Flow cytometry with closed droplet sorter with controlled aerosol content and methods of use thereof

Abstract

The invention provides a flow cytometer with a closed particle sorting module. Aspects of the flow cytometer include, in addition to the closed particle sorting module, a sample input module coupled in fluid communication with an inlet of the closed particle sorting module; a waste pool coupled in fluid communication with the first outlet of the closed particle sorting module; and a first sorted particle collection system coupled in fluid communication with a second outlet of the closed particle sorting module. Flow cytometers described herein are configured to control aerosol content within the closed particle sorting module, including a sorting chamber of the module. The invention also provides methods of use of the flow cytometer, as well as kits belonging to consumables for use in conjunction with the flow cytometer, the kits comprising one or more components of the flow cytometer.

Description

[0001] This application is a divisional application, the parent application of which is a PCT application with application number PCT / US2019 / 028512 and application date April 22, 2019, and entered the Chinese national phase on November 17, 2020, with application number 201980033089.2, entitled “Flow cytometer with closed droplet sorter with controlled aerosol content and method of use thereof”.

[0002] Cross-references to related patent applications

[0003] This patent application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 663,936, filed on April 27, 2018; the contents of which are incorporated herein by reference. Technical Field

[0004] The present application relates to a flow cytometer having a closed droplet sorter with controlled aerosol content and a method of using the same. Background Art

[0005] Flow particle sorting systems (e.g., sorting flow cytometers) are used to sort particles in a fluid sample based on at least one measured characteristic of the particles. In a flow particle sorting system, particles (e.g., molecules, microbeads bound to an analyte, or individual cells) in a fluid suspension are passed through a detection region in the form of a stream, where a sensor detects the type of particles to be sorted contained in the stream. When the sensor detects the type of particles to be sorted, it triggers a sorting mechanism that selectively separates the particles of interest.

[0006] Particle sensing is typically performed by passing a fluid medium through a detection zone where the particles are exposed to irradiation from one or more lasers and the light scattering and fluorescence properties of the particles are measured. Particles or their components are labeled with fluorescent dyes for detection, and various particles or components can be detected simultaneously by labeling with fluorescent dyes with different spectral properties. Detection is performed using one or more photoelectric sensors to independently measure the fluorescence emitted by each different fluorescent dye.

[0007] One type of flow particle sorting system is the electrostatic sorting type. In an electrostatic sorter, a fluid suspension is ejected from a nozzle, causing it to vibrate, thereby breaking the stream into uniform discrete droplets. The sorting mechanism includes a droplet charging device connected to the stream to charge droplets containing the type of particles to be sorted when the droplets separate from the jet stream. The droplet stream passes through a transverse electrostatic field established by two oppositely charged deflection plates. The deflection direction and number of charged droplets containing the type of particles to be sorted are related to the polarity and magnitude of the droplet charge, and the charged droplets will be collected in different collection containers. Uncharged droplets will not be deflected when passing through the electrostatic field and will be collected in a central container. Summary of the invention

[0008] The present invention provides a flow cytometer with a closed particle sorting module. In addition to the closed particle sorting module, aspects of the flow cytometer also include a sample input module coupled to the inlet of the closed particle sorting module in a fluid-connected manner; a waste pool coupled to the first outlet of the closed particle sorting module in a fluid-connected manner; and a first sorting particle collection system coupled to the second outlet of the closed particle sorting module in a fluid-connected manner. The flow cytometer described herein is configured to control the aerosol content within the closed particle sorting module (including the sorting chamber of the module). The present invention also provides a method for using the flow cytometer, and a kit of consumables used in combination with the flow cytometer, the kit including one or more components of the flow cytometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The most comprehensive understanding of the present invention can be obtained by reading the following detailed description in conjunction with the accompanying drawings, which include the following illustrations:

[0010] Figure 1 An illustration of a closed particle sorting module according to an embodiment of the present invention is provided.

[0011] Figure 2 A schematic diagram of a flow cytometer system is provided, in which Figure 1 The enclosed particle sorting module shown is coupled with the system to obtain the sterile flow cytometer-type cell sorter according to an embodiment of the present invention.

[0012] Figure 3 A schematic diagram of a flow cytometer-type cell sorter according to an embodiment of the present invention is provided. DETAILED DESCRIPTION

[0013] The present invention provides a flow cytometer with a closed particle sorting module. In addition to the closed particle sorting module, aspects of the flow cytometer also include a sample input module coupled to the inlet of the closed particle sorting module in a fluid-connected manner; a waste pool coupled to the first outlet of the closed particle sorting module in a fluid-connected manner; and a first sorting particle collection system coupled to the second outlet of the closed particle sorting module in a fluid-connected manner. The flow cytometer described herein is configured to control the aerosol content within the closed particle sorting module (including the sorting chamber of the module). The present invention also provides a method for using the flow cytometer, and a kit of consumables used in combination with the flow cytometer, the kit including one or more components of the flow cytometer.

[0014] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, because there will be differences in actual implementation. It should also be understood that the terms used herein are only used to describe specific embodiments, and are not intended to limit the inventive concept, and the scope of the present invention will only be limited by the appended claims.

[0015] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the range are included within the scope of the present invention. Unless the context clearly specifies otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the present invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or two of those included limits is also included within the present invention.

[0016] Certain ranges set forth herein are preceded by the term "about." The term "about" is used herein for the purpose of providing literal support for the exact number that follows, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, a close or approximately unrecited number may be a number that is substantially equivalent to the specifically recited number in the context in which it appears.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0018] 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 the purpose of incorporating by reference into this document is to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for 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 reason of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0019] It is 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 is also noted that claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of special terminology such as "solely," "only," etc. in connection with reciting claim elements or use of a "negative" limitation.

[0020] After reading the present invention, the following will be apparent to those skilled in the art that each individual embodiment described and listed herein has layered components and features that can be quickly decomposed or combined with the features of any of the other several embodiments without departing from the scope and spirit of the present invention. Any stated method can be implemented in the order of events stated or in any other order that is logically possible.

[0021] Although the apparatus and method have been or will be described for grammatical fluency and functional description, it should be clearly understood that unless otherwise expressly provided in Chapter 35 of the United States Code, the claims shall not be construed as necessarily being limited to "means" or "steps" but shall be consistent with the meaning and full scope of equivalents in accordance with the judicial doctrine of equivalents, and when the claims are expressly drafted in accordance with the provisions of Chapter 35 of the United States Code, the claims shall be fully consistent with the statutory equivalents in Chapter 35 of the United States Code.

[0022] Flow cytometry

[0023] As described above, the present invention provides a flow cytometer with a closed particle sorting module. The flow cytometer described herein includes a flow cytometer system and a closed particle sorting module operably coupled thereto. In some cases, the configuration of the flow cytometer system and the closed particle sorting module allows the closed particle sorting module to be easily operably engaged with the flow cytometer system and removed from the system. Each of the components of the flow cytometer of the present invention is described in further detail below.

[0024] An aspect of the flow cytometer is that it is configured to control the aerosol content within the closed particle sorting module (e.g., its sorting chamber). The inventors have found that in a closed particle sorting module, such as a closed particle sorting module of the flow cytometer of the present invention, aerosols tend to form in the sorting chamber, which can cause problems related to the function of the sorting module, such as arcing of the deflection plate and liquid contamination of the sensitive element. Embodiments of the present invention solve these problems by controlling the aerosol content within the closed particle sorting module (e.g., the sorting chamber of the closed particle sorting module). In some cases, controlling the aerosol content means making the number of aerosol droplets (if any) in the sorting chamber less than the number required to cause one or more of the above-mentioned problems (e.g., arcing). In some cases, the aerosol content is controlled so that it does not exceed the detectable amount. One or more different methods are used to control the aerosol content in the flow cytometer of the present invention, for example, the methods described in more detail below.

[0025] Closed particle sorting module

[0026] As described above, the flow cytometer of the present invention includes a closed particle sorting module for sorting components in a liquid sample (e.g., cells in a biological sample). In accordance with the conventional meaning, the term "sorting" used herein refers to separating components in the sample (e.g., cells, non-cellular particles (e.g., biomacromolecules)), and in some cases, as described below, the separated components are delivered to a receiving position such as a container or a collection system, for example, a container or a collection system described in more detail below. For example, the target particle sorting module can be configured to sort a single component from the remaining components of the sample, or be configured to sort samples with 2 or more components, for example, 3 or more components, 4 or more components, 5 or more components, 10 or more components, 15 or more components, including sorting samples with 25 or more components. One or more sample components can be separated from the sample and delivered to a container, for example, 2 or more sample components, 3 or more sample components, 4 or more sample components, 5 or more sample components, 10 or more sample components, including 15 or more sample components separated from the sample and delivered to a receiving position in a container.

[0027] In an embodiment, a particle sorting module comprises a closed housing having an alignment member for coupling the housing to a particle sorting system; a flow cell nozzle located at a proximal end of the housing; a sample interrogation region in fluid communication with the flow cell nozzle orifice; and a droplet deflector. The term "closed" means that each component of the particle sorting module is completely contained within the housing and that the components are sealed or isolated from the surrounding environment. In other words, the components within the closed housing are not exposed to the external environment and are not in contact with the external environment. In some embodiments, the components contained in the housing are isolated from the gas environment of the surrounding environment (i.e., not exposed to the gas outside the housing). In other embodiments, the components contained in the housing are isolated from the fluid environment of the surrounding environment (i.e., not exposed to any fluid outside the housing). In other embodiments, the components contained in the housing are sterile and isolated from live bacteria or other microorganisms present in the surrounding environment (i.e., are sterile).

[0028] The particle sorting module of interest is configured to be coupled to a particle sorting system of a flow cytometer, wherein a stream of droplets is generated in the particle sorting module and substantially one stream of droplets at a time passes through a sample interrogation region where the particles are detected and identified. A droplet deflector is located downstream of the sample interrogation region (e.g., the region within a sorting chamber) and is configured to deflect the analyzed droplets to pass through different outlets, such as a first outlet coupled to a waste reservoir or a second outlet coupled to a sorted particle receiver, such as a container or sorted particle collection system, such as the container or system described in more detail below.

[0029] A particle sorting module can include an enclosed housing having an alignment feature for coupling the housing to a particle sorting system of a flow cytometer; a flow cell nozzle located at a proximal end of the housing; a sample interrogation region in fluid communication with the flow cell nozzle orifice; and a droplet deflector, for example, a droplet deflector located on a sorting chamber of the module. In some cases, the housing has a distal end, a proximal end, and walls therebetween that combine to form an inner cavity. In embodiments, one or more outer walls of the housing have an alignment feature for coupling the housing to a particle sorting system of a flow cytometer. For example, the housing can have two or more walls having an alignment feature for coupling the housing to a particle sorting system, for example, three or more walls, including four or more walls having an alignment feature. In certain embodiments, the housing has one wall having an alignment feature for coupling the housing to a particle sorting system. Each wall having alignment elements may include 1 or more alignment elements, for example, 2 or more alignment elements, 3 or more alignment elements, 4 or more alignment elements, 5 or more alignment elements, 7 or more alignment elements, including 10 or more alignment elements. In some embodiments, the particle sorting device includes an outer wall having 3 alignment elements.

[0030] Any suitable type of alignment member can be used, for example, an alignment protrusion, an alignment track, an alignment notch, an alignment groove, an alignment slot, an alignment countersunk hole, an alignment counterbore, an alignment recess, an alignment hole, or a combination thereof. For example, in some cases, the outer wall of the shell includes one or more protrusions, such as a pin, a dowel, or a bump. In certain embodiments, the alignment member is a pin, such as a ball stud. In other cases, the outer wall of the shell includes one or more recesses, such as a hole or a notch. In some cases, the outer wall of the shell includes one or more alignment protrusions and one or more alignment recesses. For more detailed information about the alignment member and the closed particle sorting module including the alignment member, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of the patent are incorporated herein by reference.

[0031] In some embodiments, the outer wall of the shell of the particle sorting module includes one or more electrical connectors, which are configured for electrical connection between the particle sorting module and the particle sorting system. For example, the housing may include 2 or more electrical connectors, for example, 3 or more electrical connectors, 4 or more electrical connectors, 5 or more electrical connectors, including 10 or more electrical connectors. In some embodiments, the electrical connector supplies power to the droplet deflection plate. Any suitable electrical connector can be used, for example, a conductive pin, pad, wire or coil protruding from or recessed into the outer wall of the shell. In certain embodiments, the target particle sorting module includes 5 or more electrical pins. For more detailed information about electrical connectors and closed particle sorting modules including the electrical connectors, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of the patent are incorporated herein by reference.

