to the accumulator 255 by a short tube so that the pressure in the tube is equal to the pressure in the accumulator. It is desirable to include a bleed port (e.g., a small orifice that connects the inside of the tube to the outside air) in the tube connecting the transducer 231 and the accumulator 255 located near the transducer to allow a small amount of air to be drawn into the tube and through the tube under the extraction of the vacuum in the accumulator. The bleed port should be small enough so that the air flow through the pipe has no significant effect on the measurement of the static pressure drop. The small amount of air flow through the pipe in the direction from the orifice (near the transducer) toward the accumulator prevents any fluid or foam that may be present in the accumulator from entering the pipe to reach the transducer, which may affect the accuracy of the measurement.

[0061] The transducer 232 measures the pressure drop across the cuvette 102 (measured from upstream of the flow cell to the accumulator 255). This pressure drop is referred to herein as the "dynamic pressure drop". For a given total flow rate through the cuvette, the dynamic pressure drop is constant. Therefore, by modulating the power of the vacuum pump 211 to provide a constant dynamic pressure drop, a constant total flow rate through the cuvette can be maintained.

[0062] The sample fluid is drawn through the sample line 220 and enters the flow cell 200 through the sample inlet port 208. The entire sample path has a fluid resistance R0. The sheath fluid is drawn through the fluid resistance unit 222 and enters the flow cell 200 through the sheath inlet port 210. In one embodiment, the fluid resistance of the fluid resistance unit is adjustable to a plurality of discrete fluid resistance settings. In another embodiment, the fluid resistance of the fluid resistance unit is continuously adjustable within a predetermined range.

[0063] The relative proportions of sample fluid and sheath fluid drawn into the flow cell 100 depend on the ratio of the sample line fluid resistance R0 and the fluid resistance of the fluid resistance unit 222. Therefore, by modulating the fluid resistance of the fluid resistance unit 222, the ratio of sample fluid to sheath fluid can be controlled. If the total flow rate through the flow cell is kept constant by maintaining a constant dynamic pressure drop, each reduction in the effective fluid resistance of the fluid resistance unit 222 results in a decrease in the sample fluid flow rate and an increase in the sheath fluid flow rate. Therefore, while maintaining a constant flow rate through the flow cell, the desired sample flow rate can be selected by appropriately modulating the total fluid resistance of the fluid resistance unit 222.

[0064] Valve 253 can completely cut off the fluid flow through the flow cell. Flow can be suspended to allow, for example, replacement to a new sample source after each sample analysis. In this system, the flow of fluid can be suspended by closing the valve in the fluid path between the flow cell and the pump. When the valve is closed, the dynamic pressure drop drops to zero, the first feedback loop between the dynamic pressure drop and the pump is stopped, and the second feedback loop between the static pressure drop and the pump is activated. The second feedback loop can maintain the static pressure drop at a constant level during the pause state, such as the static pressure drop that exists when the system is kept in the running state before pausing the system (while under the control of the first feedback loop). When the fluid flow through the flow cell is restarted, the control of the pump is switched back to the first feedback loop, which makes it possible to maintain a constant flow across the flow cell even after the sample flow rate changes. This switching between the feedback control circuits eliminates the large fluctuations in the vacuum during the transition between the running and pause states, while maintaining the constant flow rate through the flow cell of all samples.

[0065] Valve 251 is capable of completely shutting off the sheath fluid flow. Valve 251 is used to temporarily stop the sheath fluid flow and temporarily increase ("boost") the sample fluid flow rate after the sample tube 201 is connected to the sample line 220 to shorten the time required to draw the sample fluid into the flow cell 200. When the sample fluid reaches the flow cell, valve 251 opens, the flow of sheath fluid establishes a hydrodynamic focusing flow, and the sample and sheath fluid flow rates return to the desired rates for analysis. Valves 251 and 253 can be under automatic control in a coordinated manner so that valve 253 can be opened for a predetermined time before opening valve 251 to allow a vacuum to form in the flow cell before opening the valve 251.

[0066] A controller 261 having a switching mechanism 263 controls switching between modulating the power of the vacuum pump 211 to provide a constant dynamic pressure drop and modulating the power of the vacuum pump 211 to provide a constant static pressure drop. To maintain a constant dynamic pressure drop, the controller compares the dynamic pressure drop measured by the transducer 232 with a stored desired dynamic pressure drop P D For comparison, the expected dynamic pressure drop P D The dynamic pressure drop is determined during instrument setup to provide the desired flow rate through the flow cell. To maintain a constant static pressure drop, the controller compares the dynamic pressure drop measured by transducer 231 with the stored desired static pressure drop P S For comparison, the expected static pressure drop P S is the measured static pressure drop corresponding to the instrument running at the desired flow rate through the flow cell, depending on the selected setting of the fluid retardation unit. The desired static pressure drop P can be stored S , each value corresponding to a predetermined retardation setting of the fluid retardation unit. Alternatively, the desired static pressure drop P may be stored immediately before pausing the system.S , and the system restarts with the same retardation setting of the fluid retardation unit that was in effect before the system was paused. In some embodiments, a pressure drop feedback loop (via controller 261) and automatic control of valves 251 and 253 will be provided in a coordinated manner.

[0067] A flow sensor 235 is located on the sample line 220 to provide a direct measurement of the sample fluid flow rate. Suitable high-precision liquid flow sensors and liquid flow meters with a measurement range as low as nanoliters per minute are commercially available from, for example, Sensirion Inc. (Westlake Village, California). The flow sensor 235 is optional, but it helps to set up the flow system. During instrument setup (system calibration), the fluid resistance of the fluid resistance unit 222 is adjusted to provide the desired ratio of sheath to sample fluid, and the flow sensor provides an independent measurement of the resulting sample fluid flow rate. Alternatively, the flow rate of the sample fluid can be measured by other means, such as by analyzing a sample containing a known concentration of test particles. By measuring the detection rate of the test particles, the flow rate in the sample line 220 can be inferred.

[0068] In some embodiments, the subject flow cytometer operates in conjunction with programmable logic that can be implemented in hardware, software, firmware, or any combination thereof to change the blocking state of the fluid blocking unit. For example, where the programmable logic is implemented in software, the blocking state adjustment can be implemented at least in part by a computer-readable data storage medium including a program code, the program code including instructions that, when executed, are configured to initiate the switching of a valve in a valve line from an open state to a closed state. The subject programmable logic can be implemented in any of a variety of devices, such as a specially programmed event processing computer, a wireless communication device, an integrated circuit device, and the like. In some embodiments, the programmable logic can be executed by a specially programmed processor, which can include one or more processors, such as one or more digital signal processors (DSPs), configurable microprocessors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or other equivalent integrated or discrete logic circuit systems. A combination of computing devices, such as a combination of a DSP and a microprocessor, a combination of multiple microprocessors, a combination of one or more microprocessors with a DSP core, or any other such configuration in at least partial data connectivity, can implement one or more of the features described herein.

[0069] Embodiments of the subject flow cytometer include a processor. The processor of interest is operably connected to each valve of the valve line and is configured to change the blocking state of the fluid retardation unit by initiating the switching of the valve in the valve line from an open state to a closed state or vice versa. In some cases, the processor is configured to compare the actual flow rate with the target flow rate and adjust the blocking state of the fluid retardation unit accordingly to achieve the target flow rate. In a selected version, the processor operates automatically, such as in an automatic feedback control circuit. In some cases, the processor can receive input from a user. In such cases, the processor can receive input about the target flow rate, and the processor changes the blocking state of the fluid retardation unit to achieve the target flow rate.

[0070] Figure 3 An automatic feedback control circuit is depicted for automatically modulating the sample fluid flow rate during system calibration to obtain a preselected sample fluid flow rate. The fluid resistance unit 322 is configured to be able to modulate the fluid resistance using a servo mechanism. The processor 361 compares the flow rate measured by the flow sensor 235 with the stored desired flow rate FR and modulates the fluid resistance of the variable resistance fluid resistance 322 until the measured flow rate matches the desired flow rate FR. The processor 361 is configured to open or close any of the valves in the fluid resistance unit 322, for example, via a servo mechanism.

