Flow cytometer sample injection needle

Abstract

A flow cell is provided that includes a sample injection needle for operably connecting a sample injection line to the body of the flow cell. [Solution] The sample injection needle includes a sample injection needle adapter having a sample tube adapter fixed to a needle for delivering a sample fluid from a sample injection line to the flow cell body, and a clamp for operably coupling the sample injection needle to the flow cell body. A kit including the sample injection needle and a flow cytometer having the flow cell, as well as a method of use and assembly thereof, are also provided.

Description

[Technical Field]

[0001] The characterization of biological fluid analytes is a crucial part of biological research, medical diagnosis, and the assessment of a patient's overall health and wellness. Detecting biological fluid analytes, such as human blood or blood-derived products, can yield results that can play a role in determining treatment protocols for patients with various disease conditions. [Background technology]

[0002] Flow cytometry is a technique used to characterize and frequently sort biological materials, such as cells in a blood sample or particles of interest contained in other types of biological or chemical samples. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir for containing a sheath fluid. The flow cytometer directs the sheath fluid towards a flow cell while transporting particles (including cells) in the fluid sample to the flow cell as a stream of cells. Light is irradiated into the flow stream to characterize its components. Variations in the material in the flow stream, such as morphology or the presence of fluorescent labels, can cause variations in the observed light, which enable characterization and separation. To characterize the components in the flow stream, light must strike and collect from the flow stream. The light source in a flow cytometer can vary and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0003] The isolation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles in an isolated stream that are detected to possess one or more desired characteristics are individually isolated from the sample stream by mechanical or electrical removal. A common flow sorting technique utilizes droplet sorting, in which a fluid stream containing linearly isolated particles is divided into droplets. The droplets containing the target particles are charged and deflected towards a collection tube by passing through an electric field. Typically, linearly isolated particles in the stream are characterized as they pass through an observation point located directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, it is possible to predict when the particle will reach the droplet departure point and when it will detach from the stream in the droplet. Ideally, a brief charge is applied to the fluid stream just before the droplet containing the selected particle detaches from the fluid stream, and then grounded immediately after the droplet detaches. The sorted droplet retains its charge when it detaches from the fluid stream, while all other droplets remain uncharged.

[0004] Some flow cytometer systems use pressure-driven fluid engineering to supply both the sample fluid and the sheath fluid to the flow cell. In these systems, the sample fluid and sheath fluid are delivered to a flow cell containing an interrogation region (i.e., where particles are irradiated by a light source) under pressure higher than the ambient pressure. Changes in the flow rate through the flow cell in a pressure-driven fluid engineering system are achieved by changing the pressure in the sample tube and / or sheath fluid reservoir that supplies the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is governed by both the pressure levels in the sample tube and sheath fluid reservoir and the ratio of the resistances of the sample fluid and sheath fluid paths.

[0005] Alternatively, flow cytometer systems have been implemented using a vacuum-driven fluid system, where a vacuum pump draws a vacuum downstream of the flow cell, holding the sample fluid and sheath fluid at ambient pressure. In a vacuum-driven fluid system, changes in flow rate through the flow cell are achieved by altering the vacuum drawn by the vacuum pump, and the ratio of sample fluid to sheath fluid flowing through the flow cell is governed by the ratio of the resistances of the sample fluid and sheath fluid paths.

[0006] To enable the characterization and isolation of biological materials at the individual particle level, some flow cytometers include an injection needle for introducing a sample fluid into a flow cell. Using the sample injection needle, the sample fluid may be combined with the sheath fluid in the flow cell in a manner sufficient to generate a focused core stream of particles containing the sample fluid. The core stream can then transport the particles in a linear fashion through the detection area and / or sorting means. This technique is known as hydrodynamic focusing. [Overview of the project]

[0007] The inventors have recognized that imaging particles using a flow cytometer requires precise control over the velocity and position at which the sample fluid combines with the sheath fluid within the flow cell. In particular, they have found that at imaging velocity, the core stream formed by conventional sample injection mechanisms often shifts outside the interrogation region of the flow cell and / or breaks down due to fluid "vortices" associated with perturbations of the flow stream and core stream. Furthermore, the inventors have found that by precisely setting the rotation and rotation angle of the sample injection needle within the flow cell, the sample fluid can be introduced into the sheath fluid path at a velocity and position sufficient to maintain a complete core stream within the interrogation region of the flow cell under various flow conditions (e.g., so that particles can be imaged and / or sorted). Therefore, it has been recognized that a flow cell that allows for improved positional control of the sample injection needle is desirable. Embodiments of the present disclosure satisfy this need.

[0008] Aspects of the present disclosure include a sample injection needle for operably coupling a sample injection line to the body of a flow cell. The sample injection needle in question includes a sample injection needle adapter and a clamp, the sample injection needle adapter including a needle having a passage through which a sample fluid is delivered from a sample injection line at its proximal end to a flow cell cone of the flow cell body at its distal end, and a sample tube adapter including a proximal end and a distal end, the distal end of which is fixed to the proximal end of the needle, the clamp including a distal end attached to the proximal end of the sample tube adapter and a proximal end configured to fluidly connect the sample injection line to the proximal end of the needle of the sample injection needle adapter, the clamp is configured to operably couple the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body. In some embodiments, the sample injection needle adapter is rotatably movable. In some embodiments, at least a portion of the proximal end of the sample tube adapter is configured to be positioned within a recess of the clamp. In these cases, the portion of the proximal end of the sample tube adapter may include an outer surface concentric with the inner surface of the recess.

[0009] In certain embodiments, at least a portion of the distal end of the sample tube adapter is configured to be positioned within the flow cell body. In these cases, the sample tube adapter may include a flange configured to position a portion of the distal end of the sample tube adapter within the flow cell body proximal to the flow cell cone. In some embodiments, when the sample injection needle is operably coupled to the flow cell body by the clamp, the tightening of the clamp on the sample tube adapter is configured to immobilize the sample injection needle adapter relative to the flow cell body. In these cases, the sample injection needle adapter may be immobilized such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone. In some embodiments, the fixed position allows for the maintenance of a complete core stream within the flow cell cone under flow conditions of an order of magnitude or more. In some embodiments, the fixed position is located a longitudinal distance in the range of 17 mm to 26 mm away from the sheath fluid introduction port of the flow cell body. In some embodiments, the needle of the sample injection needle adapter includes a taper at its distal end. For example, the needle of the sample injection needle adapter may include a rounded distal end.

[0010] In certain embodiments, the clamp is configured to receive a fastening member for fastening the clamp to the flow cell body. In some embodiments, the sample injection needle is configured such that, when the clamp is fastened to the flow cell body by the fastening member, the tightening of the clamp by the fastening member makes the sample injection needle adapter immobile relative to the flow cell body. In some embodiments, the fastening member includes a plurality of screws. In these cases, the clamp includes a set of holes for receiving each of the plurality of screws. In some embodiments, the set of holes is configured to align with a set of holes in the flow cell body. In some embodiments, when the clamp is fastened to the flow cell body by the plurality of screws, the sample injection needle is configured such that the inclination of the sample injection needle adapter relative to the flow cell body is adjustable by manipulating the torque of at least one of the plurality of screws. In these cases, when the clamp is fastened to the flow cell body by the plurality of screws, the sample injection needle may be configured such that, when the clamp is fastened to the flow cell body by the plurality of screws, the position of the distal end of the needle of the sample injection needle adapter within the flow cell cone is adjustable by manipulating the torque of at least one of the plurality of screws. For example, the distal end of the needle may be rotated around an axis by adjusting the torque of one of the plurality of screws. In some embodiments, the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter. In some embodiments, the proximal end of the clamp is configured to position a flow meter board connector.

[0011] Aspects of the present disclosure further include a flow cell having a sample injection needle as described above and herein, for use in a flow cytometer, for example. The flow cell includes a flow cell body having a flow cell cone at its proximal end for transporting particles in a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a passage for delivering a sample fluid from a sample injection line at the proximal end to the flow cell body at the distal end to generate a core stream, wherein the sample injection needle includes a sample injection needle adapter having a sample tube adapter to which the needle is attached, and a clamp operably coupling the sample injection needle to the flow cell body, wherein in a non-clamped configuration of the flow cell, the sample injection needle adapter rotates freely relative to the flow cell cone and clamp, and in a clamped configuration of the flow cell, the sample injection needle adapter is fixed relative to the flow cell cone and clamp. In some embodiments, the clamp operably coupling the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body. In some embodiments, the needle of the sample injection needle adapter includes a proximal end attached to the sample tube adapter and a distal end positioned within the flow cell cone. In some embodiments, in a clamp configuration, the sample injection needle adapter is secured to the flow cell cone and the clamp by tightening the clamp.

[0012] In certain embodiments, the clamp is fastened to the flow cell body by one or more fastening members. In some embodiments, the clamp is tightened by one or more fastening members. For example, the clamp may be tightened by multiple screws. In some embodiments, the clamp is tightened by three screws. In these cases, the clamp includes a set of holes to receive each of the multiple screws. In some embodiments, the flow cell body includes a set of holes aligned with the set of holes in the clamp to receive each of the multiple screws. In some embodiments, the clamp is configured such that the inclination of the sample injection needle relative to the flow cell body is adjustable by manipulating the torque of at least one of the multiple screws. In some embodiments, the clamp includes a distal end that contacts the sample tube adapter and a proximal end configured to fluidly connect the sample injection line to the sample injection needle. In some embodiments, the distal end of the clamp includes a recess into which at least a portion of the sample tube adapter is positioned and a surface that contacts the proximal end of the flow cell body. In some embodiments, the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid as it flows from the sample injection line to the sample injection needle. In some embodiments, the proximal end of the clamp is configured to position a flow meter board connector.

[0013] In certain embodiments, the sample tube adapter includes a proximal end positioned in a recess of the clamp and a distal end in contact with the proximal end of the flow cell body. In some embodiments, at least a portion of the distal end of the sample tube adapter is positioned within the flow cell body. In these cases, the sample tube adapter may include a flange in contact with the proximal end of the flow cell body. In some embodiments, the distal end of the sample tube adapter is pressed against the proximal end of the flow cell body by the clamp so that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone. In some embodiments, the needle of the sample injection needle adapter includes a taper at its distal end. For example, the needle of the sample injection needle adapter may include a rounded distal end. In some embodiments, the distal end of the needle of the sample injection needle adapter is positioned to allow the maintenance of a complete core stream within the flow cell cone under flow conditions of an order of magnitude or more.

[0014] In certain embodiments, the flow cell body includes a sheath fluid introduction port for delivering sheath fluid to the flow cell cone. In these cases, the distal end of the needle of the sample injection needle adapter may be located a predetermined longitudinal distance away from the sheath fluid introduction port, for example, a distance in the range of 17 mm to 26 mm. In some embodiments, the flow cell body includes a plurality of sheath fluid introduction ports. In these cases, the sheath fluid introduction ports may be offset from each other so that the sheath fluid swirls within the flow cell cone. In some embodiments, the distal end of the flow cell body includes a cuvette for transporting particles in the core stream through a sample interrogation region. In these cases, at least a portion of the cuvette includes an optically transparent solid configured to allow optical detection of particles in the core stream. In some embodiments, the cuvette is positioned at the distal end of the flow cell body by a clamp fixed to the flow cell body. In these cases, the cuvette may be releasably attached to the distal end of the flow cell body by a flow cell body clamp. In some embodiments, the cuvette is positioned by a flow cell body clamp so that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0015] Aspects of the present disclosure further include, for example, a method for assembling a flow cell of interest, as described above and herein. The method of interest includes operably coupling a sample injection needle to a flow cell body for transporting particles in a core stream of flowstream from a proximal end to a distal end, wherein the flow cell body includes a flow cell cone at its proximal end, and the sample injection needle includes a clamp and a sample injection needle adapter, wherein the sample injection needle adapter includes a sample tube adapter attached to the needle and having a passage through which to deliver sample fluid from a sample injection line at the proximal end to the flow cell body at the distal end to generate a core stream, and the method includes operably coupling the sample injection needle to the flow cell body using a sample injection needle clamp. In some embodiments, the method further includes operably positioning the flow cell in a flow cytometer. In other embodiments, for example, a kit including a sample injection needle and / or flow cell of interest is provided, as described above and herein.

[0016] Aspects of the present disclosure further include, for example, a flow cytometer having a flow cell of interest, as described above and herein. The flow cytometer of interest is a flow cell body for transporting particles in a core stream of a stream from a proximal end to a distal end, wherein the flow cell body includes a flow cell cone at its proximal end; and a sample injection needle having a passage for delivering a sample fluid from a sample injection line at the proximal end to the flow cell body at the distal end in order to generate a core stream, wherein the sample injection needle includes a sample injection needle adapter including a sample tube adapter to be attached to the needle; and a clamp that operably connects the sample injection needle to the flow cell body; and a light source configured to irradiate particles in the flow stream in a sample interrogation region within the flow cell; and a detector configured to collect light emitted by the irradiated particles. In another aspect, a method for analyzing a sample fluid using a flow cytometer having a flow cell of interest is provided, for example, as described above and herein. [Brief explanation of the drawing]

[0017] This disclosure can be best understood when the following detailed description is read in conjunction with the accompanying drawings. The drawings include the following figures.

[0018] [Figure 1A] Exploded view of a flow cell according to a particular embodiment. [Figure 1B] Various views of the fully assembled flow cell of FIG. 1A according to a particular embodiment. [Figure 1C] Various views of a fully assembled flow cell according to a particular embodiment. [Figure 1D] Cross-sectional view of a sample injection needle according to a particular embodiment. [Figure 1E] View showing a sample injection needle according to a particular embodiment. [Figure 1F] View showing a clamp attached to the distal end of a sample injection line and including a sample injection line connector at its proximal end according to a particular embodiment. [Figure 2] View of a flow cytometry system according to a particular embodiment. [Figure 3-1] View of an image-correlated particle sorter according to a particular embodiment. [Figure 3-2] View of an image-correlated particle sorter according to a particular embodiment. [Figure 4] Functional block diagram of a particle analysis system according to a particular embodiment. [Figure 5] Functional block diagram of an example of a control system according to a particular embodiment. [Figure 6A] Schematic view of a particle sorter system according to a particular embodiment. [Figure 6B] Schematic view of a particle sorter system according to a particular embodiment. [Figure 7] View showing aspects of a computer control system according to a particular embodiment.

MODE FOR CARRYING OUT THE INVENTION

[0019] A flow cell is provided that includes a sample injection needle for operably connecting a sample injection line to the body of the flow cell. The sample injection needle includes a sample injection needle adapter having a sample tube adapter that is fixed to the needle for delivering a sample fluid from the sample injection line to the body of the flow cell, and a clamp for operably connecting the sample injection needle to the body of the flow cell. Furthermore, the disclosure provides a kit including the sample injection needle and / or flow cell and a flow cytometer having the flow cell. A method for assembling the flow cell and a method for analyzing a sample using the flow cytometer are also provided.

[0020] Before describing this disclosure in detail, please understand that this disclosure is not limited to the specific embodiments described and is therefore naturally subject to change. Since the scope of this disclosure is limited only by the appended claims, please also understand that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0021] When a range of values ​​is presented, unless explicitly indicated in the context, it is understood that the values ​​between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other values ​​or values ​​within the stated range are included in this disclosure. These smaller upper and lower limits may independently be included in the smaller range, subject to any specifically excluded limits within the stated range, and are also included in this disclosure. If the stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0022] In this specification, certain ranges are indicated by numbers preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number preceded by the term, as well as for numbers that are close to or nearly close to the number preceded by the term. In determining whether a number is close to or approximates a specifically stated number, a number not explicitly stated as close to or approximates may, in the context in which it is presented, be a number that presents a substantial equivalent of the specifically stated number.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but representative exemplary methods and materials are described below.

[0024] All publications and patents referenced herein are incorporated herein by reference in such a way as to specifically and individually indicate that each individual publication or patent is incorporated by reference, and are incorporated herein by reference to disclose and describe the manner and / or materials by which the publications are referenced. Any reference to a publication is for the purpose of making that disclosure prior to the filing date, and this disclosure should not be construed as an acknowledgment that such publication has no prior rights by prior disclosure. Furthermore, the publication dates presented may differ from the actual publication dates and may need to be independently verified.

[0025] In this specification and the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise. Furthermore, it should be noted that claims may be written to exclude any optional element. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as "alone" and "only" in relation to the enumeration of elements in the claims or the use of "negative" limitations.