[0032] The size of the particle sorting module housing may also vary, with a length ranging from 10 cm to 100 cm, for example, 15 cm to 95 cm, 20 cm to 90 cm, 25 cm to 85 cm, 30 cm to 80 cm, 35 cm to 75 cm, including 40 cm to 60 cm. The width of the particle sorting module housing may range from 1 cm to 25 cm, for example, 2 cm to 20 cm, 3 cm to 15 cm, including 5 cm to 10 cm.

[0033] The housing can be made of any suitable material compatible with the fluid sample (e.g., a biological sample), including metal, glass (e.g., Pyrex glass, borosilicate glass), ceramic, or plastic. In some embodiments, the particle sorting module housing is made of plastic, such as a hard plastic, a polymeric material, or a thermoplastic material. For example, suitable plastics may include, but are not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of the thermoplastics, such as PETG (polyethylene terephthalate modified with ethylene glycol) and other polymeric plastic materials. In some embodiments, the particle sorting module housing is made of polyester, wherein the target polyester may include but is not limited to poly(alkylene terephthalates), such as poly(ethylene terephthalate) (PET), bottle-grade PET (copolymers based on monoethylene glycol, terephthalic acid and other comonomers (such as isophthalic acid, cyclohexene dimethanol, etc.)), poly(butylene terephthalate) (PBT) and poly(hexane terephthalate); poly(alkylene adipates), such as poly(ethylene adipate), poly(1,4-butylene ... terephthalate), poly(1 esters) and poly(hexamethylene adipate); poly(alkylene suberates), for example, poly(ethylene suberate); poly(alkylene sebacates), for example, poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalates), for example, poly(ethylene isophthalate); poly(2,6-naphthalene-dicarboxylic acid alkylene esters), for example, poly(ethylene 2,6-naphthalene-dicarboxylic acid alkylene esters); poly(sulfonyl-4,4'-dibenzoate), for example, poly(sulfonyl-4,4'-dibenzoate); esters); poly(alkylene p-phenylenedicarboxylates), for example, poly(ethylene p-phenylenedicarboxylate); poly(trans-1,4-cyclohexanediyldicarboxylate), for example, poly(ethylene trans-1,4-cyclohexanediyldicarboxylate); poly(1,4-cyclohexane-dimethylenedicarboxylate), for example, poly(ethylene 1,4-cyclohexane-dimethylenedicarboxylate); poly([2.2.2]-bicyclooctane-1,4-dimethylenedicarboxylate), for example, poly([2.2.2]-bicyclooctane- 1,4-dimethylene dicarboxylic acid ethylene glycol ester); lactic acid polymers and copolymers, for example, (S)-polylactide, (R,S)-polylactide, poly(tetramethyl glycolide) and poly(lactide-co-glycolide); and polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides, for example, poly(p-phenylene terephthalamide); polyesters, for example, polyethylene terephthalate, for example, MylarTM polyethylene terephthalate, etc.

[0034] As described above, the closed particle sorting module includes a flow pool nozzle located at the proximal end of the housing and having an orifice. Any suitable nozzle flow pool can be used to deliver the fluid sample to the sample interrogation area, wherein in some embodiments, the flow pool nozzle includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminated at a flat surface having a nozzle orifice and transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) may vary, ranging from 1mm to 15mm, for example, 1.5mm to 12.5mm, 2mm to 10mm, 3mm to 9mm, including 4mm to 8mm. The length of the distal truncated conical portion (measured along the longitudinal axis) may also vary, ranging from 1mm to 10mm, for example, 2mm to 9mm, 3mm to 8mm, including 4mm to 7mm. In some embodiments, the diameter of the flow pool nozzle cavity may vary, ranging from 1mm to 10mm, for example, 2mm to 9mm, 3mm to 8mm, including 4mm to 7mm.

[0035] In some cases, the nozzle cavity does not include a cylindrical portion, and the entire flow cell nozzle cavity is frustoconical. In these embodiments, the length of the frustoconical nozzle cavity (measured along the longitudinal axis transverse to the nozzle orifice) ranges from 1 mm to 15 mm, for example, 1.5 mm to 12.5 mm, 2 mm to 10 mm, 3 mm to 9 mm, including 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical nozzle cavity ranges from 1 mm to 10 mm, for example, 2 mm to 9 mm, 3 mm to 8 mm, including 4 mm to 7 mm.

[0036] In an embodiment, the sample fluid medium flows out of an orifice at the distal end of the flow cell nozzle. Depending on the desired properties of the fluid medium, the flow cell nozzle orifice can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to, a cross-sectional shape surrounded by straight lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a cross-sectional shape surrounded by curves (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom connected to a flat top). In certain embodiments, the flow cell nozzle of interest has a circular orifice. The size of the nozzle orifice may vary, and in some embodiments, the size ranges from 1 μm to 20,000 μm, for example, 2 μm to 17,500 μm, 5 μm to 15,000 μm, 10 μm to 12,500 μm, 15 μm to 10,000 μm, 25 μm to 7,500 μm, 50 μm to 5,000 μm, 75 μm to 1,000 μm, 100 μm to 750 μm, including 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0037] In some embodiments, the flow cell nozzle comprises an injection port configured to provide a sample to the flow cell nozzle. In embodiments, the injection system is configured to provide a suitable sample flow to the flow cell nozzle chamber. Depending on the desired properties of the fluid medium, the delivery rate of the sample to the flow cell nozzle chamber through the injection port can be 1 μL / min or more, for example, 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 15 μL / min or more, 25 μL / min or more, 50 μL / min or more, including 100 μL / min or more; in some cases, the delivery rate of the sample to the flow cell nozzle chamber through the injection port can be 1 μL / s or more, for example, 2 μL / s or more, 3 μL / s or more, 5 μL / s or more, 10 μL / s or more, 15 μL / s or more, 25 μL / s or more, 50 μL / s or more, including 100 μL / s or more.

[0038] The injection port can be an orifice located in the wall of the nozzle chamber, or a conduit located at the proximal end of the nozzle chamber. In the case where the injection port is an orifice located in the wall of the nozzle chamber, the injection port orifice can be any suitable shape, wherein the cross-sectional shape of interest includes but is not limited to: a cross-sectional shape surrounded by a straight line (e.g., a square, rectangle, trapezoid, triangle, hexagon, etc.), a cross-sectional shape surrounded by a curve (e.g., a circle, an ellipse, etc.) and an irregular shape (e.g., a parabolic bottom connected to a plane top). In certain embodiments, the injection port has a circular orifice. The size of the injection port orifice can vary according to the shape, and in certain cases, its opening range is 0.1mm to 5.0mm, for example, 0.2 to 3.0mm, 0.5mm to 2.5mm, 0.75mm to 2.25mm, 1mm to 2mm, including 1.25mm to 1.75mm, for example 1.5mm.

[0039] In some cases, the injection port is a conduit located at the proximal end of the flow cell nozzle cavity. For example, the injection port can be a conduit, and its position makes the injection port orifice and the flow cell nozzle orifice in the same straight line. In the case where the injection port is a conduit in the same straight line as the flow cell nozzle orifice, the cross-sectional shape of the injection tube can be any suitable shape, wherein the cross-sectional shape of interest includes but is not limited to: a cross-sectional shape surrounded by a straight line (e.g., a square, rectangle, trapezoid, triangle, hexagon, etc.), a cross-sectional shape surrounded by a curve (e.g., a circle, an ellipse), and an irregular shape (e.g., a parabolic bottom connected to a plane top). The orifice of the conduit can vary according to the shape, and in some cases, its opening range is 0.1mm to 5.0mm, for example, 0.2 to 3.0mm, 0.5mm to 2.5mm, 0.75mm to 2.25mm, 1mm to 2mm, including 1.25mm to 1.75mm, for example 1.5mm. The top shape of the injection port can be the same as or different from the cross-sectional shape of the injection tube. For example, the injection port orifice may include a beveled top having a bevel angle in the range of 1° to 10°, such as 2° to 9°, 3° to 8°, 4° to 7°, including a bevel angle of 5°.

[0040] In some embodiments, the flow cell nozzle also includes a sheath fluid injection port configured to provide sheath fluid to the flow cell nozzle. In embodiments, the sheath fluid injection system is configured to provide a sheath fluid flow to the flow cell nozzle chamber, for example, in combination with the sample to form a layered sheath fluid medium around the sample fluid medium. Depending on the desired properties of the fluid medium, the delivery rate of the sheath fluid to the flow cell nozzle chamber can be 25 μL / s or more, for example, 50 μL / s or more, 75 μL / s or more, 100 μL / s or more, 250 μL / s or more, 500 μL / s or more, 750 μL / s or more, 1000 μL / ms or more, including 2500 μL / s or more.

[0041] In some embodiments, the sheath fluid injection port is an orifice located in the nozzle cavity wall. The sheath fluid injection port orifice can be any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to, a cross-sectional shape surrounded by straight lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a cross-sectional shape surrounded by curves (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom connected to a flat top). The size of the injection port orifice can vary depending on the shape, and in some cases, its opening range is 0.1 mm to 5.0 mm, for example, 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0042] The closed particle sorting module also includes a sample interrogation area connected to the flow pool nozzle orifice by a fluid. The sample fluid medium flows out from the orifice located at the distal end of the flow pool nozzle, and the particles in the fluid medium can be irradiated with a light source in the sample interrogation area of ​​the particle sorting module, and a more detailed description is provided below. The size of the interrogation area of ​​the particle sorting module can vary according to the characteristics of the fluid nozzle, for example, the size of the nozzle orifice and the size of the injection port. In an embodiment, the width of the interrogation area can be 0.01 mm or more, for example, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 5 mm or more, including 10 mm or more. The length of the interrogation area of ​​the particle sorting module may also vary. In some cases, the length of the area may be 0.01 mm or more, for example, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 1.5 mm or more, 2 mm or more, 3 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 20 mm or more, 25 mm or more, including 50 mm or more.

[0043] The interrogation region on the particle sorting module can be configured to facilitate irradiation of a planar cross-section of the outflowing fluid medium, or can be configured to facilitate irradiation of a predetermined length of a diffuse field (e.g., irradiated with a diffuse laser or lamp). In some embodiments, the interrogation region on the particle sorting module includes a transparent window that facilitates irradiation of a predetermined length of the outflowing fluid medium, for example, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, including 10 mm or more. Depending on the light source used to irradiate the outflowing fluid medium (as described below), the interrogation region of the particle sorting module can be configured to allow light of a certain wavelength range to pass, the wavelength range being 100nm to 1500nm, for example, 150nm to 1400nm, 200nm to 1300nm, 250nm to 1200nm, 300nm to 1100nm, 350nm to 1000nm, 400nm to 900nm, including 500nm to 800nm. Therefore, the particle sorting module located in the interrogation area can be made of any transparent material that can pass light in the required wavelength range, including but not limited to optical glass, borosilicate glass, Pyrex glass, ultraviolet quartz, infrared quartz, sapphire and plastic, for example, any polymeric plastic material used to make the above-mentioned shell.

[0044] In some embodiments, the particle sorting module of interest includes a cuvette located within the sample interrogation region. In some cases, the cuvette is secured within the particle sorting module located within the sample interrogation region using, for example, an adhesive or a suitable mechanical securing means (e.g., with a clip or screw). In other cases, the cuvette is co-molded with the particle sorting module located within the sample interrogation region. In some cases, the cuvette is directly incorporated into the particle sorting module.The cuvette can be made of any transparent material that can pass light of the desired wavelength range, including but not limited to optical glass, borosilicate glass, pyrex glass, ultraviolet quartz, infrared quartz, sapphire and plastics, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide or copolymers of the thermoplastics, such as PETG (polyethylene terephthalate modified with ethylene glycol) and other polymeric plastic materials, including polyesters, wherein the target polyesters can include but are not limited to poly (alkylene terephthalates), such as poly (ethylene terephthalate) (PET), bottle-grade PET (based on monoethylene glycol, terephthalate), Copolymers made of diphthalic acid and other comonomers (such as isophthalic acid, cyclohexene dimethanol, etc.), poly(butylene terephthalate) (PBT) and poly(hexylene terephthalate); poly(alkylene adipates), such as poly(ethylene adipate), poly(1,4-butylene adipate) and poly(hexylene adipate); poly(alkylene suberates), such as poly(ethylene suberate); poly(alkylene sebacates), such as poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalates), such as poly(ethylene isophthalate); poly(2,6-naphthalene- dicarboxylates), for example, poly(ethylene 2,6-naphthalene-dicarboxylate); poly(sulfonyl-4,4'-dibenzoate), for example, poly(sulfonyl-4,4'-dibenzoate); poly(p-phenylene dicarboxylates), for example, poly(ethylene p-phenylene dicarboxylates); poly(trans-1,4-cyclohexanediyl dicarboxylates), for example, poly(trans-1,4-cyclohexanediyl dicarboxylates); poly(1,4-cyclohexane-dimethylene dicarboxylates), for example, poly(1,4-cyclohexane-dimethylene dicarboxylates); poly([2.2.2]-bicyclooctane-1, 4-dimethylene dicarboxylic acid alkylene esters), for example, poly([2.2.2]-bicyclooctane-1,4-dimethylene dicarboxylic acid ethylene glycol ester); lactic acid polymers and copolymers, for example, (S)-polylactide, (R,S)-polylactide, poly(tetramethyl glycolide) and poly(lactide-co-glycolide); and polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A; polyamides, for example, poly(p-phenylene terephthalamide); polyesters, for example, polyethylene terephthalate, for example, MylarTM polyethylene terephthalate, and the like.In an embodiment, the cuvette can allow light of a certain wavelength range to pass, and the wavelength range is 100nm to 1500nm, for example, 150nm to 1400nm, 200nm to 1300nm, 250nm to 1200nm, 300nm to 1100nm, 350nm to 1000nm, 400nm to 900nm, including 500nm to 800nm.

