Sample injection needle of flow cytometer

Abstract

The present disclosure provides a flow cell comprising a sample injection needle for operatively connecting a sample injection line to a flow cell body. The sample injection needle includes a sample injection needle adapter having a sample tube adapter secured to the needle for transferring a sample fluid from a sample injection line to a flow cell body; and a clamp for operatively connecting the sample injection needle to the flow cell body. The present disclosure also provides kits including the sample injection needle and a flow cytometer having the flow cell, as well as methods of their use and assembly.

Description

Technical Field

[0001] Characterization of analytes in biofluids has become an important component of biological research, medical diagnostics, and the assessment of overall patient health and well-being. Detection of analytes in biofluids, such as human blood or blood-derived products, can provide results that can play a role in determining treatment plans for patients with multiple disease states. Background Technology

[0002] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in blood samples or particles of interest in other types of biological or chemical samples. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as blood, and a sheath fluid reservoir containing sheath fluid. The flow cytometer delivers particles (including cells) from the fluid sample as a cell stream to the flow chamber, while simultaneously directing the sheath fluid into the flow chamber. To characterize the components of the flow stream, the flow stream is illuminated. Changes in the material within the flow stream (e.g., the presence of morphological or fluorescent labels) can cause changes in the observed light, and these changes can be used for characterization and separation. To characterize the components in the flow stream, light must be illuminating and collected from the flow stream. The light source of 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 irradiated particles is collected and quantified.

[0003] Biological particles can be separated by adding sorting or collection capabilities to flow cytometers. In the separated stream, particles detected to have one or more desired characteristics are separated from the sample stream by mechanical or electrical removal. A commonly used flow cytometry sorting technique employs droplet sorting, where the stream containing linearly separated particles is broken into droplets. Droplets containing particles of interest are charged and deflected into a collection tube as they pass through an electric field. Typically, particles linearly separated 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, the time it takes to reach the droplet breakup point and separate from the stream into droplets can be predicted. Ideally, a brief charge is applied to the stream just before the droplet containing the selected particles is about to detach from the fluid stream, and then the stream is grounded immediately after detachment. The droplet to be sorted remains charged as it detaches from the fluid stream, while all other droplets remain uncharged.

[0004] Some flow cytometry systems employ pressure-driven flow control technology to simultaneously deliver sample fluid and sheath fluid into the flow chamber. In these systems, the sample fluid and sheath fluid are delivered to the flow chamber, which contains the detection region (i.e., the area where particles are illuminated by the light source), under conditions above ambient pressure. The flow rate through the flow chamber of the pressure-driven flow control system can be varied by changing the pressure in the sample tube and / or sheath fluid reservoir delivered to the flow chamber. The ratio of sample fluid to sheath fluid flowing through the flow chamber is controlled by two factors: the pressure levels in the sample tube and sheath fluid reservoir, and the ratio of the resistance along the sample fluid path to the resistance along the sheath fluid path.

[0005] Alternatively, flow cytometry systems are implemented using vacuum-driven flow control technology, in which a vacuum pump evacuates the flow chamber downstream, and the sample fluid and sheath fluid are maintained at ambient pressure. In vacuum-driven flow control systems, the flow rate through the flow chamber can be altered by changing the vacuum level evacuated by the pump, and the ratio of sample fluid to sheath fluid flowing through the flow chamber is controlled by the ratio of the resistance along the sample fluid path to the resistance along the sheath fluid path.

[0006] To achieve single-particle-level characterization and separation of biological materials, some flow cytometers are equipped with injection needles to introduce sample fluid into the flow chamber. Using the injection needle, the sample fluid can be mixed with the sheath fluid in the flow chamber in the form of a central stream of particles sufficient to form a focused flow containing the sample fluid. This central stream can then transport the particles in a single-pass manner through the detection zone and / or sorting device. This technique is known as hydrodynamic focusing. Summary of the Invention

[0007] The inventors have recognized that imaging particles using flow cytometry requires precise control of the speed and position of the mixing of sample fluid and sheath fluid within the flow chamber. Specifically, it has been found that at imaging speeds, the central flow generated by conventional sample injection mechanisms often deviates from the detection area of ​​the flow chamber and / or breaks down due to fluid "vortices" generated by disturbances in the flow and central flow. Furthermore, the inventors have discovered that by precisely setting the rotation and pivot angles of the sample injection needle within the flow chamber, the sample fluid can be introduced into the sheath fluid flow path at a speed and position sufficient to maintain the integrity of the central flow within the detection area of ​​the flow chamber under various flow conditions (e.g., for particle imaging and / or sorting). Therefore, it has been recognized that a flow chamber capable of better controlling the position of the sample injection needle is essential. The embodiments of this disclosure meet this requirement.

[0008] This disclosure includes a sample injection needle for operatively connecting a sample injection line to a flow chamber body. The sample injection needle includes: a sample injection needle adapter comprising: a needle having a through-passage for conveying sample fluid from a sample injection line at a proximal end to a flow chamber body at a distal end; and a sample tube adapter including a proximal end and a distal end, wherein the distal end is secured to the proximal end of the needle; and a clamp comprising: 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, wherein the clamp is configured to operatively connect the sample injection needle adapter to the flow chamber body by pressing the sample tube adapter against the flow chamber 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, a 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 some embodiments, at least a portion of the distal end of the sample tube adapter is configured to be positioned within the flow chamber body. In these cases, the sample tube adapter may include a flange configured to position said distal portion of the sample tube adapter within the flow chamber body near the flow chamber cone structure. In some embodiments, the sample injection needle is configured such that compression of the sample tube adapter by the clamp, when operatively connected to the flow chamber body via a clamp, fixes the sample injection needle adapter relative to the flow chamber body. In these cases, the sample injection needle adapter can be fixed such that the distal end of the needle of the sample injection needle adapter is located in a fixed position within the flow chamber cone structure. In some embodiments, this fixed position ensures that a complete central flow is maintained within the flow chamber cone structure even when flow conditions change by one or more orders of magnitude. In some embodiments, the longitudinal distance between this fixed position and the sheath inlet of the flow chamber body is in the range of 17 mm to 26 mm. In one embodiment, the needle of the sample injection needle adapter is tapered at its distal end. For example, the needle of the sample injection needle adapter may include a rounded distal end.

[0010] In some embodiments, the clamp is configured to receive a fastening member for securing the clamp to the flow chamber body. In some embodiments, the sample needle is configured such that when the clamp is secured to the flow chamber body by the fastening member, compression of the clamp by the fastening member fixes the sample needle adapter relative to the flow chamber body. In some embodiments, the fastening member comprises 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 chamber body. In some embodiments, the sample needle is configured such that when the clamp is secured to the flow chamber body by the plurality of screws, the tilt of the sample needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws. In these cases, the sample needle can be configured such that when the clamp is secured to the flow chamber body by the plurality of screws, the position of the distal end of the sample needle adapter needle within the flow chamber conical structure can be adjusted by manipulating the torque of at least one of the plurality of screws. For example, adjusting the torque of one of a plurality of screws can rotate the distal end of the needle about an axis. 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 tubing to the needle of the sample injection needle adapter. In some embodiments, the proximal end of the clamp is configured to position a flowmeter plate connector.

[0011] This disclosure also relates to flow chambers having the sample injection needle, for example, flow chambers for use in flow cytometry, as described above and herein. The flow chamber of interest includes: a flow chamber body for transporting particles in a central flow of the flow from a proximal end to a distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end; and a sample injection needle having a through passage for conveying sample fluid from a sample injection line at the proximal end to the flow chamber body at the distal end to generate a central flow, wherein the sample injection needle includes: a sample injection needle adapter including a sample tube adapter attached to the needle; and a clamp for operatively connecting the sample injection needle to the flow chamber body. In a non-clamped configuration of the flow chamber, the sample injection needle adapter is freely rotatable relative to the flow chamber cone structure and the clamp, and in a clamped configuration of the flow chamber, the sample injection needle adapter is fixed relative to the flow chamber cone structure and the clamp. In some embodiments, the clamp operatively connects the sample injection needle adapter to the flow chamber body by pressing the sample tube adapter against the flow chamber 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 chamber cone structure. In some embodiments, in a clamping configuration, the sample injection needle adapter is secured relative to the flow chamber cone structure and the clamp by compression of the clamp.

[0012] In some embodiments, the clamp is fastened to the flow chamber body by one or more fastening members. In some embodiments, the clamp is pressed by one or more fastening members. For example, the clamp may be pressed by multiple screws. In some embodiments, the clamp is pressed by three screws. In these cases, the clamp includes a set of holes for receiving each of the multiple screws. In some embodiments, the flow chamber body includes a set of holes aligned with the clamp holes and for receiving each of the multiple screws. In some embodiments, the clamp is configured such that the tilt of the sample injection needle relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the multiple screws. In some embodiments, the clamp includes a distal end that contacts a sample tube adapter and a proximal end configured to fluidly connect a sample injection line to the sample injection needle. In some embodiments, the distal end of the clamp includes a recess in which at least a portion of the sample tube adapter is positioned; and a surface that contacts the proximal end of the flow chamber 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 flowmeter plate connector.

[0013] In some embodiments, the sample tube adapter includes a proximal end positioned in a clamp recess and a distal end contacting the proximal end of the flow chamber body. In some embodiments, at least a portion of the distal end of the sample tube adapter is positioned within the flow chamber body. In these cases, the sample tube adapter may include a flange contacting the proximal end of the flow chamber body. In some embodiments, the distal end of the sample tube adapter is clamped against the proximal end of the flow chamber body such that the distal end of the needle of the sample injection needle adapter is fixed in position within the flow chamber conical structure. In some embodiments, the needle of the sample injection needle adapter is tapered 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 within the flow chamber conical structure such that a complete central flow can be maintained when flow conditions vary by one or more orders of magnitude.

[0014] In some embodiments, the flow chamber body includes a sheath fluid inlet for delivering sheath fluid to the flow chamber conical structure. In these cases, the distal end of the needle of the sample injection needle adapter is separated from the sheath fluid inlet by a predetermined longitudinal distance, for example, a distance in the range of 17 mm to 26 mm. In some embodiments, the flow chamber body includes multiple sheath fluid inlets. In these cases, the sheath fluid inlets may be offset from each other, such that the sheath fluid flows through the flow chamber conical structure in a swirling manner. In some embodiments, the distal end of the flow chamber body includes a cuvette for conveying particles in the central stream through a sample detection area. In these cases, at least a portion of the cuvette includes a light-transmitting entity configured to allow optical detection of particles in the central stream. In some embodiments, the cuvette is positioned at the distal end of the flow chamber body by a clamp attached to the flow chamber body. In these cases, the cuvette can be releasably attached to the distal end of the flow chamber body by the flow chamber body clamp. In some implementations, the flow chamber body fixture positions the cuvette so that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0015] This disclosure also includes methods for assembling the flow chamber (e.g., flow chambers as described above and herein). Methods of interest include: operatively connecting a sample injection needle to a flow chamber body to deliver particles in a central flow of the flow from a proximal end to a distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end, and wherein: the sample injection needle includes a clamp and a sample injection needle adapter, the sample injection needle adapter including a sample tubing adapter attached to the needle and having a through passage for delivering sample fluid from the sample injection tubing at the proximal end to the flow chamber body at the distal end to generate a central flow, and the method includes: operatively connecting the sample injection needle to the flow chamber body using the sample injection needle clamp. In some embodiments, the method further includes: operatively positioning the flow chamber in a flow cytometer. In another aspect, kits comprising the sample injection needle and / or the flow chamber (e.g., those described above and herein) are provided.

[0016] This disclosure also includes flow cytometers having said flow chambers (e.g., flow chambers as described above and herein). Flow cytometers of interest include: a flow chamber body for transporting particles in a central flow of the flow stream from a proximal end to a distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end; and a sample injection needle having a through passage for delivering sample fluid from a sample injection line at the proximal end to the flow chamber body at the distal end to generate a central flow, wherein the sample injection needle includes: a sample injection needle adapter including a sample tube adapter attached to the needle; and a clamp operatively connecting the sample injection needle to the flow chamber body; a light source configured to irradiate particles in a flow stream in a sample detection region within the flow chamber; and a detector configured to collect light emitted by the irradiated particles. In another aspect, a method for analyzing sample fluids using a flow cytometer having said flow chambers (e.g., flow cytometers as described above and herein) is also provided. Attached Figure Description

[0017] This disclosure can be best understood by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures:

[0018] Figure 1A Exploded diagrams of the flow chamber according to certain implementation schemes are provided. Figure 1B Provided fully assembled according to certain implementation schemes Figure 1A Various views of the flow chamber. Figure 1C Various views of the fully assembled flow chamber according to certain implementation schemes are provided. Figure 1D A cross-sectional view of a sample injection needle according to certain implementation schemes is provided. Figure 1E A sample injection needle according to certain implementation schemes is depicted. Figure 1F A fixture according to certain embodiments is depicted, which includes a sample injection tubing connector at its proximal end and is attached to the distal end of a sample injection needle adapter.

[0019] Figure 2 A flow cytometry system according to certain implementation schemes is demonstrated.

[0020] Figure 3 A particle sorting instrument with image functionality according to certain embodiments is described.

[0021] Figure 4 A functional block diagram of a particle analysis system according to certain implementation schemes is depicted.

[0022] Figure 5 A functional block diagram of an example control system according to certain implementation schemes is depicted.

[0023] Figures 6A to 6BA schematic diagram of a particle sorting system according to certain implementation schemes is depicted.

[0024] Figure 7 Various aspects of a computer control system according to certain implementation schemes are described. Detailed Implementation

[0025] A flow chamber is provided, comprising a sample injection needle for operatively connecting a sample tubing to a flow chamber body. The sample injection needle includes: a sample injection needle adapter having a sample tubing adapter fixed to the needle for delivering sample fluid from the sample injection tubing to the flow chamber body; and a clamp for operatively connecting the sample injection needle to the flow chamber body. Furthermore, this disclosure also provides a kit including the sample injection needle and / or the flow chamber, and a flow cytometer having the flow chamber. Methods for assembling the flow chamber and for analyzing samples using the flow cytometer are also provided.

[0026] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, as they can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure is limited only by the appended claims.

[0027] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, and any other specified value or intermediate value within the specified range, is included in this disclosure, wherein, unless the context expressly specifies otherwise, the precision of the intermediate value is one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this disclosure, but are subject to any explicitly excluded limits within the specified range. When a specified range contains one or two limits, the range excluding one or both of the included limits is also included in this disclosure.

[0028] Certain ranges shown herein use the term “about” before the numerical value. As used herein, the term “about” provides written support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately the specifically stated number, the unstated number that is close to or approximately the number may be a number that is substantially equivalent to the specifically stated number when shown.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure, those described herein are representative and illustrative.

[0030] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually designated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publications. Any reference to a publication is for its disclosure prior to the filing date and should not be construed as an admission that this disclosure has no right to precede that publication by virtue of a prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

[0031] It should be noted that, unless the context clearly indicates otherwise, as used herein and in the appended claims, the absence of a quantifier before an element includes both singular and plural forms. It should also be noted that the claims may exclude any optional element. Therefore, this statement is intended to serve as a preliminary basis for the use of exclusive terms such as “solely” or “only” or for the use of negative limitations when relating to the elements of a claim.

[0032] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has independent components and features, which can be readily separated from or combined with features of any other several embodiments without departing from the scope or spirit of this disclosure. Any described method may be performed in the order of the described events or in any other logically possible order.

[0033] Although the system and method have been or will be described and their functions explained for grammatical fluency, it should be clearly understood that, unless expressly provided for in Chapter 35 of the United States Code, a claim shall not in any case be construed as necessarily being limited to “method” or “step”, but shall conform to the judicial principles of equivalence and the meaning and full scope of the equivalent as defined in the claim. When a claim is explicitly drafted in accordance with Section 112 of Chapter 35 of the United States Code, the claim shall be in full conformity with the legal equivalents in Section 112 of Chapter 35 of the United States Code.

[0034] Flow chamber

[0035] As described above, aspects of this disclosure include flow chambers for use in flow cytometry. The flow chamber of interest includes: a flow chamber body for transporting particles in a central flow of the flow from a proximal end to a distal end, wherein the flow chamber body includes a flow chamber cone structure located at the proximal end; and a sample injection needle having a through-through (e.g., continuously through-through) passage for transporting sample fluid from a sample injection tube at the proximal end to the flow chamber body at the distal end to generate a central flow, wherein the sample injection needle includes: a sample injection needle adapter including a sample tube adapter attached to the needle; and a clamp that operatively connects the sample injection needle to the flow chamber body. The term "central flow" as used herein refers to a fluid flow (i.e., a flow) described in its conventional sense, in which particles are focused (e.g., by hydrodynamic focusing) by a sheath fluid flow and transported through the flow chamber. Typically, particles are transported through the central flow in a single-file manner. The dimensions (e.g., diameter) of the central flow can be varied as desired.