[0071] As previously described, the flow cytometer of the present invention includes a flow cell for transporting particles in a flow stream. The flow stream can be configured to be irradiated with light from a light source at an interrogation point. The flow stream for which the flow channel is configured may include a liquid sample injected from a sample tube. In certain embodiments, the flow stream may include a narrow, fast-flowing liquid stream that is arranged so that the linearly isolated particles transported therein are separated from each other in a single-column manner. The "interrogation point" discussed herein refers to an area within the flow cell in which the particles are irradiated with light from a light source, for example, for analysis. The size of the interrogation point may vary as needed. For example, where 0 μm represents the axis of the light emitted by the light source, the interrogation point may be in the range of -100 μm to 100 μm, such as -50 μm to 50 μm, such as -25 μm to 40 μm, and including -15 μm to 30 μm.

[0072] After irradiating the particles in the flow cell, particle modulated light can be observed. "Particle modulated light" means light received from particles in the flow stream after irradiating the particles with light from a light source. In some cases, the particle modulated light is side scattered light. As discussed herein, side scattered light refers to light refracted and reflected from the surface and internal structure of the particles. In additional embodiments, the particle modulated light includes forward scattered light (i.e., light that passes through or around the particles primarily in the forward direction). In yet other cases, the particle modulated light includes fluorescence (i.e., light emitted from a fluorescent dye after irradiation with light of an excitation wavelength).

[0073] Aspects of the present invention also include a light source configured to irradiate particles passing through the flow cell at the interrogation point. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser can be any convenient laser, such as a continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some instances, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other instances, the subject flow cytometer includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In yet other examples, the laser of interest includes a metal-vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet other examples, the subject flow cytometer includes a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, an yttrium oxide laser, or a cerium-doped laser, and combinations thereof.

[0074] The laser light source according to certain embodiments may also include one or more optical adjustment components. In certain embodiments, the optical adjustment component is located between the light source and the flow cell, and may include any device capable of changing the spatial irradiation width or some other irradiation characteristic from the light source, such as, for example, irradiation direction, wavelength, beam width, beam intensity, and focus. The optical adjustment scheme may include any convenient device for adjusting one or more characteristics of the light source, including but not limited to lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation schemes, and combinations thereof. In certain embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens may be a reduction lens. In yet other embodiments, the flow cytometer of interest includes an optical fiber.

[0075] Where the optical adjustment component is configured to move, the optical adjustment component may be configured to move continuously or in discrete intervals with increments such as, for example, 0.01 µm or greater, such as 0.05 µm or greater, such as 0.1 µm or greater, such as 0.5 µm or greater, such as 1 µm or greater, such as 10 µm or greater, such as 100 µm or greater, such as 500 µm or greater, such as 1 mm or greater, such as 5 mm or greater, such as 10 mm or greater and including increments of 25 mm or greater.

[0076] Any displacement scheme can be used to move the optical adjustment component structure, which is, for example, coupled to a movable support table or directly coupled to a motor-actuated translation table, a screw translation assembly, a gear translation device, such as those using stepper motors, servo motors, brushless motors, brushed DC motors, micro-stepping drive motors, high-resolution stepper motors, and other types of motors.

[0077] The light source can be positioned at any suitable distance from the flow cell, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more and including being 100 mm or more apart from the flow cell. In addition, the light source can be positioned at any suitable angle relative to the flow cell, such as with 10 to 90 degrees, such as 15 to 85 degrees, such as 20 to 80 degrees, such as 25 to 75 degrees and including an angle positioning in the range of 30 to 60 degrees, for example, with 90 degrees.

[0078] In some embodiments, the light source of interest includes multiple lasers configured to provide lasers for discrete irradiation of the flow stream, such as 2 or more lasers, such as 3 or more lasers, such as 4 or more lasers, such as 5 or more lasers, such as 10 or more lasers, and including 15 or more lasers. Depending on the desired wavelength of light for irradiating the flow stream, each laser can have a specific wavelength of 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and including 400 nm to 800 nm. In certain embodiments, the laser of interest can include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0079] The flow cytometer of interest may also include one or more particle modulated light detectors for detecting particle modulated light intensity data. In some embodiments, the particle modulated light detector includes one or more forward scattered light detectors configured to detect forward scattered light. For example, the subject particle analyzer may include 1 forward scattered light detector or multiple forward scattered light detectors, such as 2 or more, such as 3 or more, such as 4 or more, and including 5 or more forward scattered light detectors. In certain embodiments, the particle analyzer includes 1 forward scattered light detector. In other embodiments, the particle analyzer includes 2 forward scattered light detectors.

[0080] Any convenient detector for detecting the collected light may be used in the forward scattered light detector described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured with a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10 cm 2 , such as 0.05 cm 2 Up to 9 cm 2, such as 0.1 cm 2 Up to 8 cm 2 , such as 0.5 cm 2 Up to 7 cm 2 And includes 1 cm 2 Up to 5 cm 2 range of photomultiplier tubes.

[0081] In embodiments, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some instances, the detector of interest is configured to measure the collected light continuously. In other instances, the detector of interest is configured to measure at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or measuring light at some other interval.

[0082] In additional embodiments, the one or more particle modulated light detectors may include one or more side scattered light detectors for detecting side scattered wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particles). In some embodiments, the particle analyzer includes a single side scattered light detector. In other embodiments, the particle analyzer includes multiple side scattered light detectors, such as 2 or more, such as 3 or more, such as 4 or more, and includes 5 or more side scattered light detectors.

[0083] Any convenient detector for detecting the collected light may be used in the side scattered light detector described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured with a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10 cm 2 , such as 0.05 cm 2 Up to 9 cm 2 , such as 0.1 cm 2 Up to 8 cm2 , such as 0.5 cm 2 Up to 7 cm 2 And includes 1 cm 2 Up to 5 cm 2 range of photomultiplier tubes.

[0084] In embodiments, the subject particle analyzer further comprises a fluorescence detector configured to detect light of one or more fluorescence wavelengths. In other embodiments, the particle analyzer comprises a plurality of fluorescence detectors, such as 2 or more, such as 3 or more, such as 4 or more, 5 or more, 10 or more, 15 or more, and comprises 20 or more fluorescence detectors.

[0085] Any convenient detector for detecting the collected light may be used in the fluorescence detector described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), enhanced charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, and other detectors. In certain embodiments, the collected light is measured with a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area of ​​0.01 cm per region. 2 Up to 10 cm 2 , such as 0.05 cm 2 Up to 9 cm 2 , such as 0.1 cm 2 Up to 8 cm 2 , such as 0.5 cm 2 Up to 7 cm 2 And includes 1 cm 2 Up to 5 cm 2 range of photomultiplier tubes.

[0086] Where the subject particle analyzer includes multiple fluorescence detectors, each fluorescence detector may be identical, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, where the subject particle analyzer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector (or imaging sensor) is a CMOS-type device. In other embodiments, both the first and second fluorescence detectors are CCD-type devices. In yet other embodiments, both the first and second fluorescence detectors are CMOS-type devices. In yet other embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector is a photomultiplier tube (PMT). In yet other embodiments, the first fluorescence detector is a CMOS-type device and the second fluorescence detector is a photomultiplier tube. In yet other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0087] In embodiments of the present disclosure, the photodetector of interest is configured to measure collected light at one or more wavelengths, such as at 2 or more wavelengths, such as at 5 or more different wavelengths, such as at 10 or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and including measuring light emitted by a sample in a flowing stream at 400 or more different wavelengths. In some embodiments, 2 or more detectors in a particle analyzer as described herein are configured to measure collected light of the same or overlapping wavelengths.