[0045] In some embodiments, the sample interrogation region includes one or more optical adjustment components. "Optical adjustment" refers to changing the light irradiated to the sample interrogation region or the light collected from the irradiated fluid medium as needed. In some embodiments, the sample interrogation region includes an optical adjustment component, which is used to adjust the light irradiated to the sample interrogation region by the light source. In other embodiments, the sample interrogation region includes an optical adjustment component, which is used to adjust the light originating from the irradiated fluid medium before it is transmitted to the detector for measurement. In other embodiments, the sample interrogation region includes an optical adjustment component, which is used to adjust the light irradiated to the sample interrogation region by the light source and the light originating from the irradiated fluid medium before the light is transmitted to the detector for measurement. For example, the optical adjustment can be to increase the size of the light, focus the light, or collimate the light. In some cases, the optical adjustment is an amplification scheme to increase the size of the light (e.g., beam spot), for example, to increase the size by 5% or more, 10% or more, 25% or more, 50% or more, including increasing the size by 75% or more. In other embodiments, optical conditioning includes focusing the collected light to reduce the size of the light, for example, by 5% or more, 10% or more, 25% or more, 50% or more, including reducing the beam spot size by 75% or more. In certain embodiments, optical conditioning includes collimating the light. The term "collimation" is conventionally used to refer to optical conditioning for collinearity of light propagation or reducing divergence of the light about a common propagation axis. In some cases, collimation includes narrowing the spatial cross-section of the light beam.

[0046] The optical conditioning assembly can be any suitable device or structure that causes the desired change in the collected light, and can include, but is not limited to, lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. The particle sorting module can include one or more optical conditioning assemblies, such as two or more, three or more, four or more, including five or more optical conditioning assemblies, placed in the sample interrogation area as required.

[0047] Where the particle sorting module includes one or more optical conditioning components positioned at the sample interrogation region, the optical conditioning components may be physically coupled to the particle sorting module (e.g., using an adhesive); co-molded with the housing; or directly integrated into the particle sorting module housing, wherein the optical conditioning components are positioned at the sample interrogation region. Thus, the optical conditioning components and the particle sorting module may be integrated into a single device.

[0048] In some embodiments, the optical adjustment component is a focusing lens having a magnification range of 0.1 to 0.95, for example, a magnification range of 0.2 to 0.9, a magnification range of 0.3 to 0.85, a magnification range of 0.35 to 0.8, a magnification range of 0.5 to 0.75, including a magnification range of 0.55 to 0.7, such as a magnification of 0.6. For example, in some cases, the focusing lens is a bichromatic reduction lens with a magnification of approximately 0.6. The focal length of the focusing lens can vary, and the focal length range is 5 mm to 20 mm, for example, 6 mm to 19 mm, 7 mm to 18 mm, 8 mm to 17 mm, 9 mm to 16 mm, including a focal length in the range of 10 mm to 15 mm. In some embodiments, the focusing lens has a focal length of approximately 13 mm.

[0049] In other embodiments, the optical adjustment component is a collimator. The collimator can be any suitable collimation scheme, for example, one or more mirrors or curved lenses or a combination thereof. For example, in some cases, the collimator is a single-sided collimating lens. In other cases, the collimator is a collimating mirror. In other cases, the collimator includes two lenses. In other cases, the collimator includes a mirror and a lens. In the case where the collimator includes one or more lenses, the focal length of the collimating lens can vary, and the focal length range is 5mm to 40mm, for example, 6mm to 37.5mm, 7mm to 35mm, 8mm to 32.5mm, 9mm to 30mm, 10mm to 27.5mm, 12.5mm to 25mm, including a focal length ranging from 15mm to 20mm.

[0050] In some embodiments, the optical adjustment component is a wavelength separator. According to the conventional meaning, the term "wavelength separator" used in this article refers to an optical scheme for separating polychromatic light into its component wavelengths. Wavelength separation according to some embodiments may include selectively blocking a specific wavelength or wavelength range of the polychromatic light or allowing it to pass. The purpose wavelength separation scheme that may be part of the target flow pool nozzle or used in combination with it includes but is not limited to colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators and combinations thereof, and other wavelength separation schemes. In some embodiments, the wavelength separator is a filter. For example, the filter can be a bandpass filter with a minimum bandwidth range of 2nm to 100nm, for example, 3nm to 95nm, 5nm to 95nm, 10nm to 90nm, 12nm to 85nm, 15nm to 80nm, including a bandpass filter with a minimum bandwidth range of 20nm to 50nm.

[0051] In an embodiment, the particle sorting module further comprises a droplet deflector configured to transfer droplets containing analyzed cells in a droplet stream formed by a fluid medium flowing out of the fluid nozzle to a receiving location, for example, an outlet operably coupled to a container or a sorted particle collection system. The transferred droplets may be referred to herein as sorted droplets. Transferring the target droplets to the receiving location may be achieved by a droplet deflector, which electrostatically charges the droplets and deflects the charged droplets from the fluid medium by applying an electrostatic field. The electrostatic field may be generated by a deflection plate located near the fluid medium. The term "deflection" as used herein refers to electrostatically deflecting the target droplets from the analyzed droplet fluid medium so as to identify and track cells in the fluid medium, and only the droplets containing the target cells in the fluid medium are transferred and collected in a container. In some cases, the particle sorting module comprises a droplet deflector configured to deflect a single droplet into each container.

[0052] The particle sorting module is configured to generate a stream of analyzed droplets and deflect each analyzed droplet from the stream of analyzed droplets and then enter a deflected droplet receiving position. The term "deflected droplet receiving position" as used herein refers to a downstream position of the droplet deflector, wherein the droplets can be collected at the position after the sorted droplets containing the target cells are deflected by the droplet deflection plate. The target particle sorting module can have two or more deflection plates as needed, for example, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, including 10 or more deflection plates.

[0053] The particles in the fluid medium may be deflected by any suitable deflector plate scheme, including but not limited to the cell sorting deflector plates described in the following U.S. Patents: 3,960,449; 4,347,935; 4,667,830; 5,245,318; 5,464,581; 5,483,469; 5,602,039; 5,643,796; 5,700,692; 6,372,506 and 6,809,804, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the deflector plates include charged plates for sorting cells in a fluid medium used in a flow cytometry system, such as the BD Biosciences Influx TM Cell sorter, BD Biosciences FACSAria TM III and BDFACSAria TM Fusion Cell Sorter, BD Biosciences FACSJazz TM Cell sorter, BD Biosciences FACS Melody TM Cell sorter, etc.

[0054] The deflection plates in the target particle sorting module can be configured according to the cell type to be sorted, the sorting rate, the applied voltage applied to the cells, and the number of components to be sorted in the sample. In an embodiment, the length of a suitable deflection plate ranges from 5mm to 100mm, for example, 6mm to 90mm, 7mm to 80mm, 8mm to 70mm, 9mm to 60mm, including 10mm to 50mm. The width of the deflection plate can also vary, ranging from 1mm to 25mm, for example, 2mm to 20mm, 3mm to 15mm, including 5mm to 10mm. The distance between each deflection plate can vary according to the applied voltage and the size of the particles to be sorted in the fluid medium. In some embodiments, the distance between each deflection plate can be 1mm or more, for example, 2mm or more, 3mm or more, 4mm or more, 5mm or more, including 10mm or more. For example, the distance between each deflector plate can range from 1 mm to 25 mm, such as 2 mm to 22.5 mm, 3 mm to 20 mm, 4 mm to 17.5 mm, including 5 mm to 15 mm. The deflector plates can also be oriented at an angle to each other, such as 15° to 75°, 20° to 70°, 25° to 65°, including 30° to 60°.

[0055] The voltage applied to the deflection plates to transfer the charged particles (see below) can be 10 mV or more, for example, 25 mV or more, 50 mV or more, 100 mV or more, 250 mV or more, 500 mV or more, 750 mV or more, 1000 mV or more, 2500 mV or more, 5,000 mV or more, including 10000 mV or more. In some embodiments, the voltage applied to the deflection plates ranges from 0.001 V to 6000 V, including 0.001 V to 5000 V, for example, 0.01 V to 4000 V, 0.1 V to 3000 V, 1 V to 2000 V, 5 V to 1500 V, 10 V to 1000 V, 25 V to 750 V, including 100 V to 500 V.

[0056] The deflector is configured to transfer particles in the fluid medium to a receiving location downstream of the deflector, for example, any of two or more different outlets of the sorting chamber, such as a first waste outlet or a second sorting particle outlet. In an embodiment, the deflector can transfer each particle at a different angle. In some embodiments, the deflector is configured to deflect each particle at an angle of 0.5 degrees or more to the longitudinal axis of the fluid medium, for example, 1 degree or more, 1.5 degrees or more, 2 degrees or more, 2.5 degrees or more, 3 degrees or more, 5 degrees or more, 7.5 degrees or more, including deflecting each particle at an angle of 10 degrees or more to the longitudinal axis of the fluid medium. For example, each particle can be transferred at an angle to the longitudinal axis of the fluid medium, the angle ranging from 0.1 degrees to 30 degrees, for example, 0.5 degrees to 25 degrees, 1 degree to 20 degrees, 2 degrees to 15 degrees, including 5 degrees to 10 degrees.

[0057] In some embodiments, the distal end of the target particle sorting module is configured to be coupled to one or more containers to collect deflected particle droplets from the fluid medium. For example, the distal end of the particle sorting module may be configured to be coupled to one or more containers, such as 2 or more containers, 3 or more containers, 4 or more containers, 5 or more containers, 6 or more containers, 10 or more containers, including 25 or more containers, through an outlet such as on the shell. In some cases, the distal end of the shell may include one or more alignment members for coupling the shell to the container. Suitable alignment members for coupling the distal end of the shell to the container may include, but are not limited to, alignment protrusions, alignment tracks, alignment notches, alignment grooves, alignment slots, alignment countersunk holes, alignment counterbores, alignment recesses, alignment holes, or combinations thereof. In some embodiments, the distal end of the shell also includes one or more fasteners for fixing the container to the distal end of the shell. Suitable fasteners may include, but are not limited to, magnets, hook and loop fasteners, latches, notches, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives, or combinations thereof. In certain embodiments, the housing distal end includes threads for coupling the container to the housing via a threaded connection.

[0058] In certain embodiments, the particle sorting module includes one or more containers for receiving deflected particles from the fluid medium at the distal end of the housing. For example, the particle sorting module according to these embodiments may include 2 or more containers, for example, 3 or more containers, 4 or more containers, 5 or more containers, 6 or more containers, including 10 or more containers. In some embodiments, the container is mechanically coupled to the distal end of the housing by, for example, a Luer lock connection, sterile tube welding, or a threaded connection with the housing. In other embodiments, the container is fixed to the distal end of the housing by a permanent or non-permanent adhesive. In other embodiments, the container is co-molded with the particle sorting module housing. In other embodiments, the container is integrated with the housing so that the container and the housing constitute a single device. In other cases, the container can be coupled to the housing in a fluid-connected manner by, for example, a piping system configured to transport sorted droplets, wherein the embodiments can achieve aseptic retrieval of sorted droplets, for example, by pinching and cutting the fluid transport structure (e.g., piping system). Suitable containers for collecting droplets from the fluid medium may include, but are not limited to, test tubes, conical tubes, multi-compartment containers such as microtiter plates (e.g., 96-well plates), centrifuge tubes, culture tubes, microtubes, caps, cuvettes, bottles, linear polymer containers and bags, and other types of containers.