[0036] In some cases, the diameter of the central flow can be approximately the same as the diameter of the particles being analyzed. In some cases, the diameter of the central flow is between 5 μm and 25 μm, including 10 μm to 20 μm. The diameter of the central flow can be adjusted proportionally according to the pressure (e.g., positive or negative pressure) applied when injecting the particles into the sheath fluid flow. In some cases, the flow rate of the sheath fluid remains constant. In this way, the particles are injected into the sheath fluid and hydrodynamically focused, resulting in laminar flow, and the particles move along the same axis at approximately the same speed.

[0037] A "complete" central flow can be described if the central flow associated with this disclosure maintains a relatively constant shape over the entire length of the flow chamber. In some cases, the complete central flow of this disclosure is defined by straight edges. In other words, the boundary of the complete flow appears straight when viewed in two dimensions. In selected versions, the flow and its component central flow are hydrodynamically focused and are characterized as laminar flow. In some such versions, the straight edges of the central flow are substantially parallel to each other (e.g., if the edges deviate from true parallel lines by five degrees or less, such as two degrees or less). In some cases, the complete central flow is not characterized by disturbances from the distal end of the sample injection needle to the distal end of the flow chamber. In some such cases, the complete central flow does not include vortices (e.g., vortices around the tip of the sample injection needle). In some cases, the flow chamber and its component sample injection needle reduce flow disturbance and center flow disturbance by 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, and up to 100% compared to conventional instruments.

[0038] As described above, the sample injection needle is configured and positioned to maintain intact central flow under flow conditions varying by one order of magnitude or more. The term "order of magnitude"—also known as "magnitude"—is used in its conventional sense to refer to flow conditions differing by a factor of ten or more. In some cases, the sample injection needle is configured and positioned to maintain intact central flow under flow conditions varying by two orders of magnitude or more. For example, flow conditions described herein may include sheath fluid velocity, sample fluid velocity, particle size (e.g., diameter), pressure (positive or negative), etc. In some embodiments, the sample injection needle is configured and positioned to maintain intact central flow at sheath fluid velocities varying by one order of magnitude or more. For example, in some cases, the sample injection needle is configured and positioned to maintain intact central flow at flow velocities ranging from 0.5 m / s to 10 m / s. In selected cases, the sample injection needle is configured and positioned to maintain intact central flow at sample fluid velocities varying by one order of magnitude or more. For example, in some cases, the sample injection needle is configured and positioned to maintain a complete central flow at sample fluid flow rates from 1 μL / min to 150 μL / min.

[0039] As described herein, a "flow chamber," in its conventional sense, refers to a component containing a flow channel for a liquid flow stream used to transport particles in the sheath fluid (e.g., particles in the central flow of a flow stream as described above). Any convenient flow chamber for transferring a fluid sample to a sample detection area can be used as the flow chamber described herein, wherein in some embodiments, the flow chamber is: a cylindrical flow chamber, a truncated conical flow chamber, or a flow chamber comprising a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating in a plane having an orifice transverse to the longitudinal axis. The flow chamber of interest 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 discharging fluid. Depending on the flow cytometer configuration, the fluid at the distal end of the flow chamber may flow to one or more collection containers of different types.

[0040] The flow channel is configured for a flow stream, which may include a liquid sample injected from a sample tube through a sample injection line. In some embodiments, the flow stream may include a narrow, fast-flowing liquid stream arranged such that particles transported therein, separated in a straight line, are isolated from each other in a single file. In some cases, the flow chamber includes a light-accessible flow channel. In some cases, the flow chamber is configured to receive light irradiation from a light source at one or more detection points. As used herein, a “detection point” or “detection area” refers to an area within the flow chamber where particles are irradiated by light from a light source, for example, for analysis. The size of the detection point can vary as desired. For example, 0 μm represents the optical axis of the light emitted through the light source, and detection points can be from -100 μm to 100 μm, such as -50 μm to 50 μm, such as -25 μm to 40 μm, and include -15 μm to 30 μm. Depending on certain factors (e.g., the number and arrangement of lasers), multiple irradiation points may exist within the flow chamber.

[0041] main body of the flow chamber

[0042] The flow chamber of interest includes a flow chamber body having a proximal end and a distal end, wherein the flow chamber body includes a flow chamber conical structure at its proximal end (e.g., as described below). In some embodiments, the flow chamber of this disclosure includes a cuvette at the distal end of the flow chamber body for conveying particles in the flow stream (e.g., its central flow) through a sample detection point or region. The cuvette of interest includes a passageway (i.e., a flow channel) therethrough and a light-transmitting region or portion for receiving light from a light source (i.e., such that a sample detection point or region is formed as described below). In some embodiments, the light-transmitting portion of the cuvette is configured to allow optical detection of particles in the central flow. In some embodiments, the light-transmitting portion includes a light-transmitting solid. In other words, the light-transmitting portion may include a transparent material that allows light to pass through it (i.e., such that light can reach the region or portion). The cuvette may be made of materials such as quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is made of silica (e.g., fused silica).

[0043] In one embodiment, the cuvette (e.g., as described above) is positioned at the distal end of the flow chamber body by a clamp (referred to herein as a flow chamber body clamp or cuvette clamp) attached to the flow chamber body. The cuvette clamp can be secured to the flow chamber body by any suitable means. In some embodiments, the cuvette clamp is secured to the flow chamber body by fastening members (e.g., pins, screws, etc.). In some embodiments, the cuvette is releasably attached to the distal end of the flow chamber body by the cuvette clamp. In some cases, the positioning of the cuvette (i.e., its positioning relative to the rest of the flow chamber body and / or one or more light sources) can be adjusted after it has been attached to the flow chamber body by the cuvette clamp. In some embodiments, the positioning of the cuvette can be adjusted by removing the cuvette from the flow chamber body and reattaching it to the flow chamber body via the cuvette clamp. In some embodiments, the cuvette is positioned by the flow chamber body clamp such that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0044] The flow chamber body of interest includes a conical structure at its proximal end. "Flow chamber conical structure" refers to a conical (e.g., a perfect circular cone) recess in the flow chamber body that narrows towards the analytical region (i.e., the detection zone) of the flow chamber. The flow chamber conical structure can function in hydrodynamic focusing flows. The flow chamber conical structure described herein is characterized by an angle (i.e., the angle measured relative to the generatrix of the conical structure through an imaginary axis passing through its center) of 15° to 25°, for example 18° to 22°, or for example 19° to 21°. In some cases, the flow chamber conical structure described herein may be characterized by an angle of 20°.

[0045] In some embodiments, the flow cytometer (i.e., the flow chamber described herein, which will be connected thereto, as will be described in detail below) includes a sample fluid source. The sample fluid source can be any suitable reservoir or container (e.g., having rigid or flexible walls) for containing the sample fluid. The volume of the sample fluid container can be from 1 mL to 100 mL; for example, the volume of the container can be from 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 chamber (e.g., the sample injection needle of the flow chamber as described herein) via a sample injection line (e.g., a tubing).

[0046] In some embodiments, the flow cytometer includes a sheath fluid reservoir. The sheath fluid reservoir can be any suitable reservoir or container for containing sheath fluid (e.g., having rigid or flexible walls). In some embodiments, the sheath fluid reservoir is fluidly connected to the inlet of the flow chamber (e.g., the inlet of the flow chamber body described below). The volume of the sheath fluid container can be from 1 L to 100 L; for example, the container volume can be from 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 fluidly connected to a sheath fluid line configured to deliver sheath fluid from the reservoir to the flow chamber.

[0047] In some embodiments, the flow chamber body includes a sheath fluid inlet configured to supply sheath fluid to the flow chamber. The sheath fluid inlet is fluidly connected to a sheath fluid source (i.e., a reservoir) and supplies sheath fluid to the flow chamber body proximally. In other embodiments, the sheath fluid introduction system is configured to supply a sheath fluid flow to the inner chamber of the flow chamber body (e.g., a flow chamber conical structure of the flow chamber body), for example, to generate a lamellar sheath fluid flow around the sample flow (i.e., a lamellar sheath fluid flow around a central flow). Depending on the desired flow characteristics, the sheath fluid flow rate delivered to the flow chamber cavity (i.e., the cavity of the flow chamber body, such as the flow chamber conical structure of the flow chamber body) can be 25 μL / s or higher, for example 50 μL / s or higher, for example 75 μL / s or higher, for example 100 μL / s or higher, for example 250 μL / s or higher, for example 500 μL / s or higher, for example 750 μL / s or higher, for example 1000 μL / s or higher, and includes 2500 μL / s or higher. In some embodiments, the sheath fluid flow rate delivered to the flow chamber cavity can range from 25 μL / s to 2500 μL / s, for example 50 μL / s to 1000 μL / s, and includes 75 μL / s or higher to 750 μL / s.

[0048] In some embodiments, the sheath fluid inlet is an orifice located in the wall of the inner chamber. The sheath fluid inlet orifice can be of any suitable shape, with cross-sectional shapes of interest including, but not limited to: straight cross-sectional shapes, such as squares, rectangles, irregular quadrilaterals, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ovals; and irregular shapes, such as the bottom of a parabola connected to the top of a plane. The size of the sheath fluid inlet orifice can vary depending on the shape, and in some cases, its opening is 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0049] In some cases, one or more sheath fluid inlets are located within the conical structure of the flow chamber body (i.e., at the "base" of the conical structure). In some cases, the flow chamber body includes a single sheath fluid inlet. In other cases, the flow chamber body includes multiple sheath fluid inlets. The number of sheath fluid inlets among the multiple sheath fluid inlets can vary and includes, for example, 2, 3, 4, and 5 sheath fluid inlets. In a selected version, the flow chamber body includes 2 sheath fluid inlets. In some cases where the flow chamber body of this disclosure includes multiple sheath fluid inlets, the multiple sheath fluid inlets are offset from each other, causing the sheath fluid to rotate through the conical structure of the flow chamber (e.g., in a "toilet-like" manner). In some such cases, the multiple sheath fluid inlets are offset from each other, causing the sheath fluid to rotate clockwise. In other cases, the multiple sheath fluid inlets are offset from each other, causing the sheath fluid to rotate counterclockwise. In some cases, the rotation of the sheath fluid can also provide flow stability and center flow stability.

[0050] Sample injection needle

[0051] As described above, aspects of this disclosure include: a sample injection needle for operatively connecting a sample injection line to a flow chamber body (e.g., as described above). The sample injection needle includes a sample injection needle adapter and a clamp, wherein the clamp is configured to operatively connect the sample injection needle to the flow chamber body. The sample injection needle adapter of this disclosure includes: a sample tube adapter attached to the needle. The needle of the sample injection needle adapter of this disclosure includes a through-passage for conveying sample fluid from a sample injection line at a proximal end to a flow chamber body at a distal end. In some embodiments, the sample tube adapter (i.e., the sample injection needle adapter of this disclosure) includes a proximal end and a distal end, wherein at least the distal end is secured to the proximal end of the needle. In some embodiments, the clamp (i.e., the clamp of the sample injection needle, referred to herein as a clamp or sample injection needle clamp) is configured to operatively connect the sample injection needle adapter to the flow chamber body by pressing the sample tube adapter against the flow chamber body.

[0052] A sample injection needle holder of interest may include: a distal end configured to attach to a sample injection needle adapter; and a proximal end configured to fluidly connect a sample tubing to a needle of the sample injection needle adapter, wherein the holder is configured to operatively connect the sample injection needle adapter to the flow chamber body by pressing the sample tubing adapter against the flow chamber body. In some embodiments, the distal end of the holder includes a surface configured to contact the flow chamber body (e.g., the outermost edge or outermost surface of the flow chamber body near the proximal end of the flow chamber's tapered structure) when the holder presses the sample tubing adapter against the flow chamber body. In some embodiments, the distal end of the holder includes a recess configured to receive at least a portion of the sample tubing adapter. In these cases, the recess may include an inner surface concentric with the outer surface of said portion of the sample tubing adapter. In some embodiments, the recess includes multiple depth components. For example, the recess may include: a first depth member configured to receive a flange of a sample tube adapter (i.e., wherein the first depth member is recessed into the clamp from the outermost edge of the distal end of the clamp by a first distance or a first depth), and a second depth member configured to receive a portion of the sample tube adapter (i.e., wherein the second depth member is recessed into the clamp from the outermost edge of the distal end of the clamp by a second distance or a second depth), wherein the distance by which the second depth member is recessed into the clamp is greater than that of the first depth member.

[0053] In some embodiments, the recess is configured such that when the portion of the sample tube adapter is positioned within the recess, the sample needle adapter is rotatably movable relative to the clamp. Rotatability means that when the sample needle is attached to the flow chamber body (and when the clamp is not compressed to secure or fix the sample needle adapter, as described herein), the sample needle adapter can rotate about its central axis to adjust its orientation relative to the clamp (and, for example, the flow chamber body). In some embodiments, the sample needle adapter is capable of rotating 90 degrees or more about its central axis, for example, 180 degrees or more, or 270 degrees or more, or 360 degrees. In some cases, the sample needle adapter is capable of rotating a full circle about its central axis (i.e., capable of rotating 360 degrees or freely about its central axis) to adjust its orientation relative to the clamp (and, for example, the flow chamber body). The implementation of the flow chamber (in which the sample injection needle adapter is rotatably movable relative to the clamp and the flow chamber conical structure or freely rotatable about its central axis) is referred to herein as the non-clamping configuration of the flow chamber.

[0054] In some embodiments, the clamp is configured to receive fastening members (e.g., one or more pins, screws, rivets, etc.) for securing the clamp to the flow chamber body. In some embodiments, the clamp is configured such that when the sample needle adapter is operatively connected to the flow chamber body via the clamp, the compression of the fastening members against the clamp fixes the sample needle adapter relative to the flow chamber body. Fixing means that the movement of the sample needle adapter relative to the flow chamber body (and its flow chamber conical structure) is restricted. In some embodiments, the movement of the sample needle adapter relative to the flow chamber body and the clamp is completely restricted (i.e., the sample needle adapter is fixed relative to the flow chamber body and the clamp). Embodiments of the flow chamber in which the sample needle adapter is fixed relative to the clamp and the flow chamber body (e.g., where the sample needle adapter is fixed relative to the flow chamber body and the clamp) are referred to herein as a clamping configuration of the flow chamber.

[0055] In some embodiments, the fastening member comprises a plurality of screws. In some embodiments, the clamp includes a set of holes for receiving each of the plurality of screws. In some embodiments, the clamp includes three holes. In these cases, the set of holes can be configured to align with a set of holes on the flow chamber body (e.g., located at the outermost edge or outermost surface of the proximal end of the flow chamber body). In some embodiments, the clamp is configured such that when the sample injection needle (and its sample injection needle adapter) is operatively connected to the flow chamber body via the clamp, the tilt of the sample injection needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws. In other words, the angle of the sample injection needle adapter relative to the flow chamber body can be adjusted by changing the torque of one or more of the plurality of screws (e.g., tightening or loosening). In these cases, the clamp can be configured such that when the sample injection needle is operatively connected to the flow chamber body via the clamp, the position of the distal end of the needle of the sample injection needle adapter within the flow chamber conical structure of the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws. For example, adjusting the torque of one of a plurality of screws can pivot the distal end of the needle about an axis. In some embodiments, manipulating the torque of at least one of the plurality of screws can achieve micron-level adjustment of the distal end of the needle of the sample injection needle adapter.

[0056] 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 tubing to the needle of the sample injection needle adapter. Dead volume refers to regions within the sample injection tubing or needle (e.g., the area where the sample injection tubing interfaces with the sample injection needle) where sample fluid may be trapped, stagnant, or unutilized and cannot flow effectively through the system (i.e., a flow cytometry system in which the flow chamber is operatively positioned). In these cases, the connector can reduce dead space or voids, such as dead space and voids within the joint, thereby minimizing the dead volume of the sample fluid as it flows from the sample injection tubing to the needle of the sample injection needle adapter. For example, the connector can reduce the number of joints through which the sample fluid flows. In some embodiments, the proximal end of the clamp is configured to position a flowmeter plate connector.