[0026] As will be obvious to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any of the listed methods may be performed in the order of the listed events, or in any other logically possible order.

[0027] While systems and methods are described for grammatical fluidity with functional descriptions, claims should not necessarily be interpreted as being limited by constructing a limitation of “means” or “steps” unless explicitly formulated under 35 U.S. SC § 112, and the meaning of the definitions provided by the claims and the full scope of equivalents should be given under the doctrine of equivalents, and if the claims are explicitly formulated under 35 U.S. SC § 112, it should be clearly understood that the full legal equivalents should be given under 35 U.S. SC § 112.

[0028] Flow Cell As summarized above, aspects of this disclosure include flow cells for use in flow cytometers. The flow cell in question includes a flow cell body for transporting particles in a core stream of a flow stream from a proximal end to a distal end, and a sample injection needle having a (e.g., continuously penetrating) passage for delivering a sample fluid from a sample injection line at the proximal end to the flow cell body at the distal end in order to generate a core stream. The flow cell body includes a flow cell cone at the proximal end. The sample injection needle includes a sample injection needle adapter including a sample tube adapter attached to the needle, and a clamp that operably connects the sample injection needle to the flow cell body. As used herein, “core stream” is used in its conventional sense to describe a fluid stream (i.e., flow stream) through which particles are transported by a sheath fluid stream (e.g., hydrodynamically focused). Generally, particles are transported through the core stream in a single-line manner. The size (e.g., diameter) of the core stream may vary as desired.

[0029] In some cases, the core stream may have a diameter approximately equal to the diameter of the particles being analyzed. In some cases, the core stream diameter is in the range of 5 μm to 25 μm, including 10 μm to 20 μm. The core stream diameter may be adjusted in proportion to the pressure (e.g., positive or negative pressure) applied to the particles when they are injected into the sheath fluid stream. In some cases, the flow rate of the sheath fluid remains constant. In this way, the particles are injected into the sheath fluid, a laminar flow is generated, and the particles are hydrodynamically focused so that they move along the same axis at approximately the same velocity.

[0030] Core streams relating to this disclosure may be described as “complete” if they maintain a relatively constant shape over the entire length of the flow cell. In some cases, a complete core stream in this disclosure is defined by straight edges. In other words, when viewed in two dimensions, the boundary of a complete flow stream appears straight. In selected versions, the flow stream and the core streams comprising it are hydrodynamically focused and characterized by laminar flow. In some such versions, the straight edges of the core streams are substantially parallel to each other (e.g., the edges are deviated from true parallel lines by 5 degrees or less, e.g., 2 degrees or less). In some cases, a complete core stream is not characterized by perturbations from the distal end of the sample injection needle to the distal end of the flow cell. In some such cases, a complete core stream does not contain vortices (e.g., surrounding the tip of the sample injection needle). In certain cases, the flow cells and their constituent sample injection needles reduce flowstream and corestream perturbations by 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% (including 100%) compared to conventional instruments.

[0031] As described above, the sample injection needle in question is configured and positioned to maintain a complete core stream under flow conditions of more than one order of magnitude variation. The term “order of magnitude” is also called “order of magnitude” and is used in its conventional sense to refer to flow conditions that differ by more than ten times. In certain cases, the sample injection needle in question is configured and positioned to maintain a complete core stream under flow conditions of more than two orders of magnitude variation. Flow conditions described herein may include, for example, sheath fluid flow rate, sample fluid flow rate, particle size (e.g., diameter), pressure (positive or negative), etc. In some embodiments, the sample injection needle in question is configured and positioned to maintain a complete core stream under sheath fluid flow rates that differ by more than one order of magnitude. For example, in some cases, the sample injection needle is configured and positioned to maintain a complete core stream under conditions where the flow stream flow rate is in the range of 0.5 m / s to 10 m / s. In selected cases, the sample injection needle in question is configured and positioned to maintain a complete core stream under sample fluid flow rates that differ by more than one order of magnitude. For example, in some cases, the sample injection needle is configured and positioned to maintain a complete core stream under conditions where the sample fluid flow rate is in the range of 1 μl / min to 150 μl / min.

[0032] As described herein, “flow cell” is described in its conventional sense as referring to a component comprising a channel for a liquid flow stream for transporting particles in a sheath fluid (for example, for transporting particles in the core stream of a flow stream, as described above). Any convenient flow cell for transferring a fluid sample to a sample interrogation area can be used as a flow cell as described herein, and in some embodiments, the flow cell includes a cylindrical flow cell, a frustoconical flow cell, or a flow cell comprising a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating with a flat surface having an orifice transverse the longitudinal axis. The flow cell in question has a proximal end for receiving fluid from a sample fluid source (i.e., via a sample injection needle) and a sheath fluid source, and a distal end for releasing the fluid. Depending on the configuration of the flow cytometer, the fluid at the distal end of the flow cell may be directed to one or more different types of collection containers.

[0033] The flowstream comprising the channel may include a liquid sample injected from a sample tube through a sample injection line. In certain embodiments, the flowstream may include a narrow, rapidly flowing stream of liquid arranged such that linearly isolated particles transported internally are separated from each other in a line. In certain cases, the flow cell includes a light-accessible channel. In some cases, the flow cell is configured to be illuminated from a light source at one or more interrogation points. As described herein, “interrogation point” or “interrogation region” refers to a region within the flow cell where particles are illuminated by light from a light source, for example, for analysis. The size of the interrogation points may be varied as needed. For example, if 0 μm represents the axis of light emitted by the light source, the interrogation points may be in the range of -100 μm to 100 μm, e.g., -50 μm to 50 μm, e.g., -25 μm to 40 μm, and e.g., -15 μm to 30 μm. Depending on specific considerations (e.g., the number and arrangement of lasers), multiple irradiation points may exist within the flow cell.

[0034] Flow cell body The flow cell of the subject includes a flow cell body having a proximal end and a distal end, the flow cell body including a flow cell cone at the proximal end (for example, as described below). In some embodiments, the flow cell of the subject of the disclosure includes a cuvette at the distal end of the flow cell body for transporting particles in a flow stream (e.g., and its core stream) passing through a sample interrogation point or region. The cuvette of the subject includes a passage (i.e., a channel) through the cuvette and an optically transparent region or portion for receiving light from a light source (i.e., so that a sample interrogation point or region may be formed, as described below). In some embodiments, the optically transparent portion of the cuvette is configured to allow optical detection of particles in the core stream. In some embodiments, the optically transparent portion includes an optically transparent solid. In other words, the optically transparent portion may include a transparent material that allows light to pass through (i.e., such that the region or portion is accessible to light). The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed from silica, such as fused silica.

[0035] In some embodiments, the cuvette is positioned at the distal end of the flow cell body by a clamp (hereinafter referred to as the flow cell body clamp or cuvette clamp) that is fixed to the flow cell body. The cuvette clamp may be fixed to the flow cell body by any suitable means. In some embodiments, the cuvette clamp is fixed to the flow cell body by fastening members (e.g., pins, screws, etc.). In some embodiments, the cuvette is releasably attached to the distal end of the flow cell body by the cuvette clamp. In some cases, the positioning of the cuvette (i.e., the positioning of the cuvette relative to the rest of the flow cell body and / or one or more light sources) may be adjusted after it has been attached to the flow cell body via the cuvette clamp. In some embodiments, the positioning of the cuvette may be adjusted by removing the cuvette from the flow cell body via the cuvette clamp and reattaching it. In some embodiments, the cuvette is positioned by the flow cell body clamp so that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0036] The flow cell body in question includes a flow cell cone at its proximal end. The “flow cell cone” refers to a cone-shaped (e.g., right-angle cone) recess in the flow cell body that narrows toward the analytical region (i.e., the interrogation region) of the flow cell. The flow cell cone can serve to hydrodynamically focus the flow stream. The flow cell cones described herein may be characterized by angles in the range of 15° to 25°, e.g., 18° to 22°, e.g., 19° to 21° (i.e., measured with respect to a virtual axis extending through the center of the cone to the generatrix of the cone). In some cases, the flow cell cones described herein may be characterized by an angle of 20°.

[0037] In certain embodiments, the flow cytometer (i.e., the flow cytometer to which the flow cell of the Disclosure is coupled, as described in detail below) includes a sample fluid source. The sample fluid source may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding the sample fluid. The sample fluid container may have a volume in the range of 1 mL to 100 mL. For example, the volume of the container may be in the range of 1 mL to 90 mL, 1 mL to 80 mL, 1 mL to 70 mL, 1 mL to 60 mL, 1 mL to 50 mL, 1 mL to 40 mL, 1 mL to 30 mL, 1 mL to 20 mL, or 1 mL to 10 mL. In embodiments, the sample fluid is supplied from the sample fluid source to the flow cell (e.g., the sample injection needle of the flow cell described herein) via a sample injection line (e.g., a tube).

[0038] In some embodiments, the flow cytometer includes a sheath fluid reservoir. The sheath fluid reservoir may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding the sheath fluid. In certain embodiments, the sheath fluid reservoir is fluidically coupled to the input of the flow cell (e.g., the inlet port of the flow cell body, as described below). The sheath fluid container may have a volume in the range of 1 L to 100 L. For example, the volume of the container may be in the range of 1 L to 90 L, 1 L to 80 L, 1 L to 70 L, 1 L to 60 L, 1 L to 50 L, 1 L to 40 L, 1 L to 30 L, 1 L to 20 L, or 1 L to 10 L. The sheath fluid reservoir may be fluidically connected to a sheath fluid line configured to transport the sheath fluid from the reservoir to the flow cell.

[0039] In some embodiments, the flow cell body includes a sheath fluid introduction port configured to supply sheath fluid to the flow cell. The sheath fluid introduction port in question is fluidically connected to a sheath fluid source (i.e., a reservoir) and supplies the sheath fluid to the flow cell body at its proximal end. In embodiments, the sheath fluid introduction system is configured to supply a flow of sheath fluid, for example, together with a sample, to an internal chamber of the flow cell body (e.g., the flow cell cone of the flow cell body) to generate a stacked flow stream of sheath fluid surrounding a sample flow stream (i.e., a stacked flow stream of sheath fluid surrounding a core stream). Depending on the desired characteristics of the flowstream, the flow rate of the sheath fluid delivered to the flow cell chamber (i.e., the chamber of the flow cell body, such as the flow cell cone of the flow cell body) is 25 μL / sec or more, for example 50 μL / sec or more, for example 75 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, for example 1000 μL / sec or more, and may include, for example, 2500 μL / sec or more. In some embodiments, the flow rate of the sheath fluid delivered to the flow cell chamber may be in the range of 25 μL / sec to 2500 μL / sec, for example 50 μL / sec to 1000 μL / sec, and includes 75 μL / sec or more to 750 μL / sec.

[0040] In some embodiments, the sheath fluid introduction port is an orifice positioned in the wall of the internal chamber. The sheath fluid introduction port orifice may have any suitable cross-sectional shape, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom coupled to a flat top. The size of the sheath fluid introduction port orifice may vary depending on the shape, and in certain cases it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm.

[0041] In some cases, one or more sheath fluid inlet ports are located within the flow cell cone of the flow cell body (i.e., at the “base” of the cone). In some cases, the flow cell body includes a single sheath fluid inlet port. In other cases, the flow cell body includes multiple sheath fluid inlet ports. The number of sheath fluid ports in the multiple sheath fluid inlet ports may vary, for example, including two sheath fluid inlet ports, three sheath fluid inlet ports, four sheath fluid inlet ports, and five sheath fluid inlet ports. In a selected version, the flow cell body has two sheath fluid inlet ports. In some cases where the flow cell body of this disclosure includes multiple sheath fluid inlet ports, the multiple sheath fluid inlet ports are offset from each other so that the sheath fluid swirls within the flow cell cone (e.g., in a “toilet” manner). In some such cases, the multiple sheath fluid inlet ports are offset from each other so that the sheath fluid swirls clockwise. In other cases, multiple sheath fluid inlet ports are offset from each other so that the sheath fluid swirls counterclockwise. In certain cases, the swirl of the sheath fluid results in greater stability of the flowstream and corestream.

[0042] Sample injection needle As summarized above, aspects of the present disclosure include a sample injection needle for operably coupling a sample injection line to a flow cell body (e.g., as described above). The sample injection needle in question includes a sample injection needle adapter and a clamp, the clamp being configured to operably couple the sample injection needle to the flow cell body. The sample injection needle adapter in the present disclosure includes a sample tube adapter attached to the needle. The needle of the sample injection needle adapter in the present disclosure includes a passage through which a sample fluid is delivered from the sample injection line at the proximal end to the flow cell cone of the flow cell body at the distal end. In some embodiments, the sample tube adapter (i.e., the sample tube adapter of the sample injection needle adapter in the present disclosure) includes a proximal end and a distal end, at least the distal end being fixed to the proximal end of the needle. In some embodiments, the clamp (i.e., the clamp of the sample injection needle in question, referred herein as the clamp or sample injection needle clamp) is configured to operably couple the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body.

[0043] The sample injection needle clamp may include a distal end configured to attach to a sample injection needle adapter and a proximal end configured to fluidly connect the sample injection line to the needle of the sample injection needle adapter. The clamp is configured to operably couple the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body. In some embodiments, the distal end of the clamp includes a surface configured to contact the flow cell body (e.g., the outermost edge or outermost surface of the proximal end of the flow cell body, located proximal to the flow cell cone) when the clamp presses the sample tube adapter against the flow cell body. In some embodiments, the distal end of the clamp includes a recess configured to receive at least a portion of the sample tube adapter. In these cases, the recess may include an inner surface concentric with the outer surface of the portion of the sample tube adapter. In some embodiments, the recess includes multiple depth components. For example, the recess may include a first depth component configured to receive the flange of the sample tube adapter (i.e., the first depth component recesses into the clamp by a first distance or a first depth from the outermost edge of the distal end of the clamp) and a second depth component configured to receive a portion of the sample tube adapter (i.e., the second depth component recesses into the clamp by a second distance or a second depth from the outermost edge of the distal end of the clamp), wherein the second depth component recesses into the clamp by a longer distance than the first depth component.

[0044] In some embodiments, the recess is configured such that the sample injection needle adapter is rotatably movable relative to the clamp when a portion of the sample tube adapter is positioned within the recess. Rotatably movable means that when the sample injection needle is attached to the flow cell body (as described herein, when the clamp is not tightened to immobilize or fix the sample injection needle adapter), the sample injection needle adapter can rotate around its central axis to adjust its orientation relative to the clamp and, for example, the flow cell body. In some embodiments, the sample injection needle adapter can rotate around its central axis by 90 degrees or more, for example, 180 degrees or more, 270 degrees or more, or 360 degrees. In some cases, the sample injection needle adapter can rotate completely around its central axis to adjust its orientation relative to the clamp (and, for example, the flow cell body) (i.e., it can rotate 360 ​​degrees around its central axis, or it can rotate freely around its central axis). Embodiments of a flow cell in which the sample injection needle adapter is rotatably movable or rotates freely around its central axis relative to the clamp and the flow cell cone are referred to herein as non-clamp configurations of the flow cell.

[0045] In some embodiments, the clamp is configured to receive fastening members (e.g., one or more pins, screws, rivets, etc.) for fastening the clamp to the flow cell body. In some embodiments, when the sample injection needle adapter is operably coupled to the flow cell body by the clamp, the clamp is configured such that the tightening of the clamp by the fastening members makes the sample injection needle adapter immobile relative to the flow cell body. Immobility means that the movement of the sample injection needle adapter relative to the flow cell body (and its flow cell cone) is restricted. In some embodiments, the movement of the sample injection needle adapter relative to the flow cell body and clamp is completely restricted (i.e., the sample injection needle adapter is fixed to the flow cell body and clamp). Embodiments of a flow cell in which the sample injection needle adapter is immobile relative to the clamp and flow cell body (e.g., the sample injection needle adapter is fixed to the flow cell body and clamp) are referred to herein as flow cell clamp configurations.