[0088] In some embodiments, the fluorescence detector of interest is configured to measure light collected within a certain wavelength range (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect a spectrum within a certain wavelength range. For example, a particle analyzer may include one or more detectors configured to collect a spectrum within one or more wavelength ranges in a wavelength range of 200 nm to 1000 nm. In yet other embodiments, the detector of interest is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a particle analyzer may include one or more detectors configured to measure light of one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, one or more detectors can be configured to pair with specific fluorophores, such as those used with the sample in a fluorescence assay.

[0089] In some embodiments, the particle analyzer includes one or more wavelength separators between the flow cell and the particle modulation light detector. The term "wavelength separator" is used in its conventional meaning herein and refers to an optical component configured to separate the light collected from the sample into a predetermined spectral range. In some embodiments, the particle analyzer includes a single wavelength separator. In other embodiments, the particle analyzer includes a plurality of wavelength separators, such as 2 or more wavelength separators, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more, such as 75 or more and including 100 or more wavelength separators. In some embodiments, the wavelength separator is configured to separate the light collected from the sample into a predetermined spectral range by passing light with a predetermined spectral range and reflecting light of one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to separate the light collected from the sample into a predetermined spectral range by passing light with a predetermined spectral range and absorbing the light of one or more remaining spectral ranges. In yet other embodiments, the wavelength separator is configured to diffract the light space collected from the sample into a predetermined spectral range. Each wavelength separator can be any convenient light separation scheme, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator in the subject light detection system is a dichroic mirror.

[0090] Suitable flow cytometry systems may include, but are not limited to, Ormerod (ed.), Flow Cytometry: A Practical Approach , Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols , Methods in Molecular Biology No. 91, Humana Publishing House (1997); Practical Flow Cytometry , 3rd ed., Wiley-Leese (1995); Virgo et al. (2012) Ann Clin Biochem. Jan;49(Part 1):17-28; Linden et al., Semin Throm Hemost . 2004 Oct;30(5):502-11; Alison et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of these documents are incorporated herein by reference. In certain embodiments, the flow cytometry system of interest includes the BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell Sorters, BD Biosciences Influx™ Cell Sorter, BD Biosciences Jazz™ Cell Sorter, BD Biosciences Aria™ Cell Sorter, BD Biosciences FACSAria™ II Cell Sorter, BD Biosciences FACSAria™ III Cell Sorter, BD Biosciences FACSAria™ Fusion Cell Sorter, and BD Biosciences FACSMelody™ Cell Sorter, BD Biosciences FACSymphony TMS6 cell sorter, etc.

[0091] In some embodiments, the subject system is a flow cytometric system, such as described in U.S. Pat. Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9 those described in ,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766; the disclosures of which are incorporated herein by reference in their entirety.

[0092] Figure 4 A system 400 for flow cytometry according to an illustrative embodiment of the invention is shown. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long pass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0093] The excitation lasers 415a-c emit light in the form of laser beams. Figure 4 In the exemplary system of , the wavelengths of the laser beams emitted from the excitation lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits the 488 nm light and reflects the 633 nm light. The beam splitter 445b transmits UV light (light with a wavelength in the range of 10 to 400 nm) and reflects the 488 nm and 633 nm light.

[0094] The laser beam is then directed to a focusing lens 420, which focuses the beam onto a portion of the fluid stream where particles of the sample are located within a flow cell 425. The flow cell is the portion of the fluidics system that directs particles in the stream to the focused laser beam for interrogation, typically one particle at a time. The flow cell may include a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0095] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various wavelengths depending on the properties of the particles, such as the size of the particles, the internal structure, and the presence of one or more fluorescent molecules attached to or naturally present in the particles. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be routed to one or more of a forward scatter detector 430, a side scatter detector 435, and one or more fluorescence detectors 460a-460f via one or more of beam splitters 445c-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and a fluorescence collection lens 440.

[0096] Fluorescence collection lens 440 collects light emitted from the particle-laser beam interaction and routes the light toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Therefore, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or extends from 500 nm to 520 nm. Short-pass filters transmit light with a wavelength equal to or shorter than a specified wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit light with a wavelength equal to or longer than a specified wavelength. For example, long-pass filter 455b, which is a 670 nm long-pass filter, transmits light equal to or longer than 670 nm. Filters are typically selected to optimize the specificity of the detector for a particular fluorescent dye. The filter can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0097] The forward scatter detector 430 is positioned slightly off the axis of the direct beam through the flow cell and is configured to detect diffracted light, i.e., excitation light that passes through or travels around the particle primarily in the forward direction. The intensity of the light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the surface and internal structure of the particle, which tends to increase with the increase in the complexity of the particle structure. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include a photomultiplier tube. The signals detected at the forward scatter detector 430, the side scatter detector 435, and the fluorescence detector may be converted into electronic signals (voltages) by the detectors. The data may provide information about the sample.

[0098] Those skilled in the art will recognize that a flow cytometer according to one embodiment of the present invention is not limited to Figure 4 The flow cytometer depicted in the embodiment of the present invention may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations.

[0099] In operation, the cytometer operation is controlled by the controller / processor 490, and the measurement data from the detector can be stored in the memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive the output signal from the detector, and can also be coupled to the electrical and electromechanical components of the flow cytometer 410 to control the laser, fluid flow parameters, etc. Input / output (I / O) capabilities 497 can also be provided in the system. The memory 495, the controller / processor 490, and the I / O 497 can be provided entirely as an integral part of the flow cytometer 410. In such embodiments, the display can also form part of the I / O capabilities 497 for presenting experimental data to the user of the cytometer 410. Alternatively, the memory 495 and the controller / processor 490 and some or all of the I / O capabilities can be part of one or more external devices such as a general-purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate with the cytometer 410 wirelessly or by wire. Controller / processor 490, along with memory 495 and I / O 497, may be configured to perform various functions related to the preparation and analysis of flow cytometric experiments.

[0100] Figure 4The system shown in 497 includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as "filter windows" for a given detector), as defined by the configuration of filters and / or beam splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules in the set of fluorescent dyes used for the flow cytometer experiment will emit light in their own characteristic wavelength bands. The specific fluorescent markers used for the experiment and their associated fluorescence emission bands can be selected to generally coincide with the filter windows of the detectors. I / O 497 can be configured to receive data about a flow cytometer experiment having a set of fluorescent markers and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 497 can also be configured to receive biological data that assigns one or more markers to one or more cell populations, marker density data, emission spectrum data, data that assigns markers to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as marker spectral characteristics and flow cytometer configuration data, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more assignments of markers to markers.

[0101] Figure 5 A functional block diagram of one example of a processing system for analyzing and displaying biological events is shown. The processor 500 may be configured to implement various processes for controlling the graphical display of biological events.

[0102] The flow cytometer 502 can be configured to acquire biological event data. For example, the flow cytometer can generate flow cytometric event data (e.g., particle modulated light data). The flow cytometer 502 can be configured to provide the biological event data to the processor 500. A data communication channel can be included between the flow cytometer 502 and the processor 500. The biological event data can be provided to the processor 500 via the data communication channel.

[0103] Processor 500 can be configured to receive biological event data from flow cytometer 502. The biological event data received from flow cytometer 502 can include flow cytometric event data. Processor 500 can be configured to provide a graphical display of the biological event data to display device 506. Processor 500 can be further configured to enable regions of interest to be gated around populations of biological event data displayed by display device 506. In some embodiments, a gate can be a logical combination of one or more graphical regions of interest plotted on a single parameter histogram or a bivariate graph. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0104] Processor 500 may be further configured to display the bio-event data on display device 506 inside the gate, different from other events in the bio-event data outside the gate. For example, processor 500 may be configured to distinguish the color of the bio-event data contained inside the gate from the color of the bio-event data outside the gate. Display device 506 may be implemented as a monitor, a tablet computer, a smart phone, or other electronic device configured to present a graphical interface.