[0057] As described above, the sample injection needle adapter of interest (i.e., for operatively connecting a sample injection line to a flow chamber body) may include: a needle having a through passage for delivering sample fluid from a sample injection line at a proximal end to a flow chamber body at a distal end; and a sample tube adapter including a proximal end and a distal end, the proximal end being configured to attach to a clamp, and the distal end being secured to the proximal end of the needle. In some embodiments, the sample tube adapter is configured such that when the clamp presses the sample tube adapter against the flow chamber body, the needle of the sample injection needle adapter is operatively connected to the flow chamber body. In some embodiments, the sample tube adapter is configured such that when the clamp operatively connects the sample tube adapter to the flow chamber body, the compression action of the clamp on the sample tube adapter fixes the sample injection needle adapter relative to the flow chamber body. In these cases, the sample tube adapter can be fixed such that the distal end of the needle of the sample injection needle adapter is located in a fixed position within the flow chamber cone structure. The fixed position of the distal end of the sample injection needle adapter relative to the flow chamber conical structure (e.g., and within the flow chamber body) can be described in several ways. For example, in some cases, the fixed position of the distal end of the sample injection needle adapter is described relative to the location of one or more sheath fluid inlets. For example, in some cases, the distal end of the needle is positioned within the flow chamber conical structure and separated from the sheath fluid inlet by a longitudinal distance ranging from 15 mm to 30 mm, such as 17 mm to 26 mm, including 20 mm to 22 mm. The positioning of the distal end of the needle can also be described relative to the detection point (i.e., the point where the flow chamber is illuminated by one or more light sources). In some cases, the distal end of the needle is separated from the detection point by a distance ranging from 10 mm to 20 mm, such as 13 mm to 17 mm, including 14 mm to 16 mm. In some embodiments, the distal end of the sample injection needle adapter is fixed such that a complete central flow is maintained within the flow chamber conical structure even when flow conditions change by an order of magnitude or more.

[0058] In some embodiments, the sample injection needle adapter is configured such that, when operatively connected to the flow chamber body via a clamp, the tilt of the sample injection needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of a plurality of screws that fasten the clamp to the flow chamber body (e.g., as described above). In some cases, by adjusting the tilt of the sample injection needle adapter, a complete central flow can be maintained within the conical structure of the flow chamber despite variations in flow conditions by one or more orders of magnitude.

[0059] 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 (e.g., as described above). In these cases, said 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, said portion of the proximal end of the sample tube adapter is configured such that when said portion of the sample tube adapter is positioned within the recess (e.g., as described above), the sample injection needle adapter can be rotatably moved 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 chamber body. In these cases, said portion of the distal end of the sample tube adapter may include an outer surface concentric with the inner surface of the flow chamber body's opening leading to the flow chamber cone structure. In some embodiments, the sample tube adapter includes a flange for positioning said portion of the distal end of the sample tube adapter within the flow chamber body near the flow chamber cone structure. In these cases, the inner surface of the opening of the flow chamber body may include a crossbar or step for pressing against the flange. In some embodiments, the flange is configured to position the distal portion of the sample tube adapter within the flow chamber body such that the distal end of the needle is separated from the sheath fluid inlet of the flow chamber body by a longitudinal distance ranging from 17 mm to 26 mm.

[0060] The needle of the sample injection needle adapter disclosed herein may include an elongated structure. An "elongated structure" means that the length of the needle is greater than its width. In other words, the needle of the sample injection needle adapter has a clearly distinguishable proximal and distal end. The proximal end is the end of the needle that receives sample fluid (i.e., receives sample fluid from a sample injection line connected to a sample fluid source fluid), while the distal end is the end of the sample injection needle that injects the sample into a flow chamber (e.g., a flow chamber conical structure). The elongated structure can have any convenient cross-sectional shape, wherein the cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes, such as squares, rectangles, irregular quadrilaterals, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ovals; and irregular shapes, such as the bottom of a parabola connected to the top of a plane. In embodiments, the elongated structure has a substantially circular cross-sectional shape at locations along its length. "Substantially" circular means that, in embodiments, the cross-section at one or more locations along the length of the outlet fitting may slightly deviate from a circular cross-section, while the remainder of the structure is characterized by a circular cross-section. 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 some cases, the width (e.g., cross-sectional diameter) of the elongated structure varies along the length of the outlet fitting. In other words, in these versions, the elongated structure is not a perfect cylinder, but rather the diameter of the cross-sectional shape in some regions is larger or smaller than the diameter of the cross-sectional shape in other regions. In other cases, each part of the needle of the sample injection needle adapter has a circular cross-section. In some such cases, the different parts of the needle may be characterized by different diameters. In some cases, the outer diameter of at least a portion of the needle of the sample injection needle adapter (i.e., the diameter measured from the geometric center of the sample injection needle to the outer edge) is 0.5 mm to 4 mm, such as 0.75 mm to 3 mm, such as 1 mm to 2 mm, such as 1.25 mm to 1.75 mm, and includes 1.5 mm to 1.6 mm. In some cases, the outer diameter of the needle is 1.562 mm. The needle of the sample injection needle adapter can have any convenient length, with the length of interest ranging from 15 mm to 30 mm, such as 17 mm to 26 mm, and including 20 mm to 22 mm. In some cases, the needle of the sample injection needle adapter does not flatten at the distal end, for example, forming a "duckbill" shape. In these cases, the needle has a substantially circular cross-section throughout its length.

[0061] In some cases, the needle of a sample injection needle adapter includes a tapering structure at its distal end. In some such cases, the sample injection needle has a relatively constant outer diameter over most of its length (i.e., starting from the proximal end), but the outer diameter gradually (e.g., uniformly) decreases towards the distal end. In some embodiments, the needle of the sample injection needle adapter includes a tapering structure with a radius of 2.5 mm to 12 mm over a length span of 1.75 mm to 4 mm. For example, in some versions, the tapering structure of the needle has a radius of 2.79 mm to 11.63 mm over a length span of 1.78 mm to 3.81 mm. The tapering structure may begin at different locations along the length of the needle. In some cases, the distance between the starting point of the tapering structure along the length of the needle and the distal end is 3 mm to 5 mm, such as 3.5 mm to 4.0 mm, and includes 3.8 mm to 3.9 mm. In some cases, such a distal end may be referred to as having a "super bullet" structure. In some embodiments with a super-bullet structure, the distal taper has a radius of 11.63 mm within a length span of 3.81 mm. In other cases, the distance between the starting point of the taper and the distal end along the needle length direction is 1 mm to 3 mm, such as 1.5 mm to 2 mm, and includes 1.7 mm to 1.8 mm. In some cases, such a distal end may be referred to as having a "bullet" structure. In some embodiments of the sample injection needle adapter needle having a bullet structure, the distal taper has a radius of 2.79 mm within a length span of 1.78 mm. In other cases, the sample injection needle adapter needle includes a rounded distal end.

[0062] The needle of the sample injection needle adapter disclosed herein further includes a distal opening and a through channel for conveying a sample fluid—optionally containing particles—to a flow chamber (e.g., a flow chamber conical structure). In embodiments, the opening is located at the geometric center of the cross-section of the distal outlet fitting. The opening can have any convenient cross-sectional shape, wherein the cross-sectional shapes of interest include, but are not limited to: linear cross-sectional shapes, such as squares, rectangles, irregular quadrilaterals, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ovals; and irregular shapes, such as the base of a parabola connected to the top of a plane. In some cases, the opening has a circular cross-sectional shape. In other cases, the channel similarly comprises a circular cross-section. The opening can have any suitable diameter, such as 0.1 mm to 2 mm, 0.1 mm to 1 mm, 0.2 mm to 0.4 mm, including 0.25 mm to 0.30 mm. Similarly, the channel can have any suitable diameter, such as 0.1 mm to 2 mm, 0.1 mm to 1 mm, 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-section with the same or similar diameters. In other cases, the diameter of the opening is different from (e.g., larger than) the diameter of the channel.

[0063] The flow cell of this disclosure, when assembled for use, includes a sample injection needle operatively connected to the flow cell body (e.g., the sample injection needle is connected 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 clamp hole. In some embodiments, the flow cell of this disclosure is configured after assembly such that: the distal end of the sample injection needle is in a fixed position (e.g., as described above), and / or the position of the sample injection needle relative to the flow cell body is fixed by compression of the clamp (e.g., as described above). In some cases, the assembled flow cell requires adjustment before or after operatively positioning with a flow cytometer. In some embodiments, the assembled flow cell is operatively positioned with or after the flow cytometer. For example, the torque of one or more screws used to fasten the sample injection needle to the flow cell body can be adjusted based on operator observation. In some cases, after the assembled flow cell is operatively positioned with the flow cytometer using a cuvette clamp (e.g., as described above), the cuvette of the flow cell body is aligned with a light source (i.e., forming a sample detection area).

[0064] Figure 1A Provide exploded diagrams of the flow chamber according to certain implementation schemes. For example... Figure 1AAs shown, the sample injection needle 120 includes a sample injection needle adapter (which has a sample tube adapter 122 and a needle 121) and a clamp 123. A fastening member 130 (i.e., a screw) is configured to insert into a pair of holes in the clamp 123 and the flow chamber body 110 to secure the sample injection needle to the flow chamber body. A sheath fluid inlet nozzle 111 of the sheath fluid inlet of the flow chamber body is configured to introduce sheath fluid into the flow chamber conical structure of the flow chamber body. A cuvette clamp 112 is fastened to the flow chamber body 110 and is used to position the cuvette 140 at the distal end of the flow chamber body in a manner capable of forming a sample detection area. O-rings 141 and 142 ensure fluid connection of the cuvette to the flow chamber body in a manner that allows a continuous central flow throughout the cuvette. Accessory 150 and fastening member 151 allow the flow chamber nozzle 160 to be fluidly coupled to an end of the flow chamber body, for example, for sorting particles into different containers. Figure 1B Provided fully assembled according to certain implementation schemes Figure 1A Various views of the flow chamber are shown.

[0065] Figure 1C Various views of the fully assembled flow chamber according to certain implementation schemes are provided. Figure 1D A cross-sectional view of a sample injection needle according to certain embodiments is provided, the sample injection needle having a sample injection needle adapter (sample tube adapter 122 and needle 121) and a clamp 123.

[0066] Figure 1E A sample injection needle according to certain embodiments is shown, the sample injection needle having a sample injection needle adapter (sample tube adapter 122 and needle 121) and a clamp 123. Figure 1F Also shown is a clamp according to some embodiments, having a sample injection tubing connector at its proximal end, which is attached to the distal end of a sample injection needle adapter. The connector is configured to reduce dead space and voids within the engagement to minimize the dead volume of the sample fluid as it flows from the sample injection tubing to the needle of the sample injection needle adapter.

[0067] Assembly method of flow chamber

[0068] As described above, aspects of this disclosure also include methods for assembling a flow chamber for use with a flow cytometer. Methods of interest include: operatively attaching a sample injection needle to the flow chamber body using a sample injection needle clamp (e.g., as described above). In some embodiments, the sample injection needle is operatively attached to the flow chamber body by pressing a sample tube adapter against the flow chamber body using a clamp. In some cases, the method further includes: securing the sample injection needle adapter by compressing the clamp (e.g., such that the distal end of the needle of the sample injection needle adapter is secured at a location within the flow chamber conical structure, as described above).

[0069] In some embodiments, the method further includes inserting a plurality of screws into a set of holes in a clamp and a set of holes in a flow chamber body, wherein the set of holes in the clamp is aligned with the set of holes in the flow chamber body. In some embodiments, the method further includes adjusting the tilt of the sample injection needle relative to the flow chamber body by manipulating the torque of at least one of the plurality of screws (e.g., as described above). In these cases, the position of the distal end of the sample injection needle adapter can be adjusted by pivoting the distal end of the sample injection needle adapter about an axis—e.g., by individually adjusting the torque of one of the plurality of screws. In some embodiments, the method further includes adjusting the rotational position of the sample injection needle adapter before securing it with the clamp.

[0070] In some embodiments, the assembly method further includes attaching a sample tube adapter to a clamp before operatively connecting the sample injection needle to the flow chamber body. For example, a portion of the sample tube adapter may be positioned within a recess in the clamp (e.g., as described above). In some embodiments, the method further includes positioning a cuvette of the flow chamber body at a distal end of the flow chamber body using a cuvette clamp attached to the flow chamber body (e.g., as described above). In these cases, the cuvette can be positioned by the flow chamber body clamp such that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0071] In some embodiments, the assembly method further includes operatively positioning the flow chamber (including the flow chamber body and sample injection needle as described above) into a flow cytometer. A flow cytometer suitable for operative connection with the flow chamber of this disclosure will be described in more detail below. In some cases, operatively positioning the flow chamber into the flow cytometer includes using a clamp to fluidly connect the sample injection tubing to the needle of the sample injection needle adapter. In some embodiments, operatively positioning the flow chamber into the flow cytometer includes aligning a cuvette located at the distal end of the flow chamber body for delivering particles in the central stream through the sample detection area with a light source of the flow cytometer to irradiate the particles in the central stream in the sample detection area. In some embodiments, operatively positioning the flow chamber into the flow cytometer includes optically coupling a detector of the flow cytometer, configured to collect light emitted by the irradiated particles, to the sample detection area.

[0072] Flow cytometer

[0073] This disclosure also includes aspects of flow cytometers. Flow cytometers of interest include the flow cell of this disclosure. As detailed above, the flow cell of interest includes a sample injection needle (including the sample injection needle adapter and clamp as described above), which is operatively connected to the flow cell body (including the flow cell conical structure as described above) via the sample injection needle clamp. Furthermore, the flow cytometer of this disclosure includes: a light source configured to irradiate particles in the flow stream at a detection point within the flow cell, and a detector configured to collect light emitted by the irradiated particles.

[0074] The flow cytometer disclosed herein includes a light source configured to illuminate particles in a flow stream at a detection point within a flow chamber. 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, and includes 2 to 4. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser can be any convenient laser, such as a continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer includes dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers. In still other cases, lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other cases, the flow cytometer includes a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:YCa4O(BO3)3 laser, an Nd:YCOB laser, a titanite laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, or combinations thereof.

[0075] According to some embodiments, the laser source may also include one or more optical adjustment elements. In some embodiments, the optical adjustment elements are positioned between the source and the flow chamber, and may include any device capable of altering the spatial width of illumination from the source or certain other characteristics of the illumination, such as illumination direction, wavelength, beam width, beam intensity, and focal spot. Optical adjustment schemes may include any convenient devices for adjusting one or more characteristics of the source, including but not limited to lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation schemes, and combinations thereof. In some embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens may be a reducing lens. In other embodiments, the flow cytometer of interest includes optical fibers.

[0076] When the optical adjustment element is configured to move, it can be configured to move continuously or at discrete intervals, such as increments of 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1 mm or more, such as 5 mm or more, such as increments of 10 mm or more, and including increments of 25 mm or more.

[0077] The optical adjustment element structure can be moved using any displacement scheme, such as connecting it to a movable support platform, or directly connecting it to a motor-driven translation stage, lead screw translation assembly, or gear translation device, such as using stepper motors, servo motors, brushless motors, brushed DC motors, micro-stepping drive motors, high-resolution stepper motors, etc.

[0078] The light source can be positioned at any suitable distance from the flow chamber, such as 0.005 mm or greater, 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 5 mm or greater, 10 mm or greater, 25 mm or greater, and including 100 mm or greater. Furthermore, the light source can be positioned at any suitable angle relative to the flow chamber, such as angles from 10 to 90 degrees, from 15 to 85 degrees, from 20 to 80 degrees, from 25 to 75 degrees, and including 30 to 60 degrees, such as an angle of 90 degrees.

[0079] In some embodiments, the light source of interest includes multiple lasers configured to provide laser light for discretely irradiating the flowing stream, such as two or more lasers, three or more lasers, four or more lasers, five or more lasers, ten or more lasers, and even fifteen or more lasers configured to provide laser light for discretely irradiating the flowing stream. Depending on the wavelength of the light desired for irradiating the flowing stream, the specific wavelength of each laser can vary from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, and includes variations from 400 nm to 800 nm. In some embodiments, the lasers of interest may include one or more 405 nm, 488 nm, 561 nm, and 635 nm lasers.

[0080] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some cases, the beam generator includes a laser and a radio frequency (RF) generator, the RF generator being configured to apply an RF drive signal to the acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, lasers in the beam generator of interest include those listed above.

[0081] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. In this system, the acousto-optic device is configured to generate an angle-deflected laser beam using light emitted from a laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device from any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0082] In some implementations, the controller is configured to apply radio frequency (RF) drive signals to the acousto-optic device to generate a desired number of angle-deflected laser beams in the output laser beam. For example, it is configured to apply 3 or more RF drive signals, such as 4 or more RF drive signals, such as 5 or more RF drive signals, such as 6 or more RF drive signals, such as 7 or more RF drive signals, such as 8 or more RF drive signals, such as 9 or more RF drive signals, such as 10 or more RF drive signals, such as 15 or more RF drive signals, such as 25 or more RF drive signals, such as 50 or more RF drive signals, and is configured to apply 100 or more RF drive signals.