[0046] In some embodiments, the fastening member includes a plurality of screws. In some embodiments, the clamp includes a set of holes for receiving each of the plurality of screws. In certain embodiments, the clamp includes three holes. In these cases, the set of holes may be configured to align with a set of holes in the flow cell body (e.g., on the outermost edge or outermost surface of the proximal end of the flow cell body). In some embodiments, the clamp is configured such that when the sample injection needle (and its sample injection needle adapter) is operably coupled to the flow cell body by the clamp, the inclination of the sample injection needle adapter relative to the flow cell body is adjustable by manipulating the torque of at least one of the plurality of screws. In other words, the sample injection needle adapter may be angle-adjustable relative to the flow cell body by changing (e.g., tightening or loosening) the torque of one or more of the plurality of screws. In these cases, when the sample injection needle is operably coupled to the flow cell body by the clamp, the clamp is configured such that the position of the distal end of the needle of the sample injection needle adapter within the flow cell cone of the flow cell body is adjustable by manipulating the torque of at least one of the plurality of screws. For example, the distal end of the needle may be rotated around an axis by adjusting the torque of one of several screws. In some embodiments, micron-level adjustment of the distal end of the needle of the sample injection needle adapter is possible by manipulating the torque of at least one of several screws.

[0047] In some embodiments, the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter. Dead volume refers to areas within the sample injection line or sample injection needle (or, for example, the area where the sample injection line connects to the sample injection needle) where the sample fluid may be trapped, stagnant, or remain unused and not effectively flow through the system (i.e., a flow cytometer system capable of operably positioning a flow cell inside). In these cases, when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter, the connector may reduce dead space or gaps (e.g., dead space and gaps within joints) to minimize the dead volume of the sample fluid. For example, the connector may reduce the number of joints traversed by the sample fluid. In some embodiments, the proximal end of the clamp is configured to position a flow meter board connector.

[0048] As described above, the sample injection needle adapter in question (i.e., a sample injection needle adapter for operably connecting a sample injection line to the body of a flow cell) may include a sample tube adapter including a needle having a passage for delivering a sample fluid from the sample injection line at its proximal end to the flow cell cone of the flow cell body at its distal end, a proximal end configured to be attached to a clamp, and a distal end fixed to the proximal end of the needle. In some embodiments, the sample tube adapter is configured to operably connect the needle of the sample injection needle adapter to the flow cell body when pressed against the flow cell body by a clamp. In some embodiments, when the sample tube adapter is operably connected to the flow cell body by a clamp, the tightening of the clamp on the sample tube adapter is configured to immobilize the sample injection needle adapter relative to the flow cell body. In these cases, the sample tube adapter may be immobilized such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone. The fixed position of the distal end of the needle of the sample injection needle adapter relative to the flow cell cone (e.g., generally within the flow cell body) can be described in several ways. For example, in certain cases, the fixed position of the distal end of the needle of the sample injection needle adapter is described with respect to the location of one or more sheath fluid introduction ports. For example, in some cases, the distal end of the needle is positioned within the flow cell cone and is separated from the sheath fluid introduction point by a longitudinal distance in the range of 15 mm to 30 mm, e.g., 17 mm to 26 mm, and 20 mm to 22 mm. The positioning of the distal end of the needle may also be described with respect to the location of the interrogation point (i.e., the point where the flow cell is illuminated by one or more light sources). In some cases, the distal end of the needle is separated from the interrogation point by a distance in the range of 10 mm to 20 mm, e.g., 13 mm to 17 mm, and 14 mm to 16 mm. In some embodiments, the fixed position of the distal end of the needle of the sample injection needle adapter allows for maintaining a complete core stream within the flow cell cone under flow conditions of more than an order of magnitude variation.

[0049] In some embodiments, the sample injection needle adapter is configured such that, when operably coupled to the flow cell body by a clamp, the tilt of the sample injection needle adapter relative to the flow cell body is adjustable by manipulating the torque of at least one of the multiple screws that fasten the clamp to the flow cell body (as described above, for example). In some cases, the tilt of the sample injection needle adapter is adjusted so that a complete core stream can be maintained within the flow cell cone under flow conditions of an order of magnitude or more.

[0050] In some embodiments, (for example, as described above) at least a portion of the proximal end of the sample tube adapter is configured to be positioned within a recess of the clamp. In these cases, the portion of the proximal end of the sample tube adapter may include an outer surface concentric with the inner surface of the recess. In some embodiments, (for example, as described above) when the portion of the sample tube adapter is positioned within the recess, the portion of the proximal end of the sample tube adapter is configured such that the sample injection needle adapter is rotatably movable relative to the clamp. In some embodiments, at least a portion of the distal end of the sample tube adapter is configured to be positioned within the flow cell body. In these cases, the portion of the distal end of the sample tube adapter may include an outer surface concentric with the inner surface of the opening of the flow cell body leading to the flow cell cone. In some embodiments, the sample tube adapter includes a flange for positioning the portion of the distal end of the sample tube adapter within the flow cell body proximal to the flow cell cone. In these cases, the inner surface of the opening of the flow cell body may include a projection or step for pressing the flange against. In some embodiments, the flange is configured to position a portion of the distal end of the sample tube adapter within the flow cell body such that the distal end of the needle is separated from the sheath fluid introduction port of the flow cell body by a longitudinal distance ranging from 17 mm to 26 mm.

[0051] The needle of the sample injection needle adapter of this disclosure may include an elongated structure. “Elongated structure” means that the needle has a length longer than its width. In other words, the needle of the sample injection needle adapter has a separate proximal end and a distal end. The proximal end is the end of the needle that receives the sample fluid (i.e., from a sample injection line fluidically connected to a sample fluid source), and the distal end is the end of the sample injection needle that injects the sample into a flow cell (e.g., a flow cell cone). The elongated structure may have any convenient cross-sectional shape, and the cross-sectional shapes in question include, but are not limited to, linear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes, e.g., circular, elliptical, and irregular shapes, e.g., a parabolic bottom coupled to a flat top. In embodiments, the elongated structure has a substantially circular cross-sectional shape at positions along its length. "Substantially" circular cross-section means, in embodiments, that one or more locations along the length of the outlet fitting may have a cross-section that deviates slightly from the circular cross-section characterizing the rest of the structure. For example, in some versions, the elongated structure has polygonal (e.g., hexagonal, pentagonal, etc.) cross-sections at one or more locations along its length. In certain particular cases, the width (e.g., diameter of the cross-section) of the elongated structure varies along the length of the outlet fitting. In other words, in such versions, the elongated structure is not a perfect cylinder, but rather has several regions that have a cross-sectional shape with a diameter larger or smaller than the diameter of other regions. In other cases, all parts of the needle of the sample injection needle adapter have a circular cross-section. In some such cases, different parts of the needle may be characterized by different diameters. In some cases, at least a portion of the needle of the sample injection needle adapter has an outer diameter in the range of 0.5 mm to 4 mm, e.g., 0.75 mm to 3 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm (i.e., measured from the geometric center of the sample injection needle to the outer edge), including 1.5 mm to 1.6 mm. In some cases, the needle includes an outer diameter of 1.562 mm.The needle of the sample injection needle adapter may have any convenient length, ranging from 15 mm to 30 mm, for example, from 17 mm to 26 mm, including 20 mm to 22 mm. In certain cases, the needle of the sample injection needle adapter may not be flattened at the distal end, forming, for example, a "duckbill" shape. In these cases, the needle may have a substantially circular cross-section throughout.

[0052] In some cases, the needle of the sample injection needle adapter has a taper at its distal end. In some such cases, the needle has a relatively constant outer diameter over most of its length (i.e., starting at the proximal end), but the outer diameter gradually (e.g., evenly) decreases as it approaches the distal end. In certain embodiments, the needle of the sample injection needle adapter includes a tapered range of 2.5 mm to 12 mm in radius over a length of 1.75 mm to 4 mm. For example, in some versions, the needle includes a tapered range of 2.79 mm to 11.63 mm in radius over a length of 1.78 mm to 3.81 mm. The taper may start at various positions along the length of the needle. In some cases, the taper starts at a position along the length of the needle that is only a distance in the range of 3 mm to 5 mm, including 3.8 mm to 3.9 mm, such as 3.5 mm to 4.0 mm. Such a distal end may in some cases be referred to as having a "super bullet" configuration. In some embodiments having a superbullet configuration, the distal end has a tapered range with a radius of 11.63 mm over a length of 3.81 mm. In other cases, the taper begins at a position along the length of the needle, at a distance including 1.7 mm to 1.8 mm, in the range of 1 mm to 3 mm, for example, 1.5 mm to 2 mm. Such a distal end is sometimes referred to as having a "bullet" configuration. In some embodiments where the needle of the sample injection needle adapter has a bullet configuration, the distal end has a tapered range with a radius of 2.79 mm over a length of 1.78 mm. In other cases, the needle of the sample injection needle adapter includes a rounded distal end.

[0053] The needle of the sample injection needle adapter of this disclosure also includes an opening at the distal end and a channel through which a sample fluid containing particles optionally passes to a flow cell (e.g., the flow cell cone of a flow cell). In embodiments, the opening is located at the geometric center of the cross-section of the outlet fitting at the distal end. The opening may have any convenient cross-sectional shape, which includes, but is not limited to, linear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes, e.g., circular, elliptical, and irregular shapes, e.g., a parabolic bottom coupled to a flat top. In certain cases, the opening has a circular cross-sectional shape. In additional cases, the channel also includes a circular cross-sectional shape. The opening may have any suitable diameter, which ranges from 0.1 mm to 2 mm, e.g., 0.1 mm to 1 mm, e.g., 0.2 mm to 0.4 mm, and includes 0.25 mm to 0.30 mm. Similarly, the channel may have any suitable diameter, ranging from 0.1 mm to 2 mm, for example, 0.1 mm to 1 mm, for example, 0.2 mm to 0.4 mm, and including 0.25 mm to 0.30 mm. In some cases, the opening and the channel have a circular cross-sectional shape with the same or similar diameter. In other cases, the opening has a diameter different from (e.g., larger than) the diameter of the channel.

[0054] The flow cell of this disclosure includes a sample injection needle that is operably coupled to the flow cell body when assembled for use (for example, the sample injection needle is configured to be coupled to the flow cell body by any means, as described above). In some cases, the assembled flow cell includes a fastening member inserted into a hole in a clamp. In some embodiments, the assembled flow cell of this disclosure is configured such that (for example, as described above) the distal end of the needle of the sample injection needle is in a fixed position and / or (for example, as described above) the position of the sample injection needle relative to the flow cell body is immovable by tightening the clamp. In some cases, the assembled flow cell is adjusted before or after it is operably positioned in a flow cytometer. In some embodiments, the assembled flow cell is adjusted after it has been operably positioned in a flow cytometer. For example, the torque of one or more of the screws used to fasten the sample injection needle to the flow cell body may be adjusted based on observation by an operator. In some cases, (for example, as described above) a cuvette clamp is used to position the assembled flow cell in a operable location on the flow cytometer, and then the cuvette on the flow cell body is aligned with the light source (i.e., to form a sample interrogation area).

[0055] Figure 1A is an exploded view of a flow cell according to a particular embodiment. As shown in Figure 1A, the sample injection needle 120 includes a sample injection needle adapter having a sample tube adapter 122 and a needle 121, and a clamp 123. A fastening member 130 (i.e., a screw) is configured to be inserted into the clamp 123 and a pair of holes in the flow cell body 110 to fasten the sample injection needle to the flow cell body. A sheath fluid introduction nozzle 111 of the sheath fluid introduction port of the flow cell body is configured to introduce sheath fluid into the flow cell cone of the flow cell body. A cuvette clamp 112 is fixed to the flow cell body 110 and used to position the cuvette 140 at the distal end of the flow cell body to allow the formation of a sample interrogation area. O-rings 141 and 142 ensure that the cuvette is fluidly connected to the flow cell body so that the core stream can be sustained throughout the cuvette. The attachment 150 and fastening member 151 allow the flow cell nozzle 160 to fluidly connect to the end of the flow cell body, for example, to separate particles into separate containers. Figure 1B shows various diagrams of the fully assembled flow cell of Figure 1A according to a particular embodiment.

[0056] Figure 1C shows various diagrams of a fully assembled flow cell according to a particular embodiment. Figure 1D is a cross-sectional view of a sample injection needle having a sample injection needle adapter (sample tube adapter 122 and needle 121) and a clamp 123 according to a particular embodiment.

[0057] Figure 1E shows a sample injection needle having a sample injection needle adapter (sample tube adapter 122 and needle 121) and a clamp 123 according to a particular embodiment. Figure 1F shows a clamp, according to a particular embodiment, attached to the distal end of the sample injection needle line, with a sample injection line connector at its proximal end. The connector is configured to reduce dead space and gaps within the joint in order to minimize the dead volume of sample fluid when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter.

[0058] How to assemble a flow cell As described above, aspects of the present disclosure also include methods for assembling a flow cell for use in a flow cytometer. The methods in question include operably coupling a sample injection needle to the flow cell body (for example, as described above) using a sample injection needle clamp. In some embodiments, the sample injection needle is operably coupled to the flow cell body by pressing a sample tube adapter against the flow cell body using a clamp. In some cases, the method further includes immobilizing the sample injection needle adapter by tightening the clamp (for example, so that the distal end of the needle of the sample injection needle adapter is fixed in place within the flow cell cone, as described above).

[0059] In some embodiments, the method further includes inserting a plurality of screws into a set of holes in the clamp and a set of holes in the flow cell body, the set of holes in the clamp being aligned with the set of holes in the flow cell body. In some embodiments, the method further includes adjusting the inclination of the sample injection needle relative to the flow cell body by manipulating the torque of at least one of the plurality of screws (for example, as described above). In these cases, the position of the distal end of the needle of the sample injection needle adapter may be adjusted by rotating the distal end of the needle around an axis, for example by individually adjusting the torque of one of the plurality of screws. In some cases, the method further includes adjusting the rotational position of the sample injection needle adapter before it is immobilized by the clamp.

[0060] In some embodiments, the assembly method further includes attaching the sample tube adapter to a clamp before operably coupling the sample injection needle to the flow cell body. For example, a portion of the sample tube adapter may be positioned within a recess of the clamp (as described above, for example). In some embodiments, the method further includes positioning the cuvette of the flow cell body at the distal end of the flow cell body using a cuvette clamp fixed to the flow cell body (as described above, for example). In these cases, the cuvette may be positioned by the flow cell body clamp so that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0061] In some embodiments, the assembly method further includes operably positioning the flow cell (including the flow cell body and sample injection needle as described above) to a flow cytometer. Suitable flow cytometers for operably coupling with the flow cell of this disclosure are described in detail below. In some cases, operably positioning the flow cell to a flow cytometer includes fluidly coupling the sample injection line to the needle of a sample injection needle adapter using a clamp. In some embodiments, operably positioning the flow cell to a flow cytometer includes aligning the cuvette at the distal end of the flow cell body for transporting particles in the core stream through the sample interrogation region with the light source of the flow cytometer for illuminating particles in the core stream in the sample interrogation region. In some embodiments, operably positioning the flow cell to a flow cytometer includes optically coupling the detector of the flow cytometer, configured to collect light emitted by the illuminated particles, to the sample interrogation region.

[0062] Flow cytometer Aspects of the present disclosure also include flow cytometers. The flow cytometer in question includes a flow cell of the present disclosure. As described in detail above, the flow cell in question includes a sample injection needle (including a sample injection needle adapter and clamp, as described above) which is operably coupled to the flow cell body (including a flow cell cone, as described above) by a sample injection needle clamp. Furthermore, the flow cytometer of the present disclosure includes a light source configured to irradiate particles in the flow stream at an interrogation point within the flow cell, and a detector configured to collect light emitted by the irradiated particles.

[0063] The flow cytometers of this disclosure include light sources configured to irradiate particles in the flow stream at an interrogation point within a flow cell. The number of light sources in the flow cytometer can vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, the flow cytometer may include multiple light sources in some cases. In some such cases, the number of light sources ranges from 2 to 10, for example, 2 to 5, or for example, 2 to 4. Any convenient light source may be used as a light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer in question includes dye lasers such as a stilbene laser, coumarin laser, or rhodamine laser. In yet another case, the laser in question includes metal vapor lasers such as a helium-cadmium (HeCd) laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser, copper laser, or gold laser, and combinations thereof. In other cases, the flow cytometers in question include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, trim YAG lasers, ytterbium YAG lasers, ytterbium 2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0064] A laser light source according to a particular embodiment may also include one or more optical tuning components. In a particular embodiment, the optical tuning component may include any device located between the light source and the flow cell that can alter the spatial width of the irradiation, or any other characteristics of the irradiation from the light source, such as the direction of irradiation, wavelength, beam width, beam intensity, and focus. The optical tuning protocol may include, but is not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof, any convenient device for tuning one or more characteristics of the light source. In a particular embodiment, the flow cytometer in question includes one or more focusing lenses. The focusing lenses may, in one example, be reduction lenses. In yet another embodiment, the flow cytometer in question includes optical fibers.

