A flow cytometer with a variable depth sample line injection tube and its method of use.

Abstract

In flow cytometers, the design of the sample injection tube does not take into account the bottom structure of the sample container, resulting in some sample remaining in the container that is not drawn into the flow cytometer, thus wasting sample. Therefore, this invention provides a flow cytometer that reduces this dead volume. [Solution] A flow cytometer having a variable depth sample line injection tube, comprising a flow cell and a sample injection tube (SIT) assembly having a sample line operably coupled to the flow cell, wherein the SIT assembly is configured to provide a variable sample line depth within the sample container.

Description

[Technical Field]

[0001] Cross-reference of related applications In accordance with 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 718,451, filed on 8 November 2024, the disclosures of which are incorporated herein by reference in their entirety. [Background technology]

[0002] The characterization of analytes in biological fluids is a crucial part of biological research, medical diagnosis, and the assessment of a patient's overall health and wellness. Detecting analytes in biological fluids such as human blood and blood-derived products can yield results that can play a role in determining treatment protocols for patients with various disease conditions.

[0003] 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 a blood sample, and a sheath reservoir for containing sheath fluid. The flow cytometer transports particles (including cells) in the fluid sample as a flow of cells into the flow cell while directing the sheath fluid towards the flow cell. Light is irradiated into the flow stream to characterize its components. Variations in the material in the flow stream, such as morphology or the presence of fluorescent labels, can cause variations in the observed light, which enable characterization and separation. To characterize the components in the flow stream, light must strike and collect the flow stream. The light source in the flow cytometer can vary and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0004] The isolation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles in an isolated stream that are detected to possess one or more desired characteristics are individually isolated from the sample stream by mechanical or electrical removal. A common flow sorting technique utilizes droplet sorting, in which a fluid stream containing linearly isolated particles is divided into droplets. The droplets containing the particles of interest are charged and deflected towards the collection tube by passing through an electric field. Typically, linearly isolated particles in the stream are characterized as they pass through an observation point located directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, the time it takes for the particle to reach the droplet departure point and detach from the stream in the droplet can be predicted. Ideally, a brief charge is applied to the fluid stream just before the droplet containing the selected particles detaches from the fluid stream, and then grounded immediately after the droplet detaches. The droplet to be sorted retains its charge when detaching from the fluid stream, while all other droplets remain uncharged.

[0005] Samples to be analyzed in a flow cytometer may initially be provided in a sample container, such as a tube or a well in a multiwell plate. A sample injection tube may be used to introduce the sample from the sample container into the flow cell of the flow cytometer. Figure 1A shows a diagram of a sample injection tube found in a flow cytometer currently in use. As shown in Figure 1A, the sample injection tube 10 includes a sample line 25 fixed to the first end 40 of a support arm 30 by a sample line screw 15. Since the sample line screw fixes the sample line at the end 40 of the support arm 30, the sample line 25 does not move in the z direction relative to the end 40 of the support arm 30. An outer sleeve 20, which serves as part of a droplet containment system, is also shown. The support arm 30 is coupled at its end 45 to a first actuator 35, which moves the arm in the z direction relative to the outer sleeve 20 so that the distal end 25a of the sample line 25 can be moved into the outer sleeve 20 for cleaning and out of the outer sleeve to draw the sample from the sample container. A second actuator 50, which is a motor-driven actuator, is also shown, which moves the entire assembly up and down in the z-direction. [Overview of the project]

[0006] The inventors have recognized that the currently used sample injection tube design, as shown in Figure 1A, does not take into account the various bottom configurations of sample containers. Therefore, the currently used sample injection tube configuration results in varying amounts of dead volume (i.e., sample in the container that is not drawn into the flow cytometer), and this dead volume differs among many different sample container types and manufacturers. In a given workflow, the dead volume can vary in some cases from 5 to 30 mL, depending on the type and height of the bottom of the sample container device.

[0007] The embodiments described herein address the dead volume problem that occurs in sample injection tubes currently in use. In the embodiments, the sample injection tube (i.e., SIT) is configured such that the sample line is adjustable in the z direction relative to the support arm, thereby reducing the sample dead volume to approximately 0 ml. By providing the ability to adjust the sample line in the z direction relative to the support arm, the sample injection tube of the embodiments of the present invention can be used with a variety of different sample containers such that the dead volume is substantially reduced, if not eliminated.

[0008] A flow cytometer according to a particular embodiment includes a flow cell and a sample injection tube (SIT) assembly having a sample line operably coupled to the flow cell, the SIT assembly being configured to provide a variable sample line depth within the sample container. Methods for cytologically processing samples, for example, in analytical and / or sorting applications are also provided.

[0009] A flow cytometer is provided. An embodiment of the flow cytometer provided includes a flow cell and a sample injection tube (SIT) assembly having a sample line operably coupled to the flow cell, wherein the SIT assembly is configured to provide a variable sample line depth in the sample container. In some cases, the flow cytometer provided includes a flow cell, a sample container housing area, a sample injection tube (SIT) assembly having a sample line fluidly coupled to the flow cell, and an actuator coupled to the SIT assembly, configured to move the SIT assembly, wherein the SIT assembly is configured to provide a variable sample line depth in the sample container located in the sample container housing area. In some cases, the SIT assembly includes an arm having a first proximal end and a second distal end having a through-hole coupled to the actuator, and a sample line subassembly installed in the through-hole, wherein the sample line subassembly is reversibly movable in the z direction with respect to the xy plane of the second end. In the embodiment, the sample line subassembly is reversibly movable in the z direction by a predetermined displacement distance relative to the xy plane of the second end, and in some cases the predetermined displacement distance is in the range of 1 mm to 2 mm, for example, 1.5 mm.