[0083] In some cases, in order to generate an intensity distribution of an angle-deflected laser beam in the output laser beam, the controller is configured to apply an RF drive signal with varying amplitude, 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, including from about 5V to about 25V. In some embodiments, the frequency of each applied radio frequency drive signal is from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and includes from about 5 MHz to about 50 MHz.

[0084] In some embodiments, the controller has a processor with a memory operatively connected to the processor such that the memory contains instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with angled deflections of a desired intensity distribution. For example, the memory may contain instructions for generating two or more (e.g., three or more, four or more, five or more, ten or more, twenty-five or more, fifty or more) angled laser beams of equal intensity; the memory may contain instructions for generating one hundred or more angled laser beams of equal intensity. In other embodiments, the memory may include instructions for generating two or more (e.g., three or more, four or more, five or more, ten or more, twenty or more, fifty or more) angle-deflected laser beams with different intensities, and the memory may contain instructions for generating 100 or more angle-deflected laser beams with different intensities.

[0085] In some embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity gradually increases from the edge along the horizontal axis towards the center. In these cases, the range of intensity of the angularly deflected laser beam at the center of the output beam can be from 0.1% to about 99%, for example 0.5% to about 95%, for example 1% to about 90%, for example 2% to about 85%, for example 3% to about 80%, for example 4% to about 75%, for example 5% to about 70%, for example 6% to about 65%, for example 7% to about 60%, for example 8% to about 55%, and includes about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge along the horizontal axis. In other embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity gradually increases from its edge along a horizontal axis towards its center. In these cases, the range of angular deflection laser beam intensity at the edge of the output beam can be from 0.1% to 99% of the intensity of the deflection laser beam at the center of the output laser beam along the horizontal axis, for example, 0.5% to 95%, for example, 1% to 90%, for example, 2% to 85%, for example, 3% to 80%, for example, 4% to 75%, for example, 5% to 70%, for example, 6% to 65%, for example, 7% to 60%, for example, 8% to 55%, and includes 10% to 50% of the intensity of the deflection laser beam at the center of the output laser beam along the horizontal axis. In some other embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a Gaussian intensity profile along a horizontal axis. In still other embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a flat-topped intensity profile along a horizontal axis.

[0086] In an embodiment, the beam generator of interest can be configured to generate spatially separated, angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired output laser beam illumination profile, the spacing between the angle-deflected laser beams can be 0.001 μm or greater than 0.001 μm, for example 0.005 μm or greater, for example 0.01 μm or greater, for example 0.05 μm or greater, for example 0.1 μm or greater, for example 0.5 μm or greater, for example 1 μm or greater, for example 5 μm or greater, for example 10 μm or greater, for example 100 μm or greater, for example 500 μm or greater, for example 1000 μm or greater, and includes 5000 μm or greater. In some embodiments, the system is configured to generate angle-deflected laser beams in the output laser beam, which overlap, for example, with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap (e.g., spot overlap) between adjacent angle-deflected laser beams can be 0.001 μm or greater, such as 0.005 μm or greater, 0.01 μm or greater, 0.05 μm or greater, 0.1 μm or greater, 0.5 μm or greater, 1 μm or greater, 5 μm or greater, 10 μm or greater, and includes overlaps of 100 μm or greater.

[0087] In some cases, beam generators configured to produce two or more frequency-shifted beams include those described in 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, and 10,684,211. Laser excitation modules as described in U.S. Patent Nos. 10,845,295, 10,935,482, 10,935,485, 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; the disclosures of these U.S. Patents are incorporated herein by reference.

[0088] After the particles are irradiated in the flow chamber, particle-modulated light can be observed. "Particle-modulated light" refers to the light received from the particles in the flow stream after they have been irradiated by light from a light source. In some cases, particle-modulated light is side-scattered light. As described herein, side-scattered light refers to light refracted and reflected from the surface and internal structure of the particles. In other embodiments, particle-modulated light includes forward-scattered light (i.e., light that passes through or around the particles primarily in a forward direction). In still other cases, particle-modulated light includes fluorescence (i.e., light emitted from fluorescein after irradiation with light of the excitation wavelength).

[0089] As described above, a flow cytometer includes detectors configured to collect light emitted by irradiated particles (i.e., particle-modulated light). These photodetectors are configured to detect the particle-modulated light transmitted by an fiber optic light collection element and generate a signal based on the characteristics of the light, such as intensity. For example, one or more particle-modulated photodetectors may include one or more side-scatter detectors for detecting wavelengths of side-scattered 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 detector. In other embodiments, the flow cytometer includes multiple side-scatter detectors, such as two or more, three or more, four or more, and including five or more.

[0090] In some embodiments, the detectors for one or more particle-modulated light include one or more forward-scattered light detectors configured to detect forward-scattered light. For example, the flow cytometer may include one or more forward-scattered light detectors, such as two or more, three or more, four or more, and including five or more. In some embodiments, the flow cytometer includes one forward-scattered light detector. In other embodiments, the flow cytometer includes two forward-scattered light detectors.

[0091] In the implementation, the forward-scattering light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector of interest is configured to continuously measure the collected light. In other cases, the detector of interest is configured to measure 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 including every 1000 milliseconds or some other interval.

[0092] The side-scattering light detector described herein can be any convenient detector used to detect the collected light. Detectors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-connected devices (CCDs), enhancement-mode charge-connected devices (ICCDs), light-emitting diodes, photon counters, radiometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or combinations thereof, and other detectors. In some embodiments, the collected light is measured using a charge-connected device (CCD), semiconductor charge-connected device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, for example, with an active detection surface area of ​​0.01 cm² per region. 2 Up to 10cm 2 For example, 0.05cm 2 Up to 9cm 2 For example, 0.1cm 2 Up to 8cm 2 For example, 0.5cm 2 Up to 7cm 2 And including 1cm 2 up to 5cm 2 Photomultiplier tubes.

[0093] In one embodiment, the flow cytometer further includes a fluorescence detector configured to detect light of one or more fluorescence wavelengths. In other embodiments, the flow cytometer includes multiple fluorescence detectors, such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, including twenty or more.

[0094] The fluorescence detector described herein can be any convenient detector used to detect the collected light. Detectors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-connected devices (CCDs), enhancement-mode charge-connected devices (ICCDs), light-emitting diodes, photon counters, radiometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or combinations thereof, and other detectors. In some embodiments, the collected light is measured using a charge-connected device (CCD), semiconductor charge-connected device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, for example, with an active detection surface area of ​​0.01 cm² per region. 2 Up to 10cm 2 For example, 0.05cm 2 Up to 9cm 2 For example, 0.1cm 2 Up to 8cm 2 For example, 0.5cm 2 Up to 7cm 2 And including 1cm 2 up to 5cm 2 Photomultiplier tubes.

[0095] When the flow cytometer includes multiple fluorescence detectors, each fluorescence detector can be identical, or the collection of fluorescence detectors can be a combination of detectors of different types. For example, when the flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector (or imaging sensor) is a CMOS-type device. In other embodiments, both the first and second fluorescence detectors are CCD-type devices. In other embodiments, both the first and second fluorescence detectors are CMOS-type devices. In other embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector is a photomultiplier tube (PMT). In other embodiments, the first fluorescence detector is a CMOS-type device and the second fluorescence detector is a photomultiplier tube. In other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0096] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure one or more wavelengths of light collected, such as two or more wavelengths, five or more wavelengths, ten or more wavelengths, 25 or more wavelengths, 50 or more wavelengths, 100 or more wavelengths, 200 or more wavelengths, 300 or more wavelengths, and includes measuring 400 or more wavelengths of light emitted by a sample in a flowing stream. In some embodiments, two or more detectors in the module described herein are configured to measure collected light of the same or overlapping wavelengths.

[0097] In some embodiments, the detector of interest is configured to measure collected light within a wavelength range (e.g., 200 nm to 1000 nm). In some embodiments, the detector of interest is configured to collect the spectrum of light within a wavelength range. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light within a wavelength range of 200 nm to 1000 nm. In other embodiments, the detector of interest is configured to measure light emitted by a sample in a flowing 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 some implementations, one or more detectors may be configured to pair with a specific fluorophore, such as a fluorophore used with the sample in a fluorescence assay.

[0098] Flow cytometers may include any suitable mechanisms for supplying sheath fluid and sample fluid to the sample fluid inlet connector and the sheath fluid inlet connector. For example, the sample fluid inlet connector may be fluidly connected to a sample fluid line (e.g., a conduit) that is in turn fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid inlet connector may be fluidly connected to a sheath fluid line that is in turn fluidly connected to a sheath fluid reservoir. Similarly, flow cytometers may include any suitable mechanisms for handling waste fluid from the flowing stream. A fluid outlet connector may be fluidly connected to a waste fluid line that is in turn fluidly connected to a waste fluid reservoir. A fluid management system suitable for the flow cytometer is provided in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0099] In some embodiments, the flow cytometer includes one or more wavelength separators positioned between the flow chamber and one or more detectors of particle-modulated light. As used herein, the term "wavelength separator" refers in its conventional sense to an optical element 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, such as two or more, 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, seven-five or more, seven-five or more, and even one hundred or more wavelength separators. In some embodiments, the wavelength splitter is configured to separate the light collected from the sample into a predetermined spectral range by passing light having a predetermined spectral range and reflecting light with one or more remaining spectral ranges. In other embodiments, the wavelength splitter is configured to separate the light collected from the sample into a predetermined spectral range by passing light having a predetermined spectral range and absorbing light with one or more remaining spectral ranges. In other embodiments, the wavelength splitter is configured to spatially diffract the light collected from the sample into the predetermined spectral range. Each wavelength splitter can be any convenient light separation scheme, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength splitter is a prism. In other embodiments, the wavelength splitter is a diffraction grating. In some embodiments, the wavelength splitter in the light detection system is a dichroic mirror.

[0100] Suitable flow cytometry systems may include, but are not limited to, those described in the following literature: 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 Drug Carrier Syst. 24(3):203-255; its published content is incorporated herein by reference. In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto. TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BDBiosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BDBiosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BDBiosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TMFlow cytometer and BD Biosciences FACSCalibur TM Cell sorting instrument, BDBiosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorting instrument, BDBiosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorting instrument, BD Biosciences FACSAria TM II Cell Sorter, BD Biosciences FACSAria TM III Cell Sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorting instrument, BDBiosciences FACSymphony TM S6 cell sorter, BD Biosciences FACSDiscover™ cell sorter, etc.

[0101] In some embodiments, the system is a flow cytometry system, such as the flow cytometry systems described in the following documents: 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; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,0 No. 97,640; No. 9,095,494; No. 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. 6,8 No. 13,017; No. 6,809,804; No. 6,372,506; No. 5,700,692; No. 5,643,796; No. 5,627,040; No. 5,620,842; No. 5,602,039; No. 4,987,086; No. 4,498,766; the contents of which are incorporated herein by reference in their entirety.

[0102] In some implementations, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the system is a flow cytometry system configured to image particles in the flowing stream using fluorescence imaging using radiofrequency tagged emission (FIRE), as described in Diebold et al., Nature Photonics. Vol. 7(10); 806-810(2013) and as described in 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 U.S. Patent Nos. 10,845,295, 10,935,482, 10,935,485, 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, the disclosures of which are incorporated herein by reference. In some embodiments, when the flow cytometer is a particle sorter, the particle sorter is an image-assisted particle sorter. Image-assisted particle sorting devices are described in U.S. Patent Nos. 10,324,019, 10,620,111, 11,105,728, 11,774,343, and U.S. Patent Application Nos. 18 / 537,103, 18 / 657,618, 18,657,623, and 18 / 657,633; the entire contents of these patents and patent applications are incorporated herein by reference.

[0103] Figure 2 A flow cytometry system 200 according to one illustrative embodiment of the present disclosure is shown. System 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at a detection point 215 within a flow chamber 210. Although Figure 2The example shows a single laser, but it should be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to focusing lens 202, which focuses the beam onto the fluid flow portion containing sample particles 211 within flow chamber 210. Flow chamber 210 is part of a flow control system that directs particles (typically one at a time) in a flow to the focused laser beam for detection. Alternatively, if the flow cytometer is an air flow cytometer, a nozzle can be used.

[0104] like Figure 2 As shown, the flow chamber 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid inlet 208 via a conduit (i.e., a sheath fluid line) 207. Furthermore, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., a sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample syringe 213 (e.g., a sample injection needle) configured to introduce particles 211 into the interior of the flow chamber body 210. The particles 211 are hydrodynamically focused via the sheath fluid entering from the sheath fluid inlet 208, thereby forming a flow stream 214 downstream of the conical portion 212 of the flow chamber 210. Particles discharged from the distal end of the flow chamber body 210 can be disposed of and / or collected using any suitable method. For example, depending on the type of flow cytometry being performed, particles can be collected at the distal end of the flow chamber body 210, for example, via a waste liquid line. Alternatively, the particles can be sorted.

[0105] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at several different wavelengths depending on the characteristics of the particles, such as size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be directed to one or more detectors. Specifically, forward scattered light (FSC) is directed to forward scattered light detector 223. Forward scattered light detector 223 is positioned slightly off-center from the axis of the direct beam passing through flow cell 210 and is configured to detect diffracted light, i.e., excitation light that passes through or around the particles primarily in the forward direction. The intensity of the light detected by forward scattered light detector 223 depends on the overall size of the particles. The forward scattered light detector may include, for example, a photodiode. Optical filters 221a and scattering blocks 222 are arranged between the forward scattered light detectors 223. The optical filter 221a can be configured to filter out non-FSC light of at least one wavelength, while the scattering blocker 222 can be configured to prevent the incident beam (i.e. non-scattered light) from the laser 201 from being detected by the forward scattering detector 223.

[0106] Furthermore, side-scattered light (SSC) is detected by side-scattered light detector 224. In other words, side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of particle 211, the intensity of which tends to increase with increasing particle structure complexity. Figure 2 In the example, the flow cytometer 200 includes: a dichroic mirror 220a configured to reflect SSC light to a side-scatter light detector 224 while allowing non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to block non-SSC light of at least one wavelength from being detected by the side-scatter light detector 224. Fluorescence detectors 225a-225c are also shown in the figure, each configured to detect fluorescence of different wavelengths. 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 while allowing light of other wavelengths to pass through. The optical filter 221c may be configured to block light of at least one wavelength (which does not correspond to the first wavelength (or wavelength range)) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect fluorescence corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b, while allowing light of a third wavelength (or wavelength range) to be detected by the fluorescence detector 225c. The optical filter 221d is configured to block light of at least one wavelength (which does not correspond to the second wavelength (or wavelength range)) from being detected by the fluorescence detector 225b. Furthermore, the optical filter 221e is configured to block light of at least one wavelength (which does not correspond to the third wavelength (or wavelength range)) from being detected by the fluorescence detector 225c.

[0107] Those skilled in the art will recognize that the flow cytometer according to one embodiment of this disclosure is not limited to... Figure 2 The flow cytometer shown may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors, which may have different wavelengths and different configurations. For example, although Figure 2 The illustrated embodiment shows three fluorescence detectors for ease of explanation, but it should be understood that any suitable number of fluorescence detectors can be used.

[0108] During operation, the cytometer is controlled by a controller / processor 290, and measurement data from the detector can be stored in memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is connected to the detector to receive signals output by it, and can be connected to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. Input / output (I / O) capabilities 297 may also be provided in the system. Memory 295, controller / processor 290, and I / O 297 may be provided as an integral part of the flow cytometer. In such embodiments, a display for showing experimental data to the user of the cytometer 200 may also constitute part of the I / O capabilities 297. Alternatively, some or all of the memory 295 and controller / processor 290, as well as the I / O capabilities, 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 may be in wireless or wired communication with the cytometer 210. The controller / processor 290, together with memory 295 and I / O 297, can be configured to perform a variety of functions related to the preparation and analysis of flow cytometry experiments.

[0109] Different fluorescent molecules in the luciferin pairing scheme used in flow cytometry experiments emit light at their respective characteristic wavelengths. Specific fluorescent labels used in the experiment and their associated fluorescence emission wavelengths can be selected to approximately match the detector's filter window. I / O 297 can be configured to receive data regarding a flow cytometry experiment, having a set of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 297 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 flow cytometry configuration data. Flow cytometry experiment data (e.g., label spectral characteristics) and flow cytometry configuration data can also be stored in memory 295. Controller / processor 290 can be configured to evaluate the label assignment to one or more markers.