[0105] The processor 500 can be configured to receive a door selection signal identifying a door from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the processor 500, identifying a door to be displayed on the display device 506 or manipulated via the display device (e.g., by clicking on the door when the cursor is located at the desired door). In some implementations, the first device can be implemented as a keyboard 508 or other device for providing input signals to the processor 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such implementations, the input functions can each be considered an input device. For example, Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0106] A triggering event may cause processor 500 to change the way data is displayed, which portions of the data are actually displayed on display device 506, and / or provide input for further processing, such as selecting a population of interest for particle sorting.

[0107] In some embodiments, the processor 500 can be configured to detect when a gate selection is initiated by the mouse 510. The processor 500 can be further configured to automatically modify the map visualization to facilitate the gating process. The modification can be based on a particular distribution of bio-event data received by the processor 500. In some embodiments, the processor 500 expands the first gate such that a second gate is generated (e.g., as discussed above).

[0108] Processor 500 may be connected to storage device 504. Storage device 504 may be configured to receive and store bio-event data from processor 500. Storage device 504 may also be configured to receive and store flow cytometry event data from processor 500. Storage device 504 may further be configured to allow processor 500 to retrieve bio-event data, such as flow cytometry event data.

[0109] The display device 506 can be configured to receive display data from the processor 500. The display data can include graphs of biological event data and gates for sections of the overview graph. The display device 506 can be further configured to change the information presented based on input received from the processor 500 and input from the flow cytometer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0110] Methods for analyzing samples

[0111] Aspects of the invention also include methods of analyzing a sample. Methods of interest include: (a) introducing a particle sample into a flow cytometer having a flow cell for transporting particles in a flow stream, a sheath fluid line for fluidically coupling to a sheath fluid reservoir, and a fluidic block unit located between the sheath fluid line and the flow cell, and (b) performing a flow cytometric analysis on the sample. The fluidic block unit used in the subject method is as described above and includes an inlet fluidically coupled to the sheath fluid line, a valve line fluidically coupled to the inlet, a blocker line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the blocker line, and an outlet fluidically coupling the fluidic block unit to the flow cell.

[0112] In some embodiments, the method further comprises changing the blocking state of the fluid blocking unit by switching a valve of the valve line from an open state to a closed state or vice versa. In such embodiments, the practitioner of the method can initiate the switching of one or more valves, for example, by inputting a desired flow rate into a processor of the flow cytometer.

[0113] In some instances, the sample analyzed in the present method is a biological sample. The term "biological sample" is used in its conventional sense to refer to a subset of a complete organism, plant, fungus or animal tissue, cell or component, which in some instances can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal secretions and semen. Therefore, "biological sample" refers to a subset of a natural organism or its tissue, and refers to a homogenate, lysate or extract prepared from an organism or its tissue subset, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, gastrointestinal tract, cardiovascular and urogenital tract sections, tears, saliva, milk, blood cells, tumors, organs. Biological samples can be any type of organic tissue, including healthy and diseased tissues (e.g., cancerous, malignant, necrotic tissues, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc., wherein in some instances, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger stick (wherein the blood may or may not be mixed with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0114] In certain embodiments, the source of the sample is "mammal" or "mammalian", wherein these terms are widely used to describe organisms in the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs and rats) and Primates (e.g., humans, chimpanzees and monkeys). In some instances, the subject is a human. These methods can be applied to samples obtained from human subjects of male and female and any developmental stage (i.e., newborns, infants, teenagers, adolescents, adults), wherein in certain embodiments, the human subject is a teenager, adolescent or adult. Although the present invention can be applied to samples from human subjects, it should be understood that these methods can also be implemented on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

[0115] Cells of interest can be targeted for characterization based on a variety of parameters, such as phenotypic properties identified via attachment of specific fluorescent markers to cells of interest. In some embodiments, the system is configured to deflect droplets analyzed that are determined to contain target cells. A variety of cells can be characterized using the subject methods. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells with convenient cell surface markers or antigens that can be captured or labeled with convenient affinity reagents or conjugates thereof. For example, the target cell may include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cell is selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0116] When practicing the subject method, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module can vary, for example, in the range of 0.001 mL to 1000 mL, such as 0.005 mL to 900 mL, such as 0.01 mL to 800 mL, such as 0.05 mL to 700 mL, such as 0.1 mL to 600 mL, such as 0.5 mL to 500 mL, such as 1 mL to 400 mL, such as 2 mL to 300 mL and including 5 mL to 100 mL sample.

[0117] Methods according to embodiments of the present disclosure include counting and optionally sorting labeled particles (e.g., target cells) in a sample. When practicing the subject method, a fluid sample containing particles is first introduced into the flow nozzle of the system. When leaving the flow nozzle, the particles pass through the sample interrogation area substantially one at a time, each of the particles is irradiated by a light source in the sample interrogation area, and for each particle, the measurement of the light scattering parameter is recorded separately, and in some cases, the fluorescence emission is recorded as needed (e.g., the measurement of two or more light scattering parameters and one or more fluorescence emissions is recorded). Depending on the nature of the interrogated flow stream, 0.001 mm or more of the flow stream can be irradiated with light, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more and including 1 mm or more of the flow stream can be irradiated with light. In certain embodiments, the method includes irradiating a plane cross section of the flow stream in the sample interrogation area, such as with a laser (as described above). In other embodiments, the method includes irradiating a predetermined length of the flow stream in the sample interrogation area, such as corresponding to the irradiation profile of a diffuse laser beam or lamp.

[0118] In certain embodiments, the method includes irradiating the flow stream at or near the flow cell nozzle orifice. For example, the method can include irradiating the flow stream at a position about 0.001 mm or greater from the nozzle orifice, such as 0.005 mm or greater, such as 0.01 mm or greater, such as 0.05 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater and including 1 mm or greater from the nozzle orifice. In certain embodiments, the method includes irradiating the flow stream adjacent to the flow cell nozzle orifice.

[0119] In embodiments of these methods, a detector such as a photomultiplier tube (PMT) is used to record light passing through each particle (in some cases referred to as forward light scatter), light reflected orthogonal to the direction of flow of the particle through the sensing region (in some cases referred to as orthogonal or side light scatter), and fluorescence emitted from the particle when the particle passes through the sensing region and is illuminated by an energy source in the case of labeling with a fluorescent marker. Each of forward light scatter (FSC), side scatter (SSC), and fluorescence emission comprises a separate parameter for each particle (or each "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers.

[0120] The data recorded for each particle is analyzed in real time or stored in a data storage and analysis device such as a computer as needed. U.S. Patent No. 4,284,412 describes the configuration and use of a flow cytometer of interest equipped with a single light source, while U.S. Patent No. 4,727,020 describes the configuration and use of a flow cytometer equipped with two light sources.

[0121] In certain embodiments, particles are detected and uniquely identified by exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels, as desired. The fluorescence emitted in the detection channel for identifying the particle and the binding complex associated therewith can be measured after excitation with a single light source, or can be measured separately after excitation with different light sources. If a separate excitation light source is used to excite the particle labels, the labels can be selected so that all labels can be excited by each of the excitation light sources used.

[0122] The method in some embodiments also includes data acquisition, analysis and recording such as using a computer, wherein a plurality of data channels record the data of the light scattering from each detector and the fluorescence emitted by each particle when it passes through the sample interrogation area of ​​the particle sorting module. In these embodiments, the analysis includes classifying and counting the particles so that each particle exists as a set of digital parameter values. The subject system can be set to trigger under the selected parameters to distinguish the particles of interest from the background and noise. "Trigger" refers to the preset threshold value for detecting the parameters, and can be used as a means for detecting the passage of particles through the light source. The detection of events exceeding the threshold value of the selected parameters triggers the acquisition of the light scattering and fluorescence data of the particles. For particles or other components in the assay medium that cause a response below the threshold, no data is acquired. The trigger parameter can be the detection of the forward scattered light caused by the particle passing through the light beam. Then, the flow cytometer detects and collects the light scattering and fluorescence data of the particles.