[0065] If the optical adjustment component is configured to move, it may be configured to move continuously or at discrete intervals in increments including, for example, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1 mm or more, 5 mm or more, 10 mm or more, and 25 mm or more.

[0066] Using any displacement protocol, a motor-driven translation stage, a lead screw translation assembly, a geared translation device, and an optical adjustment component structure, such as an optical adjustment component structure employing a stepper motor, servo motor, brushless electric motor, brushed DC motor, microstepped motor, or high-resolution stepper motor, may be moved, either coupled to or directly from a movable support stage.

[0067] The light source may be positioned at any appropriate distance from the flow cell, for example, the light source and the flow cell are separated by 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 25 mm or more, and include a distance of, for example, 100 mm or more. Furthermore, the light source may be positioned at any appropriate angle with respect to the flow cell, within a range of angles including, for example, 10 to 90 degrees, e.g., 15 to 85 degrees, e.g., 20 to 80 degrees, e.g., 25 to 75 degrees, e.g., 30 to 60 degrees, e.g., 90 degrees.

[0068] In some embodiments, the light source of interest includes a plurality of lasers configured to provide laser light for discrete irradiation of a flowstream, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, and e.g., fifteen or more lasers configured to provide laser light for discrete irradiation of a flowstream. Depending on the desired wavelength of light for irradiating the flowstream, each laser may have a specific wavelength including, for example, 400 nm to 800 nm, which varies from 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm. In certain embodiments, the laser of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0069] In certain embodiments, the light source is a light beam generator configured to produce two or more frequency-shifted light beams. In some cases, the light beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optical device to produce two or more angle-deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. For example, the laser in the light beam generator in question may include the above.

[0070] The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the system of interest is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital combiner (DDS), arbitrary waveform generator (AWG), or electric pulse generator.

[0071] In the embodiment, the controller is configured to apply high-frequency drive signals to an acousto-optical device to generate a desired number of angle-deflected laser beams within the output laser beam, and includes being configured to apply, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals, and being configured to apply one hundred or more high-frequency drive signals.

[0072] In some cases, to create an intensity profile of an angularly deflected laser beam within the output laser beam, the controller is configured to apply a high-frequency drive signal having an amplitude including, for example, about 5V to about 25V, which varies, for example, from about 0.001V to about 500V, for example, from about 0.005V to about 400V, for example, from about 0.01V to about 300V, for example, from about 0.05V to about 200V, for example, from about 0.1V to about 100V, for example, from about 0.5V to about 75V, for example, from about 1V to 50V, for example, from about 2V to 40V, for example, from 3V to about 30V. In some embodiments, each of the applied high-frequency drive signals has a frequency of approximately 0.001 MHz to approximately 500 MHz, for example approximately 0.005 MHz to approximately 400 MHz, for example approximately 0.01 MHz to approximately 300 MHz, for example approximately 0.05 MHz to approximately 200 MHz, for example approximately 0.1 MHz to approximately 100 MHz, for example approximately 0.5 MHz to approximately 90 MHz, for example approximately 1 MHz to approximately 75 MHz, for example approximately 2 MHz to approximately 70 MHz, for example approximately 3 MHz to approximately 65 MHz, for example approximately 4 MHz to approximately 60 MHz, and for example approximately 5 MHz to approximately 50 MHz.

[0073] In certain embodiments, the controller has a processor having memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an angle-deflected laser beam having a desired intensity profile. For example, the memory may contain instructions for generating two or more angle-deflected laser beams having the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may contain instructions for generating 100 or more angle-deflected laser beams having the same intensity. In other embodiments, the memory may contain instructions for generating two or more angle-deflected laser beams having various intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may contain instructions for generating 100 or more angle-deflected laser beams having different intensities.

[0074] In certain embodiments, the controller has a processor having memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that decreases from the edges toward the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edges of the output laser beam along the horizontal axis, and may also include a range of about 10% to about 50%, such as 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%. In other embodiments, the controller has a processor having memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, and may also include a range of about 10% to about 50%, such as 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%. In yet another embodiment, the controller has a processor having a memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis.In yet another embodiment, the controller has a processor having a memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis.

[0075] In some embodiments, the target light beam generator may be configured to generate spatially separated angle-deflected laser beams within the output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, or may include 5000 μm or more. In some embodiments, the system is configured to generate angle-deflected laser beams within the output laser beam that overlap with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) may be an overlap of 0.001 μm or more, for example, an overlap of 0.005 μm or more, for example, an overlap of 0.01 μm or more, for example, an overlap of 0.05 μm or more, for example, an overlap of 0.1 μm or more, for example, an overlap of 0.5 μm or more, for example, an overlap of 1 μm or more, for example, an overlap of 5 μm or more, for example, an overlap of 10 μm or more, or it may include an overlap of 100 μm or more.

[0076] In certain cases, a light beam generator configured to produce two or more frequency-shifted light beams is referred to in U.S. Patents No. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and 10,684,21 This includes laser excitation modules described in No. 1, No. 10,845,295, No. 10,935,482, No. 10,935,485, No. 11,105,728, No. 11,280,718, No. 11,327,016, No. 11,366,052, No. 11,371,937, No. 11,692,926, No. 11,630,053, No. 11,774,343, No. 11,940,369, and No. 11,946,851, the disclosures of which are incorporated herein by reference.

[0077] Particle-modulated light may be observed after particles have been irradiated within a flow cell. "Particle-modulated light" refers to the light received by particles in a flow stream after they have been irradiated with light from a light source. In some cases, particle-modulated light is side-scattered light. As described herein, side-scattered light refers to light that has been refracted and reflected from the surface and internal structure of the particles. In further embodiments, particle-modulated light includes forward-scattered light (i.e., light that travels through or around the particles, primarily in a forward direction). In yet another case, particle-modulated light includes fluorescence (i.e., light emitted from a fluorescent dye after irradiation with excitation wavelength light).

[0078] As described above, a flow cytometer includes a detector configured to collect light emitted by an irradiated particle (i.e., particle-modulated light). The photodetector is configured to detect the particle-modulated light carried by an optical fiber focusing element and to generate a signal based on the characteristics of the light (e.g., intensity). For example, one or more particle-modulated photodetectors may include one or more side-scatter photodetectors for detecting the side-scatter wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particle). In some embodiments, the flow cytometer includes a single side-scatter photodetector. In other embodiments, the flow cytometer includes a plurality of side-scatter photodetectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more.

[0079] In some embodiments, the particle-modulated photodetector includes one or more forward-scattered photodetectors configured to detect forward-scattered light. For example, the flow cytometer in question may include one or more forward-scattered photodetectors, e.g., two or more, e.g., three or more, e.g., four or more, and e.g., five or more. In a particular embodiment, the flow cytometer includes one forward-scattered photodetector. In other embodiments, the flow cytometer includes two forward-scattered photodetectors.

[0080] In some embodiments, the forward scatter light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector is configured to continuously acquire measurements of the collected light. In other cases, the detector is configured to perform measurements at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and, for example, every 1000 milliseconds, or any other interval.

[0081] Any convenient detector for detecting the collected light may be used as the side-scattered light detector described herein. Examples of suitable detectors include, but are not limited to, 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 (PMT), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the collected light is measured by a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector has an active detection surface area in each region within a range including, for example, 0.01 cm 2 ~10 cm 2 , for example, 0.05 cm 2 ~9 cm 2 , for example, 0.1 cm 2 ~8 cm 2 , for example, 0.5 cm 2 ~7 cm 2 of, for example, 1 cm 2 ~5 cm 2 and is a photomultiplier tube such as a photomultiplier tube having an active detection surface area in each region within the range.

[0082] In embodiments, the flow cytometer of interest also includes a fluorescence detector configured to detect one or more fluorescence wavelengths of light. In other embodiments, the flow cytometer includes a plurality of fluorescence detectors, for example, two or more, for example, three or more, for example, four or more, five or more, ten or more, fifteen or more, twenty or more.

[0083] Any convenient detector for detecting the collected light may be used in the fluorescence detector described herein. Examples of such detectors include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the collected light is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is 0.01 cm². 2 ~10cm 2 For example, 0.05 cm 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 1cm 2 ~5cm 2 This is a photomultiplier tube, such as a photomultiplier tube, having an activity detection surface area in each region within the range including [specific region].

[0084] If the flow cytometer in question includes multiple fluorescence detectors, each fluorescence detector may be the same, or the array of fluorescence detectors may be a combination of different types of detectors. For example, if the flow cytometer in question includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD device and the second fluorescence detector (or imaging sensor) is a CMOS device. In other embodiments, both the first and second fluorescence detectors are CCD devices. In yet another embodiment, both the first and second fluorescence detectors are CMOS devices. In yet another embodiment, the first fluorescence detector is a CCD device and the second fluorescence detector is a photomultiplier tube (PMT). In yet another embodiment, the first fluorescence detector is a CMOS device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment, both the first and second fluorescence detectors are photomultiplier tubes.

[0085] In embodiments of the present disclosure, the fluorescence detector in question is configured to measure the collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths, and includes measuring the light emitted by the sample in the flow stream at 400 or more different wavelengths. In some embodiments, two or more detectors in the module described herein are configured to measure the same or overlapping wavelengths of the collected light.

[0086] In some embodiments, the fluorescence detector in question is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector in question is configured to collect the spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In yet another embodiment, the detector in question is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a module may include one or more detectors configured to measure light at one or more of the following wavelengths: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm and any combination thereof. In certain embodiments, one or more detectors may be configured to pair with a specific fluorophore, such as one used with a sample in a fluorescence assay.

[0087] The flow cytometer may include any suitable mechanism for supplying the sheath fluid and sample fluid to a sample fluid inlet coupler and the sheath fluid inlet coupler. For example, the sample fluid inlet coupler may be fluidically connected to a sample fluid line (e.g., a tube) fluidically connected to a sample fluid reservoir. Similarly, the sheath fluid inlet coupler may be fluidically connected to a sheath fluid line fluidically connected to a sheath fluid reservoir. Likewise, the flow cytometer may include any suitable mechanism for managing waste from the flow stream. A fluid discharge coupler may be fluidically connected to a waste line fluidically connected to a waste reservoir. A fluid management system that may be adapted for use with the flow cytometer in question is described in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0088] In some embodiments, the flow cytometer includes one or more wavelength separators positioned between the flow cell and the particle-modulated photodetector. The term “wavelength separator” is used herein in its conventional sense and refers to an optical component configured to separate light collected from a sample into a predetermined spectral range. In some embodiments, the flow cytometer includes a single wavelength separator. In other embodiments, the flow cytometer includes multiple wavelength separators, e.g., two or more wavelength separators, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty-five or more, fifty or more, seven-five or more, and one hundred or more wavelength separators. In some embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by allowing light having a predetermined spectral range to pass through and reflecting one or more remaining spectral ranges of the light. In other embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by allowing light having a predetermined spectral range to pass through and absorbing one or more remaining spectral ranges of the light. In further embodiments, the wavelength separator is configured to spatially diffract the light collected from the sample into a predetermined spectral range. Each wavelength separator may be any convenient optical separation protocol, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator of the photodetector system in question is a dichroic mirror.

[0089] Appropriate flow cytometry systems include, but are not limited to, those disclosed herein by reference: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49 (pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig, et al. (2007) Crit Rev Ther This may include those described in Drug Carrier Syst. 24(3):203-255.In certain cases, the flow cytometry systems in question include BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Accuri® C6 Plus flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortessa® X-20 flow cytometer, BD Biosciences FACSPresto® flow cytometer, BD Biosciences FACSVia® flow cytometer, and BD Biosciences FACSCalibur® cell sorter, BD Biosciences FACSCount® cell sorter, BD Biosciences FACSLyric® cell sorter, and BD This includes Biosciences Via® cell sorters, BD Biosciences Influx® cell sorters, BD Biosciences Jazz® cell sorters, BD Biosciences Aria® cell sorters, BD Biosciences FACSAria® II cell sorters, BD Biosciences FACSAria® III cell sorters, BD Biosciences FACSAria® Fusion cell sorters, and BD Biosciences FACSMelody® cell sorters, BD Biosciences FACSymphony® S6 cell sorters, BD Biosciences FACSDiscover® cell sorters, etc.

[0090] In some embodiments, the system in question is specified in U.S. Patent Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, and 10,302,54 No. 5, No. 10,145,793, No. 10,113,967, No. 10,006,852, No. 9,952,076, No. 9,933,341, No. 9 , 726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034 , No. 8,975,595, No. 8,753,573, No. 8,233,146, No. 8,140,300, No. 7,544,326, No. 7,201 ,875, No. 7,129,505, No. 6,821,740, No. 6,813,017, No. 6,809,804, No. 6,372,506, No. 5 Flow cytometry systems such as those described in Patent Nos. 700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766 (these disclosures are incorporated herein in their entirety by reference).

[0091] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in a particular case, the system in question is based on Diebold, et al. Nature Photonics. Vol.7(10), 806-810(2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, and Flow cytometry systems configured to image particles in a flow stream by fluorescence imaging using high-frequency tagged emission (FIRE), such as those described in Patent Nos. 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851, are incorporated herein by reference. In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in U.S. Patent Nos. 10,324,019, 10,620,111, 11,105,728, and 11,774,343, and U.S. Patent Applications Nos. 18 / 537,103, 18 / 657,618, 18,657,623, and 18 / 657,633, the disclosures of which are incorporated herein by reference in their entirety.

[0092] Figure 2 shows a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. Although the example in Figure 2 shows a single laser, it is understood that multiple lasers may also be used. The laser beam from laser 201 is directed to a focusing lens 202, which focuses the beam onto a portion of the fluid stream where the particles 211 of the sample in the flow cell 210 are located. The flow cell 210 is part of a fluid system that guides particles in the stream to the focused laser beam, typically one at a time, for interrogation. Alternatively, a nozzle top may be used if the flow cytometer is a stream-in-air cytometer.

[0093] As shown in Figure 2, the flow cell 210 is fluidically connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. The sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid inlet port 208 via a conduit (i.e., sheath fluid line) 207. Furthermore, the sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., sample fluid line) 205. The sample injection port 206 is fluidically connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce the particles 211 into the flow cell body 210. The particles 211 are hydrodynamically focused via the sheath fluid entering from the sheath fluid inlet port 208 so that a flowstream 214 is formed downstream of the tapered portion 212 of the flow cell 210. Particles released at the distal end of the flow cell body 210 can be disposed of and / or collected via any suitable protocol. For example, depending on the type of flow cytometry performed, the particles may be collected at the distal end of the flow cell body 210, for example, via a waste line. Alternatively, the particles may be sorted.

[0094] Light from the laser beam interacts with particles 211 in the sample by diffraction, refraction, reflection, scattering, and absorption, with re-emission at various different wavelengths, depending on the particle's characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be sent to one or more detectors. In particular, forward scatter (FSC) is sent to a forward scatter detector 223. The forward scatter detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect the diffracted light, i.e., the excitation light that travels mainly forward through or around the particle. The intensity of the light detected by the forward scatter detector 223 depends on the overall size of the particle. The forward scatter detector may include, for example, a photodiode. An optical filter 221a and a scattering bar 222 are positioned between the forward scatter detector 223 and the beam. The optical filter 221a may be configured to remove non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scatter light detector 223.

[0095] Furthermore, side-scattered light (SSC) is detected by the side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the complexity of the particle structure increases. In the example in Figure 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 and allow non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scattered light detector 224. Fluorescence detectors 225a to 225c, each configured to detect fluorescence of various wavelengths, are also shown. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a and allow light of other wavelengths to pass through. The optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to a first wavelength (or wavelength range) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b and to allow light of a third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. The optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to a second wavelength (or wavelength range) from being detected by the fluorescence detector 225b. Furthermore, the optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to a third wavelength (or wavelength range) from being detected by the fluorescence detector 225c.