[0059] The destination container may also be a flexible sample container, such as a bag, for example, a sterile bag. "Flexible" refers to bending or flexing the sample container compared to its original shape without any significant structural changes, such as tearing, rupture, perforation, etc. For example, a flexible sample container may be flexed and / or deformed compared to its original shape while still maintaining a sealing layer state to prevent the fluid in the sample container from contacting the surrounding environment. In some cases, the flexible sample container is made of a flexible material having a Young's modulus not exceeding 1 GPa, for example, 0.7 GPa or less, including 0.5 GPa or less, for example, 0.3 GPa or less, 0.1 GPa or less, 0.05 GPa or less, 0.01 GPa or less. In certain embodiments, the fluid in the flexible sample container is sterile, that is, free of or substantially free of viable bacteria or other microorganisms. In certain embodiments, a buffer may be contained in a container (e.g., a cryogenic container). Containers of interest include ethylene vinyl acetate (EVA) based containers, e.g., EVA cryo bags, CRYOCTYTE™ cryo bags (Baxter Healthcare Corporation, Deerfield, IL), Cryo bags (Charter Medical, Winston-Salem, NC), ORIGEN CRYOSTORE™ bags (OriGen BioMedical, Austin, TX).

[0060] For more detailed information about the container and the closed particle sorting module including the container, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of this patent are incorporated herein by reference.

[0061] In some cases, the container is a sorted particle collection system, which includes a collection container having a sorting tube in droplet receiving relationship with the outlet of the closed particle sorting module; a sample output end that aligns the cell collection position of the collection container with a vacuum receiving container (e.g., For more detailed information about the sorted particle collection system, please refer to the following publications: U.S. patent application serial number 62 / 663,792 filed on April 27, 2018 (attorney docket number: BECT-157PRV; P-15882@); and U.S. patent application serial number 16 / 390,376 filed on April 22, 2019 and claiming corresponding priority; the contents of the patents are incorporated herein by reference.

[0062] Any fluid connections associated with the sorting module (e.g., within the sorting module and / or between the sorting module and other aspects of the system (e.g., receiving containers (e.g., bags), input tubing, etc.)) can be made by sterile tube welding as desired. Any suitable sterile tube welding system and materials can be used.

[0063] Figure 1 An illustration of a closed particle sorting module according to an embodiment of the present invention is provided. Figure 1 As shown, the closed particle sorting module 100 includes a housing 105 that surrounds the various components of the sorting module, such as a sample interrogation area 140 and a sorting chamber 125. A sample input 110 is located at the top or proximal end of the sorting module. Also shown are a sheath fluid input 120 and a waste reservoir connector 115. A waste outlet 130 and a sorted particle outlet 135 are located at the bottom of the module and are responsible for providing outlets for the sorting chamber.

[0064] Flow Cytometry System

[0065] As described above, aspects of the present invention also include a flow cytometer system, and the closed particle sorting module can be operably coupled to the system to obtain a flow cytometer configured to sort particle components in a sample (e.g., cells in a biological sample), that is, a cell sorter-type flow cytometer. According to certain embodiments, the system includes one or more closed particle sorting modules (e.g., a sorting module as described above); a sample input module coupled to an inlet in a fluid-connected manner at the proximal end of the particle sorting module; and a waste pool, such as a trough or other container, coupled to an outlet of the particle sorting module in a fluid-connected manner. In an embodiment, the system is configured to be coupled to one or more of the above-mentioned closed particle sorting modules. In order to connect the particle sorting module, the system may include an aligner configured to be coupled to an alignment member on the particle sorting module housing. The aligner may include one or more alignment members that are complementary to the alignment members on the particle sorting system housing. For example, the aligner can include 2 or more alignment members, for example, 3 or more alignment members, 4 or more alignment members, 5 or more alignment members, 7 or more alignment members, including 10 or more alignment members. In some embodiments, the particle sorting system aligner includes 3 alignment members. For more detailed information about the alignment members and the flow cytometer system including the alignment members, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of the patent are incorporated herein by reference.

[0066] As described above, the system further comprises a sample input module coupled in fluid communication with an inlet at a proximal end of the particle sorting module. In an embodiment, the sample input module is configured to provide a suitable sample flow to the flow cell nozzle chamber in the particle sorting module. Depending on the desired characteristics of the fluid medium flowing out of the fluid nozzle, the delivery rate of the sample to the particle sorting module through the sample input module can be 1 μL / min or more, for example, 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 15 μL / min or more, 25 μL / min or more, 50 μL / min or more, including 100 μL / min or more; in some cases, the flow rate can be 1 μL / s or more, for example, 2 μL / s or more, 3 μL / s or more, 5 μL / s or more, 10 μL / s or more, 15 μL / s or more, 25 μL / s or more, 50 μL / s or more, including 100 μL / s or more.

[0067] In an embodiment, the sample fluid input end includes a container, a cover, and one or more ports entering the inner cavity of the container. The container has a distal end, a proximal end, and a wall between the distal end and the proximal end, and the wall combination forms the inner cavity of the container. In some embodiments, the outer wall and the inner cavity of the container have the same cross-sectional shape, wherein the cross-sectional shape of the purpose includes but is not limited to a cross-sectional shape surrounded by a curve (e.g., a circle, an ellipse), a cross-sectional shape surrounded by a straight line (e.g., a square, a rectangle, a trapezoid, a triangle, a hexagon, etc.) and an irregular shape (e.g., a parabolic bottom is connected to a plane top). For example, the outer wall and the inner cavity of the container can both have a circular or elliptical cross section, or the outer wall and the inner cavity of the container can both have a polygonal (e.g., an octagonal) cross section. In other embodiments, the outer wall and the inner cavity of the container have different cross-sectional shapes (e.g., a container with a polygonal cross section and an inner cavity with a circular cross section). In certain embodiments, the container is a tube, and the cross-sectional shapes of the outer wall and the inner wall are both circular.

[0068] The inner cavity size of the container can vary according to the sample size and the size of the particle sorting module, wherein, in some cases, the inner cavity length of the container can range from 1 cm to 25 cm, for example, 2.5 cm to 22.5 cm, 5 cm to 20 cm, 7.5 cm to 17.5 cm, including 10 cm to 15 cm; and the inner cavity width of the container can range from 1 cm to 20 cm, for example, 2 cm to 17.5 cm, 3 cm to 15 cm, 4 cm to 12.5 cm, including 5 cm to 10 cm. In the case where the inner cavity of the container has a cylindrical cross-section, in some embodiments, the diameter may vary, and the diameter ranges from 1 cm to 10 cm, for example, 2 cm to 9 cm, 3 cm to 8 cm, including 4 cm to 7 cm. Thus, the volume of the container may vary, ranging from 1 to 500 cm3, for example, 5 to 250 cm3, 10 to 200 cm3, 15 to 150 cm3, 20 to 125 cm3, including 25 to 100 cm3. In some embodiments, the container of the sample input module is a tube, and its volume ranges from 1 mL to 500 mL, for example, 2 mL to 400 mL, 3 mL to 300 mL, 4 mL to 200 mL, 5 mL to 150 mL, including 10 mL to 100 mL.

[0069] The container may be made of any suitable material, including but not limited to glass, metal or plastic (eg, flexible or rigid plastic), polymeric material or thermoplastic material (eg, as described above).

[0070] In an embodiment, the container of the sample input module also includes a cover configured to close the proximal end of the container. For example, the cover can be a screw cap, a snap cap, or a cover bonded to the container by a permanent, semi-permanent or non-permanent adhesive. In some cases, the cover forms a liquid seal with the wall of the container. The cover can be an integral part of the container, including molding the cover with the container, soldering, welding, or bonding the cover to the container using a permanent adhesive. In other embodiments, the cover is releasably attached to the container. "Releasably" means that the cover can be freely removed from the proximal end of the container and reattached to the proximal end of the container. In the case where the cover is releasably attached to the container, the cover can be non-permanently fastened to the container by any suitable attachment scheme, including but not limited to hook and loop buckles, latches, notches, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives, threaded screws or combinations thereof. In some cases, the container includes a threaded outer wall and is threadedly connected to the inner wall of the cover.

[0071] The cover may include one or more ports that are connected to the inner cavity of the container, for example, 2 or more ports, 3 or more ports, 4 or more ports, including 5 or more ports. In certain embodiments, the cover includes two ports. The port can be any suitable port configured to be connected to the inner cavity of the container by fluid or gas. In certain embodiments, the cover includes a port, which is configured to deliver gas to the container to generate a positive pressure in the container, and deliver the sample fluid in the container to the particle sorting module through a second port. In some cases, the container includes a third opening in the cover to exhaust air.

[0072] Any suitable port configuration can be adopted according to the required function of the port, wherein port examples include channels, orifices, channels with check valves, Luer taper joints, ports with frangible seals (e.g., disposable ports), and other types of ports. In certain embodiments, the port is configured with a Luer taper joint, such as a Luer twist lock or a Luer slide lock. The port in the cover of the sample input module can be of any suitable shape, wherein the cross-sectional shape of the destination port includes, but is not limited to: a cross-sectional shape surrounded by straight lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a cross-sectional shape surrounded by curves (e.g., circular, elliptical, etc.), and an irregular shape (e.g., a parabolic bottom connected to a flat top). The size of the port may vary, and in some embodiments, the size of the port ranges from 1 mm to 100 mm, for example, 2 mm to 95 mm, 3 mm to 90 mm, 4 mm to 80 mm, 5 mm to 70 mm, 6 mm to 60 mm, including 10 mm to 50 mm. In some embodiments, the port is a circular orifice, and the diameter of the port ranges from 1 mm to 100 mm, for example, 2 mm to 90 mm, 4 mm to 80 mm, 5 mm to 70 mm, 6 mm to 60 mm, including 10 mm to 50 mm. Thus, depending on the shape of the port, the port in the cover can be an opening with an area ranging from 0.01 mm2 to 250 mm2, for example, 0.05 mm2 to 200 mm2, 0.1 mm2 to 150 mm2, 0.5 mm2 to 100 mm2, 1 mm2 to 75 mm2, 2 mm2 to 50 mm2, including 5 mm2 to 25 mm2.

[0073] For more detailed information about the sample input module and the system including the sample input module, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of this patent are incorporated herein by reference.

[0074] As described above, the target particle sorting system also includes a waste tank coupled to the outlet of the particle sorting module in a fluid-connected manner. In some embodiments, the waste tank includes one or more ports, for example, a port for discharging the accumulated gas pressure in the particle sorting module; a port for collecting waste from the particle sorting module; and a port for discharging the accumulated gas pressure in the waste tank; or any combination thereof. In some embodiments, the waste tank may be in fluid communication with the particle sorting module through one or more ports. For example, the waste tank may include 2 or more ports, for example, 3 or more ports, 4 or more ports, including 5 or more ports. In certain embodiments, the waste tank includes two ports. The port may be any suitable port configured to communicate with the inner cavity of the waste tank through fluid or gas. In some embodiments, the waste tank includes a port configured to discharge (i.e., release) the accumulated gas pressure in the waste tank. In other embodiments, the waste tank includes a port for receiving waste liquid from the particle sorting module. In other embodiments, the waste reservoir includes a port configured to vent accumulated gas pressure in the particle sorting module, for example, to vent a flow cell portion of the particle sorting module.

[0075] Any suitable port configuration can be adopted according to the required function of the port, wherein port examples include channels, orifices, channels with check valves, Luer taper joints, ports with frangible seals (e.g., disposable ports), and other types of ports. In certain embodiments, the port is configured with a Luer taper joint, such as a Luer twist lock or a Luer slide lock. The waste tank port can be of any suitable shape, wherein the cross-sectional shape of the destination port includes, but is not limited to: a cross-sectional shape surrounded by a straight line (e.g., a square, rectangle, trapezoid, triangle, hexagon, etc.), a cross-sectional shape surrounded by a curve (e.g., a circle, an ellipse, etc.), and an irregular shape (e.g., a parabolic bottom connected to a plane top). The size of the port may vary, and in some embodiments, the size range of the port is 1 mm to 100 mm, for example, 2 mm to 95 mm, 3 mm to 90 mm, 4 mm to 80 mm, 5 mm to 70 mm, 6 mm to 60 mm, including 10 mm to 50 mm. In some embodiments, the port is a circular orifice, and the diameter of the port ranges from 1 mm to 100 mm, for example, 2 mm to 90 mm, 4 mm to 80 mm, 5 mm to 70 mm, 6 mm to 60 mm, including 10 mm to 50 mm. Thus, depending on the shape of the port, the port in the waste tank can be an opening with an area ranging from 0.01 mm2 to 250 mm2, for example, 0.05 mm2 to 200 mm2, 0.1 mm2 to 150 mm2, 0.5 mm2 to 100 mm2, 1 mm2 to 75 mm2, 2 mm2 to 50 mm2, including 5 mm2 to 25 mm2.

[0076] In some embodiments, the waste tank can be coupled to the particle sorting module in fluid communication via one or more conduits. For example, the particle sorting module can be coupled to the waste tank in fluid communication via 2 or more conduits, for example, 3 or more conduits, including 5 or more conduits. The conduit coupling the particle sorting module to the waste tank includes a proximal end connected to the particle sorting module and a distal end connected to the waste tank.