[0123] Then, based on the data collected for the entire population, a specific subpopulation of interest is further analyzed by "gating". In order to select the appropriate gate, the data is plotted to obtain the best separation of possible subpopulations. This procedure can be performed by plotting forward light scatter (FSC) and side (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles (i.e., those cells within the gate) is selected, and particles that are not within the gate are excluded. If necessary, a gate can be selected by drawing a line around the desired subpopulation using a cursor on the computer screen. Then, only those particles within the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If necessary, the above analysis can be configured to produce a count of particles of interest in the sample.

[0124] The method of interest may further include using particles in research, laboratory testing or treatment. In some embodiments, the subject method includes obtaining single cells prepared from a target fluid or tissue biological sample. For example, the subject method includes obtaining cells from a fluid or tissue sample to be used as a research or diagnostic specimen for a disease such as cancer. Similarly, the subject method includes obtaining cells from a fluid or tissue sample for treatment. Cell therapy protocols are protocols that can prepare living cell materials including, for example, cells and tissues and introduce them into a subject as a therapeutic treatment. Conditions that can be treated by administering flow cytometry sorting samples include, but are not limited to, blood disorders, immune system disorders, organ damage, etc.

[0125] A typical cell therapy protocol may include the following steps: sample collection, cell separation, genetic modification, in vitro culture and expansion, cell harvesting, sample reduction and washing, biological preservation, storage, and introduction of cells into a subject. The protocol may begin with the collection of living cells and tissues from the subject's source tissue to produce a sample of cells and / or tissues. The sample may be collected via any suitable procedure, including, for example, administering a cell mobilization agent to the subject, drawing blood from the subject, removing bone marrow from the subject, etc. After collecting the sample, cell enrichment may be performed via several methods, including, for example, centrifugation-based methods, filtration-based methods, elutriation, magnetic separation, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene editing. Genetically modified cells may be cultured, activated, and expanded in vitro. In some cases, cells are preserved, such as cryopreserved, and stored for future use, in which the cells are thawed and then administered to the patient, such as cells may be infused into the patient.

[0126] Methods for assembling a flow cytometer

[0127] As discussed above, aspects of the invention also include methods of assembling a flow cytometer. The method of interest includes fluidly coupling a fluid retarder unit to a flow cell for transporting particles in a flow stream and a sheath fluid line for fluidly coupling to a sheath fluid reservoir. The fluid retarder unit for the subject method includes an inlet fluidically coupled to the sheath fluid line, a valve line fluidically coupled to the inlet, a retarder line fluidically coupled to the inlet, a plurality of connectors fluidically coupling the valve line to the retarder line, and a fluid retarder unit fluidically coupled to an outlet of the flow cell. As discussed above, the valve line of interest includes a series of fluidically coupled valves, and the retarder line of interest includes a series of fluidically coupled retarders.

[0128] Embodiments of the subject method include operably connecting a processor to each valve of a valve line. As discussed above, the processor of interest is configured to change the blocking state of the fluid blocking unit by initiating a switch of the valve in the valve line from an open state to a closed state or vice versa. The processor can be operably connected to, for example, a servomechanism that is configured to initiate a switch from an open state to a closed state or vice versa. The methods of the present invention can also involve connecting a sheath fluid reservoir to the sheath fluid line. As discussed above, the sheath fluid reservoir can be any suitable reservoir or container for holding sheath fluid (e.g., having rigid or flexible walls). In certain embodiments, the method includes fluidly coupling the sheath fluid reservoir to an input of a flow cell.

[0129] Computer control system

[0130] Aspects of the present invention further include computer control systems, wherein these systems include one or more computers for full automation or partial automation. In some embodiments, the system includes a computer having a non-transitory computer-readable storage medium having a computer program stored thereon, wherein when loaded on the computer, the computer program includes instructions for receiving a target or desired flow rate and initiating a change in the blocking state in the fluid blocking unit to achieve the target or desired flow rate. The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access a memory having instructions for performing the steps of the subject method stored thereon. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device and an input-output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or it may be one of the other processors that are available or will become available. The processor executes an operating system, and the operating system interfaces with the firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs, which can be written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system, typically in cooperation with the processor, coordinates and executes the functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0131] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard drive or tape, optical media such as read and write optical disks, flash memory devices, or other memory storage devices. The memory storage device may be any of a variety of known or future devices, including an optical disk drive, a tape drive, or a floppy disk drive. This type of memory storage device typically reads from and / or writes to a program storage medium (not shown) such as an optical disk. Any of these program storage media, or other program storage media currently in use or that may be developed later, may be considered a computer program product. It should be understood that these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0132] In some embodiments, a computer program product is described that includes a computer usable medium having control logic (computer software program, including program code) stored therein. When executed by a processor computer, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are primarily implemented in hardware using, for example, a hardware state machine. It is obvious to a person skilled in the relevant art to implement a hardware state machine to perform the functions described herein.

[0133] The memory may be any suitable device in which the processor can store and retrieve data, such as a magnetic, optical or solid-state storage device (including a disk or optical disk or tape or RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor that is appropriately programmed by a computer-readable medium carrying the necessary program code. The program may be provided to the processor remotely through a communication channel, or pre-stored in a computer program product, such as a memory or some other portable or fixed computer-readable storage medium, using any of those devices connected to the memory. For example, a disk or optical disk may carry the programming and may be read by a disk writer / reader. The system of the present invention also includes programming, algorithms, such as in the form of a computer program product, for practicing the above methods. The programming according to the present invention may be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. This medium includes, but is not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0134] The processor may also access a communication channel to communicate with a user at a remote location. A remote location means that the user does not interact directly with the system, but rather relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smart phone).

[0135] In some embodiments, a system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0136] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port, to allow data communications between the subject system and other external devices, such as computer terminals configured for similar supplemental data communications (e.g., in a doctor's office or in a hospital environment).

[0137] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or any other communication device that a user may use in conjunction with it.

[0138] In one embodiment, the communication interface is configured to provide a connection for data transmission using the Internet Protocol (IP) via a cellular telephone network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0139] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, for example using a wireless communication protocol such as 802.11 or Bluetooth. ®The server device may be another portable device, such as a smartphone, a personal digital assistant (PDA), or a notebook computer, or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0140] In some embodiments, the communication interface is configured to automatically or semi-automatically transfer data stored in the subject system, such as data stored in the optional data storage unit, with a network or server device using one or more of the above-described communication protocols and / or mechanisms.

[0141] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether a person or a machine, whether local or remote. If one of the display devices provides visual information, the information may generally be logically and / or physically organized as an array of image elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The functional elements of the computer may communicate with each other via a system bus. In an alternative embodiment, some of these communications may be accomplished using a network or other types of remote communications. According to known techniques, the output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, a telephone, or a satellite network. The data presentation of the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include an Internet URL address so that a user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, but they generally belong to a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. They may be co-located or physically separated. A variety of operating systems may be employed on any of the computer platforms, which may depend on the type and / or brand of computer platform chosen. Suitable operating systems include Windows ® NT ® , Windows ® XP, Windows ® 7. Windows® 8. Windows ® 10. iOS ® , macOS ® , Linux ® , Ubuntu ® , Fedora ® 、OS / 400 ® 、i5 / OS ® 、IBM i ® 、Android™、SGI IRIX ® , Oracle Solaris ® wait.

[0142] Figure 6 Depicted is the overall architecture of an exemplary computing device 600 in accordance with certain embodiments. Figure 6 The overall architecture of the computing device 600 depicted in the figure includes an arrangement of computer hardware and software components. However, in order to provide an achievable disclosure, it is not necessary to show all of these generally conventional elements. As shown, the computing device 600 includes a processing unit 610, a network interface 620, a computer-readable medium drive 630, an input / output device interface 640, a display 650, and an input device 660, all of which can communicate with each other via a communication bus. The network interface 620 can provide a connection to one or more networks or computing systems. The processing unit 610 can therefore receive information and instructions from other computing systems or services via the network. The processing unit 610 can also communicate with the memory 670 and further provide output information to the optional display 650 via the input / output device interface 640. For example, analysis software (e.g., data analysis software or program, such as FlowJo) stored as executable instructions in the non-temporary memory of the analysis system ® ) can display flow cytometry event data to the user. The input / output device interface 640 can also accept input from an optional input device 660, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope or other input device.