[0110] In some embodiments, the system is a particle sorting system configured to sort particles using a closed particle sorting module, such as the particle sorting system described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as the sorting decision module described in U.S. Patent Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, the system for sorting sample components includes a particle sorting module with deflection plates, such as the one described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0111] In some implementations, the system is a fluorescence imaging particle sorter that utilizes radio frequency tag emission imaging technology, such as... Figure 3 As shown, the particle sorter 300 includes an illumination component 300a containing a light source 301 (e.g., a 488nm laser). The light source 301 generates an output beam 301a, which is split into beams 302a and 302b by a beam splitter 302. Beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a, which includes beams deflected at one or more angles. In some cases, the output beam 303a generated by the acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a, which includes beams deflected at one or more angles. In some cases, the output beam 304a generated by the acousto-optic device 304 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 303a and 304a, generated by acousto-optic devices 303 and 304 respectively, are combined by beam splitter 305 to generate output beam 305a, which is transmitted via optical element 306 (e.g., objective lens) to illuminate particles in flow chamber 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into multiple beams, each beam having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam and then overlaps it with the beam array at beam combiner 305. In some embodiments, the light illumination system having a light source and acousto-optic devices may also include the systems described in Schraivogel et al. (“High-Speed ​​Fluorescence Imaging Cell Sorting”, Science (2022), 375(6578):315-320) and U.S. Patent Publication No. 2021 / 0404943, the disclosure of which is incorporated herein by reference.

[0112] Output beam 305a irradiates sample particles 308 flowing through flow chamber 307 (e.g., together with sheath fluid 309) in irradiation region 310. As shown, in irradiation region 310, multiple beams (e.g., angle-deflected radio frequency offset beams represented by dots in irradiation region 310) overlap with the reference local oscillator beam (represented by shaded lines in irradiation region 310). Due to their different optical frequencies, the overlapping beams exhibit beat frequency characteristics, resulting in each beam operating at a different frequency f. 1-n Carrying sinusoidal modulation.

[0113] Light from the irradiated sample is transmitted to a light detection system 300b, which includes multiple photodetectors. The light detection system 300b includes a forward-scattering photodetector 311 for generating a forward-scattering image 311a and a side-scattering photodetector 312 for generating a side-scattering image 312a. The light detection system 300b also includes a bright-field photodetector 313 for generating a light loss image 313a. In some embodiments, the forward-scattering detector 311 and the side-scattering 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 photodetectors 314-317. In some cases, photodetectors 314-317 are photomultiplier tubes. The light beam from the irradiated sample is directed by a beam splitter 320 to the side-scattering detection channel 312 and the fluorescence detection channels 314-317. The optical detection system 300b includes bandpass optics 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optics 321 has a 534 nm / 40 nm bandpass. In some cases, optics 322 has a 586 nm / 42 nm bandpass. In some cases, optics 323 has a 700 nm / 54 nm bandpass. In some cases, optics 324 has a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm to each side of the center of the spectral band, or from 500 nm to 520 nm.

[0114] 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 digitally processed in real time by processors 350 and 351. Based on the data signals generated in processors 350 and 351, images 311a-317a can be generated in each light detection channel. Image-assisted sorting is triggered by a sorting signal generated in sorting trigger 352. Sorting component 300c includes deflection plate 331 for deflecting particles into sample container 332 or waste stream 333. In some cases, sorting component 300c is configured to sort particles using a closed particle sorting module, such as the module described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0115] In some implementations, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4 It can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection container. Figure 4 A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a flow system. Particle analysis system 401 includes a flow control system 402. Flow control system 402 may include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, in which particles 403 (e.g., cells) in the sample move along a common sample path 409.

[0116] The particle analysis system 401 includes a detection system 404 configured to collect signals generated from each particle as it passes through one or more detection stations along a common sample path. Detection station 408 typically refers to a monitored area 407 along the common sample path. In some embodiments, detection may include detecting light or one or more other properties as particle 403 passes through monitored area 407. Figure 4 A detection station 408 and a monitored area 407 are shown. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor more than one area.

[0117] Each signal is assigned a signal value, thus forming a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multi-dimensional data point, containing values ​​of various attributes measured for the particle. The detection system 404 is configured to continuously acquire such data points within a first time interval.

[0118] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system shown may be operationally associated with the flow control system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the number of data points collected by the detection system 404 within the first time interval. The control system 406 is also configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. Furthermore, the control system 406 may compare the experimental signal frequency with the calculated signal frequency or a predetermined signal frequency.

[0119] Figure 5 A functional block diagram of a particle analyzer control system (e.g., a flow cytometer control system) is shown, including an analysis controller (i.e., 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.

[0120] The particle analysis 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 may be included between the particle analysis or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via this data communication channel.

[0121] Analysis controller 500 may be configured to receive biological event data from particle analysis or sorting system 502. The biological event data received from particle analysis or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display, including a first graph of the biological event data, to display device 506. For example, analysis controller 500 may also be configured to plot regions of interest in a gated manner around a population of biological event data displayed on display device 506 and overlay them on the first graph. In some embodiments, the gating may be a logical combination of one or more graphical regions of interest plotted on a single-parameter histogram or bivariate graph. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0122] The analysis controller 500 can also be configured to display the gated biological event data on the display device 506 in a manner different from other events in the gated biological event data. For example, the analysis controller 500 can be configured to distinguish the color of the gated biological event data from the color of the gated biological event data. The display device 506 can be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to display a graphical interface.

[0123] The analysis controller 500 can be configured to receive a gating selection signal from a first input device that identifies a gating. For example, the first input device can be implemented as a mouse 510. The mouse 510 can generate a gating selection signal for the analysis controller 500 that identifies a gating to be displayed on or operated via the display device 506 (e.g., by clicking when the cursor is positioned on or within the desired gating). In some embodiments, the first device can be implemented as a keyboard 508 or other means of providing input signals to the analysis controller 500 (such as a touchscreen, stylus, optical detector, or voice recognition system). Some input devices may include multiple input functions. In such embodiments, each input function can be considered an input device. For example, such as... Figure 5 As shown, the mouse 510 can include a right mouse button and a left mouse button, and each button can generate a trigger event.

[0124] Triggering events can cause the analysis controller 500 to change the way the data is displayed, change the portion of the data actually displayed on the display device 506, and / or provide input for further processing (such as selecting groups of interest for particle sorting).

[0125] In some implementations, the analysis controller 500 may be configured to detect when the mouse 510 initiates gating selection. The analysis controller 500 may also be configured to automatically modify the graphical visualization to facilitate the gating process. This modification may be based on the specific distribution of the biological event data received by the analysis controller 500.

[0126] The analysis controller 500 can be connected to a 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 also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0127] Display device 506 can be configured to receive display data from analysis controller 500. The display data may include graphs of biological event data and gating of outlines circling portions of the graphs. Display device 506 can also be configured to change the displayed information based on input received from analysis controller 500 and input from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0128] In some implementations, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include controls for receiving sample events or sample images. Sample events, images, or sample gating may be provided before collecting event data from the sample, or based on an initial set of events from a portion of the sample.

[0129] Figure 6A This is a schematic diagram of a particle sorting system 600 (e.g., particle analysis or sorting system 502) according to some embodiments shown herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6A As shown, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is connected to a fluid conduit 601, which may be connected to, include, or be a nozzle 603. Within the fluid conduit 601, sheath fluid 604 is hydrodynamically focused into a sample fluid 606 (containing particles 609) within a moving fluid column 608 (e.g., a flow). Within the moving fluid column 608, particles 609 (e.g., cells) align in a single file to pass through a monitoring region 611 (e.g., a laser-flow intersection) and are irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to break into multiple droplets 610, some of which contain particles 609.

[0130] In operation, a detection station 614 (e.g., an event detector) identifies a particle (or cell) of interest as it crosses a monitoring area 611. The detection station 614 inputs to a timing circuit 628, which in turn inputs to a flash charging circuit 630. At the droplet break point, a timing droplet delay (Δt) notification allows flash charging of the moving fluid column 608 to charge the droplet of interest. The droplet of interest 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 it into a container, such as a collection tube, a porous sample plate, or a microporous sample plate, where pores or micropores can be associated with the droplet of particular interest. Figure 6A As shown, the droplets can be collected into the drain container 638.

[0131] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest passes through a monitoring region 611. Exemplary droplet boundary detectors are 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 position of each detected particle within the droplet. The detection system 616 may be input to an amplitude signal 620 and / or a phase signal 618, which are then input (via amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. An amplitude control circuit 626 and / or a frequency control circuit 624 may be included in the control system.

[0132] In some embodiments, sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be connected to a memory configured to store detected events and sorting decisions based thereon. The event data of the particles may include sorting decisions. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or as a communication connection, such that one of the detection system 616 or detection station 614 collects event measurements and provides them to non-collecting elements.

[0133] Figure 6B This is a schematic diagram of a particle sorting system based on some of the embodiments shown in this article. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via a current-charged wire in the barbs. This forms a droplet stream 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. The particle information is analyzed by sorting electronics or other detection systems. Figure 6B (Not shown in the image). Deflecting plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby guiding the droplets toward a target collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to guide particles along a first path 662 toward container 674 or along a second path 668 toward container 678. If a particle is not of interest (e.g., no scattering or irradiation information is displayed within a specified sorting range), the deflector plates can allow the particle to continue along flow path 664. Such uncharged droplets can enter a waste container, for example, via a suction device 670.

[0134] The sorting electronics can be included to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to induce particle sorting. Figure 6B Example implementations of the illustrated scheme include BDFACS Aria provided by Becton, Dickinson and Company (Franklin Lakes, NJ). TM A series of flow cytometers.

[0135] Sample analysis methods

[0136] As described above, this disclosure also includes methods for analyzing samples. Methods of interest include introducing a fluid sample (i.e., a sample containing particles) into the aforementioned flow cytometer to analyze the sample fluid. In some embodiments, the method further includes flow cytometry sorting of particles in the fluid sample. As described above, the flow cytometer includes: a flow chamber for conveying particles in a central flow of the flow stream from a proximal end to a distal end, having a flow chamber body, wherein the flow chamber body includes a flow chamber cone structure at the proximal end; and a sample injection needle for conveying sample fluid from a sample injection line at the proximal end to the flow chamber body at the distal end to generate a central flow, wherein the sample injection needle includes: a sample injection needle adapter including a sample tube adapter attached to the needle; and a clamp operatively connecting the sample injection needle to the flow chamber body.

[0137] In some cases, the samples analyzed in this method are biological samples. The term "biological sample," used in its conventional sense, refers to a whole organism, plant, fungus, or a subset of animal tissue, animal cells, or animal components, which in some cases can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers to a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from organisms or subsets of their tissues, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory sections, gastrointestinal sections, cardiovascular sections, and urogenital sections, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be tissues of any type of organism, including healthy tissues and diseased tissues (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc. In some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger puncture (wherein the blood may be combined with any reagent or not before testing, such as preservatives, anticoagulants, etc.).

[0138] In some embodiments, the source of the sample is "mammal" or "milk," terms that are widely used to describe organisms within 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 object is a human. The methods can be applied to samples obtained from human objects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, teenagers, and adults), wherein in some embodiments, the human object is an adolescent, teenager, or adult. Although this disclosure can be applied to samples from human objects, it is to be understood that these methods can also be applied to samples from other animal objects (i.e., "non-human objects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0139] Cells of interest can be targeted for characterization based on a variety of parameters, such as phenotypic features identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect the analyzed droplet determined to contain the target cell. The method can be used to characterize a variety of cells. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells with readily available cell surface markers or antigens that can be captured or labeled by their readily available affinity agents or conjugates. 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 α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialiacoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells derived from whole blood, bone marrow, or umbilical cord blood, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+).

[0140] In practicing the method, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module can vary, for example, from 0.001 mL to 1000 mL, such as 0.005 mL to 900 mL, 0.01 mL to 800 mL, 0.05 mL to 700 mL, 0.1 mL to 600 mL, 0.5 mL to 500 mL, 1 mL to 400 mL, 2 mL to 300 mL, and includes samples from 5 mL to 100 mL.

[0141] The method according to embodiments of this disclosure includes counting and optionally sorting labeled particles (e.g., target cells) in a sample. In practicing the method, a fluid sample containing the particles is first introduced into a flow nozzle of the system. Upon exiting the flow nozzle, the particles pass substantially one-by-one through a sample detection zone, where each particle is illuminated by a light source, and measurements of light scattering parameters and, in some cases, fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded individually for each particle. Depending on the nature of the detected flow, flow rates of 0.001 mm or greater, such as 0.005 mm or greater, 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, and including flow rates of 1 mm or greater, can be illuminated. In some embodiments, the method includes irradiating a planar cross-section of the flow in the sample detection region, for example, with a laser (as described above). In other embodiments, the method includes irradiating a predetermined length of the flow in the sample detection region, which corresponds, for example, to the irradiation profile of a diffuse laser beam or lamp.

[0142] In some embodiments, the method includes irradiating the flow at or near the flow chamber nozzle orifice. For example, the method may include irradiating the flow at a location approximately 0.001 mm or more than 0.001 mm from the nozzle orifice (e.g., 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, and including 1 mm or more). In some embodiments, the method includes irradiating the flow immediately adjacent to the flow chamber nozzle orifice.

[0143] In an embodiment of the method, as particles pass through a sensing region and are illuminated by an energy source, a detector (such as a photomultiplier tube (PMT)) records the light passing through each particle (in some cases referred to as forward scattering), the light reflected in a direction orthogonal to the direction in which the particles flow through the sensing region (in some cases referred to as orthogonal scattering or side scattering), and, if the particles are labeled with one or more fluorescent markers, the fluorescence emitted by the particles is also recorded. Each forward scattering (FSC), side scattering (SSC), and fluorescence emission contains independent 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. Depending on the desired outcome, the recorded data for each particle is analyzed in real time or stored in data storage and analysis tools (such as a computer).

[0144] In some implementations, particle detection and unique identification are desired by exposing particles to excitation light in one or more detection channels and measuring the fluorescence of each particle. In the detection channels, the fluorescence emitted to identify the particles and their associated binding complexes can be measured after excitation with a single light source, or after excitation with different light sources individually. If separate excitation sources are used to excite the particle markers, the markers can be selected such that each excitation source used excites all the markers.

[0145] Some implementations of the method also include data acquisition, analysis, and recording, for example using a computer, where multiple data channels record light scattering and fluorescence data emitted by each particle from each detector as it passes through the sample detection zone of the particle sorting module. In these implementations, the analysis includes classifying and counting the particles, such that each particle is displayed as a set of digitized parameter values. The system can be configured to be triggered by selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for the detection parameter and can be used to detect the particle's passage through the light source. Detecting an event exceeding the selected parameter threshold triggers the acquisition of light scattering and fluorescence data for the particle. No data is acquired for particles or other components in the measured medium that elicit a response below the threshold. The trigger parameter can be the detected value of forward scattered light caused by the particle passing through the light beam. Flow cytometry then detects and collects the particle's light scattering and fluorescence data.

[0146] Subsequently, based on the data collected for the entire population, specific subpopulations of interest are further analyzed using "gating." To select an appropriate gating point, the data is plotted to obtain the best possible subpopulation separation. This process can be performed by plotting a contrast between lateral (i.e., orthogonal) light scattering (SSC) and forward light scattering (FSC) on a two-dimensional scatter plot. Subpopulations of particles (i.e., those cells within the gating point) are then selected, and particles not within the gating point are excluded. If desired, the gating point can be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Those particles within the gating point are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis can be configured to determine the count of particles of interest in the sample.

[0147] Methods of interest may also include the use of particles in research, laboratory testing, or treatment. In some embodiments, the method includes obtaining individual cells prepared from a target fluid biological sample or tissue biological sample. For example, the method includes obtaining cells from a fluid sample or tissue sample for use as a research or diagnostic sample for diseases such as cancer. Similarly, the method includes obtaining cells from a fluid sample or tissue sample for therapeutic purposes. Cell therapy protocols are those in which live cellular material comprising, for example, cells and tissues can be prepared and introduced into the body of a subject as a therapeutic treatment. Conditions that can be treated by applying samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, etc.