[0077] The length of each catheter may vary, and the length of each catheter may independently be 5 cm or more, for example, 7 cm or more, 10 cm or more, 25 cm or more, 30 cm or more, 50 cm or more, 75 cm or more, 100 cm or more, 250 cm or more, including 500 cm or more. The lumen diameter of each catheter may vary, and the diameter may be 0.5 mm or more, for example, 0.75 mm or more, 1 mm or more, 1.5 mm or more, 2 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, including 50 mm or more. For example, the lumen diameter may range from 0.5 mm to 50 cm, for example, 1 mm to 25 mm, including 5 mm to 15 mm.

[0078] Each conduit can be made of a thin material, for example, the thickness of the conduit wall is 5 mm or less, for example, 3 mm or less, 2 mm or less, including 1 mm or less or 0.5 mm or less, for example, 0.4 mm or less, 0.3 mm or less, 0.2 mm or less, including 0.1 mm or less. In some embodiments, the conduit is made of a flexible material with a Young's modulus not exceeding 1 GPa, for example, 0.9 GPa or less, 0.8 GPa or less, 0.7 GPa or less, 0.6 GPa or less, 0.5 GPa or less, 0.4 GPa or less, 0.3 GPa or less, 0.2 GPa or less, 0.1 GPa or less, including 0.01 GPa or less. In some embodiments, the conduit is made of a polymeric material, for example, but not limited to, such as materials as described above, including but not limited to: polyvinyl chloride (PVC), ethyl vinyl acetate (EVA), polyethylene, polypropylene, combinations thereof, etc.

[0079] The waste pool may include one or more chambers. In some embodiments, the waste pool has a single chamber for collecting all waste components from the particle sorting module. In other embodiments, the waste pool has more than one chamber, for example, 2 or more chambers, 3 or more chambers, including 4 or more chambers. Each chamber in a multi-chamber waste tank may have one or more inlet and outlet conduits. For example, the two or more chambers may be in fluid communication with a single conduit. The inner cavities of the two or more chambers may be connected together by a Y-type connector, a valve (e.g., a pinch valve), etc.

[0080] Where the waste reservoir comprises more than one chamber, each different chamber may be configured to receive the same or different fluids. For example, a first waste chamber may collect and contain uncharged and undeflected particles from the fluid medium, while a second waste chamber may collect and contain deflected but uncollected particles from the fluid medium. In other embodiments, a first waste chamber may collect and contain excess sheath fluid and discarded excess sample fluid from the fluid medium, while a second waste chamber may collect sorted but undesirable sample fluid components from the fluid medium.

[0081] In some embodiments, the waste pool includes one or more ports, such as a port for venting the accumulated gas pressure in the particle sorting module; a port for collecting waste from the particle sorting module; and a port for venting the accumulated gas pressure in the waste tank; or any combination thereof. The waste stream from the particle sorting module can be delivered to the waste tank by a conduit. The conduit can be coupled to the waste tank by a connector, such as a Luer lock connector or a threaded fitting connector.

[0082] For more detailed information about the waste pool and the system including the waste pool, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of this patent are incorporated herein by reference.

[0083] The particle sorting system according to certain embodiments also includes a sheath liquid delivery subsystem for delivering sheath liquid to the flow pool nozzle of the closed particle sorting module. According to the conventional meaning, the term "sheath liquid" used in this article refers to a fluid delivered by a catheter (e.g., a catheter in a flow cytometer), which is used to form an annular flow coaxial with the fluid containing the sample, so that the sample fluid containing particles in the center of the sheath liquid flow is formed into a concentrated flow in a fluid dynamic manner. The target sheath liquid can be any suitable buffer composition (e.g., a buffer composition for a flow cytometer), and can include one or more salts, including but not limited to potassium phosphate, potassium chloride, sodium phosphate, sodium chloride, preservatives and chelating agents, such as disodium ethylenediaminetetraacetic acid (EDTA). In an embodiment, the sheath liquid distribution system includes a reservoir containing sheath liquid; and a catheter, the proximal end of which is connected to the sheath liquid pool by fluid, and the distal end of which is connected to the sheath liquid input end of the particle sorting module by fluid.

[0084] In some embodiments, the sheath fluid delivery subsystem includes a pressurized housing having a flexible container, the flexible container having a sheath fluid reservoir located within the housing. In other embodiments, the sheath fluid delivery subsystem includes a housing and a first flexible container and a second flexible container located within the housing. The first flexible container includes a fluid reservoir and a catheter having a proximal end and a distal end, wherein the proximal end is coupled to the fluid reservoir in a fluid-connected manner, and the distal end is configured to couple the catheter to the particle sorting module; the second flexible container includes a gas reservoir and a port connected to the gas reservoir by gas. In these embodiments, the second flexible container and the first flexible container are both located within the housing, and the second flexible container is configured to apply pressure to the fluid reservoir of the first flexible container to deliver the sheath fluid from the distal end of the catheter to the particle sorting module.

[0085] In certain embodiments, the particle sorting system of interest includes a sheath fluid delivery subsystem for delivering sheath fluid to the particle sorting module, such as the sheath fluid delivery subsystem described in co-pending PCT patent application No. PCT / US2016 / 048433, published as WO 2017 / 040151, filed on October 24, 2016; and U.S. patent application No. 14 / 365,602, now issued as U.S. Patent No. 9,551,643, the contents of which are incorporated herein by reference in their entirety. For more detailed information about the sheath fluid delivery subsystem and systems including the sheath fluid delivery subsystem, please refer to U.S. patent application No. US 2017-0299493, application serial number 15 / 472,020; the contents of which are incorporated herein by reference.

[0086] As described above, any fluid connections associated with the sorting module (e.g., within the sorting module and / or between the sorting module and other aspects of the system (e.g., receiving containers (e.g., bags), input tubing, etc.)) can be made by sterile tube welding as desired. Any suitable sterile tube welding system and materials can be used.

[0087] As described above, the system of the present invention is configured to control the aerosol content within the closed particle sorting module or at least one region thereof (e.g., a sorting chamber). In some embodiments, the system is configured to reduce the generation of aerosols within the sorting chamber. "Reducing the generation of aerosols" means that the generated aerosols are reduced by 2 times or more, for example, 5 times or more, including 10 times or more, compared to suitable controls (e.g., the device described in the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020).

[0088] One or more aerosol reduction methods can be used to reduce the generation of aerosols, for example, as described below. In some cases, the closed particle sorting module is configured to minimize the contact of the waste stream with the inner surface of the particle sorting module. In these cases, the internal structure or design configuration of the closed particle sorting module (including its sorting chamber) is selected or manufactured so that the waste stream and the sorting stream (in some cases) avoid contact with its solid surface. In some cases, the specific sorting module is configured so that the waste stream is discharged through a first outlet (i.e., an outlet that couples the sorting chamber to the waste pool in a fluid communication manner) and does not contact the first outlet wall. For example, the outlet may be axially aligned with the longitudinal axis of the waste stream so that the waste stream passes through the center of the outlet when passing through the outlet. In this case, the size of the outlet may vary, wherein, in some cases, the diameter of the outlet ranges from 2.5 to 25 mm, for example, 5 to 10 mm. In some cases, the waste pool is coupled to the first outlet in a fluid communication manner via a flexible pipeline (e.g., a pipeline as described above). Although the size of the flexible line may vary, in some cases, the inner diameter of the flexible line ranges from 2.5 mm to 25 mm, for example, 5 mm to 10 mm. If desired, the flexible line is configured so that the waste stream first contacts the inner wall of the flexible line at a predetermined distance from the outlet. Although the distance may vary, the distance can be selected to reduce the probability of aerosol droplets formed by contact flowing back into the sorting chamber. In some cases, the longitudinal axis of the flexible line is aligned with the center of the outlet, and the alignment distance is 5 mm or more, for example, 10 mm or more, including 25 mm or more, including 50 mm or more. In order to minimize the generation of aerosols, in some cases, the flexible line is configured so that the waste stream first contacts the inner wall of the flexible line at a smaller angle. Although the contact angle may vary in this case, in some embodiments, the contact angle ranges from 0.5 to 25°, for example, 0.5 to 15°, including 0.5 to 5°.

[0089] In addition or in addition to reducing aerosol formation by suitable sorting chamber and / or waste line construction (e.g., as described above), the flow cytometer system of the present invention can be configured to maintain the temperature within the sorting chamber of the closed particle sorting module above the dew point. In accordance with the conventional meaning, the term "dew point" used herein refers to a temperature (varying according to pressure and humidity) below which water droplets begin to condense and dew can be formed. The system can be configured to provide a dew point in the sorting chamber ranging from 1 to 30°C, for example, 2 to 25°C, including 3 to 15°C. The system of the present invention can be configured to maintain the temperature within the sorting chamber above the dew point using a variety of different methods. In some cases, the system includes a heater configured to maintain the temperature above the dew point. The heater can be any suitable type of heater, for example, a resistive heater, a visible light radiation heater, an infrared radiation heater, etc. The purpose resistive heater includes but is not limited to a heater having a metal heating element, a ceramic heating element, a polymer PTC heating element, a combination heating element, a composite heating element, and the like. The heating element can be configured to provide radiant heating for the sorting chamber of the closed particle sorting module. The heating element can be integrated into the flow cytometer system and / or the closed particle sorting module as needed. Therefore, in some cases, the heating element is an integral part of the flow cytometer system rather than a part of the closed particle sorting module. In other cases, the heating element is integrated into the closed particle sorting module. A given heater can be made of a single heating element or two or more heating elements as needed. In some cases, the heating element is configured to provide a temperature range within the sorting chamber of the closed particle sorting module of 5 to 40°C, for example, 10 to 35°C, including 15 to 30°C.

[0090] Alternatively or additionally, the flow cytometer may be configured to control the humidity within the sorting chamber. In this case, the flow cytometer may be configured to make the relative humidity within the sorting chamber less than 75%, for example, less than 60%, including less than 50%; in some cases, the relative humidity provided may range from 1 to 50%, for example, 5 to 25%, including 5 to 15%. Any suitable method may be used to control the humidity of the sorting chamber. In some cases, the system includes a closed recirculating airflow duct located between the sorting chamber and the waste pool. A closed recirculating airflow duct refers to a recirculating gas line provided between the waste pool and the closed particle sorting module that is not connected to the external environment, which allows gases such as air to circulate between the interior of the waste pool and the interior of the closed particle sorting chamber without being contaminated by the external environment. The closed recirculating airflow duct is composed of one or more different fluid pipelines, for example, pipelines as described above, which are connected to the interior of the waste pool and the closed particle sorting module. The airflow rate through the closed recirculating airflow conduit may vary, and in some cases, the flow rate is 5 to 500 mL / min, for example, 10 to 200 mL / min. Any suitable method may be used to provide the desired airflow, wherein in some cases, a pump such as a peristaltic pump may be used to provide the desired airflow. If desired, the recirculating airflow conduit may include a desiccant, for example, placed in one or more regions or locations of the closed recirculating airflow conduit. Any suitable desiccant may be used, such as, but not limited to, a dry clay material, such as montmorillonite clay, silica gel, etc.

[0091] As described above, the target system is configured to sort the particle components in the sample (e.g., biological sample). In some embodiments, the system further includes a light detection system configured to irradiate and identify the particle components of the sample in the fluid medium. In these embodiments, the system includes one or more light sources for irradiating the sample in the fluid medium. The light source can be a broadband light source, which emits light with a wide range of wavelengths, for example, a span of 50nm or more, 100nm or more, 150nm or more, 200nm or more, 250nm or more, 300nm or more, 350nm or more, 400nm or more, including a span of 500nm or more. For example, a suitable broadband light source emits light with a wavelength range of 200nm to 1500nm. Another example of a suitable broadband light source includes a light source that emits light with a wavelength range of 400nm to 1000nm. Any suitable broadband light source solution can be used, for example, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent diodes, semiconductor light emitting diodes, wide-spectrum LED white light sources, multiple LED integrated light sources and other broadband light sources or any combination of the above.

[0092] In other embodiments, the light source is a narrowband light source that emits light of a specific wavelength or a narrow range of wavelengths. In some cases, the narrowband light source emits light with a narrow range of wavelengths, for example, the wavelength is 50nm or less, 40nm or less, 30nm or less, 25nm or less, 20nm or less, 15nm or less, 10nm or less, 5nm or less, 2nm or less, and includes a light source that emits a specific wavelength of light (i.e., monochromatic light). Any suitable narrowband light source scheme can be used, for example, a narrow wavelength LED, a laser diode, or a broadband light source coupled to one or more optical bandpass filters, a diffraction grating, a monochromator, or any combination thereof.