[0143] Memory 670 may contain computer program instructions (grouped into modules or components in some embodiments) that are executed by processing unit 610 to implement one or more embodiments. Memory 670 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 670 may store an operating system 672 that provides computer program instructions used by processing unit 610 in general management and operation of computing device 600. Data may be stored in data storage device 690. Memory 670 may also include computer program instructions and other information for implementing various aspects of the present disclosure.

[0144] Practicality

[0145] The subject fluid retardation cells, flow cytometers, and methods can be used in a variety of applications where it is desired to analyze the composition of a sample in a fluid medium. The present invention is particularly useful in situations where it is desired to improve sample flow rate control in vacuum-driven fluidics systems. For example, the present fluid retardation cells, flow cytometers, and methods can be used to increase the number of retardation states relative to previously employed mechanisms for producing retardation in vacuum-driven fluidics.

[0146] Embodiments of the present invention can be used in applications where cells prepared from biological samples may be needed for research, laboratory testing, or for treatment. In some embodiments, subject methods and apparatus can help obtain single cells prepared from target fluids or tissue biological samples. For example, subject methods and systems help obtain cells from fluids or tissue samples to be used as research or diagnostic specimens for diseases such as cancer. Likewise, subject methods and systems can help obtain cells from fluids or tissue samples for treatment.

[0147] Reagent test kit

[0148] Aspects of the present disclosure also include a kit. A kit of interest includes a fluid retardation unit. The fluid retardation unit for the subject kit is described above and includes a plurality of valves and a plurality of retarders, wherein each of the plurality of retarders is fluidically coupled to a different valve in the plurality of valves and co-located with a different valve in the plurality of valves. In one embodiment, the fluid retardation unit includes an inlet for receiving a fluid, a valve line fluidly coupled to the inlet, a retarder line fluidly coupled to the inlet, a plurality of connectors fluidly coupling the valve line to the retarder line, and an outlet for discharging the fluid.

[0149] In addition to the above-mentioned components, the subject kit may also include (in some embodiments) instructions, such as instructions for installing the fluid retardation unit of the present invention. These instructions may be present in the subject kit in a variety of forms, wherein one or more of these forms may be present in the kit. One form in which these instructions may exist is printed information on a suitable medium or substrate, such as one or more sheets of paper in the packaging of the kit, in a package insert, etc. The other form of these instructions is a computer-readable medium having information recorded thereon, such as a disk, a compact disk (CD), a portable flash drive, etc. The other form in which these instructions may exist is a website address, which can be used via the Internet to access information at the removed site.

[0150] The following examples are provided by way of illustration and not limitation.

[0151] experiment

[0152] according to Figure 1A A series of implementation schemes construct a fluid blocking unit. Fig. 7A The specifications of the constructed fluid retardation unit are depicted. Fig. 7A As shown, the retainers in the retainer line have a constant inner diameter of 0.030 inches (0.0762 cm) and successively increasing lengths. The length of retainer R1 is 2 inches (5.08 cm), the length of retainer R2 is 4 inches (10.16 cm), the length of retainer R3 is 8 inches (20.32 cm), and the diameter of retainer R4 is 16 inches (40.64 cm). The fluidic retainer unit includes 4 retainer-valve pairs and thus includes 4 2 = 16 blocking states. The fluid is then driven through the fluid blocking unit in each of the 16 blocking states from the highest blocking level to the lowest blocking level. At the highest blocking level, all valves are closed. At the second highest blocking level, all valves except V1 are closed. At the third highest blocking level, all valves except V2 are closed, and so on, through all 16 blocking states. The sample fluid flow rate (μL / min) is then measured in each of the 16 blocking states. Figure 7B As shown in , the highest levels of resistance produce the highest flow rates. The flow rate decreases as the resistance decreases. Figure 7B , the flow rates achieved using a previous mechanism for producing fluid retardation are added for comparison. The conventional mechanism shown only allows three sample flow rates, while the subject fluid retardation unit allows 16 sample flow rates.

[0153] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses:

[0154] 1. A flow cytometer, comprising:

[0155] a flow cell for transporting particles in a flow stream;

[0156] a sheath fluid line for fluid coupling to a sheath fluid reservoir; and

[0157] A fluid retardation unit, the fluid retardation unit is located between the sheath fluid pipeline and the flow cell, wherein the fluid retardation unit comprises:

[0158] an inlet fluidly coupled to the sheath fluid line;

[0159] a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves;

[0160] a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders;

[0161] a plurality of connectors fluidly coupling the valve lines to the retarder lines; and

[0162] An outlet fluidically couples the fluid retardation unit to the flow cell.

[0163] 2. A flow cytometer according to clause 1, wherein the valve line comprises 2 to 6 valves.

[0164] 3. A flow cytometer according to clause 1 or 2, wherein the blocker line comprises 2 to 6 blocks.

[0165] 4. A flow cytometer according to any of the preceding clauses, wherein the fluidic retention unit comprises an equal number of retainers and valves.

[0166] 5. A flow cytometer according to clause 4, wherein the fluid blocking unit comprises x 2 blocking states, where x is the number of valves.

[0167] 6. A flow cytometer according to any of the preceding clauses, wherein each valve of the valve line is individually switchable between an open position in which fluid is not obstructed from passing through the valve and a closed position in which fluid is obstructed from passing through the valve.

[0168] 7. A flow cytometer according to clause 6, wherein in said closed position, fluid flow through said valve is partially blocked.

[0169] 8. A flow cytometer according to clause 6, wherein in said closed position, fluid flow through said valve is completely blocked.

[0170] 9. A flow cytometer according to any one of clauses 6 to 8, further comprising a processor operably connected to each valve of the valve line, wherein the processor is configured to change the blocking state of the fluid blocking unit by initiating switching of the valves in the valve line from an open state to a closed state or vice versa.

[0171] 10. A flow cytometer according to any of the preceding clauses, wherein each retainer in the retainer line comprises an inner diameter in the range of 0.025 cm to 1.25 cm.

[0172] 11. The flow cytometer according to clause 10, wherein the inner diameters of the blockers of the blocker line increase sequentially.

[0173] 12. The flow cytometer of clause 10, wherein the inner diameter of the blocker of the blocker line is constant.

[0174] 13. A flow cytometer according to any preceding clause, wherein each of the retainers of the retainer line has a length in the range of 1.5 cm to 90 cm.

[0175] 14. A flow cytometer according to clause 13, wherein the lengths of the blockers increase sequentially.

[0176] 15. A flow cytometer according to any preceding clause, wherein the number of connectors in the plurality of connectors is in the range of 2 to 5.

[0177] 16. A flow cytometer according to any of the preceding clauses, wherein each connector of the plurality of connectors fluidically couples a portion of the valve line between two valves in the series of valves to a portion of the blocker line between two blockers in the series of blockers.

[0178] 17. A flow cytometer according to any preceding clause, wherein the fluid retention unit comprises a plastic tube.

[0179] 18. A flow cytometer according to any preceding clause, wherein the fluid retention unit comprises a metal tube.

[0180] 19. A flow cytometer according to any preceding clause, further comprising a light source configured to irradiate the flow cell at an interrogation point.

[0181] 20. A flow cytometer according to any preceding clause, further comprising a detector configured to collect particle modulated light from the flow cell.

[0182] 21. A flow cytometer according to any preceding clause, further comprising a sheath fluid reservoir.

[0183] 22. A method for analyzing a sample, the method comprising:

[0184] (a) introducing a particle sample into a flow cytometer, the flow cytometer comprising:

[0185] a flow cell for transporting particles in a flow stream;

[0186] a sheath fluid line for fluid coupling to a sheath fluid reservoir; and

[0187] A fluid retardation unit, the fluid retardation unit is located between the sheath fluid pipeline and the flow cell, wherein the fluid retardation unit comprises:

[0188] an inlet fluidly coupled to the sheath fluid line;

[0189] a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves;

[0190] a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders;

[0191] a plurality of connectors fluidly coupling the valve lines to the retarder lines; and

[0192] an outlet fluidically coupling the fluid retardation unit to the flow cell; and

[0193] (b) performing flow cytometric analysis on the sample.