[0148] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification, culture, and in vitro expansion; cell collection, sample reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with the collection of live cells and tissues from the subject's source tissue to produce cell and / or tissue samples. Samples can be collected using any suitable procedure, including, for example, administration of cell mobilizing agents to the subject, blood aspiration from the subject, bone marrow extraction from the subject, etc. After sample collection, cells can be enriched using several methods, including, for example, centrifugation-based methods, filtration-based methods, centrifugation, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells can be genetically modified using any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and expanded in vitro. In some cases, cells are preserved (e.g., cryopreserved) and stored for future use, thawed upon use, and subsequently administered to the patient, for example, by infusion of cells into the patient.

[0149] Computer control system

[0150] This disclosure also includes a computer control system, wherein the system comprises one or more fully or partially automated computers. In some embodiments, the system includes a computer having a non-transitory computer-readable storage medium thereon storing a computer program, wherein the computer program, when loaded onto the computer, includes: instructions for receiving target or desired flow conditions (e.g., sheath fluid flow rate, sample fluid flow rate) and instructions for initiating a change in resistance state to achieve the target or desired flow conditions. Because the system described herein includes the flow chamber of this disclosure, the resulting central flow can remain intact.

[0151] 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 storing instructions for performing the steps of the method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, a memory storage device, and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor, or may be one of other available or soon-to-be-available processors. The processor executes the operating system, which interacts with firmware and hardware in well-known ways and facilitates the processor's coordination and execution of various computer programs written in various programming languages ​​known in the art, such as Java, Perl, C++, Python, other high-level languages ​​or low-level languages, and combinations thereof. The operating system typically works with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file management and data management, memory management, and communication control and related services according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0152] System memory can be any known or future memory storage device. Examples include any generally available random access memory (RAM), magnetic media such as internal hard disks or magnetic tapes, optical media such as those for reading and writing compact optical discs, flash memory devices, or other memory storage devices. Memory storage devices can be any known or unknown device, including compact optical disc drives, magnetic tape drives, or floppy disk drives. These types of memory storage devices typically read and / or write to program storage media (not shown), such as compact optical discs. Any such program storage media or other media in use or that may be developed in the future can be considered computer program products. It will be understood that these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0153] In some embodiments, a computer program product is described, comprising a computer-usable medium containing control logic (computer software program, including program code) stored therein. When the control logic is executed by a computer processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using a hardware state machine. The implementation of the hardware state machine used to perform the functions described herein will be apparent to those skilled in the art.

[0154] The memory can be any suitable device in which a processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including a disk or optical disc or magnetic tape or RAM, or any other suitable fixed or portable device). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel or pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, any of which is connected to the memory for use. For example, a disk or optical disc may carry the programming and can be read by a disk writer / reader. The systems of this disclosure also include programming, for example, in the form of a computer program product, i.e., an algorithm for practicing the methods described above. The programming according to this disclosure can be recorded on a computer-readable medium, such as 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 ROM; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0155] The processor can also access communication channels to communicate with users in remote locations. A remote location refers to a user who does not have direct contact with the system and forwards input information from external devices, such as computers connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones), to the input manager.

[0156] In some embodiments, the system 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., RFID, Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), communication protocols, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0157] In some implementations, the communication interface is configured to include one or more communication ports, such as physical ports or physical interfaces, such as USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the system and other external devices, such as computer terminals (e.g., computer terminals in a physician's office or hospital environment), which are configured for similar complementary data communication.

[0158] In some implementations, the communication interface is configured for infrared communication, wireless communication, or any other suitable wireless communication protocol to enable the system to communicate with other devices, such as computer terminals and / or networks, mobile phones with communication capabilities, personal digital assistants, or any other communication devices that the user can use in combination.

[0159] In some implementations, the communication interface is configured to provide connectivity for data transmission using the Internet Protocol (IP) via mobile phone networks, short message service (SMS), wireless connection of a personal computer (PC) connected to a local area network (LAN) connected to the Internet, or Wi-Fi connected to the Internet at a Wi-Fi hotspot.

[0160] In some embodiments, the system is configured to wirelessly communicate with a server device via a communication interface, for example using common standards such as 802.11, RF protocols, or IrDA infrared protocols. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, appliance, etc. 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.

[0161] In some implementations, the communication interface is configured to communicate automatically or semi-automatically with data stored in the system, such as data stored in an optional data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0162] The output controller may include controllers for any of the various known display devices used to present information to a user, whether human or machine, whether local or remote. If one of the display devices provides visual information, that 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 used to provide a graphical input and output interface between the system and the user, and to process user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished in alternative implementations using networks or other types of remote communication. The output manager may also provide information generated by the processing module to a user located at a remote location, such as via the Internet, telephone, or satellite networks, according to known technologies. The presentation of data by the output manager may be implemented according to various known technologies. For example, the data may include SQL documents, HTML documents, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses, allowing the user to retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the system may be any type of known computer platform or a type developed in the future, although they typically belong to a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They can be connected via any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. They may be located in the same place or physically separated. Different operating systems can be used on any computer platform, depending on the type and / or model of the computer platform chosen. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android™, SGI IRIX®, Oracle Solaris®, and others.

[0163] Figure 7 A general architecture of an example computing device 700 according to certain implementation schemes is described. Figure 7The general architecture of the computing device 700 described herein includes the layout of computer hardware components and computer software components. However, it is not necessary to show all these generally conventional components for the purpose of providing a disclosure that can be implemented. 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 can provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 can receive information and instructions from other computing systems or computing services via the network. The processing unit 710 can also communicate with and from a memory 770, and can also provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or programs such as FlowJo®) stored as executable instructions in the non-transitory memory of an analysis system can display flow cytometry event data to a user. The input / output device interface 740 can also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input device.

[0164] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments), which processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772, which provides computer program instructions for use by processing unit 710 in the general management and general operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may also contain computer program instructions for implementing aspects of this disclosure and other information.

[0165] kit

[0166] This disclosure also includes kits. Kits of interest include one or more sample injection needles, as described above (e.g., assembled or unassembled, with the sample injection needle adapter and clamp not attached). 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 super-bullet structure as described above, a sample injection needle having a bullet structure as described above, and a sample injection needle having a circular structure, or a combination of sample injection needles having these structures. In some cases, the kit also includes a flow chamber body (e.g., as described above). In some cases, the kit also includes: a package configured to house the sample injection needle adapter, clamp, and / or flow chamber body.

[0167] In addition to the components described above, the kit (in some embodiments) may also include instructions, such as those for mounting the sample injection needle of this disclosure into a flow cytometer. These instructions may be present in the kit in various forms, one or more of which may be present within the kit. One form of these instructions may be as printed information on a suitable medium or substrate, such as one or more sheets of paper with the information printed on them, in the kit's packaging, in a packaging insert, etc. Another form of these instructions is as computer-readable media, such as floppy disks, compact optical discs (CDs), portable flash drives, etc., on which the information is already recorded. Yet another form of these instructions may be as a URL, which can be used to access information on a remote website via the Internet.

[0168] practicality

[0169] The sample injection needle, the flow chamber, the flow cytometer, and the method described herein are suitable for a variety of applications where it is desirable to analyze components in a sample within a fluid medium. This disclosure is particularly applicable to improving the quality of the central flow in flow control systems. For example, the sample injection needle, flow cytometer, and method can be used to increase the degree to which the central flow remains intact. In some cases, this disclosure can be used to reduce the formation of vortices within the conical structure of the flow chamber.

[0170] The embodiments of this disclosure are applicable to applications of preparing cells from biological samples that may be intended for research, laboratory testing, or therapeutic use. In some embodiments, the methods and apparatus can facilitate the acquisition and / or analysis of individual cells prepared from a target fluid biological sample or a target tissue biological sample. For example, the methods and systems facilitate the acquisition of cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the methods and systems can facilitate the acquisition of cells from fluid or tissue samples for therapeutic purposes.

[0171] Despite the appended claims, this disclosure may also be described by the following terms:

[0172] 1. A flow chamber for use in a flow cytometer, the flow chamber comprising:

[0173] A flow chamber body for conveying particles in the center flow of a flow stream from the proximal end to the distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end;

[0174] A sample injection needle having a through passage for delivering sample fluid from a proximal sample injection tube to a distal flow chamber body to generate a central flow, wherein the sample injection needle comprises:

[0175] A sample injection needle adapter, comprising a sample tube adapter attached to the needle; and

[0176] A clamp that operatively connects the sample injection needle to the flow chamber body;

[0177] In the non-clamping configuration of the flow chamber, the sample injection needle adapter is freely rotatable relative to the flow chamber conical structure and the clamp; and in the clamping configuration of the flow chamber, the sample injection needle adapter is fixed relative to the flow chamber conical structure and the clamp.

[0178] 2. The flow chamber according to Clause 1, wherein 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 conical structure of the flow chamber.

[0179] 3. The flow chamber according to Clause 2, wherein, in the clamping configuration, the sample injection needle adapter is secured relative to the flow chamber conical structure and the clamp by compression of the clamp.

[0180] 4. The flow chamber according to Clause 2 or Clause 3, wherein the clamp is compressed by one or more fastening members.

[0181] 5. The flow chamber according to Clause 4, wherein the clamp is compressed by a plurality of screws.

[0182] 6. The flow chamber according to Clause 5, wherein the clamp is compressed by three screws.

[0183] 7. The flow chamber according to Clause 6, wherein the clamp includes a set of holes for receiving each of the plurality of screws.

[0184] 8. The flow chamber according to Clause 7, wherein the flow chamber body includes: a set of holes aligned with a set of clamping holes and receiving each of the plurality of screws.

[0185] 9. The flow chamber according to any one of Clauses 5 to 8, wherein the clamp is configured to allow adjustment of the tilt of the sample injection needle relative to the flow chamber body by manipulating the torque of at least one of the plurality of screws.

[0186] 10. The flow chamber according to any one of Clauses 2 to 9, wherein the clamp includes a distal end that contacts the sample tube adapter and a proximal end configured to fluidly connect a sample injection line to the sample injection needle.

[0187] 11. The flow chamber according to Clause 10, wherein the distal end of the clamp includes a recess and a surface, at least a portion of the sample tube adapter is positioned within the recess, and the surface contacts the proximal end of the flow chamber body.

[0188] 12. The flow chamber according to Clause 10 or Clause 11, wherein the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid as the sample fluid flows from the sample injection line to the sample injection needle.

[0189] 13. The flow chamber 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.

[0190] 14. The flow chamber according to any one of clauses 2 to 13, wherein the sample tube adapter includes a proximal end positioned in a recess of the clamp and a distal end contacting the proximal end of the flow chamber body.

[0191] 15. The flow chamber according to Clause 14, wherein at least a portion of the distal end of the sample tube adapter is located within the flow chamber body.

[0192] 16. The flow chamber according to clause 14 or 15, wherein the sample tube adapter includes a flange that contacts the proximal end of the flow chamber body.

[0193] 17. The flow chamber according to any one of clauses 14 to 16, wherein the distal end of the sample tube adapter is pressed against the proximal end of the flow chamber body by a clamp, such that the distal end of the needle of the sample injection needle adapter is fixed in position within the conical structure of the flow chamber.

[0194] 18. The flow chamber according to any one of clauses 2 to 17, wherein the needle of the sample injection needle adapter is tapered at the distal end.

[0195] 19. The flow chamber according to Clause 18, wherein the needle of the sample injection needle adapter includes a rounded distal end.

[0196] 20. The flow chamber 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 conical structure of the flow chamber such that a complete central flow is maintained even when the flow conditions change by one or more orders of magnitude.

[0197] 21. The flow chamber according to any one of clauses 2 to 20, wherein the flow chamber body includes: a sheath fluid inlet for conveying sheath fluid to the conical structure of the flow chamber.

[0198] 22. The flow chamber according to Clause 21, wherein the longitudinal distance between the distal end of the needle of the sample injection needle adapter and the sheath fluid inlet is in the range of 17 mm to 26 mm.

[0199] 23. The flow chamber according to clause 21 or 22, wherein the main body of the flow chamber includes a plurality of sheath fluid inlets.

[0200] 24. The flow chamber according to Clause 23, wherein the sheath fluid inlets are staggered from one another, such that the sheath fluid flows through the conical structure of the flow chamber in a swirling manner.

[0201] 25. The flow chamber according to any one of clauses 2 to 24, wherein the distal end of the flow chamber body comprises: a cuvette for conveying particles in the central flow through the sample detection area.

[0202] 26. The flow chamber according to Clause 25, wherein at least a portion of the cuvette comprises a light-transmitting solid.

[0203] 27. The flow chamber according to Clause 26, wherein the light-transmitting portion of the cuvette is configured to allow optical detection of particles in the central flow.

[0204] 28. The flow chamber according to any one of clauses 25 to 27, wherein the cuvette is positioned at a distal end of the flow chamber body by a clamp fixed to the flow chamber body.

[0205] 29. The flow chamber according to Clause 28, wherein the cuvette is releasably attached to the distal end of the flow chamber body via a flow chamber body clamp.

[0206] 30. The flow cell according to Clause 28 or 29, wherein the cuvette is positioned by means of the flow cell body clamp such that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0207] 31. A clamp for operatively connecting a sample injection needle adapter to a flow chamber body, the clamp comprising:

[0208] The distal end is configured to attach to the sample injection needle adapter, the sample injection needle adapter including a sample tube adapter fixed to the needle; and

[0209] The proximal end is configured to connect the sample injection tubing to the needle of the sample injection needle adapter.

[0210] The clamp is configured to operatively connect the sample injection needle adapter to the flow chamber body by pressing the sample tube adapter against the flow chamber body.

[0211] 32. The clamp according to Clause 31, wherein the distal end of the clamp includes a surface configured to contact the flow chamber body when the clamp presses the sample tube adapter against the flow chamber body.

[0212] 33. The clamp according to clause 32, wherein the distal end of the clamp includes a recess configured to receive at least a portion of the sample tube adapter.

[0213] 34. The clamp according to clause 32 or 33, wherein the recess includes an inner surface concentric with the outer surface of the portion of the sample tube adapter.

[0214] 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 the portion of the sample tube adapter is positioned within the recess.

[0215] 36. The clamp according to any one of clauses 31 to 33, wherein the clamp is configured to receive a fastening member for fastening the clamp to the flow chamber body.

[0216] 37. The clamp according to clause 36, wherein the clamp is configured such that when the sample injection needle adapter can be operatively connected to the flow chamber body via the clamp, compression of the clamp by the fastening member secures the sample injection needle adapter relative to the flow chamber body.

[0217] 38. The clamp according to clause 36 or 37, wherein the fastening member comprises a plurality of screws.

[0218] 39. The clamp according to clause 38, wherein the clamp includes a set of holes for receiving each of the plurality of screws.

[0219] 40. The clamp according to clause 39, wherein the set of holes is configured to align with a set of holes in the flow chamber body.

[0220] 41. The clamp according to any one of clauses 38 to 40, wherein the clamp is configured such that when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, the tilt of the sample injection needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0221] 42. The clamp according to clause 39, wherein the clamp is configured such that, when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, the position of the distal end of the needle of the sample injection needle adapter within the flow chamber conical structure of the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0222] 43. The clamp according to clause 42, wherein adjusting the torque of one of the plurality of screws causes the distal end of the needle to pivot about an axis.

[0223] 44. The clamp according to any one of clauses 31 to 43, wherein the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid as the sample fluid flows from the sample injection line to the needle of the sample injection needle adapter.

[0224] 45. The clamp according to any one of clauses 31 to 44, wherein the proximal end of the clamp is configured to position the flow meter board connector.

[0225] 46. ​​A sample injection needle adapter for operatively connecting a sample injection line to a flow chamber body, the sample injection needle adapter comprising:

[0226] A needle having a through-hole for delivering sample fluid from a sample injection line at the proximal end to a flow chamber conical structure at the distal end; and

[0227] A sample tube adapter includes: a proximal end configured to attach to a clamp, and a distal end fixed to the proximal end of the needle.

[0228] The sample tube adapter is configured to operatively connect the needle to the flow chamber body when pressed against the flow chamber body by the clamp.

[0229] 47. The sample injection needle adapter according to clause 46, wherein the sample tube adapter is configured such that: when the sample tube adapter is operatively connected to the flow chamber body via the clamp, compression of the sample tube adapter by the clamp fixes the sample injection needle adapter relative to the flow chamber body.

[0230] 48. The sample injection needle adapter according to Clause 47, wherein the sample tube adapter is fixed such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow chamber conical structure.

[0231] 49. The sample injection needle adapter according to Clause 48, wherein the fixed position enables the maintenance of a complete central flow within the flow chamber conical structure when flow conditions vary by an order of magnitude or more.

[0232] 50. A sample injection needle adapter according to any one of clauses 46 to 49, wherein the sample injection needle adapter is configured such that, when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, the tilt of the sample injection needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of a plurality of screws that fasten the clamp to the flow chamber body.