[0093] In certain embodiments, the light source is a laser. In some cases, the target system includes 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 fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser or a xenon fluoride (XeF) excimer laser or a combination thereof. In other cases, the target system includes a dye laser, such as a stilbene, coumarin or rhodamine laser. In other cases, the target laser 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 a combination thereof. In other cases, the target system 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 thulim YAG laser, a Ytterbium YAG laser, an Ytterbium trioxide laser, or a cerium-doped laser, and combinations thereof.

[0094] The target system may include one or more light sources as needed, for example, two or more light sources, three or more light sources, four or more light sources, five or more light sources, including ten or more light sources. The light source may include any combination of light source types. For example, in some embodiments, the target system includes a laser array, for example, an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers. In other cases, when two light sources are used, the first light source may be a broadband white light source (e.g., a broadband white light LED); and the second light source may be a broadband near-infrared light source (e.g., a broadband near-infrared LED). In other cases, when two light sources are used, the first light source may be a broadband white light source (e.g., a broadband white light LED); and the second light source may be a narrow spectrum light source (e.g., a near-infrared LED or laser). In some cases, the light sources are multiple narrow-band light sources each emitting light of a specific wavelength, for example, two or more lasers, three or more lasers, including five or more lasers. In other cases, the light source is an array of two or more LEDs, such as an array of three or more LEDs, an array of five or more LEDs, including an array of ten or more LEDs.

[0095] In some embodiments, the light source emits light with a wavelength range of 200nm to 1500nm, for example, 250nm to 1250, 300nm to 1000nm, 350nm to 900nm, including 400nm to 800nm. For example, the light source may include a broadband light source that emits light with a wavelength range of 200nm to 900nm. In other cases, the light source includes a plurality of narrowband light sources that emit light with a wavelength range of 200nm to 900nm. For example, the light source may be a plurality of narrowband LEDs (1nm–25nm), each of which emits light with a wavelength between 200nm and 900nm. In some embodiments, the narrowband light source is one or more narrowband lamps that emit light with a wavelength range of 200nm to 900nm, for example, a narrowband cadmium lamp, a cesium lamp, a helium lamp, a mercury lamp, a mercury-cadmium lamp, a potassium lamp, a sodium lamp, a neon lamp, a zinc lamp, or any combination thereof. In other embodiments, the narrowband light source includes one or more lasers emitting light in the wavelength range of 200 nm to 1000 nm, such as gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers as described above.

[0096] The light source is arranged at a certain angle relative to the fluid medium, for example, the angle range is 10° to 90°, 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°. In some embodiments, the light source is arranged at a 90° angle relative to the sample.

[0097] In these embodiments, the target system also includes one or more detectors for detecting and measuring light from the fluid medium. The target detector may include, but is not limited to, an optical sensor or a photodetector, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge coupled device (CCD), an enhanced charge coupled device (ICCD), a light emitting diode, a photon counter, a bolometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or a photodiode, and combinations thereof, and other photodetectors. In certain embodiments, a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS), an image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor is used to measure the transmitted light. In some embodiments, the imaging sensor is a CCD camera. For example, the camera may be an electron multiplying CCD (EMCCD) camera or an enhanced CCD (ICCD) camera. In other embodiments, the imaging sensor is a CMOS-type camera. In the case of measuring fluorescence or scattered light with a CCD, the effective detection surface area of ​​the CCD may vary, for example, the surface area ranges from 0.01 cm2 to 10 cm2, 0.05 cm2 to 9 cm2, 0.1 cm2 to 8 cm2, 0.5 cm2 to 7 cm2, including 1 cm2 to 5 cm2. The number of photodetectors in the target system may vary as needed, for example, 1 or more, 2 or more, 3 or more, 5 or more, including 10 or more photodetectors. In the case where the target system includes more than one photodetector, each photodetector may be the same, or a set of two or more photodetectors may be a combination of different photodetectors.

[0098] Depending on the type of irradiation light source and sample characteristics (e.g., the particle size of the particles in the sample), the detector can be placed at a distance from the fluid medium. For example, the detector can be placed at a distance of 0.01 mm or more from the sample, for example, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, including 50 mm or more from the sample. The detector can also be placed at an angle relative to the sample, and the angle can change. For example, the detector can be placed at an angle relative to the fluid medium, and the angle range is 10° to 90°, for example, 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°. In some embodiments, the detector is placed at a 90° angle relative to the fluid medium. In some embodiments, the system includes a detector placed to detect forward scattered light from the fluid medium. In other embodiments, the system comprises a detector positioned to detect side scattered light from the fluid medium. In other embodiments, the system comprises a detector positioned to detect fluorescence from the fluid medium.

[0099] For more detailed information about the flow cytometer system and its components, please refer to the U.S. patent application with publication number US2017-0299493 and application serial number 15 / 472,020; the contents of which are incorporated herein by reference.

[0100] Figure 2 Provides a schematic diagram of a flow cytometer system configured to Figure 1 The enclosed particle sorting module shown can be operably coupled to obtain a sterile flow cytometer-type cell sorter. Figure 2 As shown, system 200 includes doors 205 and 210, which, when opened, reveal a location 215 that can be operably engaged with a closed particle sorting module. A sample input module 220 is also shown.

[0101] Figure 3 A schematic diagram of a flow cytometer type cell sorter according to an embodiment of the present invention is provided. Figure 3As shown, the cell sorter 300 includes a closed particle sorting module 305 operably coupled to a sample input module 310 via a pipeline 315, wherein the sample input module 310 includes a sample tube 316, a sample pipeline 317 and an air filter 318. The closed particle sorting module 305 is also coupled to a sheath liquid subsystem 320 via a sheath liquid pipeline 321, wherein the sheath liquid subsystem includes a sheath liquid bag 322 for containing a certain amount of sheath liquid and a sheath liquid filter 323. Heating elements 307a and 307b are located near the sorting chamber 306 and are in a radiative heating relationship therewith. The first central output end 308 of the sorting chamber is axially aligned with the waste stream and is in fluid communication with a waste pool 330 via a pipeline 331. In the case of being far away from the outlet 308, the longitudinal axis of the pipeline 331 is aligned with the center of the outlet 308, so that the waste stream avoids contact with the inner wall of the pipeline 331 at a position close to the sorting chamber. In addition, the pipeline 331 is gradually bent at a position far from the output end 308, so that the waste flow is carried out at a smaller angle when contacting the inner wall, thereby minimizing the formation of aerosols. In order to provide sufficient airflow in the sorting chamber to achieve the required low humidity, the pipeline 331 is an integral part of the closed recirculating airflow duct, which also includes a waste bag 335 and a return line 337, wherein the return line 337 is connected to the sorting chamber at the entry position 309, so that the waste bag 335 is in gas communication with the sorting chamber. The return line 337 includes a desiccant chamber 338, which removes water vapor from the gas before the gas is reintroduced into the sorting chamber through the entry position 309. An air filter 339 is also shown in the figure. In order to provide the required airflow in the closed recirculating airflow duct, a peristaltic pump 340 is provided. The figure shows a sorted particle collection system 350 in a particle receiving relationship with the sorted particle output end 304 of the closed particle sorting module. The sorted particle collection system 350 includes a collection bag 351, a culture medium input line 352, a sample output end 353 having a mating element 354, and a Blood collection tube 355.

[0102] Method for sorting particle components of a sample in a fluid medium

[0103] Aspects of the invention also include methods for sorting particles in a sample (e.g., cells in a biological sample). Methods according to certain embodiments include irradiating a sample containing particles in a fluid medium within an interrogation region of a particle sorting module; detecting light (e.g., fluorescence) from the sample; and sorting the particles in the sample into two or more sample collection containers. In certain embodiments, the sample is a biological sample, and the method includes sorting and collecting two or more different types of cells.

[0104] In some embodiments, the sample is a biological sample. According to the conventional meaning, the term "biological sample" refers to a subset, cell or component of a whole organism, plant, fungus or animal tissue, and in some cases, may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen. Therefore, "biological sample" refers to both a natural organism or a subset of its tissues, and a homogenate, lysate or extract prepared using a subset of the organism or its tissues, including but not limited to: plasma; serum; cerebrospinal fluid; lymph; skin, respiratory tract, gastrointestinal tract, cardiovascular and urogenital tract sections; tears; saliva; milk; blood cells; tumors; organs, etc. A biological sample can be any type of biological tissue, including healthy tissue and pathological tissue (e.g., cancer tissue, malignant tissue, necrotic tissue, 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 certain cases, the sample is a blood sample, including a whole blood sample, such as blood obtained by venipuncture or finger stick (wherein, the blood may or may not be mixed with any reagents (e.g., preservatives, anticoagulants, etc.) prior to analysis).

[0105] In certain embodiments, the sample source is "mammal", which is a term widely used to describe organisms belonging to the class of mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method is applicable to samples obtained from male and female subjects at any stage of development (i.e., newborns, infants, teenagers, adolescents, adults), in which certain embodiments, the human subjects are teenagers, adolescents, or adults. Although the present invention is applicable to samples taken from human subjects, it should be understood that the method can also be implemented on blood taken from other animal subjects (i.e., in "non-human subjects"), including but not limited to birds, mice, rats, dogs, cats, livestock, and horses.

[0106] According to various parameters, for example, by attaching a specific fluorescent marker to the target cell and determining the phenotypic characteristics, the target cell can be separated from the fluid medium. In some embodiments, the system is configured to deflect the analyzed droplets determined to include the target cell. Various cells can be sorted using a standard method. The target cells 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. The target cells include cells with suitable cell surface markers or antigens, which can be captured or labeled by suitable affinity agents or their conjugates. 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 cells containing HIV, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0107] When implementing the target method, the particle sorting module is coupled to the particle sorting system. In order to couple the particle sorting module to the particle sorting system, an alignment piece located on the outer wall of the particle sorting module housing is appropriately arranged so that it contacts the alignment piece on the particle sorting system aligner. If any, the alignment piece on the outer wall of the particle sorting module housing contacts the alignment piece on the particle sorting system aligner so that one or more fasteners can be engaged when the particle sorting module is fixed to the particle sorting system. Depending on the sample being analyzed, the particle sorting module can be in contact with the particle sorting system for any desired duration, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 60 minutes or more, 120 minutes or more, 240 minutes or more, including 480 minutes or more.

[0108] After coupling the particle sorting module to the particle sorting system, a certain amount of fluid sample is injected into the particle sorting module. The amount of sample injected into the particle sorting module may vary, for example, the sample amount ranges from 0.001 mL to 1000 mL, for example, 0.005 mL to 900 mL, 0.01 mL to 800 mL, 0.05 mL to 700 mL, 0.1 mL to 600 mL, 0.5 mL to 500 mL, 1 mL to 400 mL, 2 mL to 300 mL, including 5 mL to 100 mL sample.

[0109] The method according to an embodiment of the present invention includes counting and sorting labeled particles (e.g., target cells) in a sample. When implementing the target method, a fluid sample including the particles is first introduced into the fluid nozzle of the particle sorting module. After flowing out of the fluid nozzle, the particles pass through the sample interrogation area in a manner of one particle at a time, in which each particle is irradiated by a light source; and the light scattering parameters and fluorescence emission measurements of each particle (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded separately as needed. The particles in the fluid medium pass through the sample interrogation area in the particle sorting module through the flow path in a manner of one particle at a time, in which each particle is irradiated by a light source. Depending on the properties of the fluid medium being interrogated, the fluid medium of 0.001 mm or more can be irradiated with light, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, including irradiating 1 mm or more of the fluid medium with light. In certain embodiments, methods include irradiating a planar cross-section of the fluid medium within the sample interrogation region, e.g., with a laser (as described above). In other embodiments, methods include irradiating a predetermined length of the fluid medium within the sample interrogation region, e.g., corresponding to an irradiation profile of a diffuse laser beam or lamp.

[0110] In some embodiments, the method includes irradiating the fluid medium at or near the flow cell nozzle orifice. For example, the method can include irradiating the fluid medium at a distance of about 0.001 mm or more from the nozzle orifice, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, including 1 mm or more from the nozzle orifice. In some embodiments, the method includes irradiating the fluid medium in close proximity to the flow cell nozzle orifice.

[0111] As it passes through the sensing region, a detector (e.g., a photomultiplier tube (or "PMT")) is used to record light that passes through each particle (in some cases, referred to as forward light scatter); light that reflects in a direction orthogonal to the direction of particle flow through the sensing region (in some cases, referred to as orthogonal or side light scatter); and fluorescence emitted by particles labeled with fluorescent markers as the particles pass through the sensing region and are illuminated by the energy source. Each of forward light scatter (or FSC), orthogonal light scatter (SSC), and fluorescence emission (FL1, FL2, etc.) comprises a separate parameter for each particle (or each "event"). Thus, for example, two, three, or four parameters may be collected (recorded) for particles labeled with two different fluorescent markers.