[0194] 23. The method according to clause 22, wherein the valve line comprises 2 to 6 valves.

[0195] 24. A method according to clause 22 or 23, wherein the retarder line comprises 2 to 6 retarders.

[0196] 25. A method according to any one of clauses 22 or 24, wherein the fluid retardation unit comprises an equal number of retarders and valves.

[0197] 26. A method according to clause 25, wherein the fluid retardation unit comprises x 2blocking states, where x is the number of valves.

[0198] 27. A method according to any one of clauses 22 to 26, wherein each valve of the valve line is individually switchable between an open position in which fluid is not obstructed from passing through the valve and a closed position in which fluid is obstructed from passing through the valve.

[0199] 28. The method of clause 27, wherein in the closed position, fluid flow through the valve is partially blocked.

[0200] 29. The method of clause 27, wherein in the closed position, fluid flow through the valve is completely blocked.

[0201] 30. The method according to any of clauses 27 to 29, further comprising changing the blocking state of the fluid blocking unit by switching a valve of the valve line from an open state to a closed state or vice versa.

[0202] 31. The method of any of clauses 22 to 30, wherein each of the retainers of the retainer line comprises an inner diameter in the range of 0.025 cm to 1.25 cm.

[0203] 32. The method according to clause 31, wherein the inner diameters of the retainers of the retainer line increase sequentially.

[0204] 33. The method of clause 31, wherein the inner diameter of the retainer of the retainer line is constant.

[0205] 34. A method according to any of clauses 22 to 33, wherein each retainer of the retainer line has a length in the range of 1.5 cm to 90 cm.

[0206] 35. The method according to clause 34, wherein the lengths of the resistors of the resistor line increase sequentially.

[0207] 36. A method according to any one of clauses 22 to 35, wherein the number of connectors in the plurality of connectors is in the range of 2 to 5.

[0208] 37. The method of any one of clauses 22 to 36, wherein each connector of the plurality of connectors fluidly couples a portion of the valve line between two valves in the series of valves to a portion of the blocker line between two blockers in the series of blockers.

[0209] 38. A method according to any one of clauses 22 to 37, wherein the fluid retardation unit comprises a plastic pipe.

[0210] 39. A method according to any one of clauses 22 to 38, wherein the fluid retardation unit comprises a metal pipe.

[0211] 40. A method according to any one of clauses 22 to 39, wherein the sample is a biological sample.

[0212] 41. A method according to any one of clauses 22 to 40, wherein the sample comprises cells.

[0213] 42. The method of any one of clauses 22 to 41, wherein the flow cytometer further comprises a sheath fluid reservoir, the sheath fluid reservoir being fluidically coupled to the sheath fluid line.

[0214] 43. A fluid retardation unit, the fluid retardation unit comprising:

[0215] an inlet for receiving a fluid;

[0216] a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves;

[0217] a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders;

[0218] a plurality of connectors fluidly coupling the valve lines to the retarder lines; and

[0219] An outlet is provided for discharging the fluid.

[0220] 44. A fluid retardation unit according to clause 43, wherein the valve line comprises 2 to 6 valves.

[0221] 45. A fluid retardation unit according to clause 44, wherein the valve line comprises 4 valves.

[0222] 46. ​​A fluid retardation unit according to any of clauses 43 to 45, wherein the retardation line comprises 2 to 6 retarders.

[0223] 47. A fluid retardation unit according to clause 46, wherein the retardation line comprises 4 retarders.

[0224] 48. A fluid retardation unit according to any of clauses 43 to 47, wherein the fluid retardation unit comprises an equal number of retarders and valves.

[0225] 49. A fluid retardation unit according to clause 48, wherein the fluid retardation unit comprises x 2 blocking states, where x is the number of valves.

[0226] 50. A fluid retardation unit according to any one of clauses 43 to 49, wherein each valve of the valve line is individually switchable between an open position in which fluid is not blocked from passing through the valve and a closed position in which fluid is blocked from passing through the valve.

[0227] 51. The fluid retardation unit of clause 50, wherein in the closed position, fluid flow through the valve is partially blocked.

[0228] 52. The fluid retardation unit of clause 50, wherein in said closed position, fluid is completely blocked from passing through said valve.

[0229] 53. A fluidic resistance unit according to any of clauses 43 to 52, wherein each resistance of the resistance line comprises an inner diameter in the range of 0.025 cm to 1.25 cm.

[0230] 54. A fluid retardation unit according to clause 53, wherein the inner diameters of the retarders of the retardation line increase sequentially.

[0231] 55. The fluid retention unit of clause 53, wherein the inner diameter of the retainer of the retainer line is constant.

[0232] 56. A fluid retarder unit according to any of clauses 43 to 55, wherein each retarder of the retarder line has a length in the range of 1.5 cm to 90 cm.

[0233] 57. A fluid retardation unit according to clause 56, wherein the lengths of the retarders of the retardation line increase sequentially.

[0234] 58. A fluid retardation unit according to any of clauses 43 to 57, wherein the number of connectors in the plurality of connectors is in the range of 2 to 5.

[0235] 59. A fluid retardation unit according to clause 58, wherein the fluid retardation unit comprises 3 connectors.

[0236] 60. A fluidic resistance unit according to any one of clauses 43 to 59, wherein each connector of the plurality of connectors fluidly couples a portion of the valve line between two valves in the series of valves to a portion of the resistor line between two resistors in the series of resistors.

[0237] 61. A fluid retardation unit according to any one of clauses 43 to 60, wherein the fluid retardation unit comprises a plastic pipe.

[0238] 62. A fluid retardation unit according to any one of clauses 43 to 61, wherein the fluid retardation unit comprises a metal pipe.

[0239] 63. A method of assembling a flow cytometer, the method comprising fluidically coupling a fluid retardation unit to:

[0240] a flow cell for transporting particles in a flow stream; and

[0241] a sheath fluid line for fluid coupling to a sheath fluid reservoir, wherein the fluid retention unit comprises:

[0242] an inlet for receiving a fluid;

[0243] a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves;

[0244] a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders;

[0245] a plurality of connectors fluidly coupling the valve lines to the retarder lines; and

[0246] An outlet is provided for discharging the fluid.

[0247] 64. A method according to clause 63, wherein the valve pipeline includes 2 to 6 valves.

[0248] 65. A method according to clause 63 or 64, wherein the retarder line comprises 2 to 6 retarders.

[0249] 66. A method according to any of clauses 63 or 65, wherein the fluid retardation unit comprises an equal number of retarders and valves.

[0250] 67. A method according to clause 66, wherein the fluid retardation unit comprises x 2 blocking states, where x is the number of valves.

[0251] 68. A method according to any one of clauses 63 to 67, wherein each valve of the valve line is individually switchable between an open position in which fluid is not obstructed from passing through the valve and a closed position in which fluid is obstructed from passing through the valve.

[0252] 69. The method of clause 68, wherein in the closed position, fluid flow through the valve is partially obstructed.

[0253] 70. The method of clause 68, wherein in the closed position, fluid flow through the valve is completely blocked.

[0254] 71. A method according to any one of clauses 68 to 70, further comprising operably connecting a processor to each valve in the valve line, wherein the processor is configured to change the blocking state of the fluid blocking unit by initiating switching of the valves in the valve line from an open state to a closed state or vice versa.

[0255] 72. The method of any one of clauses 63 to 71, wherein each of the retainers of the retainer line comprises an inner diameter in the range of 0.025 cm to 1.25 cm.

[0256] 73. The method according to clause 72, wherein the inner diameters of the retainers of the retainer line increase sequentially.