[0096] Those skilled in the art will recognize that the flow cytometer according to the embodiments of the present disclosure is not limited to the flow cytometer shown in Figure 2, but may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers of various wavelengths and various different configurations, beam splitters, filters, and detectors. For example, the embodiment in Figure 2 shows three fluorescence detectors for illustrative purposes, but it will be understood that any suitable number of fluorescence detectors may be used.

[0097] During operation, the cytometer's operation is controlled by the controller / processor 290, and measurement data from the detector is stored in memory 295 for processing by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detector to receive output signals from it, and may also be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function 297 may also be provided within the system. The memory 295, controller / processor 290, and I / O 297 may be provided as a single integrated part of the flow cytometer. In such embodiments, a display may also form part of the I / O function 297 for presenting experimental data to the user of the cytometer 200. Alternatively, some or all of the memory 295, controller / processor 290, and I / O functions may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and controller / processor 290 can communicate with the cytometer 200 wirelessly or via a wired connection. The controller / processor 290 can be configured to work in conjunction with the memory 295 and I / O 297 to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0098] The various fluorescent molecules in the fluorescent dye panel used in flow cytometry experiments emit light in their own characteristic wavelength bands. The specific fluorescent labels used in the experiment, and their associated fluorescence emission bands, may be selected to substantially match the detector's filter window. I / O297 can be configured to receive data for flow cytometry experiments with a panel of fluorescent labels, and for multiple cell populations having multiple markers, where each cell population has a subset of multiple markers. I / O297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometry experiment data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 295. The controller / processor 290 can be configured to evaluate the assignment of one or more labels to the markers.

[0099] In some embodiments, the system in question is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, whose disclosure is incorporated herein by reference. In some embodiments, the system for sorting components of a sample includes a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference.

[0100] In certain embodiments, the system is fluorescence imaging using a high-frequency tagged emission image-enabled particle sorter, as shown in Figures 3-1 and 3-2. The particle sorter 300 includes an optical illumination component 300a, which includes a light source 301 (e.g., a 488 nm laser) that generates an output optical beam 301a, which is split into beam 302a and beam 302b using a beam splitter 302. The optical beam 302a is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a having one or more angle-deflected optical beams. In some cases, the output beam 303a generated from the acousto-optical device 303 includes a local oscillator beam and multiple high-frequency comb beams. The optical beam 302b is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a having one or more angle-deflected optical beams. In some cases, the output beam 304a generated from the acousto-optic device 304 includes a local oscillator beam and multiple high-frequency comb beams. The output beams 303a and 304a generated from the acousto-optic devices 303 and 304, respectively, are combined with a beam splitter 305 to generate an output beam 305a, which is then transported through an optical component 306 (e.g., an objective lens) to irradiate particles in the flow cell 307. In certain embodiments, the acousto-optic device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in a beam combiner 305. In certain embodiments, the light irradiation system having a light source and an acoustic-optical device may also include those described in Schraivogel, et al. ("High-speed fluorescence image-enabled cell sorting," Science (2022), 375(6578):315-320) and U.S. Patent Application Publication No. 2021 / 0404943, which are incorporated herein by reference.

[0101] The output beam 305a irradiates sample particles 308 propagating through the flow cell 307 (e.g., together with the sheath fluid 309) in the irradiation area 310. As shown in the irradiation area 310, multiple beams (e.g., angle-deflected high-frequency shifted light beams shown as dots across the irradiation area 310) are superimposed on the reference local oscillator beam (indicated by diagonal lines across the irradiation area 310). Due to their various optical frequencies, the overlapping beams exhibit pulsating behavior, thereby giving each beamlet a distinct frequency f 1-n This is used to carry a sine wave modulation signal.

[0102] Light from the irradiated sample is delivered to a photodetector system 300b, which includes multiple photodetectors. The photodetector system 300b includes a forward scatter photodetector 311 for generating a forward scatter image 311a and a side scatter photodetector 312 for generating a side scatter image 312a. The photodetector system 300b also includes a bright-field photodetector 313 for generating an optical loss image 313a. In some embodiments, the forward scatter detector 311 and the side scatter detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the irradiated sample is also detected by fluorescence detectors 314-317. In some cases, the photodetectors 314-317 are photomultiplier tubes. Light from the irradiated sample is directed through a beam splitter 320 to the side scatter detection channel 312 and the fluorescence detection channels 314-317. The photodetector system 300b includes bandpass optical components 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optical component 321 is 534 nm / 40 nm bandpass. In some cases, optical component 322 is 586 nm / 42 nm bandpass. In some cases, optical component 323 is 700 nm / 54 nm bandpass. In some cases, optical component 324 is 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number indicates the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on both sides of the center of the spectral band, i.e., from 500 nm to 520 nm.

[0103] Data signals generated in response to light detected by scattered light detection channels 311 and 312, bright-field light detection channel 313, and fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-responsive sorting is performed in response to sorting signals generated by sorting trigger 352. The sorting component 300c includes deflection plates 331 for deflecting particles into the sample container 332 or into the waste stream 333. In some cases, the sorting component 300c is configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0104] In some embodiments, the system is a particle analyzer and can analyze and characterize particles using the particle analysis system 401 (Figure 4), whether or not the particles are physically sorted into a collection container. Figure 4 shows a functional block diagram of the particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid system 402. The fluid system 402 includes or can include a sample tube 405 and a moving fluid column in the sample tube through which sample particles 403 (e.g., cells) move along a common sample path 409.

[0105] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting light or one or more other properties of particle 403 as the particle passes through the monitoring area 407. Figure 4 shows one detection station 408 with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas.

[0106] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be called event data. The data points can be multidimensional data points containing the values ​​of each characteristic measured for each particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0107] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuits and / or frequency control circuits. The illustrated control system may be operably associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during a first time interval. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may further compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0108] Figure 5 shows a functional block diagram of an example of a particle analyzer control system (i.e., a flow cytometer control system), such as an analysis controller (i.e., a processor) 500, for analyzing and displaying biological events. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0109] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0110] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. The analysis controller 500 can be configured to provide a display device 506 with a graphic display including a first plot of the biological event data. The analysis controller 500 can be further configured to render a region of interest overlaid on the first plot, for example, as a gate around a set of biological event data shown by the display device 506. In some embodiments, the gate may be a logical combination of one or more graphic regions of interest depicted in a histogram or bivariate plot of a single parameter. In some embodiments, a display may be used to show particle parameters or saturation detector data.

[0111] The analysis controller 500 can be further configured to display biological event data on the display device 506 within the gate in a different manner from other events in the biological event data outside the gate. For example, the analysis controller 500 can be configured to render the colors of the biological event data contained within the gate differently from the colors of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0112] The analysis controller 500 can be configured to receive gate selection signals from a first input device to identify gates. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 to identify gates that are displayed on the display device 506 or operated via the display device (for example, by clicking on a desired gate when the cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 508, or as other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, photodetector, or voice recognition system. Some input devices can include multiple input functions. In such implementations, each of those input functions can be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0113] The trigger event can cause the analysis controller 500 to change how the data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting a target population for particle sorting.

[0114] In some embodiments, the analysis controller 500 can be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a specific distribution of biological event data received by the analysis controller 500.

[0115] The analysis controller 500 can be connected to the storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to enable the analysis controller 500 to acquire biological event data, such as flow cytometry event data.

[0116] The display device 506 can be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that outline sections of the plots. The display device 506 can be further configured to change the information presented according to the input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the memory device 504, the keyboard 508, and / or the mouse 510.

[0117] In some implementations, the analysis controller 500 can generate a user interface for receiving exemplary events for selection. For example, the user interface may include controls for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates may be provided before the collection of event data for the sample, or based on an initial set of events for a portion of the sample.

[0118] Figure 6A is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to a nozzle 603, may include a nozzle 603, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a line across a monitoring area 611 (e.g., where laser streams intersect) irradiated by an irradiation source 612 (e.g., a laser). The vibration of the droplet-forming transducer 602 divides the moving fluid column 608 into multiple droplets 610, some of which contain particles 609.

[0119] During operation, a detection station 614 (e.g., an event detector) identifies a target particle (or target cell) as it crosses the monitoring area 611. The detection station 614 supplies power to a timing circuit 628, which supplies power to a flash charge circuit 630. A flash charge can be applied to the moving fluid column 608 so that the target droplet becomes charged at the droplet departure point, which is indicated by a timed droplet delay (Δt). The target droplet may contain one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a collection tube or a container such as a multi-well or microwell sample plate, and the wells or microwells can be associated with specific target droplets. As shown in Figure 6A, the droplets can be collected in a drain receptacle 638.

[0120] The detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet driving signal as the target particle passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 enables the instrument to accurately calculate the location of each detected particle in the droplet. The detection system 616 may supply amplitude signals 620 and / or phase signals 618, which are supplied (via amplifier 622) to amplitude control circuits 626 and / or frequency control circuits 624. The amplitude control circuits 626 and / or frequency control circuits 624 control the droplet formation transducer 602. The amplitude control circuits 626 and / or frequency control circuits 624 may be included in a control system.

[0121] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit, or they can be communicatively coupled so that either the detection system 616 or the detection station 614 can collect event measurements and provide them to non-collecting elements.

[0122] Figure 6B is a schematic diagram of a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in Figure 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream-charging wire in a barb. This creates a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronics or other detection systems (not shown in Figure 6B). The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, guiding the droplets toward a target collection receptacle (e.g., one of 672, 674, 676, or 678). As shown in Figure 6B, deflection plates 652 and 654 can be used to direct particles toward receptacle 674 along the first path 662 or toward receptacle 678 along the second path 668. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within the specified sorting range), the deflection plates may allow the particles to continue along the flow path 664. Such uncharged droplets may enter the waste receptacle via an aspirator 670 or the like.

[0123] Sorting electronics may be included to initiate measurement data collection, receive fluorescence signals from particles, and determine how to adjust the deflection plates to sort the particles. An exemplary implementation of the embodiment shown in Figure 6B includes the BD FACSAria® line of flow cytometers commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0124] How to analyze a sample As described above, aspects of the present disclosure also include methods for analyzing a sample. The method in question includes introducing a fluid sample (i.e., containing particles) into the flow cytometer described above for analysis of the sample fluid. In some embodiments, the method further includes sorting the particles of the fluid sample by flow cytometry. As described above, the flow cytometer in question includes a flow cell and a sample injection needle, wherein the flow cell has a flow cell body for transporting particles in a core stream of a flow stream from a proximal end to a distal end, the flow cell body includes a flow cell cone at the proximal end, and the sample injection needle has a passage through which it delivers the sample fluid from a sample injection line at the proximal end to the flow cell body at the distal end to generate a core stream, and the sample injection needle includes a sample injection needle adapter including a sample tube adapter to which the needle is attached, and a clamp that operably connects the sample injection needle to the flow cell body.

[0125] In some cases, the sample analyzed in the method is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, a whole plant, a whole fungus, or, in certain cases, a subset of animal tissue, cells, or components that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to, but is not limited to, both a whole organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a whole organism or a subset of its tissues, including, for example, plasma, serum, cerebrospinal fluid, lymph, skin, respiratory, gastrointestinal, cardiovascular and urogenital tract sections, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of living tissue, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some cases, the sample is a blood sample containing whole blood, such as blood obtained from a venipuncture or fingertip puncture (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0126] In certain embodiments, the source of the sample is “mammal” or “mammal,” and these terms are used broadly to describe organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs and rats), and Primates (e.g., humans, chimpanzees and monkeys). In some cases, the subject is human. The method may also be applied to samples obtained from human subjects of both sexes and any developmental stage (i.e., neonatal, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is juvenile, adolescent, or adult. While this disclosure may be applied to samples derived from human subjects, it should be understood that the method may also be applied to samples from other animal subjects (i.e., “non-human subjects”), such as birds, mice, rats, dogs, cats, livestock and horses, but is not limited to these.

[0127] The target cells may be targeted for characterization by various parameters, such as phenotypic features identified by attaching specific fluorescent labels to the target cells. In some embodiments, the system is configured to deflect analyzed droplets determined to contain target cells. Various cells may be characterized using the method of the subject. Target cells of the subject include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of the subject include cells having favorable cell surface markers or antigens that can be captured or labeled by favorable affinity factors or their coupling. For example, target cells may contain 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 alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0128] When performing the method in question, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module may vary, and may be in the range of 5 mL to 100 mL, such as 0.001 mL to 1000 mL, 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, or 2 mL to 300 mL.

[0129] Methods according to embodiments of the present disclosure include counting labeled particles (e.g., target cells) in a sample and selectively sorting them. When performing the method in question, a fluid sample containing particles is first introduced into a flow nozzle of the system. Exiting the flow nozzle, the particles pass through a sample interrogation area substantially one at a time, where each particle is irradiated with a light source, and light scattering parameters and, in some cases, desired fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded separately for each particle. Depending on the characteristics of the interrogated flowstream, the light may be irradiated to a length of 0.001 mm or more of the flowstream, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more of the flowstream. In certain embodiments, the method includes irradiating a planar cross section of the flowstream within the sample interrogation area with a laser or the like (as described above). In other embodiments, the method includes irradiating a predetermined length of flowstream within a sample interrogation region, for example, a length corresponding to the irradiation profile of a diffuse laser beam or lamp.

[0130] In certain embodiments, the method includes irradiating a flowstream at or near the nozzle orifice of the flow cell. For example, the method may include irradiating a flowstream at a position of about 0.001 mm or more from the nozzle orifice, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, and e.g., 1 mm or more. In certain embodiments, the method includes irradiating a flowstream immediately adjacent to the flow cell nozzle orifice.

[0131] In embodiments of the method, a detector such as a photomultiplier tube (PMT) is used to record light passing through each particle (in certain cases called forward scattering), light reflected perpendicular to the direction of particle flow through the detection area (in some cases called orthogonal or side scattering), and, if the particles are labeled with a fluorescent marker, fluorescence emitted from the particles when they pass through the detection area and are illuminated by an energy source. Each of forward scattering (FSC), side scattering (SSC), and fluorescence emission involves distinct parameters for each particle (or each “event”). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle can, if desired, be analyzed in real time or stored in data storage and analysis means such as a computer.

[0132] In certain embodiments, particles are detected and uniquely identified, as desired, by exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted in the detection channels used to identify the particles and associated binding complexes may be measured after excitation by a single light source or separately after excitation by individual light sources. If separate excitation light sources are used to excite particle labels, the labels may be selected so that all labels are excitable by each of the excitation light sources used.

[0133] The method, in certain embodiments, includes data acquisition, analysis, and recording using a computer or the like, with multiple data channels recording data from each detector about the light scattering and fluorescence emitted by each particle as it passes through the sample interrogation area of ​​the particle sorting module. In these embodiments, the analysis includes sorting and counting the particles so that each particle is presented as a set of digitized parameter values. The system under consideration may be set up with triggers on selected parameters to distinguish the target particles from background and noise. A “trigger” refers to a preset threshold for detecting a parameter and may be used as a means to detect the passage of particles through a light source. Detection of an event exceeding the threshold of the selected parameter triggers the acquisition of light scattering and fluorescence data for the particles. For particles or other components in the medium being assayed that cause a response below the threshold, no data is acquired. The trigger parameter may be the detection of forward scattered light caused by the passage of particles through a light beam. The flow cytometer then detects and collects the light scattering and fluorescence data for the particles.

[0134] Next, a specific subpopulation of interest is further analyzed by “gating” based on data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible separation of the subpopulation. This procedure may be carried out by plotting forward light scattering (FSC) versus side (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles is selected (i.e., their cells in the gate), and particles not in the gate are excluded. If desired, the gate may be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Then, only those particles in the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to yield a count of the target particles in the sample.

[0135] The methods in question may further include the use of particles in research, laboratory testing, or treatment. In some embodiments, the methods in question include obtaining individual cells prepared from a biological sample of a target fluid or tissue. For example, the methods in question include obtaining cells from a fluid or tissue sample used as a research or diagnostic specimen for a disease such as cancer. Similarly, the methods in question include obtaining cells from a fluid or tissue sample used in treatment. A cell therapy protocol is a protocol in which viable cellular material, including cells and tissues, is prepared and can be introduced into a subject as a therapeutic procedure. Conditions that can be treated by administration of samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, and organ damage.