[0112] As described above, suitable light detection schemes include, but are not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge coupled devices (CCD), intensified charge coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, and other photodetectors. In certain embodiments, a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS), an image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor is used to measure light from the irradiated fluid medium in the sample interrogation region of the particle sorting module. In certain embodiments, light is measured using a charge coupled device (CCD). When using CCD to measure the light from the irradiated fluid medium in the sample interrogation area of ​​the particle sorting module, the effective detection surface area of ​​the CCD may change. For example, the surface area ranges from 0.01cm2 to 10cm2, 0.05cm2 to 9cm2, 0.1cm2 to 8cm2, 0.5cm2 to 7cm2, including 1cm2 to 5cm2.

[0113] The recorded data of each particle is analyzed in real time or stored in a data storage and analysis tool (e.g., a computer) as needed. U.S. Patent No. 4,284,412 describes the configuration and use of a flow cytometer 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.

[0114] In embodiments of the present disclosure according to certain embodiments, the particles are exposed to excitation light and the fluorescence of each particle is measured in one or more detection channels as needed, so that the particles are detected and uniquely identified. The fluorescence emitted in the detection channel for identifying the particles and the binding complexes 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 markers, the markers can be selected so that all markers are excitable for each excitation light source used.

[0115] The method in some embodiments also includes data acquisition, analysis and recording, such as using a computer, wherein multiple data channels record data from each detector, the data being data about light scattering and fluorescence emitted when each particle 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 in the form of a set of digitized parameter values. The target system can be set to trigger at selected parameters to distinguish the target particles from background and noise. "Trigger" refers to a preset threshold for detecting a parameter and can be used as a means for detecting particles passing through the light source. Detection of an event exceeding the selected parameter threshold triggers the light scattering and fluorescence data acquisition of the particles. Data of particles or other components to be analyzed in the medium that cause the response to be below the threshold is not collected. The trigger parameter can be the detection of 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.

[0116] Then, based on the data collected for the entire population, a specific sub-population of interest is further analyzed by "setting a gate". In order to select a suitable gate, the data is plotted into a graph to achieve optimal separation of potential sub-populations. This step can be performed by plotting the relationship between forward light scatter (FSC) and lateral (i.e., orthogonal) light scatter (SSC) on a two-dimensional dot matrix. Then, a sub-population of particles (i.e., cells within the gate) is selected to exclude particles that are not within the gate. If necessary, the gate can be selected by drawing a line around the desired sub-population using a cursor on a computer interface. Then, the particles within the gate are further analyzed by plotting other parameters of the particles (e.g., fluorescence) into a graph. If necessary, the above analysis can be configured to give a count of the particles of interest in the sample.

[0117] In some embodiments, the system is operated to determine a time period during which one or more containers located at the distal end of the particle sorting module are aligned with the deflected droplet receiving position. In some cases, the deflection signal includes an initial deflection sub-signal and a final deflection sub-signal; and the system is operated to generate the deflection signal by sending the initial deflection sub-signal at the beginning of the time period, the deflection signal configuring the deflector to deflect the analyzed droplet (if any). In some cases, the method includes sending a final deflection sub-signal to the particle sorting module at the end of the time period, the deflection sub-signal configuring the deflector to avoid deflecting the analyzed droplet. In some embodiments, the method includes sending a final deflection sub-signal to the particle sorting module after a single analyzed droplet is deflected during the time period, wherein the final deflection sub-signal configures the deflector to avoid deflecting the analyzed droplet.

[0118] In some cases, the method may include maintaining a desired temperature within a sorting chamber of the sorting module. For example, the method may include operating one or more heating elements, which may be integrated with the system or module, to maintain a desired temperature within the sorting chamber, such as a temperature as described above, wherein the desired temperature within the sorting chamber may be above the dew point.

[0119] In some embodiments, the method includes disengaging the particle sorting module from the particle sorting system, which can be achieved by disengaging the alignment member (and fastener, if any). In some cases, the method further includes reattaching the second particle sorting module to the particle sorting system after removing the first particle sorting module. The first particle sorting module can be washed and sterilized for future use (e.g., sterilized with an autoclave), or it can be discarded. Therefore, in some embodiments, the particle sorting modules described herein are disposable, for example after being used once.

[0120] Computer control system

[0121] Aspects of the invention further include a computer controlled system for implementing the subject method, wherein the system further includes one or more computers to fully automate or partially automate the system for implementing the method described herein. In some embodiments, the system includes a computer having a computer-readable storage medium having a computer program stored therein, wherein the computer program includes instructions for irradiating a sample in a fluid medium in a sample interrogation region of a particle sorting module when loaded onto the computer; an algorithm for detecting light from the sample and determining the light detected at one or more wavelengths; and an algorithm for sorting particles in the sample into two or more sample collection containers.

[0122] In an embodiment, the system includes an input module, a processing module, and an output module. In some embodiments, the target system may include an input module, the input content such as: parameters or information about each fluid sample, the intensity and wavelength (discrete or range) of the light source used, the characteristics of the particle sorting module (including the flow cell nozzle cavity size), the nozzle orifice size, the size of the particle sorting module sample interrogation area, the applied voltage of the deflection plate, the position of the container at the far end of the particle sorting module, the duration of irradiation with the light source, the number of different light sources, the distance between the light source and the fluid medium in the particle sorting module sample interrogation area, the focal length of any optical adjustment component, the refractive index of the fluid medium (e.g., sheath fluid), the presence of any wavelength separator, the characteristics of the wavelength separator (including the bandpass width), opacity, grating spacing, and the characteristics and sensitivity of the photodetector.

[0123] The processing module includes a memory having a plurality of instructions for executing target method steps, for example, irradiating a sample in a fluid medium within a sample interrogation region of a particle sorting module; detecting light from the sample in the fluid medium; measuring the detected light at one or more wavelengths; and sorting particles in the sample into two or more sample collection containers, the containers being located at a distal end of the particle sorting module.

[0124] After the processing module performs one or more steps of the subject method, the output module communicates the results to a user, for example, by displaying them on a display or by printing a report.

[0125] The target system may include hardware and software components, where the hardware components may be one or more platforms, such as a server, which enables the functional elements of the system (i.e., the elements of the system that perform specific tasks (e.g., managing the input and output of information, processing information, etc.)) to function by executing software applications on one or more computer platforms equipped by the system.

[0126] The system may include a display and an operator input device. The operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory having instructions stored thereon for performing the steps of the subject method, for example, irradiating a sample in a fluid medium in a sample interrogation region of a particle sorting module; detecting light from the sample in the fluid medium; determining the light detected at one or more wavelengths; and sorting particles in the sample into two or more sample collection containers, the containers being located at a distal end of the particle sorting module.

[0127] The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input-output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or any other processor that is or will be available. As is well known in the art, the processor executes an operating system, which is connected to the firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs, which can be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages ​​and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, communication control and related services in accordance with known techniques.

[0128] The system memory may be any of a variety of known or later-introduced memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media (e.g., resident hard disk or tape), optical media (e.g., read-write optical disk), flash memory device, or other memory storage device. The memory storage device may be any of a variety of known or later-introduced devices, including an optical disk drive, a tape drive, a removable hard disk drive, or a floppy disk drive. The type of memory storage device typically reads content from and / or writes content to a program storage medium (not shown), which includes an optical disk, a tape, a removable hard disk, or a floppy disk, etc. Any of these program storage media, or other media currently in use or that may be developed in the future, may be considered a computer program product. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0129] In some embodiments, a computer program product is described that includes a computer usable medium storing control logic (computer software program, including program code). When the control logic is executed by a computer processor, the processor can perform the functions described herein. In other embodiments, some functions are mainly implemented in hardware using, for example, a hardware state machine. It will be apparent to a person skilled in the relevant art that the hardware state machine is enabled to perform the functions described herein.

[0130] The memory may be any suitable device in which the processor can store and retrieve data, for example, a magnetic, optical or solid-state storage device (including a disk, optical disk or tape or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor that has been appropriately programmed based on a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely through a communication channel, or it may be pre-stored in a computer program product using any device connected to the memory, for example, a memory or some other portable or fixed computer-readable storage medium. 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 a programming for implementing the above method, for example, a computer program product, an algorithm. The programming according to the present invention may be recorded in a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. The media include, but are 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; hybrids of the above categories, such as magnetic / optical storage media.

[0131] The processor may also access a communication channel to communicate with a user at a remote location, where the user has no direct contact with the system but instead forwards input information to the input manager from an external device (e.g., 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 smartphone)).

[0132] In some embodiments, the system according to the present invention can 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 can 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, WiFi, infrared communication, wireless universal serial bus (USB), ultra-wideband (UWB), Communication protocols and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0133] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces (e.g., USB ports, RS-232 ports) or any other suitable electrical connection ports to enable data communications between the target system and any external device (e.g., a computer terminal configured to implement similar complementary data communications (e.g., in a physician's office or hospital environment)).

[0134] In one embodiment, the communication interface is configured for infrared communication, communications or any other suitable wireless communications protocol to enable the subject system to communicate with other devices, such as a computer terminal and / or network, a communications-enabled mobile phone, a personal digital assistant, or any other communications device that a user may use in conjunction with managing treatment for a condition (e.g., HIV, AIDS, or anemia).

[0135] In one embodiment, the communication interface is configured to provide a data transmission connection using the Internet Protocol (IP) through a mobile phone network, a short message service (SMS); provide a wireless connection to a personal computer (PC) within a local area network (LAN) connected to the Internet; or provide a WiFi connection at a WiFi hotspot to connect to the Internet.

[0136] In one embodiment, the subject system is configured to communicate wirelessly with the server device via a communication interface, for example, using a common standard such as 802.11 or RF protocol or IrDA infrared protocol. The server device can be another portable device, such as a smart phone, a personal digital assistant (PDA) or a laptop; or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device includes a display (e.g., a liquid crystal display (LCD)) and an input device (e.g., a button, a keyboard, a mouse or a touch screen).

[0137] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the target system (e.g., stored in an optional data storage unit) with a network or server device using one or more of the above-mentioned communication protocols and / or mechanisms.

[0138] The output controller may include a controller for any of a variety of known display devices to provide information to a local or remote user (whether human or machine). If any of the display devices provide visual information, the information may generally be logically and / or physically organized into an array of image elements. A graphical user interface (GUI) controller may include any of a variety of known or later developed software programs to provide a graphical input and output interface between the system and the user, and to process user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be implemented using a network or other type of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location according to known techniques, for example, via the Internet, telephone or satellite network. The output manager may implement data display according to various known techniques. In some examples, the data may include SQL, HTML or XML documents, emails or other files or other forms of data. The data may include Internet URL addresses so that users can retrieve other SQL, HTML, XML or other documents or data from remote sources. One or more platforms in the target system can be any type of known computer platform or a computer platform to be developed in the future, although it usually belongs to a certain type of computer (usually called a server). However, the platform can also be a mainframe computer, workstation or other computer type. They can be connected by any known or later developed cable or other communication system (including wireless systems connected by networking or other means). They can be located in the same location or physically separated. Various operating systems can be used on any computer platform, which may depend on the type and / or brand of computer platform selected. Suitable operating systems include Windows NT □ , WindowsXP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGIIRIX, Siemens Reliant Unix, etc.

[0139] Kits

[0140] Aspects of the present invention further include a kit, wherein the kit includes one or more closed particle sorting modules, such as the sorting modules described herein. In some embodiments, the kit also includes one or more sample input modules and one or more waste tanks, such as bags. The kit may also include one or more conduits that couple the sample input module and the waste tank to the particle sorting module in a fluid-connected manner. In some cases, the kit also includes a connector for coupling the components of the target system together, such as a connector for coupling the sample input module to the particle sorting module; a connector for coupling the waste tank to the particle sorting module; and a connector for coupling the sheath fluid delivery subsystem to the particle sorting module. The kit may include a connector, such as a Luer lock connector, a threaded fitting connector, and a connector connecting two components with a frangible seal. The kit may include one or more sorted particle receiving containers, such as a sorted particle collection assembly and its components, such as a vacuum receiving container as described above. In some cases, the kit may include one or more assay components (e.g., labeled reagents, buffers, etc. as described above). In some cases, the kit may further include a sample collection device, such as a lancet or needle configured to pierce the skin to obtain a whole blood sample, a pipette, etc., as desired.

[0141] The various analytical components of the kit can be contained in separate containers, some or all of which can be pre-assembled. For example, in some cases, one or more components of the kit (e.g., particle sorting module, sample input module, waste tank) are contained in a sealed bag, e.g., sterile packaging, such as a sterile foil bag or envelope.

[0142] In addition to the above components, the subject kit may further include (in some embodiments) instructions for implementing the subject method. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may exist is printed information printed on a suitable medium or substrate (e.g., a piece of paper or several pieces of paper with information printed thereon), kit packaging, package instructions, etc. Another form in which these instructions exist is a computer-readable medium with information recorded thereon, such as a floppy disk, a compact disk (CD), a portable flash drive, etc. Another form in which these instructions may exist is a website address, which can be used to access information on a remote website via the Internet.