[0257] 74. The method of clause 72, wherein the inner diameter of the retainer of the retainer line is constant.

[0258] 75. A method according to any of clauses 63 to 74, wherein each retainer of the retainer line has a length in the range of 1.5 cm to 90 cm.

[0259] 76. The method of clause 75, wherein the lengths of the resistors of the resistor line increase sequentially.

[0260] 77. A method according to any of clauses 63 to 76, wherein the number of connectors in the plurality of connectors is in the range of 2 to 5.

[0261] 78. The method of any one of clauses 63 to 77, wherein each connector of the plurality of connectors fluidly couples a portion of the valve line between two valves in the series of valves to a portion of the blocker line between two blockers in the series of blockers.

[0262] 79. A method according to any one of clauses 63 to 78, wherein the fluid retardation unit comprises a plastic pipe.

[0263] 80. A method according to any one of clauses 63 to 79, wherein the fluid retardation unit comprises a metal pipe.

[0264] 81. The method of any one of clauses 63 to 80, further comprising fluidly connecting a sheath fluid reservoir to the sheath fluid line.

[0265] 82. A fluid retardation unit, the fluid retardation unit comprising:

[0266] a plurality of valves; and

[0267] A plurality of retarders, wherein each retarder of the plurality of retarders is fluidly coupled to and co-located with a different valve of the plurality of valves.

[0268] 83. A fluid retardation unit according to clause 82, wherein the fluid retardation unit comprises 2 to 6 valves.

[0269] 84. A fluid retardation unit according to clause 83, wherein the fluid retardation unit comprises 4 valves.

[0270] 85. A fluid retardation unit according to any of clauses 82 to 84, wherein the fluid retardation unit comprises 2 to 6 retarders.

[0271] 86. A fluid retardation unit according to clause 85, wherein the fluid retardation unit comprises 4 retarders.

[0272] 87. A fluid retardation unit according to any of clauses 82 to 86, wherein the fluid retardation unit comprises an equal number of retarders and valves.

[0273] 88. A fluid retardation unit according to clause 87, wherein the fluid retardation unit comprises x 2 blocking states, where x is the number of valves.

[0274] 89. A fluid retardation unit according to any one of clauses 82 to 88, wherein each valve of the plurality of valves is individually switchable between an open position in which fluid is not blocked from passing through the valve and a closed position in which fluid is blocked from passing through the valve.

[0275] 90. A fluid retardation unit according to clause 89, wherein in the closed position, fluid is partially blocked from passing through the valve.

[0276] 91. A fluid retardation unit according to clause 89, wherein in the closed position, fluid is completely blocked from passing through the valve.

[0277] 92. A fluidic resistance unit according to any of clauses 82 to 91, wherein each resistance of the resistance line comprises an inner diameter in the range of 0.025 cm to 1.25 cm.

[0278] 93. A fluidic resistance unit according to clause 92, wherein the inner diameter of the resistance of the resistance line is constant.

[0279] 94. A fluid retarder unit according to any of clauses 82 to 93, wherein each retarder of the retarder line has a length in the range of 1.5 cm to 90 cm.

[0280] 95. A fluid retardation unit according to any of clauses 82 to 94, wherein the fluid retardation unit comprises a plastic pipe.

[0281] 96. A fluid retardation unit according to any one of clauses 82 to 94, wherein the fluid retardation unit comprises a metal pipe.

[0282] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0283] Therefore, the above merely illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design a variety of layouts, although such layouts are not explicitly described or shown here, but embody the principle of the present invention and are included in the spirit and scope of the present invention.In addition, all examples and conditional language recorded herein are mainly intended to help readers understand the principle of the present invention and the concept that the inventor contributes to advance the prior art, and should be interpreted as not being limited to these specifically described examples.In addition, all statements of the principles, aspects, and embodiments of the present invention and its specific examples recorded herein are intended to cover the equivalents of its structure and function.In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, that is, any element of the development that performs the same function, regardless of the structure.In addition, any content disclosed herein is not intended to be dedicated to the public, regardless of whether this disclosure is clearly stated in the claims.

[0284] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 USC §112 (f) or 35 USC §112 (6) is expressly defined to be incorporated into a limitation in a claim only if the exact phrase "means for..." or the exact phrase "step for..." is cited at the beginning of a limitation in the claim; if the exact phrase is not used in a limitation in a claim, 35 USC §112 (f) or 35 USC §112 (6) is not incorporated into the limitation.

Claims

1. A flow cytometer, comprising: a flow cell for transporting particles in a flow stream; a sheath fluid line for fluid coupling to a sheath fluid reservoir; as well as A fluid retardation unit, the fluid retardation unit is located between the sheath fluid pipeline and the flow cell, wherein the fluid retardation unit comprises: an inlet fluidly coupled to the sheath fluid line; a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves; a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders; a plurality of connectors fluidly coupling the valve lines to the retarder lines; and An outlet fluidically couples the fluid retardation unit to the flow cell.

2. The flow cytometer according to claim 1, wherein the valve line comprises 2 to 6 valves.

3. The flow cytometer according to claim 1 or 2, wherein the blocker line comprises 2 to 6 blocks.

4. A flow cytometer according to any one of the preceding claims, wherein the fluidic retention unit comprises an equal number of retainers and valves.

5. The flow cytometer according to claim 4, wherein the fluid blocking unit comprises x 2 blocking states, where x is the number of valves.

6. A flow cytometer according to any of the preceding claims, wherein each valve of the valve line is capable of being individually switched between an open position in which fluid is not obstructed from passing through the valve and a closed position in which fluid is obstructed from passing through the valve.

7. The flow cytometer according to claim 6 further comprises a processor operably connected to each valve of the valve line, wherein the processor is configured to change the blocking state of the fluid blocking unit by initiating switching of the valve in the valve line from an open state to a closed state or vice versa.

8. A flow cytometer according to any of the preceding claims, wherein each of the plurality of connectors couples a portion of the fluid of the valve line between two valves in the series of valves to a portion of the blocker line between two blockers in the series of blockers.

9. The flow cytometer of any of the preceding claims, further comprising a light source configured to irradiate the flow cell at an interrogation point.

10. The flow cytometer of any of the preceding claims, further comprising a detector configured to collect particle modulated light from the flow cell.

11. The flow cytometer of any of the preceding claims, further comprising a sheath fluid reservoir.

12. A method for analyzing a sample, the method comprising: (a) introducing a particle sample into a flow cytometer, the flow cytometer comprising: a flow cell for transporting particles in a flow stream; a sheath fluid line for fluid coupling to a sheath fluid reservoir; and A fluid retardation unit, the fluid retardation unit is located between the sheath fluid pipeline and the flow cell, wherein the fluid retardation unit comprises: an inlet fluidly coupled to the sheath fluid line; a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves; a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders; a plurality of connectors fluidly coupling the valve lines to the retarder lines; and an outlet fluidically coupling the fluid retardation unit to the flow cell; and (b) performing flow cytometric analysis on the sample.

13. A fluid retardation unit, comprising: an inlet for receiving a fluid; a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves; a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders; a plurality of connectors fluidly coupling the valve lines to the retarder lines; as well as An outlet is provided for discharging the fluid.

14. A method of assembling a flow cytometer, the method comprising fluidically coupling a fluid retardation unit to: a flow cell for transporting particles in a flow stream; and a sheath fluid line for fluid coupling to a sheath fluid reservoir, wherein the fluid retention unit comprises: an inlet for receiving a fluid; a valve line fluidly coupled to the inlet, wherein the valve line includes a series of fluidly coupled valves; a retarder line fluidly coupled to the inlet, wherein the retarder line includes a series of fluidly coupled retarders; a plurality of connectors fluidly coupling the valve lines to the retarder lines; and An outlet is provided for discharging the fluid.

15. A fluid retardation unit, comprising: Multiple valves; and A plurality of retarders, wherein each retarder of the plurality of retarders is fluidly coupled to and co-located with a different valve of the plurality of valves.

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