[0233] 51. The sample injection needle adapter according to Clause 50, wherein the sample injection needle adapter is configured such that, when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, the position of the distal end of the needle within the flow chamber conical structure can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0234] 52. The sample injection needle adapter according to Clause 51, wherein adjusting the torque of one of the plurality of screws causes the distal end of the needle to pivot about an axis.

[0235] 53. The sample injection needle adapter according to any one of 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.

[0236] 54. The sample injection needle adapter according to Clause 53, wherein the portion of the proximal end of the sample tube adapter includes an outer surface concentric with the inner surface of the recess.

[0237] 55. The sample injection needle adapter according to clause 53 or 54, wherein 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 when the portion of the sample tube adapter is positioned within the recess.

[0238] 56. The sample injection needle adapter according to any one of 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 chamber body.

[0239] 57. The sample injection needle adapter according to clause 56, wherein the sample tube adapter includes a flange for positioning the distal portion of the sample tube adapter within the flow chamber body near the flow chamber conical structure.

[0240] 58. The sample injection needle adapter according to Clause 57, wherein the flange is configured to position the distal portion of the sample tube adapter within the flow chamber body such that the longitudinal distance separating the distal end of the needle from the sheath fluid inlet of the flow chamber body is in the range of 17 mm to 26 mm.

[0241] 59. A sample injection needle adapter according to any one of clauses 46 to 58, wherein the needle is tapered at its distal end.

[0242] 60. The sample injection needle adapter according to Clause 59, wherein the needle includes a circular distal end.

[0243] 61. A sample injection needle for operatively connecting a sample injection line to a flow chamber body, the sample injection needle comprising:

[0244] Sample injection needle adapter, comprising:

[0245] A needle having a passageway therethrough for delivering sample fluid from a sample injection line at a proximal end to the flow chamber body at a distal end; and a flow chamber conical structure.

[0246] A sample tube adapter, comprising a proximal end and a distal end, wherein the distal end is secured to the proximal end of the needle; and

[0247] The fixture includes:

[0248] The distal end is attached to the proximal end of the sample tube adapter; and

[0249] The proximal end is configured to fluidly connect the sample injection tubing to the proximal end of the needle of the sample injection needle adapter.

[0250] The clamp is configured to operatively connect the sample injection needle adapter to the flow chamber body by pressing the sample tube adapter against the flow chamber body.

[0251] 62. The sample injection needle according to Clause 61, wherein the sample injection needle adapter is freely rotatable relative to the flow chamber conical structure and the clamp.

[0252] 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.

[0253] 64. The sample injection needle according to Clause 63, wherein the portion of the proximal end of the sample tube adapter includes an outer surface concentric with the inner surface of the recess.

[0254] 65. The 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 chamber body.

[0255] 66. The sample injection needle according to Clause 65, wherein the sample tube adapter includes a flange configured to position the distal portion of the sample tube adapter within the flow chamber body near the flow chamber conical structure.

[0256] 67. The sample injection needle according to any one of clauses 62 to 66, wherein the sample injection needle is configured such that: when operatively connected to the flow chamber body via the clamp, compression of the sample tube adapter by the clamp fixes the sample injection needle adapter relative to the flow chamber body.

[0257] 68. The sample injection needle according to Clause 67, wherein the sample injection needle adapter is fixed such that the distal end of the needle of the sample injection needle adapter is in a fixed position within the flow chamber conical structure.

[0258] 69. The sample injection needle according to Clause 68, wherein the fixed position enables the maintenance of a complete central flow within the flow chamber conical structure when flow conditions vary by one or more orders of magnitude.

[0259] 70. The sample injection needle according to clause 68 or 69, wherein the longitudinal distance between the fixed position and the sheath fluid inlet of the flow chamber body is in the range of 17 mm to 26 mm.

[0260] 71. The sample injection needle according to any one of clauses 61 to 70, wherein the clamp is configured to receive a fastening member for securing the clamp to the flow chamber body.

[0261] 72. The sample injection needle according to Clause 71, wherein the sample injection needle is configured such that: when the clamp is fastened to the flow chamber body by the fastening member, compression of the clamp by the fastening member fixes the sample injection needle adapter relative to the flow chamber body.

[0262] 73. The sample injection needle according to clause 71 or 72, wherein the fastening member comprises a plurality of screws.

[0263] 74. The sample injection needle according to Clause 73, wherein the clamp includes a set of holes for receiving each of the plurality of screws.

[0264] 75. The sample injection needle according to Clause 74, wherein the set of orifices is configured to align with a set of orifices of the flow chamber body.

[0265] 76. The sample injection needle according to any one of clauses 73 to 75, wherein the sample injection needle is configured such that: when the clamp is fastened to the flow chamber body by the plurality of screws, the tilt of the sample injection needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0266] 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 chamber body by a plurality of screws, the position of the distal end of the needle of the sample injection needle adapter within the flow chamber conical structure can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0267] 78. The sample injection needle according to Clause 77, wherein adjusting the torque of one of the plurality of screws causes the distal end of the needle to pivot about an axis.

[0268] 79. The sample injection needle according to any one of clauses 61 to 78, wherein the needle of the sample injection needle adapter is tapered at the distal end.

[0269] 80. The sample injection needle according to Clause 79, wherein the needle of the sample injection needle adapter includes a rounded distal end.

[0270] 81. The sample injection needle according to any one of clauses 61 to 80, wherein the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid as the sample fluid flows from the sample injection tube to the needle of the sample injection needle adapter.

[0271] 82. The 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.

[0272] 83. A method of assembling a flow chamber for use in a flow cytometer, the method comprising: operatively connecting a sample injection needle to a flow chamber body to deliver particles in a central flow of the flow from a proximal end to a distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end, and wherein:

[0273] The sample injection needle includes a clamp and a sample injection needle adapter. The sample injection needle adapter includes a sample tubing adapter attached to the needle and has a through passage for conveying sample fluid from the proximal sample injection tubing to the distal flow chamber body to generate a central flow.

[0274] The method includes operatively connecting the sample injection needle to the flow chamber body using the sample injection needle clamp.

[0275] 84. The method according to Clause 83, wherein the needle of the sample injection needle adapter comprises: a proximal end attached to the sample tube adapter and a distal end positioned within the flow chamber conical structure, wherein the sample tube adapter is attached to the clamp.

[0276] 85. The method according to clause 84, wherein the sample injection needle is operatively connected to the flow chamber body by pressing the sample tube adapter against the flow chamber body using the clamp.

[0277] 86. The method according to clause 85, wherein the method further comprises securing the sample injection needle adapter by compressing the clamp.

[0278] 87. The method according to Clause 86, wherein the sample injection needle adapter is secured such that the distal end of the needle of the sample injection needle adapter is secured in a suitable position within the flow chamber conical structure.

[0279] 88. The clamp according to clause 86 or 87, wherein the clamp is compressed by a fastening member received by the clamp and the flow chamber body.

[0280] 89. The method according to Clause 88, wherein the fastening member comprises a plurality of screws.

[0281] 90. The method according to clause 89, wherein the method further comprises inserting the plurality of screws into a set of holes in the clamp and a set of holes in the flow chamber body, wherein the set of holes in the clamp is aligned with the set of holes in the flow chamber body.

[0282] 91. The method according to clause 89 or 90, wherein the method further comprises: being able to adjust the tilt of the sample injection needle relative to the flow chamber body by manipulating the torque of at least one of the plurality of screws.

[0283] 92. The method according to Clause 91, wherein the method further comprises: being able to adjust the position of the distal end of the needle of the sample injection needle adapter within the flow chamber conical structure by manipulating the torque of at least one of the plurality of screws.

[0284] 93. The method according to Clause 92, wherein adjusting the position of the distal end of the needle of the sample injection needle adapter comprises: pivoting the distal end of the needle about an axis by individually adjusting the torque of one of the plurality of screws.

[0285] 94. The method according to any one of clauses 91 to 93, wherein the distal end of the needle of the sample injection needle adapter is positioned within the conical structure of the flow chamber such that a complete central flow can be maintained when the flow conditions change by one or more orders of magnitude.

[0286] 95. The method according to any one of clauses 86 to 94, wherein the sample injection needle adapter is rotatable relative to the flow chamber conical structure and the clamp before being secured by the clamp.

[0287] 96. The method according to clause 95, wherein the method further comprises: adjusting the rotational position of the sample injection needle adapter before securing it with the clamp.

[0288] 97. The method according to any one of clauses 85 to 96, wherein the method further comprises: attaching the sample tube adapter to the clamp before operatively connecting the sample injection needle to the flow chamber body.

[0289] 98. The method according to Clause 97, wherein a portion of the sample tube adapter is positioned within a recess of the clamp before attaching the sample tube adapter to the clamp.

[0290] 99. The method according to any one of clauses 83 to 98, wherein the clamp comprises: a distal end in contact with the sample tube adapter, and a proximal end configured to fluidly connect the sample injection line to the sample injection needle.

[0291] 100. The method according to Clause 99, wherein the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid as the sample fluid flows from the sample injection line to the sample injection needle.

[0292] 101. The method according to any one of clauses 83 to 100, wherein the method further comprises: using the proximal end of the clamp to position the flow meter board connector.

[0293] 102. The method according to any one of clauses 83 to 100, wherein the flow chamber body includes: a sheath fluid inlet for conveying sheath fluid to the conical structure of the flow chamber.

[0294] 103. The method according to any one of clauses 83 to 101, wherein the needle of the sample injection needle adapter is tapered at the distal end.

[0295] 104. The method according to Clause 103, wherein the needle of the sample injection needle adapter includes a rounded distal end.

[0296] 105. The method according to any one of clauses 83 to 104, wherein the distal end of the flow chamber body includes a cuvette for conveying particles in the central flow through the sample detection area.

[0297] 106. The method according to Clause 105, wherein at least a portion of the cuvette comprises a light-transmitting entity.

[0298] 107. The method according to Clause 106, wherein the light-transmitting portion of the cuvette is configured to allow optical detection of particles in the central stream.

[0299] 108. The method according to any one of clauses 105 to 107, wherein the method further comprises: positioning the cuvette at a distal end of the flow chamber body using a clamp fixed to the flow chamber body.

[0300] 109. The method according to Clause 108, wherein the cuvette is releasably attached to the distal end of the flow chamber body via the flow chamber body clamp.

[0301] 110. The method according to clause 108 or 109, wherein the cuvette is positioned by the flow chamber body fixture such that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0302] 111. The method according to any one of clauses 83 to 110, wherein the method further comprises operatively positioning the flow chamber into a flow cytometer.

[0303] 112. The method according to Clause 111, wherein operatively positioning the flow chamber in the flow cytometer comprises: using the clamp to fluidly connect the sample injection tubing to the needle of the sample injection needle adapter.

[0304] 113. The method according to Clause 112, wherein operatively positioning the flow chamber in the flow cytometer comprises: fluidly connecting the sheath fluid inlet of the flow chamber body to a sheath fluid source to deliver sheath fluid to the conical structure of the flow chamber.

[0305] 114. The method according to any one of clauses 111 to 113, wherein operatively positioning the flow chamber in the flow cytometer comprises: aligning a cuvette at a distal end of the flow chamber body to deliver particles in the central stream through a sample detection region, and aligning it with a light source of the flow cytometer to illuminate particles in the central stream of the sample detection region.

[0306] 115. The method according to Clause 114, wherein operatively positioning the flow chamber within the flow cytometer comprises: optically coupling a detector of the flow cytometer configured to collect light emitted by irradiated particles to the sample detection region.

[0307] 116. A flow cytometer, comprising:

[0308] The flow chamber includes:

[0309] A flow chamber body for conveying particles in the center flow of a flow stream from the proximal end to the distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end;

[0310] A sample injection needle having a through passage for delivering sample fluid from a proximal sample injection line to a distal flow chamber body to generate the central flow, wherein the sample injection needle comprises:

[0311] Sample injection needle adapter, comprising a sample tube adapter attached to the needle; and

[0312] A clamp that operatively connects the sample injection needle to the flow chamber body;

[0313] In the non-clamping configuration of the flow chamber, the sample injection needle adapter is free to rotate relative to the flow chamber conical structure and the clamp, and in the clamping configuration of the flow chamber, the sample injection needle adapter is fixed relative to the flow chamber conical structure and the clamp.

[0314] A light source, configured to irradiate particles in a flow stream within the sample detection region of the flow chamber; and

[0315] The detector is configured to collect the light emitted by the irradiated particles.

[0316] 117. The flow cytometer according to Clause 116, wherein 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 conical structure of the flow chamber.

[0317] 118. The flow cytometer according to Clause 117, wherein, in the clamping configuration, the sample injection needle adapter is secured relative to the flow chamber conical structure and the clamp by compression of the clamp.

[0318] 119. The flow cytometer according to clause 117 or 118, wherein the clamp is compressed by one or more fastening members.

[0319] 120. The flow cytometer according to Clause 119, wherein the clamp is compressed by a plurality of screws.

[0320] 121. The flow cytometer according to Clause 120, wherein the clamp is compressed by three screws.

[0321] 122. The flow cytometer according to clause 120 or 121, wherein the clamp includes a set of holes for receiving each of the plurality of screws.

[0322] 123. The flow cytometer according to Clause 122, wherein the flow chamber body includes: a set of orifices aligned with a set of clamp orifices for receiving each of the plurality of screws.

[0323] 124. The flow chamber according to any one of clauses 120 to 123, wherein the clamp is configured such that the tilt of the sample injection needle relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0324] 125. The flow cytometer according to any one of clauses 117 to 124, wherein the clamp comprises: a distal end in contact with the sample tube adapter, and a proximal end fluidly connecting the sample injection tubing to the sample injection needle.

[0325] 126. The flow cytometer according to Clause 125, wherein the distal end of the clamp includes a recess and a surface, at least a portion of the sample tube adapter is positioned in the recess, and the surface contacts the proximal end of the flow chamber body.

[0326] 127. The flow cytometer according to clause 125 or 126, wherein 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.

[0327] 128. The flow cytometer according to any one of clauses 125 to 127, wherein the clamp has a proximal positioning flow meter plate connector.

[0328] 129. The 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 chamber body.

[0329] 130. The flow cytometer according to Clause 129, wherein at least a portion of the distal end of the sample tube adapter is located within the flow chamber body.

[0330] 131. The flow cytometer according to clauses 129 or 130, wherein the sample tube adapter includes a flange that contacts the proximal end of the flow chamber body.

[0331] 132. The flow cytometer according to any one of clauses 129 to 131, wherein the distal end of the sample tube adapter is pressed against the proximal end of the flow chamber body by the clamp, such that the distal end of the needle of the sample injection needle adapter is fixed in position within the conical structure of the flow chamber.

[0332] 133. The flow cytometer according to any one of clauses 117 to 132, wherein the needle of the sample injection needle adapter is tapered at the distal end.

[0333] 134. The flow cytometer according to Clause 133, wherein the needle of the sample injection needle adapter includes a rounded distal end.

[0334] 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 conical structure of the flow chamber such that a complete central flow is maintained when the flow conditions change by one or more orders of magnitude.

[0335] 136. The flow cytometer according to any one of clauses 117 to 135, wherein the flow chamber body includes: a sheath fluid inlet for delivering sheath fluid to the conical structure of the flow chamber.

[0336] 137. The flow cytometer according to Clause 136, wherein the longitudinal distance between the distal end of the needle of the sample injection needle adapter and the sheath fluid inlet is in the range of 17 mm to 26 mm.

[0337] 138. The flow cytometer according to clause 136 or 137, wherein the flow chamber body includes a plurality of sheath fluid inlets.

[0338] 139. The flow cytometer according to Clause 138, wherein the sheath fluid inlets are staggered, such that the sheath fluid flows through the conical structure of the flow chamber in a swirling manner.

[0339] 140. The flow cytometer according to any one of clauses 117 to 139, wherein the distal end of the flow chamber body includes a cuvette for conveying particles in the central flow through the sample detection region.

[0340] 141. The flow cytometer according to Clause 140, wherein at least a portion of the cuvette comprises a light-transmitting entity.

[0341] 142. The flow cytometer according to Clause 141, wherein the light-transmitting portion of the cuvette is configured to allow optical detection of particles in the central stream.

[0342] 143. The flow cytometer according to any one of clauses 140 to 142, wherein the cuvette is positioned at the distal end of the flow chamber body by a clamp fixed to the flow chamber body.

[0343] 144. The flow cytometer according to Clause 143, wherein the cuvette is releasably connected to the distal end of the flow chamber body via the flow chamber body clamp.