[0136] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification, culture and in vitro growth, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with the collection of viable cells and tissues from the subject's source tissues to generate cell and / or tissue samples. Samples may be collected by any appropriate procedure, including, for example, administering a cell recruiter to the subject, drawing blood from the subject, or removing bone marrow from the subject. After sample collection, cell enrichment may be performed by several methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, and fluorescence-activated cell sorting (FACS). In some cases, enriched cells may be genetically modified by any convenient method, for example, nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and grown in vitro. In some cases, cells may be stored, for example by cryopreservation, and then thawed and stored for future use, and subsequently administered to a patient, for example, the cells may be injected into the patient.

[0137] Computer control system Aspects of the present disclosure further include a computer-controlled system, the system including one or more computers for full or partial automation. In some embodiments, the system includes a computer having a non-temporary computer-readable storage medium for storing a computer program, the computer program, once loaded into the computer, receiving target or desired flow conditions (e.g., sheath fluid flow rate, sample fluid flow rate) and including instructions for initiating resistance state changes to achieve the target or desired flow conditions. Since the system described herein includes the flow cell of the present disclosure, the resulting core stream may remain intact.

[0138] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access memory containing instructions for carrying out steps of the method in question. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of several other processors that are available or will become available. The processor runs the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system usually works 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, and communication control and related services, according to all known technologies. In some embodiments, the processor includes analog electronic equipment that provides feedback control, such as negative feedback control.

[0139] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as permanent hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or floppy disk drives. Such types of memory storage devices typically read from and / or write to program storage media such as compact disks (not shown). Any of these program storage media, or others currently in use or to be developed in the future, may be considered computer program products. As is understood, 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 program storage devices used in conjunction with system memory and / or memory storage devices.

[0140] In some embodiments, a computer program product is described that includes a computer-usable medium on which control logic (a computer software program including program code) is stored. When the control logic is executed by the computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of a hardware state machine for performing the functions described herein will be obvious to those skilled in the art.

[0141] Memory may be any suitable device on which the processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including magnetic or optical disks, or tapes or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium having the necessary program code. The programming may be provided to the processor remotely via a communication channel, or it may be pre-stored in a computer program product such as memory or some other portable or fixed computer-readable storage medium using any of the memory-related devices. For example, a magnetic or optical disk may have a program that can be read by a disk writer / reader. The system of this disclosure also includes, for example, programming in the form of a computer program product, algorithms for use in carrying out the methods described above. The programming according to this disclosure may be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROMs, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0142] The processor may also access communication channels to communicate with users in remote locations. Remote location means that the user is not in direct contact with the system, but relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0143] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0144] In one embodiment, the communication interface is configured to include one or more communication ports, such as a USB port, a USB-C port, an RS-232 port, or any other suitable physical port or interface that enables data communication between the system in question and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0145] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the system in question to communicate with computer terminals and / or networks, other devices such as mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with it.

[0146] In one embodiment, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP), Short Message Service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0147] In one embodiment, the system in question is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or electrical appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD), and input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0148] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in a target system, such as an optional data storage unit, with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0149] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a remote user, for example, via the Internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email, or other files, or data in other forms. The data may also include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present in the system under consideration may be any type of known computer platform or a type to be developed in the future, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, whether networked or not. They may be located in the same place or physically separated. Depending on the type and / or manufacturer of the selected computer platform, various operating systems may be employed on any computer platform.Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android®, SGI IRIX®, Oracle Solaris®, and others.

[0150] Figure 7 shows a general architecture of an exemplary computing device 700 according to a particular embodiment. The general architecture of the computing device 700 shown in Figure 7 includes the configuration of computer hardware and software components. However, it is not necessary to illustrate all of these generally conventional elements in order to provide an implementable disclosure. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may also communicate with memory 770 and further provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (such as data analysis software or programs like FlowJo®) stored as executable instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 740 may also receive input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, speech recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0151] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Data may be stored in a data storage device 790. Memory 770 may further include computer program instructions and other information for implementing embodiments of the present disclosure.

[0152] kit Embodiments of the present disclosure further include kits. The kits in question include one or more sample injection needles (e.g., assembled or unassembled, without the sample injection needle adapter and clamp attached) as described above. In one embodiment, the kit includes a single sample injection needle. In other embodiments, the kit includes multiple sample injection needles. If the kit includes multiple sample injection needles, the sample injection needles may be the same or different. For example, in one case, the kit includes a sample injection needle having a superbullet configuration as described above, a bullet configuration as described above, and a rounded configuration, or any combination of these configurations. In some cases, the kit further includes a flow cell body (e.g., as described above). In some cases, the kit further includes packaging configured to hold the sample injection needle adapter, clamp and / or flow cell body.

[0153] In addition to the components described above, the kit may further include (in some embodiments) instructions for, for example, how to install the sample injection needle of this disclosure into a flow cytometer. These instructions may be present in the kit in various forms, or one or more of them may be present in the kit. One possible form of these instructions is information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the kit packaging, or an accompanying document. Yet another form of these instructions is a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), or a portable flash drive. Yet another possible form of these instructions is a website address that can be used via the Internet to access the information at a remote site.

[0154] usefulness The sample injection needle, flow cell, flow cytometer, and method described in this invention are found to be used in a variety of applications where it is desirable to analyze the components in a sample in a fluid medium. The invention is particularly found to be used to improve the quality of the core stream in a fluid system. For example, the sample injection needle, flow cytometer, and method can be used to increase the degree to which the core stream remains intact. In some cases, the invention can be used to reduce the formation of vortices within the flow cell cone.

[0155] Embodiments of this disclosure are used in applications where cells prepared from biological samples may be desired for use in research, laboratory testing, or therapeutic settings. In some embodiments, the methods and devices of the subject may facilitate the acquisition and / or analysis of individual cells prepared from biological samples of target fluids or tissues. For example, the methods and systems of the subject may facilitate the acquisition of cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods and systems of the subject may facilitate the acquisition of cells from fluid or tissue samples used in therapeutic settings.

[0156] Notwithstanding the attached claims, this disclosure is also defined by the following clauses:

[0157] 1. A flow cell for use in a flow cytometer, A flow cell body for transporting particles in the core stream of a flowstream from the proximal end to the distal end, the flow cell body having a flow cell cone at the proximal end, To generate a core stream, a sample injection needle has a passage for delivering the sample fluid from the sample injection line at the proximal end to the flow cell body at the distal end, The sample injection needle is equipped with A sample injection needle adapter equipped with a sample tube adapter that can be attached to a needle, A clamp that movably connects the sample injection needle to the flow cell body. Equipped with, In a non-clamped flow cell configuration, the sample injection needle adapter rotates freely relative to the flow cell cone and clamp, while in a clamped flow cell configuration, the sample injection needle adapter is fixed relative to the flow cell cone and clamp.

[0158] 2. The flow cell according to Clause 1, wherein the needle of the sample injection needle adapter comprises a proximal end that is attached to the sample tube adapter and a distal end that is positioned within the flow cell cone.

[0159] 3. In the clamp configuration, the sample injection needle adapter is fixed to the flow cell cone and clamp by tightening the clamp, as described in Clause 2.

[0160] 4. A flow cell as described in Clause 2 or 3, wherein the clamp is fastened by one or more fastening members.

[0161] 5. A flow cell as described in Clause 4, where the clamp is fastened by multiple screws.

[0162] 6. The flow cell as described in Clause 5, with the clamp fastened by three screws.

[0163] 7. A flow cell as described in Clause 6, wherein the clamp comprises a set of holes for receiving each of a plurality of screws.

[0164] 8. The flow cell according to Clause 7, wherein the flow cell body is equipped with a set of holes for clamps and a set of holes for receiving each of a plurality of screws.

[0165] 9. A flow cell according to any one of clauses 5 to 8, wherein the clamp is configured such that the inclination of the sample injection needle relative to the flow cell body can be adjusted by manipulating the torque of at least one of several screws.

[0166] 10. A flow cell according to any one of Clauses 2 to 9, wherein the clamp comprises a distal end that contacts a sample tube adapter and a proximal end configured to fluidly connect the sample injection line to a sample injection needle.

[0167] 11. The flow cell according to Clause 10, wherein the distal end of the clamp comprises a recess into which at least a portion of the sample tube adapter is positioned and a surface that contacts the proximal end of the flow cell body.

[0168] 12. The flow cell according to Clause 10 or 11, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the sample injection needle.

[0169] 13. A flow cell according to any one of clauses 10 to 12, wherein the proximal end of the clamp is configured to position the flow meter board connector.

[0170] 14. A flow cell according to any one of clauses 2 to 13, wherein the sample tube adapter comprises a proximal end that is positioned in a recess of the clamp and a distal end that contacts the proximal end of the flow cell body.

[0171] 15. The flow cell according to Clause 14, wherein at least a portion of the distal end of the sample tube adapter is positioned within the flow cell body.

[0172] 16. The flow cell according to clause 14 or 15, wherein the sample tube adapter comprises a flange that contacts the proximal end of the flow cell body.

[0173] 17. A flow cell according to any one of clauses 14 to 16, wherein the distal end of a sample tube adapter is pressed against the proximal end of the flow cell body by a clamp, such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

[0174] 18. A flow cell according to any one of clauses 2 to 17, wherein the needle of the sample injection needle adapter is tapered at its distal end.

[0175] 19. The flow cell according to Clause 18, wherein the needle of the sample injection needle adapter has a rounded distal end.

[0176] 20. A flow cell according to any one of Clauses 2 to 19, wherein the distal end of the needle of the sample injection needle adapter is positioned within the flow cell cone in a manner that enables the maintenance of a complete core stream under flow conditions of more than one order of magnitude.

[0177] 21. A flow cell according to any one of clauses 2 to 20, wherein the flow cell body comprises a sheath fluid introduction port for delivering sheath fluid to the flow cell cone.

[0178] 22. The flow cell as described in Clause 21, wherein the distal end of the needle of the sample injection needle adapter is separated from the sheath fluid introduction port by a longitudinal distance in the range of 17 mm to 26 mm.

[0179] 23. The flow cell according to clause 21 or 22, wherein the flow cell body comprises a plurality of sheath fluid introduction ports.

[0180] 24. A flow cell as described in Clause 23, wherein the sheath fluid introduction ports are offset from each other so that the sheath fluid swirls within the flow cell cone.

[0181] 25. A flow cell according to any one of clauses 2 to 24, wherein the distal end of the flow cell body comprises a cuvette for transporting particles in the core stream through a sample interrogation region.

[0182] 26. The flow cell according to Clause 25, wherein at least a portion of the cuvette comprises an optically transparent solid.

[0183] 27. The flow cell according to Clause 26, wherein the optically transparent portion of the cuvette is configured to allow optical detection of particles in the core stream.

[0184] 28. A flow cell according to any one of clauses 25 to 27, wherein the cuvette is positioned at the distal end of the flow cell body by a clamp that is fixed to the flow cell body.

[0185] 29. The flow cell according to Clause 28, wherein the cuvette is releasably attached to the distal end of the flow cell body by a flow cell body clamp.

[0186] 30. A flow cell according to clause 28 or 29, wherein the cuvette is positioned by a flow cell body clamp so that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0187] 31. A clamp for operably connecting a sample injection needle adapter to the body of a flow cell, A distal end configured to be attached to a sample injection needle adapter, which includes a sample tube adapter that is fixed to the needle, The proximal end of the sample injection line is configured to fluidly connect to the needle of the sample injection needle adapter. Equipped with, A clamp configured to operably connect the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body.

[0188] 32. The clamp according to Clause 31, wherein the distal end of the clamp has a surface configured to contact the flow cell body when the clamp is pressing the sample tube adapter against the flow cell body.

[0189] 33. The clamp according to clause 32, wherein the distal end of the clamp has a recess configured to receive at least a portion of a sample tube adapter.

[0190] 34. The clamp according to clause 32 or 33, wherein the recess has an inner surface concentric with the outer surface of a portion of the sample tube adapter.

[0191] 35. The clamp according to clause 33 or 34, wherein the recess is configured such that the sample injection needle adapter is rotatably movable relative to the clamp when a portion of the sample tube adapter is positioned within the recess.

[0192] 36. A clamp according to any one of the clauses 31 to 33, wherein the clamp is configured to receive a fastening member for fastening the clamp to a flow cell body.

[0193] 37. The clamp according to Clause 36, wherein when the sample injection needle adapter is operably coupled to the flow cell body by the clamp, the clamp is configured such that the tightening of the clamp by the fastening member secures the sample injection needle adapter to the flow cell body.

[0194] 38. A clamp as described in Clause 36 or 37, wherein the fastening member includes multiple screws.

[0195] 39. The clamp according to Clause 38, wherein the clamp comprises a set of holes for receiving each of a plurality of screws.

[0196] 40. The clamp according to Clause 39, wherein the set of holes is configured to align with the set of holes in the flow cell body.

[0197] 41. The clamp according to any one of the clauses 38 to 40, wherein when the sample injection needle adapter is operably coupled to the flow cell body by the clamp, the inclination of the sample injection needle adapter relative to the flow cell body is adjustable by manipulating the torque of at least one of the multiple screws.

[0198] 42. The clamp according to Clause 39, wherein when the sample injection needle adapter is operably coupled to the flow cell body by the clamp, the position of the distal end of the needle of the sample injection needle adapter within the flow cell cone of the flow cell body is adjustable by manipulating the torque of at least one of a plurality of screws.

[0199] 43. A clamp as described in Clause 42, which rotates the distal end of a needle around an axis by adjusting the torque of one of several screws.

[0200] 44. A clamp according to any one of the clauses 31 to 43, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter.

[0201] 45. A clamp according to any one of the clauses 31 to 44, wherein the proximal end of the clamp is configured to position a flow meter board connector.

[0202] 46. ​​A sample injection needle adapter for operably connecting a sample injection line to the body of a flow cell, A needle having a passage that penetrates to deliver the sample fluid from the sample injection line at the proximal end to the flow cell cone of the flow cell body at the distal end, A sample tube adapter comprising a proximal end configured to be attached to a clamp and a distal end fixed to the proximal end of a needle. Equipped with, A sample injection needle adapter is configured such that when the sample tube adapter is pressed against the flow cell body by a clamp, the needle is operably coupled to the flow cell body.

[0203] 47. The sample injection needle adapter according to Clause 46, wherein when the sample tube adapter is operably coupled to the flow cell body by a clamp, the tightening of the clamp on the sample tube adapter is configured to make the sample injection needle adapter immovable relative to the flow cell body.

[0204] 48. The sample injection needle adapter according to Clause 47, wherein the sample tube adapter is immobilized such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

[0205] 49. A sample injection needle adapter as described in Clause 48, which allows the fixed position to maintain a complete core stream within the flow cell cone under flow conditions where the flow changes by more than one order of magnitude.

[0206] 50. A sample injection needle adapter according to any one of the clauses 46 to 49, wherein, when the sample injection needle adapter is operably coupled to the flow cell body by a clamp, the inclination of the sample injection needle adapter relative to the flow cell body is adjustable by manipulating the torque of at least one of the multiple screws that fasten the clamp to the flow cell body.

[0207] 51. The sample injection needle adapter according to Clause 50, wherein when the sample injection needle adapter is operably coupled to the flow cell body by a clamp, the position of the distal end of the needle in the flow cell cone is adjustable by manipulating the torque of at least one of a plurality of screws.

[0208] 52. A sample injection needle adapter as described in Clause 51, wherein the distal end of the needle is rotated around an axis by adjusting the torque of one of several screws.

[0209] 53. A sample injection needle adapter according to any one of the clauses 46 to 52, wherein at least a portion of the proximal end of the sample tube adapter is configured to be positioned within a recess of the clamp.

[0210] 54. The sample injection needle adapter according to Clause 53, wherein a portion of the proximal end of the sample tube adapter has an outer surface concentric with the inner surface of a recess.

[0211] 55. The sample injection needle adapter according to Clause 53 or 54, wherein a portion of the proximal end of the sample tube adapter is configured such that the sample injection needle adapter is rotatably movable relative to the clamp when a portion of the sample tube adapter is positioned within the recess.

[0212] 56. A sample injection needle adapter according to any one of the clauses 46 to 55, wherein at least a portion of the distal end of the sample tube adapter is configured to be positioned within the flow cell body.

[0213] 57. The sample injection needle adapter according to Clause 56, wherein the sample tube adapter is provided with a flange for positioning a portion of the distal end of the sample tube adapter within the flow cell body proximal to the flow cell cone.