[0143] utility

[0144] The subject particle sorting modules, particle sorting systems, methods, and computer systems can be used in various applications where it is necessary to analyze and sort the particle components of a sample (e.g., a biological sample) within a fluid medium. Embodiments of the present invention can also be used to improve the sterility of a particle sorting system, which can facilitate the collection of higher purity samples and reduce the incidence of cross contamination between analyzed samples, for example, in research and high-throughput laboratory testing. Embodiments of the present invention can also be used in situations where it is necessary to provide a flow cytometer that meets the following requirements: improved cell sorting accuracy; enhanced particle collection capability; improved particle charging efficiency; more accurate particle charging; and enhanced particle deflection during cell sorting.

[0145] Embodiments of the present invention may also be used in applications where cells prepared using biological samples may need to be used for research, laboratory testing, or for treatment. In some embodiments, the subject methods and devices help to obtain a single cell prepared using a target fluid or tissue biological sample. For example, the subject methods and systems help to obtain cells from a fluid or tissue sample for use as a research or diagnostic specimen for diseases such as cancer. Similarly, the subject methods and systems help to obtain cells for treatment from a fluid or tissue sample. Compared to traditional flow cytometry systems, the methods and devices of the present invention can separate and collect cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with higher efficiency and lower cost.

[0146] Embodiments of the present invention provide closed sorting equipment and methods, which can reduce (e.g., cannot eliminate) one or more of the following: the risk of contamination of the sample to be processed; the risk of operator exposure to sample components, which is critical when handling biohazardous samples; and the like.

[0147] The flow cytometry sorted samples obtained using the systems and methods described herein can be administered to subjects in cell therapy protocols or any application requiring sterile live cells to be infused into subjects. Conditions that can be treated by administering flow cytometry sorted samples include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0148] Therefore, the systems and methods described herein can be used for cell therapy programs. Cell therapy programs are programs that may require the preparation of living cell materials (including, for example, cells and tissues) and their introduction into the subject as a therapeutic agent. Typical cell therapy programs may include the following steps: sample collection, cell separation, genetic modification, in vitro culture and amplification, cell harvesting, reducing sample volume and washing, biological preservation, storage, and introducing cells into the subject. The program may begin with the collection of living cells and tissues from the source tissue of the subject to produce cell and / or tissue samples. The sample can be collected by any suitable step, including, for example, administering a cell mobilization agent to the subject; extracting the subject's blood; extracting the subject's bone marrow, etc. After collecting the sample, cell enrichment can be performed by several methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic sorting, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells can be genetically modified by any suitable method (e.g., nuclease-mediated gene editing). The genetically modified cells can be cultured, activated, and amplified in vitro. In some cases, the cells are preserved (eg, cryopreserved) and stored for later use, at which time the cells are thawed and then administered to a patient, eg, the cells can be infused into a patient.

[0149] Although the claims are appended hereto, the scope of the present invention is further defined by the following clauses:

[0150] 1. A flow cytometer, comprising:

[0151] a closed particle sorting module having a sorting chamber including a droplet deflector;

[0152] a sample input module coupled in fluid communication with an inlet of the closed particle sorting module;

[0153] a waste reservoir coupled in fluid communication with the first outlet of the closed particle sorting module; and

[0154] a first sorting particle collection system coupled in fluid communication with a second outlet of the closed particle sorting module;

[0155] Wherein the flow cytometer is configured to control the aerosol content within the sorting chamber.

[0156] 2. A flow cytometer according to claim 1, wherein the system is configured to reduce the generation of aerosols within the sorting chamber.

[0157] 3. A flow cytometer according to any one of clauses 1 to 2, wherein the closed particle sorting module is configured to minimize contact of the waste stream with the inner surface of the particle sorting module.

[0158] 4. A flow cytometer according to clause 3, wherein the specific sorting module is configured to allow the waste stream to be discharged through the first outlet without contacting the first outlet wall.

[0159] 5. A flow cytometer according to clause 4, wherein the diameter of the first outlet is in the range of 5 to 10 mm.

[0160] 6. A flow cytometer according to clause 5, wherein the waste reservoir is coupled to the first outlet in fluid communication via a flexible tubing having an inner diameter ranging from 5 to 10 mm.

[0161] 7. The flow cytometer according to clause 6, wherein the flexible pipeline is configured so that the waste stream first contacts the inner wall of the flexible pipeline at a distance of 5 mm or more from the first outlet.

[0162] 8. A flow cytometer according to clause 7, wherein the flexible tubing is configured so that the waste stream first contacts an inner wall of the flexible tubing at a small angle.

[0163] 9. A flow cytometer according to any of the preceding clauses, wherein the flow cytometer is configured to maintain the temperature within the sorting chamber above the dew point.

[0164] 10. A flow cytometer according to clause 9, wherein the flow cytometer comprises a heater.

[0165] 11. A flow cytometer according to clause 10, wherein the heater is not integrated into the closed particle sorting module.

[0166] 12. A flow cytometer according to clause 10, wherein the heater is integrated into the closed particle sorting module.

[0167] 13. A flow cytometer according to any one of clauses 9 to 12, wherein the flow cytometer is configured to control the humidity within the sorting chamber.

[0168] 14. A flow cytometer according to clause 13, wherein the system comprises a closed recirculating gas flow conduit located between the sorting chamber and the waste reservoir.

[0169] 15. A flow cytometer according to clause 14, wherein the closed recirculating gas flow conduit comprises a peristaltic pump.

[0170] 16. A flow cytometer according to any one of clauses 14 and 15, wherein the closed recirculating gas flow conduit comprises a desiccant.

[0171] 17. A flow cytometer according to any of the preceding clauses, wherein the sorted particle collection system comprises:

[0172] (a) a collection vessel having a sorting tube in droplet receiving relationship with said second outlet; and

[0173] (b) a sample output port that operably couples the cell collection location of the collection container to a mating connector of a vacuum receiving container.

[0174] 18. A flow cytometer according to any of the preceding clauses, wherein the closed particle sorting module further comprises:

[0175] A housing includes a proximal end, a distal end, and a wall therebetween, the wall including an alignment feature for aligning the housing within the flow cytometer.

[0176] a flow cell nozzle at a proximal end of the housing, the flow cell nozzle comprising an orifice; and

[0177] A sample interrogation region is in fluid communication with the flow cell nozzle orifice.

[0178] 19. The flow cytometer according to any of the preceding clauses, further comprising a second collection system coupled in fluid communication with the third outlet of the closed particle sorting module.

[0179] 20. A flow cytometer according to any of the preceding clauses, wherein the flow cytometer is sterile.

[0180] 21. A flow cytometry sample processing method, the method comprising:

[0181] introducing the sample into a closed particle sorting module of a flow cytometer according to any one of clauses 1 to 20; and

[0182] The particles introduced into the sample are sorted.

[0183] 22. The method of clause 21, wherein the method comprises maintaining the temperature within the sorting chamber above the dew point.

[0184] 23. A method according to any one of clauses 21 and 22, wherein the particles are cells.

[0185] 24. A method according to any one of clauses 21 to 23, wherein the method is performed under sterile conditions.

[0186] 25. A closed particle sorting module, comprising:

[0187] a housing comprising a proximal end and a distal end;

[0188] a flow cell nozzle at a proximal end of the housing, the flow cell nozzle comprising an orifice;

[0189] a sample interrogation region in fluid communication with the flow cell nozzle orifice;

[0190] a sorting chamber in droplet receiving relationship with the interrogation region, the sorting chamber comprising:

[0191] Droplet Deflector

[0192] a first outlet configured to couple the waste stream in fluid communication with the waste reservoir; and

[0193] a second outlet configured to couple the sorted particles in fluid communication with a collection system;

[0194] Wherein the particle sorting module is configured to minimize contact of the waste stream with interior surfaces of the particle sorting module.

[0195] 26. A closed particle sorting module according to clause 25, wherein the particular sorting module is configured to allow the waste stream to be discharged through the first outlet without contacting the first outlet wall.

[0196] 27. A closed particle sorting module according to clause 26, wherein the diameter of the first outlet is in the range of 5 to 10 mm.

[0197] 28. A closed particle sorting module according to any one of clauses 25 to 27, wherein the closed particle sorting module further comprises a heater.

[0198] 29. A closed particle sorting module according to any one of clauses 25 to 28, wherein the particle sorting module further comprises an alignment member.

[0199] 30. A closed particle sorting module according to any one of clauses 25 to 29, further comprising a cuvette located within the sample interrogation region.

[0200] 31. A method according to any one of clauses 25 to 30, wherein the module is sterile.

[0201] 32. A kit comprising:

[0202] A closed particle sorting module according to any one of clauses 25 to 31; and

[0203] Sterile packaging for the closed particle sorting module.

[0204] 33. The kit of claim 32, wherein the kit further comprises a sorted particle collection system.

[0205] 34. The kit of clause 33, wherein the sorted particle collection system comprises:

[0206] (a) a collection vessel having a sorting tube in droplet receiving relationship with said second outlet; and

[0207] (b) a sample output port that operably couples the cell collection location of the collection container to a mating connector of a vacuum receiving container.

[0208] 35. The kit according to clause 35, wherein the kit further comprises a vacuum receiving vessel.

[0209] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, in view of the teachings of the invention, it will be apparent to one skilled in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0210] Therefore, the foregoing only illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various structures, although not explicitly stated or shown here, but these designs reflect the principles of the present invention and do not exceed the spirit and scope of the present invention. In addition, all examples and conditional language listed herein are mainly to help readers understand the principles of the present invention and the ideas provided by the inventors to further expand the art, and should be interpreted as not being limited by these specific examples and conditions. Moreover, all statements citing the principles, aspects and embodiments of the present invention and their specific examples herein are intended to cover their equivalents in structure and function. In addition, the equivalents are intended to include currently known equivalents and equivalents to be developed in the future, that is, any functionally identical originals developed and have nothing to do with the structure. Moreover, regardless of whether the disclosure of the present invention is clearly described in the claims, none of the contents therein will be disclosed to the public.

[0211] Therefore, the scope of the present invention is not 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, only when the phrase "means for..." or "step for..." is explicitly used at the beginning of the limitation content of the claim, "35 U.S.C. Section 112(f)" or "35 U.S.C. Section 112(6)" is explicitly defined as being cited; if the phrase is not used in the limitation content of the claim, "35 U.S.C. Section 112(f)" or "35 U.S.C. Section 112(6)" is not cited.

Claims

1. A flow cytometry sample processing method, the method comprising: The sample is introduced into a closed particle sorting module of a flow cytometer, the flow cytometer comprising: a closed particle sorting module having a sorting chamber including a droplet deflector; a sample input module coupled in fluid communication with an inlet of the closed particle sorting module; a waste reservoir coupled in fluid communication with the first outlet of the closed particle sorting module; as well as a first sorting particle collection system coupled in fluid communication with a second outlet of the closed particle sorting module; wherein the flow cytometer is configured to control the aerosol content within the sorting chamber, and The particles in the introduced sample are sorted.

2. The method of claim 1, wherein the method comprises maintaining the temperature within the sorting chamber above the dew point.

3. The method according to any one of the preceding claims, wherein the particles are cells.

4. The method according to any one of the preceding claims, wherein the method is performed under sterile conditions.

5. A particle sorting module, comprising: A sorting chamber, the sorting chamber comprising: Droplet deflectors; a first outlet configured to couple the waste stream in fluid communication with the waste reservoir; and a second outlet configured to couple the sorted particles in fluid communication with a collection system; Wherein the particle sorting module is configured to minimize contact of the waste stream with an inner surface of the particle sorting module.

6. The particle sorting module according to claim 5, wherein the specific sorting module is configured to allow the waste stream to be discharged through the first outlet without contacting the wall of the first outlet.

7. The particle sorting module according to claim 6, wherein the diameter of the first outlet is in the range of 5 to 10 mm.

8. The particle sorting module according to any one of claims 5 to 7, wherein the closed particle sorting module further comprises a heater.

9. The particle sorting module according to any one of claims 5 to 8, wherein the particle sorting module further comprises an alignment member.

10. A particle sorting module according to any one of claims 5 to 9, wherein the module is sterile.

11. A kit comprising: A particle sorting module according to any one of claims 5 to 10; and Sterile packaging for the closed particle sorting module.

12. The kit of claim 11, wherein the kit further comprises a sorted particle collection system.

13. The kit of claim 12, wherein the sorted particle collection system comprises: (a) a collection vessel having a sorting tube in droplet receiving relationship with said second outlet; and (b) a sample output port that operably couples the cell collection location of the collection container to a mating connector of a vacuum receiving container.

14. The kit of claim 13, wherein the kit further comprises a vacuum receiving vessel.

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