[0344] 145. A flow cytometer according to clause 143 or 144, wherein the cuvette is positioned by the flow chamber body clamp such that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0345] 146. A method for analyzing a sample fluid, the method comprising:

[0346] (a) Introducing sample fluid into a flow cytometer, the flow cytometer comprising:

[0347] The flow chamber includes:

[0348] A flow chamber body for transporting particles in the center flow of a flow stream from a proximal end to a distal end, wherein the flow chamber body includes a flow chamber conical structure at the proximal end; and

[0349] A sample injection needle having a through-flow for delivering sample fluid from a proximal sample injection line to a distal flow chamber body to generate the central flow, wherein the sample injection needle comprises:

[0350] A sample injection needle adapter, comprising a sample tube adapter attached to the needle; and

[0351] A clamp that operatively connects the sample injection needle to the flow chamber body;

[0352] In the non-clamping configuration of the flow chamber, the sample injection needle adapter is free to rotate relative to the flow chamber conical structure and the clamp, and in the clamping configuration of the flow chamber, the sample injection needle adapter is fixed relative to the flow chamber conical structure and the clamp.

[0353] A light source, configured to irradiate particles in the flow stream at the sample detection point within the flow chamber; and

[0354] A detector configured to collect light emitted by irradiated particles; and

[0355] (b) Irradiate the particles in the flow to analyze the sample fluid.

[0356] 147. The method according to Clause 146, wherein 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 chamber conical structure.

[0357] 148. The method according to Clause 147, wherein, in the clamping configuration, the sample injection needle adapter is secured relative to the flow chamber conical structure and the clamp by compression of the clamp.

[0358] 149. The method according to clause 147 or 148, wherein the clamp is compressed by one or more fastening members.

[0359] 150. The method according to Clause 149, wherein the clamp is compressed by a plurality of screws.

[0360] 151. The method according to Clause 150, wherein the clamp is compressed by three screws.

[0361] 152. The method according to clause 151 or 152, wherein the clamp includes a set of holes for receiving each of a plurality of screws.

[0362] 153. The method according to Clause 152, wherein the flow chamber body includes: a set of holes aligned with a set of clamping holes for receiving each of the plurality of screws.

[0363] 154. The method according to any one of clauses 150 to 153, wherein the clamp is configured such that the tilt of the sample injection needle relative to the flow chamber body can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0364] 155. The method according to any one of clauses 147 to 154, wherein the clamp comprises: a distal end in contact with the sample tube adapter, and a proximal end fluidly connecting the sample injection line to the sample injection needle.

[0365] 156. The method according to Clause 155, wherein the distal end of the clamp includes a recess and a surface, at least a portion of the sample tube adapter is positioned within the recess, and the surface contacts the proximal end of the flow chamber body.

[0366] 157. The method according to clause 155 or 156, wherein the proximal end of the clamp includes a connector configured to minimize the dead volume of the sample fluid as the sample fluid flows from the sample injection line to the sample injection needle.

[0367] 158. The method according to any one of clauses 155 to 157, wherein the clamp is used to position the flow meter board connector at its proximal end.

[0368] 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 a proximal end of the flow chamber body.

[0369] 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 chamber body.

[0370] 161. The method according to clause 159 or 160, wherein the sample tube adapter includes a flange that contacts the proximal end of the flow chamber body.

[0371] 162. The method according to any one of clauses 159 to 161, wherein the distal end of the sample tube adapter is pressed against the proximal end of the flow chamber body by the clamp, such that the distal end of the needle of the sample injection needle adapter is fixed in position within the conical structure of the flow chamber.

[0372] 163. The method according to any one of clauses 157 to 162, wherein the needle of the sample injection needle adapter is tapered at the distal end.

[0373] 164. The method according to Clause 163, wherein the needle of the sample injection needle adapter includes a rounded distal end.

[0374] 165. The method according to any one of clauses 147 to 164, wherein the distal end of the needle of the sample injection needle adapter is positioned within the conical structure of the flow chamber such that a complete central flow can be maintained when the flow conditions change by an order of magnitude or more.

[0375] 166. The method according to any one of clauses 147 to 165, wherein the flow chamber body includes: a sheath fluid inlet for conveying sheath fluid to the conical structure of the flow chamber.

[0376] 167. The method according to Clause 166, wherein the longitudinal distance between the distal end of the needle of the sample injection needle adapter and the sheath fluid inlet is in the range of 17 mm to 26 mm.

[0377] 168. The method according to clause 166 or 167, wherein the flow chamber body includes a plurality of sheath fluid inlets.

[0378] 169. The method according to Clause 168, wherein the sheath fluid inlets are staggered from one another, such that the sheath fluid flows through the conical structure of the flow chamber in a swirling manner.

[0379] 170. The method according to any one of clauses 147 to 169, wherein the distal end of the flow chamber body includes a cuvette for conveying particles in the central flow through the sample detection area.

[0380] 171. The method according to Clause 170, wherein at least a portion of the cuvette comprises a light-transmitting entity.

[0381] 172. The method according to Clause 171, wherein the light-transmitting portion of the cuvette is configured to allow optical detection of particles in the central stream.

[0382] 173. The method according to any one of clauses 170 to 172, wherein the cuvette is positioned at a distal end of the flow chamber body by a clamp fixed to the flow chamber body.

[0383] 174. The method according to Clause 173, wherein the cuvette is releasably attached to the distal end of the flow chamber body via the flow chamber body clamp.

[0384] 175. The method according to clause 173 or 174, wherein the cuvette is positioned by the flow chamber body fixture such that the sample detection area is optimally aligned with the cuvette for optical detection of particles in the central flow.

[0385] 176. The method according to any one of clauses 146 to 175, wherein the sample is a biological sample.

[0386] 177. The method according to Clause 176, wherein the sample comprises cells.

[0387] 178. The method according to any one of clauses 146 to 177, wherein the method further comprises: sorting the sample by flow cytometry.

[0388] 179. A kit comprising:

[0389] A sample injection needle adapter for fluidly connecting a sample injection tubing to the flow chamber body of a flow cytometer, the sample injection needle adapter including a sample tubing adapter attached to the needle; and

[0390] A clamp for operatively connecting the sample injection needle adapter to the flow chamber body.

[0391] 180. The kit according to Clause 179, wherein the needle of the sample injection needle adapter includes a through-passage for conveying sample fluid from a sample injection line at a proximal end to a flow chamber conical structure of a flow chamber body at a distal end, and

[0392] The sample tube adapter includes a proximal end and a distal end, the proximal end being configured to attach to a clamp, and the distal end being fixed to the proximal end of the needle.

[0393] 181. The kit according to Clause 180, wherein the clamp is configured to operatively connect the sample injection needle adapter to the flow chamber body by pressing the sample tube adapter against the flow chamber body.

[0394] 182. The kit according to Clause 181, wherein the distal end of the clamp includes a surface configured to contact the flow chamber body when the clamp presses the sample tube adapter against the flow chamber body.

[0395] 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 chamber body.

[0396] 184. The kit according to Clause 183, wherein the sample tube adapter includes a flange for positioning the distal portion of the sample tube adapter within the flow chamber body near the flow chamber conical structure.

[0397] 185. The kit according to Clause 184, wherein the flange is configured to position the distal portion of the sample tube adapter within the flow chamber body such that the longitudinal distance separating the distal end of the needle from the sheath fluid inlet of the flow chamber body is in the range of 17 mm to 26 mm.

[0398] 186. The kit according to any one of clauses 181 to 185, wherein the sample tube adapter and the clamp are configured such that: when the clamp operatively connects the sample injection needle adapter to the flow chamber body, compression of the sample tube adapter by the clamp fixes the sample injection needle adapter relative to the flow chamber body.

[0399] 187. The kit according to Clause 186, wherein the sample tube adapter is secured such that the distal end of the needle of the sample injection needle adapter is fixed in position within the flow chamber conical structure.

[0400] 188. The kit according to Clause 187, wherein the fixed position enables the maintenance of a complete central flow within the flow chamber conical structure under varying flow conditions by an order of magnitude or more.

[0401] 189. The kit according to any one of clauses 181 to 188, wherein the clamp is configured to receive: a fastening member for securing the clamp to the flow chamber body.

[0402] 190. The kit according to Clause 189, wherein the clamp is configured such that when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, compression of the clamp by the fastening member fixes the sample injection needle adapter relative to the flow chamber body.

[0403] 191. The kit according to clauses 189 or 190, wherein the fastening member comprises a plurality of screws.

[0404] 192. The kit according to Clause 191, wherein the clamp includes a set of holes for receiving each of a plurality of screws.

[0405] 193. The kit according to Clause 192, wherein the set of holes is configured to align with a set of holes in the flow chamber body.

[0406] 194. The kit according to any one of clauses 191 to 193, wherein the sample tube adapter and the clamp are configured such that: when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, the tilt of the sample injection needle adapter relative to the flow chamber body can be adjusted by manipulating the torque of at least one of a plurality of screws that fasten the clamp to the flow chamber body.

[0407] 195. The kit according to Clause 194, wherein the sample tube adapter and the clamp are configured such that: when the sample injection needle adapter is operatively connected to the flow chamber body via the clamp, the position of the distal end of the needle within the flow chamber conical structure can be adjusted by manipulating the torque of at least one of the plurality of screws.

[0408] 196. The kit according to Clause 195, wherein adjusting the torque of one of the plurality of screws causes the distal end of the needle to pivot about an axis.

[0409] 197. The kit according to any one of clauses 192 to 196, wherein the kit further comprises a plurality of screws.

[0410] 198. The kit according to any one of 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.

[0411] 199. The kit according to Clause 198, wherein the portion of the proximal end of the sample tube adapter includes an outer surface concentric with the inner surface of the recess.

[0412] 200. The kit according to clause 198 or 199, wherein the portion of the proximal end of the sample tube adapter and the recess of the clamp are configured such that the sample injection needle adapter is rotatably movable relative to the clamp when the portion of the sample tube adapter is positioned within the recess.

[0413] 201. The kit according to any one of clauses 181 to 200, wherein the needle is tapered at the distal end.

[0414] 202. The kit according to Clause 201, wherein the needle includes a circular distal end.

[0415] 203. The kit according to any one of clauses 181 to 202, wherein 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 tubing to the needle of the sample injection needle adapter.

[0416] 204. The kit according to Clause 203, wherein the proximal end of the clamp is configured to position the flow meter board connector.

[0417] 205. The kit according to any one of clauses 181 to 204, wherein the kit further comprises: packaging configured to receive the sample injection needle adapter and the clamp.

[0418] 206. The kit according to any one of clauses 181 to 205, wherein the kit further comprises a flow chamber body.

[0419] 207. The kit according to Clause 206, wherein the flow chamber body is configured to transport particles in the center flow of the flow stream from the proximal end to the distal end, wherein the flow chamber body includes a flow chamber conical structure at the proximal end.

[0420] 208. The kit according to Clause 207, wherein the flow chamber body includes: a sheath fluid inlet for delivering sheath fluid to the conical structure of the flow chamber.

[0421] 209. The kit according to Clause 208, wherein the flow chamber body includes a plurality of sheath fluid inlets.

[0422] 210. The kit according to Clause 209, wherein the sheath fluid inlets are staggered from one another, such that the sheath fluid flows through the conical structure of the flow chamber in a swirling manner.

[0423] 211. The kit according to any one of clauses 207 to 209, wherein the kit further comprises:

[0424] A cuvette for conveying particles in the central flow through a sample detection area at the distal end of the flow chamber body; and

[0425] A clamp is configured to be fixed to the flow chamber body for positioning the cuvette at the distal end of the flow chamber body.

[0426] 212. The kit according to Clause 211, wherein at least a portion of the cuvette comprises a light-transmitting entity.

[0427] 213. The kit according to Clause 212, wherein the light-transmitting portion of the cuvette is configured to allow optical detection of particles in the central stream.

[0428] 214. The kit according to any one of clauses 211 to 213, wherein the flow chamber body clamp is configured to releasably attach the cuvette to the distal end of the flow chamber body.

[0429] 215. The kit according to Clause 214, wherein the flow chamber body fixture and the cuvette are configured such that the flow chamber body fixture can optimally align the cuvette with the sample detection area for optical detection of particles in the central flow.

[0430] 216. The kit according to any one of clauses 206 to 215, wherein the kit further comprises: packaging configured to receive the sample injection needle adapter, the clamp, and the flow chamber body.

[0431] Although the foregoing disclosure has been described in detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art that some changes and modifications may be made therein without departing from the spirit or scope of the appended claims, based on the teachings of this disclosure.

[0432] Therefore, the foregoing merely illustrates the principles of this disclosure. It will be understood that those skilled in the art will be able to devise various arrangements that, while not expressly described or shown herein, embody the principles of this disclosure and are included within its spirit and scope. Furthermore, all instances and conditional language recorded herein are primarily intended to aid the reader in understanding the principles of this disclosure and the concepts contributed by the discloser to advance the art, and should be construed as not being limited to these specifically recorded instances and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of this disclosure, as well as specific examples thereof, are intended to include equivalents of their structure and function. Furthermore, it is intended that such equivalents include both currently known equivalents and those developed in the future, i.e., any developed element performing the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly stated in the claims.

[0433] Therefore, 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.S.C. 112(f) or 112(6) expressly provides that the foregoing clause is invoked only if a claim begins with the precise expression “meansfor” or “step for”; and if a claim does not use the precise expression, then 35 U.S.C. 112(f) or 112(6) is not invoked.

Claims

1. A flow cytometer, comprising: The flow chamber includes: A flow chamber body for conveying particles in the center flow of a flow stream from the proximal end to the distal end, wherein the flow chamber body includes a flow chamber cone structure at the proximal end; A sample injection needle having a through passage for delivering sample fluid from a proximal sample injection line to a distal flow chamber body to generate the central flow, wherein the sample injection needle comprises: Sample injection needle adapter, comprising a sample tube adapter attached to the needle; and A clamp that operatively connects the sample injection needle to the flow chamber body; In the non-clamping configuration of the flow chamber, the sample injection needle adapter is free to rotate relative to the flow chamber conical structure and the clamp, and in the clamping configuration of the flow chamber, the sample injection needle adapter is fixed relative to the flow chamber conical structure and the clamp. A light source, configured to irradiate particles in a flow stream within the sample detection region of the flow chamber; and The detector is configured to collect the light emitted by the irradiated particles.

2. The flow cytometer according to claim 1, wherein, 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 chamber conical structure.

3. The flow cytometer according to claim 2, wherein, In the clamping configuration, the sample injection needle adapter is secured relative to the flow chamber conical structure and the clamp by compression of the clamp.

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

5. The flow cytometer according to claim 4, wherein, The clamp is compressed by multiple screws.

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

7. The flow cytometer according to claim 6, wherein, The flow chamber body includes a set of holes that align with a set of clamping holes and receive each of the plurality of screws.

8. The flow cytometer according to any one of claims 5 to 7, wherein, The clamp is configured such that the tilt of the sample injection needle relative to the flow chamber 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 includes a distal end that contacts the sample tube adapter and a proximal end that fluidly connects the sample injection tubing to the sample injection needle.

10. The flow cytometer according to claim 9, wherein, The distal end of the clamp includes a recess and a surface, at least a portion of the sample tube adapter is positioned within the recess, and the surface contacts the proximal end of the flow chamber body.

11. The flow cytometer according to claim 9 or 10, wherein, 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.

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

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

14. The flow cytometer according to any one of claims 11 to 13, wherein, The distal end of the sample tube adapter is pressed against the proximal end of the flow chamber body by the clamp, so that the distal end of the needle of the sample injection needle adapter is fixed in the conical structure of the flow chamber.

15. The flow cytometer according to any one of claims 2 to 14, wherein, The needle of the sample injection needle adapter is tapered at the 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 conical structure of the flow chamber, so that a complete central flow can be maintained when the flow conditions change by one or more orders of magnitude.

17. The flow cytometer according to any one of claims 2 to 16, wherein, The main body of the flow chamber includes a sheath fluid inlet for conveying sheath fluid to the conical structure of the flow chamber.

18. The flow cytometer according to claim 17, wherein, The longitudinal distance between the distal end of the needle of the sample injection needle adapter and the sheath fluid inlet is 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 chamber body includes a cuvette for conveying particles in the central flow through the sample detection area, wherein the cuvette is positioned at the distal end of the flow chamber body by a clamp fixed to the flow chamber body.

20. The flow cytometer according to claim 19, wherein, The cuvette is positioned by the main fixture of the flow chamber, so that the sample detection area is optimally aligned with the cuvette, so as to perform optical detection of particles in the central flow.

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