[0214] 58. The sample injection needle adapter according to Clause 57, wherein the flange is configured to position a portion of the distal end of the sample tube adapter within the flow cell body such that the distal end of the needle is separated from the sheath fluid introduction port of the flow cell body by a longitudinal distance in the range of 17 mm to 26 mm.

[0215] 59. A sample injection needle adapter according to any one of the clauses 46 to 58, wherein the needle is tapered at its distal end.

[0216] 60. A sample injection needle adapter according to Clause 59, wherein the needle has a rounded distal end.

[0217] 61. A sample injection needle for operably connecting a sample injection line to the body of a flow cell, A sample injection needle adapter, A needle having a passage for delivering the sample fluid from the sample injection line at the proximal end to the flow cell cone of the flow cell body at the distal end, and A sample tube adapter comprising a proximal end and a distal end, the distal end of which is fixed to the proximal end of a needle. A sample injection needle adapter equipped with, It is a clamp, The distal end attached to the proximal end of the sample tube adapter, and The proximal end of the sample injection needle adapter is configured to fluidly connect the sample injection line to the proximal end of the needle. A clamp and Equipped with, A sample injection needle, wherein the clamp is configured to operably connect the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body.

[0218] 62. The sample injection needle according to Clause 61, wherein the sample injection needle adapter rotates freely relative to the flow cell cone and clamp.

[0219] 63. The sample injection needle according to Clause 62, wherein at least a portion of the proximal end of the sample tube adapter is configured to be positioned within a recess of the clamp.

[0220] 64. A sample injection needle according to Clause 63, wherein a portion of the proximal end of the sample tube adapter has an outer surface concentric with the inner surface of a recess.

[0221] 65. A sample injection needle according to any one of clauses 62 to 64, wherein at least a portion of the distal end of the sample tube adapter is configured to be positioned within the flow cell body.

[0222] 66. The sample injection needle according to Clause 65, wherein the sample tube adapter comprises a flange configured to position a portion of the distal end of the sample tube adapter within the flow cell body proximal to the flow cell cone.

[0223] 67. A sample injection needle according to any one of clauses 62 to 66, wherein when the sample injection needle is operably coupled to the flow cell body by a clamp, the tightening of the clamp on the sample tube adapter is configured to make the sample injection needle adapter immovable relative to the flow cell body.

[0224] 68. The sample injection needle according to Clause 67, wherein the sample injection needle adapter is immobilized such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

[0225] 69. A sample injection needle as described in Clause 68, which allows the fixed position to maintain a complete core stream within the flow cell cone under flow conditions where the flow changes by more than one order of magnitude.

[0226] 70. A sample injection needle as described in Clause 68 or 69, wherein the fixed position is located a longitudinal distance of 17 mm to 26 mm away from the sheath fluid introduction port of the flow cell body.

[0227] 71. A sample injection needle according to any one of the clauses 61 to 70, wherein the clamp is configured to receive a fastening member for fastening the clamp to the flow cell body.

[0228] 72. The sample injection needle according to Clause 71, wherein when the clamp is fastened to the flow cell body by the fastening member, the tightening of the clamp by the fastening member prevents the sample injection needle adapter from moving relative to the flow cell body.

[0229] 73. A sample injection needle as described in Clause 71 or 72, wherein the fastening member includes multiple screws.

[0230] 74. A sample injection needle as described in Clause 73, wherein the clamp has a set of holes for receiving each of a plurality of screws.

[0231] 75. A sample injection needle as described in Clause 74, wherein the set of holes is configured to align with the set of holes in the flow cell body.

[0232] 76. A sample injection needle according to any one of the clauses 73 to 75, wherein the sample injection needle is configured such that, when the clamp is fastened to the flow cell body by multiple screws, the inclination of the sample injection needle adapter relative to the flow cell body can be adjusted by manipulating the torque of at least one of the multiple screws.

[0233] 77. The sample injection needle according to Clause 76, wherein the sample injection needle is configured such that, when the clamp is fastened to the flow cell body by multiple screws, the position of the distal end of the needle of the sample injection needle adapter within the flow cell cone is adjustable by manipulating the torque of at least one of the multiple screws.

[0234] 78. A sample injection needle as described in Clause 77, wherein the distal end of the needle is rotated around an axis by adjusting the torque of one of several screws.

[0235] 79. A sample injection needle according to any one of the clauses 61 to 78, wherein the needle of the sample injection needle adapter is tapered at its distal end.

[0236] 80. The sample injection needle according to Clause 79, wherein the needle of the sample injection needle adapter has a rounded distal end.

[0237] 81. A sample injection needle according to any one of the clauses 61 to 80, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter.

[0238] 82. A sample injection needle according to any one of clauses 61 to 81, wherein the proximal end of the clamp is configured to position the flow meter board connector.

[0239] 83. A method for assembling a flow cell for use in a flow cytometer, To transport particles in the core stream of a flowstream from the proximal end to the distal end, the sample injection needle is operably coupled to a flow cell body having a flow cell cone at its proximal end. The sample injection needle comprises a clamp and a sample tube adapter that is attached to the needle, and the sample injection needle adapter has a passage for delivering the sample fluid from the sample injection line at the proximal end to the flow cell body at the distal end in order to generate a core stream. A method comprising operably coupling a sample injection needle to a flow cell body using a sample injection needle clamp.

[0240] 84. The method according to Clause 83, wherein the needle of the sample injection needle adapter comprises a proximal end that is attached to the sample tube adapter and a distal end that is positioned within the flow cell cone, and the sample tube adapter is attached to a clamp.

[0241] 85. The method according to clause 84, wherein the sample injection needle is operably coupled to the flow cell body by pressing the sample tube adapter against the flow cell body using a clamp.

[0242] 86. The method according to clause 85, further comprising immobilizing the sample injection needle adapter by tightening a clamp.

[0243] 87. The method according to clause 86, wherein the sample injection needle adapter is made immovable such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

[0244] 88. A clamp as described in Clause 86 or 87, wherein the clamp is fastened by a fastening member that is received by the clamp and the flow cell body.

[0245] 89. The method according to Clause 88, wherein the fastening member includes multiple screws.

[0246] 90. The method according to Clause 89, further comprising inserting a set of screws into a set of holes in a clamp and a set of holes in the flow cell body, wherein the set of holes in the clamp is aligned with the set of holes in the flow cell body.

[0247] 91. The method according to clause 89 or 90, further comprising adjusting the inclination of the sample injection needle relative to the flow cell body by manipulating the torque of at least one of a plurality of screws.

[0248] 92. The method according to clause 91, further comprising adjusting the position of the distal end of the needle of the sample injection needle adapter within the flow cell cone by manipulating the torque of at least one of a plurality of screws.

[0249] 93. The method according to Clause 92, wherein adjusting the position of the distal end of the needle of the sample injection needle adapter includes rotating the distal end of the needle around an axis by individually adjusting the torque of one of a plurality of screws.

[0250] 94. The method according to any one of the clauses 91 to 93, wherein the distal end of the needle of the sample injection needle adapter is positioned within the flow cell cone in a manner that allows a complete core stream to be maintained under flow conditions of more than one order of magnitude.

[0251] 95. The method according to any one of the clauses 86 to 94, wherein the sample injection needle adapter rotates freely relative to the flow cell cone and clamp before being immobilized by the clamp.

[0252] 96. The method of Clause 95, further comprising adjusting the rotational position of the sample injection needle adapter before it is immobilized by a clamp.

[0253] 97. The method according to any one of the clauses 85 to 96, further comprising attaching the sample tube adapter to the clamp before operably coupling the sample injection needle to the flow cell body.

[0254] 98. The method according to Clause 97, wherein pre-attaching the sample tube adapter to the clamp includes positioning a portion of the sample tube adapter within a recess in the clamp.

[0255] 99. The method according to any one of the clauses 83 to 98, wherein the clamp comprises a distal end that contacts a sample tube adapter and a proximal end configured to fluidly connect the sample injection line to a sample injection needle.

[0256] 100. The method according to Clause 99, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the sample injection needle.

[0257] 101. The method according to any one of the clauses 83 to 100, further comprising using the proximal end of the clamp to position the flow meter board connector.

[0258] 102. The method according to any one of the clauses 83 to 100, wherein the flow cell body comprises a sheath fluid introduction port for delivering sheath fluid to the flow cell cone.

[0259] 103. The method according to any one of the clauses 83 to 101, wherein the needle of the sample injection needle adapter is tapered at its distal end.

[0260] 104. The method according to clause 103, wherein the needle of the sample injection needle adapter has a rounded distal end.

[0261] 105. The method according to any one of clauses 83 to 104, wherein the distal end of the flow cell body comprises a cuvette for transporting particles in the core stream through the sample interrogation region.

[0262] 106. The method according to clause 105, wherein at least a portion of the cuvette comprises an optically transmissible solid.

[0263] 107. The method according to clause 106, wherein the optically transmissible portion of the cuvette is configured to enable optical detection of particles in the core stream.

[0264] 108. The method according to any one of clauses 105 to 107, further comprising positioning a cuvette at the distal end of the flow cell body using a clamp fixed to the flow cell body.

[0265] 109. The method according to clause 108, wherein the cuvette is releasably attached to the distal end of the flow cell body by a flow cell body clamp.

[0266] 110. The method according to clause 108 or 109, wherein the cuvette is positioned by a flow cell body clamp such that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0267] 111. The method according to any one of clauses 83 to 110, further comprising operably positioning the flow cell on a flow cytometer.

[0268] 112. The method according to clause 111, wherein operably positioning the flow cell on a flow cytometer comprises fluidly coupling a sample injection line to the needle of a sample injection needle adapter using a clamp.

[0269] 113. The method according to clause 112, wherein operably positioning the flow cell in the flow cytometer includes fluidly coupling a sheath fluid introduction port of the flow cell body for sending the sheath fluid to the flow cell cone to a sheath fluid source.

[0270] 114. The method according to any one of clauses 111 to 113, wherein operably positioning the flow cell in the flow cytometer includes aligning a cuvette at a distal end of the flow cell body for transporting particles in the core stream through the sample interrogation region with a light source of the flow cytometer for irradiating particles in the core stream in the sample interrogation region.

[0271] 115. The method according to clause 114, wherein operably positioning the flow cell in the flow cytometer includes optically coupling a detector of the flow cytometer configured to collect light emitted by the irradiated particles to the sample interrogation region.

[0272] 116. A flow cell body for transporting particles in the core stream of the flow stream from the proximal end to the distal end, the flow cell body having a flow cell cone at the proximal end, and a sample injection needle having a passage for sending a sample fluid from a sample injection line at the proximal end to the flow cell body at the distal end to generate a core stream comprising a flow cell, a light source configured to irradiate particles in the flow stream in the sample interrogation region within the flow cell, a detector configured to collect light emitted by the irradiated particles, and comprising, the sample injection needle comprises a sample injection needle adapter having a sample tube adapter attached to the needle, and a clamp for operably coupling the sample injection needle to the flow cell body comprises, In the non-clamp configuration of the flow cell, the sample injection needle adapter rotates freely relative to the flow cell cone and clamp. In a flow cell clamp configuration, the sample injection needle adapter is fixed to the flow cell cone and clamp, in a flow cytometer.

[0273] 117. The flow cytometer according to Clause 116, wherein the needle of the sample injection needle adapter comprises a proximal end that is attached to the sample tube adapter and a distal end that is positioned within the flow cell cone.

[0274] 118. In a clamp configuration, the sample injection needle adapter is secured to the flow cell cone and the clamp by tightening the clamp, as described in Clause 117.

[0275] 119. A flow cytometer according to Clause 117 or 118, wherein the clamp is fastened by one or more fastening members.

[0276] 120. A flow cytometer as described in Clause 119, wherein the clamp is fastened by multiple screws.

[0277] 121. A flow cytometer as described in Clause 120, with a clamp fastened by three screws.

[0278] 122. A flow cytometer according to clause 120 or 121, wherein the clamp comprises a set of holes for receiving each of a plurality of screws.

[0279] 123. A flow cytometer as described in Clause 122, wherein the flow cell body is equipped with a set of holes for clamping and a set of holes for receiving each of a plurality of screws.

[0280] 124. A flow cell according to any one of the clauses 120 to 123, wherein the clamp is configured such that the inclination of the sample injection needle relative to the flow cell body can be adjusted by manipulating the torque of at least one of several screws.

[0281] 125. A flow cytometer according to any one of Clauses 117 to 124, wherein the clamp comprises a distal end that contacts a sample tube adapter and a proximal end that fluidly connects a sample injection line to a sample injection needle.

[0282] 126. The flow cytometer according to Clause 125, wherein the distal end of the clamp comprises a recess into which at least a portion of the sample tube adapter is positioned and a surface that contacts the proximal end of the flow cell body.

[0283] 127. A flow cytometer according to Clause 125 or 126, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the sample injection needle.

[0284] 128. A flow cytometer according to any one of clauses 125 to 127, wherein the proximal end of the clamp positions the flow meter board connector.

[0285] 129. A flow cytometer according to any one of Clauses 117 to 128, wherein the sample tube adapter comprises a proximal end positioned in a recess of the clamp and a distal end in contact with the proximal end of the flow cell body.

[0286] 130. A flow cytometer according to Clause 129, wherein at least a portion of the distal end of the sample tube adapter is positioned within the flow cell body.

[0287] 131. A flow cytometer according to clause 129 or 130, wherein the sample tube adapter comprises a flange that contacts the proximal end of the flow cell body.

[0288] 132. The flow cytometer according to any one of clauses 129 to 131, wherein the distal end of the needle of the sample injection needle adapter is pressed against the proximal end of the flow cell body by a clamp so that the distal end of the sample tube adapter is in a fixed position within the flow cell cone.

[0289] 133. The flow cytometer according to any one of clauses 117 to 132, wherein the needle of the sample injection needle adapter has a taper at the distal end.

[0290] 134. The flow cytometer according to clause 133, wherein the needle of the sample injection needle adapter has a rounded distal end.

[0291] 135. The flow cytometer according to any one of clauses 117 to 134, wherein the distal end of the needle of the sample injection needle adapter is positioned within the flow cell cone in a manner that allows a complete core stream to be maintained under flow conditions that vary by one digit or more.

[0292] 136. The flow cytometer according to any one of clauses 117 to 135, wherein the flow cell body includes a sheath fluid introduction port for sending sheath fluid to the flow cell cone.

[0293] 137. The flow cytometer according to clause 136, wherein the distal end of the needle of the sample injection needle adapter is separated from the sheath fluid introduction port by a longitudinal distance in the range of 17 mm to 26 mm.

[0294] 138. The flow cytometer according to clause 136 or 137, wherein the flow cell body includes a plurality of sheath fluid introduction ports.

[0295] 139. The flow cytometer according to clause 138, wherein the sheath fluid introduction ports are offset from each other so that the sheath fluid swirls within the flow cell cone.

[0296] 140. A flow cytometer according to any one of Clauses 117 to 139, wherein the distal end of the flow cell body is equipped with a cuvette for transporting particles in the core stream through a sample interrogation region.

[0297] 141. The flow cytometer according to Clause 140, wherein at least a portion of the cuvette is made of an optically transparent solid.

[0298] 142. The flow cytometer according to Clause 141, wherein the optically transparent portion of the cuvette is configured to allow optical detection of particles in the core stream.

[0299] 143. A flow cytometer according to any one of clauses 140 to 142, wherein the cuvette is positioned at the distal end of the flow cell body by a clamp that is fixed to the flow cell body.

[0300] 144. A flow cytometer according to Clause 143, wherein the cuvette is releasably attached to the distal end of the flow cell body by a flow cell body clamp.

[0301] 145. A flow cytometer according to clause 143 or 144, wherein the cuvette is positioned by a flow cell body clamp so that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0302] 146. A method for analyzing a sample fluid, (a) Introducing the sample fluid into a flow cytometer, A flow cytometer, A flow cell body for transporting particles in the core stream of a flow stream from the proximal end to the distal end, the flow cell body comprising a flow cell cone at the proximal end, and A sample injection needle having a passage through which the sample fluid is delivered from the sample injection line at the proximal end to the flow cell body at the distal end in order to generate a core stream. A flow cell equipped with, A light source configured to irradiate particles in the flow stream at a sample interrogation point within a flow cell, A detector configured to collect light emitted by irradiated particles and The sample injection needle is equipped with A sample injection needle adapter equipped with a sample tube adapter that can be attached to a needle, A clamp that movably connects the sample injection needle to the flow cell body. Equipped with, In the non-clamp configuration of the flow cell, the sample injection needle adapter rotates freely relative to the flow cell cone and clamp. In the flow cell clamp configuration, the sample injection needle adapter is fixed to the flow cell cone and clamp, and is introduced. (b) A method comprising irradiating particles in a flow stream for analysis of a sample fluid.

[0303] 147. The method according to Clause 146, wherein the needle of the sample injection needle adapter comprises a proximal end that is attached to the sample tube adapter and a distal end that is positioned within the flow cell cone.

[0304] 148. The method according to Clamp 147, wherein the sample injection needle adapter is secured to the flow cell cone and clamp by tightening the clamp.

[0305] 149. The method according to clause 147 or 148, wherein the clamp is fastened by one or more fastening members.

[0306] 150. The method according to Clause 149, wherein the clamp is fastened by multiple screws.

[0307] 151. The clamp is fastened by three screws, as described in Clause 150.

[0308] 152. The method according to clause 151 or 152, wherein the clamp comprises a set of holes for receiving each of a plurality of screws.

[0309] 153. The method according to Clause 152, wherein the flow cell body is provided with a set of holes for clamping and a set of holes for receiving each of a plurality of screws.

[0310] 154. The method according to any one of the claims 150 to 153, wherein the clamp is configured such that the inclination of the sample injection needle relative to the flow cell body can be adjusted by manipulating the torque of at least one of the multiple screws.

[0311] 155. The method according to any one of Clauses 147 to 154, wherein the clamp comprises a distal end that contacts a sample tube adapter and a proximal end that fluidly connects a sample injection line to a sample injection needle.

[0312] 156. The method according to Clause 155, wherein the distal end of the clamp comprises a recess into which at least a portion of the sample tube adapter is positioned and a surface that contacts the proximal end of the flow cell body.

[0313] 157. The method according to clause 155 or 156, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the sample injection needle.

[0314] 158. The method according to any one of the clauses 155 to 157, wherein the proximal end of the clamp positions the flowmeter board connector.

[0315] 159. The method according to any one of Clauses 156 to 158, wherein the sample tube adapter comprises a proximal end positioned in a recess of the clamp and a distal end in contact with the proximal end of the flow cell body.

[0316] 160. The method according to clause 159, wherein at least a portion of the distal end of the sample tube adapter is positioned within the flow cell body.

[0317] 161. The method according to clause 159 or 160, wherein the sample tube adapter comprises a flange that contacts the proximal end of the flow cell body.

[0318] 162. The method according to any one of clauses 159 to 161, wherein the distal end of a sample tube adapter is pressed against the proximal end of the flow cell body by a clamp, such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

[0319] 163. The method according to any one of the clauses 157 to 162, wherein the needle of the sample injection needle adapter is tapered at its distal end.

[0320] 164. The method according to Clause 163, wherein the needle of the sample injection needle adapter has a rounded distal end.

[0321] 165. The method according to any one of the clauses 147 to 164, wherein the distal end of the needle of a sample injection needle adapter is positioned within a flow cell cone in a manner that allows a complete core stream to be maintained under flow conditions of more than one order of magnitude.

[0322] 166. The method according to any one of the clauses 147 to 165, wherein the flow cell body comprises a sheath fluid introduction port for delivering sheath fluid to the flow cell cone.

[0323] 167. The method according to clause 166, wherein the distal end of the needle of the sample injection needle adapter is separated from the sheath fluid introduction port by a longitudinal distance in the range of 17 mm to 26 mm.

[0324] 168. The method according to clause 166 or 167, wherein the flow cell body comprises a plurality of sheath fluid introduction ports.

[0325] 169. The method according to Clause 168, wherein the sheath fluid introduction ports are offset from each other so that the sheath fluid swirls within the flow cell cone.

[0326] 170. The method according to any one of the clauses 147 to 169, wherein the distal end of the flow cell body is equipped with a cuvette for transporting particles in the core stream through a sample interrogation region.

[0327] 171. The method according to clause 170, wherein at least a portion of the cuvette is made of an optically transparent solid.

[0328] 172. The method according to clause 171, wherein an optically transparent portion of the cuvette is configured to allow optical detection of particles in the core stream.

[0329] 173. The method according to any one of the clauses 170 to 172, wherein the cuvette is positioned at the distal end of the flow cell body by a clamp that is fixed to the flow cell body.

[0330] 174. The method according to clause 173, wherein the cuvette is releasably attached to the distal end of the flow cell body by a flow cell body clamp.

[0331] 175. The method according to clause 173 or 174, wherein the cuvette is positioned by a flow cell body clamp so that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

[0332] 176. The method described in any one of the clauses 146 to 175, wherein the sample is a biological sample.

[0333] 177. The method according to Clause 176, wherein the sample contains cells.

[0334] 178. The method described in any one of the clauses 146 to 177, further comprising sorting the sample by flow cytometry.

[0335] 179. A sample injection needle adapter for fluidly connecting a sample injection line to the body of a flow cell of a flow cytometer, comprising a sample tube adapter attached to a needle, A clamp for operably connecting the sample injection needle adapter to the body of the flow cell and A kit that includes the following:

[0336] 180. The needle of the sample injection needle adapter has a passage through which it delivers the sample fluid from the sample injection line at the proximal end to the flow cell cone of the flow cell body at the distal end. The kit according to Clause 179, comprising a sample tube adapter having a proximal end configured to be attached to a clamp and a distal end fixed to the proximal end of a needle.

[0337] 181. The kit as described in Clause 180, wherein the clamp is configured to operably connect the sample injection needle adapter to the flow cell body by pressing the sample tube adapter against the flow cell body.

[0338] 182. The kit according to Clause 181, wherein the distal end of the clamp has a surface configured to contact the flow cell body when the clamp presses the sample tube adapter against the flow cell body.

[0339] 183. The kit according to clause 181 or 182, wherein at least a portion of the distal end of the sample tube adapter is configured to be positioned within the flow cell body.

[0340] 184. The kit according to Clause 183, wherein the sample tube adapter is provided with a flange for positioning a portion of the distal end of the sample tube adapter within the flow cell body proximal to the flow cell cone.

[0341] 185. The kit according to Clause 184, wherein the flange is configured to position a portion of the distal end of the sample tube adapter within the flow cell body such that the distal end of the needle is separated from the sheath fluid introduction port of the flow cell body by a longitudinal distance in the range of 17 mm to 26 mm.

[0342] 186. The kit according to any one of Clauses 181 to 185, wherein the sample tube adapter and clamp are configured such that when the clamp operably connects the sample injection needle adapter to the flow cell body, the tightening of the clamp on the sample tube adapter makes the sample injection needle adapter immobile relative to the flow cell body.

[0343] 187. The kit as described in Clause 186, wherein the sample tube adapter is immobilized so that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

[0344] 188. The kit described in Clause 187, which enables the fixed position to maintain a complete core stream within the flow cell cone under flow conditions where the flow changes by more than one order of magnitude.

[0345] 189. A kit according to any one of Clauses 181 to 188, wherein the clamp is configured to receive a fastening member for fastening the clamp to the flow cell body.

[0346] 190. The kit according to Clause 189, wherein when the sample injection needle adapter is operably coupled to the flow cell body by the clamp, the clamp is configured such that the tightening of the clamp by the fastening member makes the sample injection needle adapter immobile relative to the flow cell body.

[0347] 191. A kit as described in Clause 189 or 190, wherein the fastening member includes multiple screws.

[0348] 192. The kit described in Clause 191, wherein the clamp has a set of holes for receiving each of a number of screws.

[0349] 193. The kit described in Clause 192, wherein the set of holes is configured to align with the set of holes in the flow cell body.

[0350] 194. The kit according to any one of Clauses 191 to 193, wherein the sample tube adapter and clamp are configured such that when the sample injection needle adapter is operably coupled to the flow cell body by a clamp, the inclination of the sample injection needle adapter relative to the flow cell body is adjustable by manipulating the torque of at least one of the multiple screws that fasten the clamp to the flow cell body.

[0351] 195. The sample tube adapter and clamp are configured such that when the sample injection needle adapter is operably coupled to the flow cell body by a clamp, the position of the distal end of the needle in the flow cell cone is adjustable by manipulating the torque of at least one of several screws, as described in Clause 194.

[0352] 196. The kit described in Clause 195, which rotates the distal end of a needle around an axis by adjusting the torque of one of several screws.

[0353] 197. A kit further comprising multiple screws, as described in any one of clauses 192 to 196.

[0354] 198. The kit according to any one of the clauses 181 to 197, wherein at least a portion of the proximal end of the sample tube adapter is configured to be positioned within a recess of the clamp.

[0355] 199. The kit according to Clause 198, wherein a portion of the proximal end of the sample tube adapter has an outer surface concentric with the inner surface of a recess.

[0356] 200. The kit according to Clause 198 or 199, wherein a portion of the proximal end of the sample tube adapter and a recess in the clamp are configured such that the sample injection needle adapter is rotatably movable relative to the clamp when a portion of the sample tube adapter is positioned within the recess.

[0357] 201. A kit according to any one of the clauses 181 to 200, wherein the needle has a taper at its distal end.

[0358] 202. The kit according to Clause 201, wherein the needle has a rounded distal end.

[0359] 203. The kit according to any one of Clauses 181 to 202, wherein the proximal end of the clamp is equipped with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the needle of the sample injection needle adapter.

[0360] 204. The kit as described in Clause 203, wherein the proximal end of the clamp is configured to position the flow meter board connector.

[0361] 205. The kit according to any one of clauses 181 to 204, further comprising packaging configured to hold a sample injection needle adapter and a clamp.

[0362] 206. A kit according to any one of the clauses 181 to 205, further comprising a flow cell body.

[0363] 207. The kit according to Clause 206, wherein the flow cell body is configured to transport particles in the core stream of a flow stream from a proximal end to a distal end, and the flow cell body comprises a flow cell cone at its proximal end.

[0364] 208. The kit as described in Clause 207, wherein the flow cell body is equipped with a sheath fluid introduction port for delivering sheath fluid to the flow cell cone.

[0365] 209. The kit described in Clause 208, wherein the flow cell body is equipped with multiple sheath fluid introduction ports.

[0366] 210. The kit as described in Clause 209, wherein the sheath fluid introduction ports are offset from each other so that the sheath fluid swirls within the flow cell cone.

[0367] 211. A cuvette for transporting particles in the core stream through the sample interrogation region at the distal end of the flow cell body, A clamp configured to fix the cuvette to the flow cell body in order to position it at the distal end of the flow cell body, A kit further comprising any one of clauses 207 to 209.

[0368] 212. The kit according to Clause 211, wherein at least a portion of the cuvette comprises an optically transparent solid.

[0369] 213. The kit according to Clause 212, wherein the optically transparent portion of the cuvette is configured to allow optical detection of particles in the core stream.

[0370] 214. The kit according to any one of clauses 211 to 213, wherein the flow cell body clamp is configured to releasably attach a cuvette to the distal end of the flow cell body.

[0371] 215. The kit according to Clause 214, wherein the flow cell body clamp and cuvette are configured such that the flow cell body clamp can optimally align the cuvette with the sample interrogation region for optical detection of particles in the core stream.

[0372] 216. The kit according to any one of clauses 206 to 215, further comprising packaging configured to hold a sample injection needle adapter, a clamp, and a flow cell body.

[0373] While the foregoing disclosure has been described in some detail as examples and illustrations to clarify understanding, it will be readily apparent to those skilled in the art that several changes and modifications can be made in light of the teachings of this disclosure without departing from the spirit or scope of the attached claims.

[0374] Therefore, the foregoing is merely illustrative of the principles of the present disclosure. Those skilled in the art will understand that various configurations embodying the principles of the present disclosure and that fall within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional statements described herein are intended primarily to help the reader understand the principles of the present disclosure and the concepts to which the inventors have contributed to the advancement of the art, and should be interpreted as not being limited to such specifically described examples and conditions. Furthermore, all descriptions herein listing the principles, aspects, and embodiments of the present disclosure, and specific examples thereof, are intended to encompass both structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any developed elements that perform the same function regardless of their structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether such disclosure is expressly described in the claims or not.

[0375] Accordingly, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) are expressly defined as applying to the limitation in the claims only if the exact phrase “means” or the exact phrase “step” is stated at the beginning of such limitation in the claims, and if such exact phrase is not used in the limitation of the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) does not apply.

Claims

1. A flow cell body for transporting particles in the core stream of a flow stream from a proximal end to a distal end, the flow cell body comprising a flow cell cone at the proximal end, and A sample injection needle having a passage for delivering the sample fluid from the sample injection line at the proximal end to the flow cell body at the distal end in order to generate the core stream. A flow cell equipped with, A light source configured to irradiate the particles in the flow stream in the sample interrogation region within the flow cell, A detector configured to collect light emitted by the irradiated particles, Equipped with, The aforementioned sample injection needle A sample injection needle adapter equipped with a sample tube adapter that can be attached to a needle, A clamp that movably connects the sample injection needle to the flow cell body. Equipped with, In the non-clamp configuration of the flow cell, the sample injection needle adapter rotates freely relative to the flow cell cone and the clamp. In the clamp configuration of the flow cell, the sample injection needle adapter is fixed to the flow cell cone and the clamp, in a flow cytometer.

2. The flow cytometer according to claim 1, wherein the needle of the sample injection needle adapter comprises a proximal end that is attached to the sample tube adapter and a distal end that is positioned within the flow cell cone.

3. The flow cytometer according to claim 2, wherein the sample injection needle adapter is fixed to the flow cell cone and the clamp by tightening the clamp.

4. The flow cytometer according to claim 2 or 3, wherein the clamp is fastened by one or more fastening members.

5. The flow cytometer according to claim 4, wherein the clamp is tightened by a plurality of screws.

6. The flow cytometer according to claim 5, wherein the clamp comprises a set of holes for receiving each of the plurality of screws.

7. The flow cytometer according to claim 6, wherein the flow cell body comprises a set of holes that are aligned with the set of holes in the clamp and that receive each of the plurality of screws.

8. The flow cell according to any one of claims 5 to 7, wherein the clamp is configured such that the inclination of the sample injection needle relative to the flow cell body can be adjusted by manipulating the torque of at least one of the plurality of screws.

9. The flow cytometer according to any one of claims 2 to 8, wherein the clamp comprises a distal end that contacts the sample tube adapter and a proximal end that fluidly connects the sample injection line to the sample injection needle.

10. The flow cytometer according to claim 9, wherein the distal end of the clamp comprises a recess into which at least a portion of the sample tube adapter is positioned, and a surface that contacts the proximal end of the flow cell body.

11. The flow cytometer according to claim 9 or 10, wherein the proximal end of the clamp is provided with a connector configured to minimize the dead volume of the sample fluid when the sample fluid is flowing from the sample injection line to the sample injection needle.

12. The flow cytometer according to any one of claims 2 to 11, wherein the sample tube adapter comprises a proximal end positioned in a recess of the clamp and a distal end in contact with the proximal end of the flow cell body.

13. The flow cytometer according to claim 11 or 12, wherein the sample tube adapter comprises a flange that contacts the proximal end of the flow cell body.

14. The flow cytometer according to any one of claims 11 to 13, wherein the distal end of the sample injection needle adapter is pressed against the proximal end of the flow cell body by the clamp, such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow cell cone.

15. The flow cytometer according to any one of claims 2 to 14, wherein the needle of the sample injection needle adapter has a taper at its distal end.

16. The flow cytometer according to any one of claims 2 to 15, wherein the distal end of the needle of the sample injection needle adapter is positioned within the flow cell cone in such a manner that a complete core stream is maintained under flow conditions of an order of magnitude or more.

17. The flow cytometer according to any one of claims 2 to 16, wherein the flow cell body is provided with a sheath fluid introduction port for sending sheath fluid to the flow cell cone.

18. The flow cytometer according to claim 17, wherein the distal end of the needle of the sample injection needle adapter is separated from the sheath fluid introduction port by a longitudinal distance in the range of 17 mm to 26 mm.

19. The flow cytometer according to any one of claims 2 to 18, wherein the distal end of the flow cell body is provided with a cuvette for transporting particles in the core stream through the sample interrogation region, and the cuvette is positioned at the distal end of the flow cell body by a clamp fixed to the flow cell body.

20. The flow cytometer according to claim 19, wherein the cuvette is positioned by a clamp fixed to the flow cell body such that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

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