[0010] In embodiments, the sample line subassembly includes a threaded bush, at least a portion of which is located within a through hole, having a threaded hole and a sample line hole through which it passes, and a sample line thread coupled to the threaded hole, having an inner bore for receiving the sample line. In some cases, the sample line subassembly includes a threaded bush in the form of a sliding bush installed within a through hole. The sliding bush may include an upper wall having threads and a bottom having a central hole. Furthermore, the sample line subassembly may include a sample line thread screwed into the sliding bush, and a sample line passing through the central axis of the sample line thread and the central hole of the sliding bush. In embodiments, the sample line subassembly further includes a ferrule in the threaded hole, through which the sample line passes; for example, the ferrule is positioned between the sample line thread and the bottom of the sliding bush, and the sample line passes through the ferrule.

[0011] In embodiments, the SIT assembly may further include a spring configured to compress the sample line subassembly onto an arm. In some cases, the spring includes an elongated, flat structure including a return bend, for example, an opening at a first end through which the sample line thread of the sample line subassembly is coupled to the arm. If desired, the SIT assembly may further include a restraint member configured to restrict the movement of the sample line subassembly in the z direction relative to the xy plane at a second end. In embodiments, the flow cytometer may further include a sensor for measuring the displacement of the return bend, for example, the flow cytometer is configured to provide an evaluation of the positioning of the sample line in the sample container based on the output from the sensor. In some cases, the SIT is coupled to an actuator, such as a dual guide rod cylinder actuator.

[0012] The flow cytometer of the embodiment further includes a light source configured to irradiate the flow cell at an interrogation point and a detector configured to collect particle-modulated light from the flow cell. The flow cytometer may be a particle analyzer and may further be a particle sorter. In some cases, the flow cytometer is an imaging flow cytometer.

[0013] Aspects of the present disclosure further include a method of analyzing a sample, the method comprising introducing a particulate sample from a sample container into a flow cytometer using a sample injection tube (SIT) assembly operatively coupled to a flow cell, the SIT assembly being configured to provide a variable sample line depth within the sample container, and analyzing the sample by flow cytometry.

Brief Description of the Drawings

[0014] The present disclosure can be best understood when the following detailed description is read in conjunction with the accompanying drawings. The drawings include the following figures.

[0015] [Figure 1A] The figure showing a sample injection tube (SIT) assembly used in a currently used flow cytometer. [Figure 1B] The figure showing the SIT assembly according to a specific embodiment. [Figure 1C] The figure showing the SIT assembly according to a specific embodiment. [Figure 1D] The figure showing the SIT assembly according to a specific embodiment. [Figure 1E] The figure showing the SIT assembly according to a specific embodiment. [Figure 2] The figure showing a flow cytometry system according to a specific embodiment. [Figure 3-1] The figure showing an image-corresponding particle sorter according to a specific embodiment. [Figure 3-2] The figure showing an image-corresponding particle sorter according to a specific embodiment (continuation of FIG. 3-1). [Figure 4]Functional block diagram of a particle analysis system according to a particular embodiment. [Figure 5] Functional block diagram of an example of a control system according to a particular embodiment. [Figure 6A] Schematic diagram of a particle sorter system according to a particular embodiment. [Figure 6B] Schematic diagram of a particle sorter system according to a particular embodiment. [Figure 7] Aspect of a computer control system according to a particular embodiment. **Modes for Carrying Out the Invention**

[0016] Aspects of the present disclosure include a flow cytometer having a variable depth sample line injection tube. A flow cytometer according to a particular embodiment includes a flow cell and a sample injection tube (SIT) assembly having a sample line operably coupled to the flow cell, and the SIT assembly is configured to provide a variable sample line depth within a sample container. For example, a method of cytometrically processing a sample in an analytical and / or sorting application is also provided.

[0017] Before explaining the present disclosure in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, and thus may of course vary. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0018] Where a range of values ​​is presented, it should be understood that each value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless explicitly indicated otherwise in the context, and any other stated or intermediate values ​​within that stated range are included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller range, except for any specifically excluded limits within the stated range, and are also included in this disclosure. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0019] In this specification, certain ranges are presented with numbers preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number preceded by the term, as well as for numbers that are close to or nearly close to the number preceded by the term. In determining whether a number is close to or approximates a specifically stated number, the close or approximate unstated number may, in the context in which it is presented, represent a substantial equivalent of the specifically stated number.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but representative exemplary methods and materials are described here.

[0021] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials to which the publications are relatedly cited. Any citation of a publication is for the purpose of making that disclosure prior to the filing date, and this disclosure should not be construed as an acknowledgment that such publication has no prior rights by prior disclosure. Furthermore, the publication dates presented may differ from the actual publication dates and may need to be independently verified.

[0022] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. It should also be noted that claims may be written to exclude any optional element. Therefore, this statement is intended to serve as a precedent for the use of exclusive terms such as “alone” and “only” in relation to the enumeration of elements in the claims or the use of “negative” limitations.

[0023] As will be obvious to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of this disclosure. Any method of description may be carried out in the order of the described events or in any other logically possible order.

[0024] While systems and methods are described for grammatical fluidity with functional descriptions, claims should not necessarily be interpreted as being limited by constructing a limitation of “means” or “steps” unless explicitly formulated under 35 U.S. SC § 112, and should be given the meaning of the definitions and the full scope of equivalents provided by the claims under the doctrine of equivalents, and if the claims are explicitly formulated under 35 U.S. SC § 112, it should be clearly understood that a full set of statutory equivalents should be given under 35 U.S. SC § 112.

[0025] Device / System Section As summarized above, aspects of the present disclosure include sample injection tube (SIT) assemblies with variable sample line depth. The SIT assemblies disclosed herein are used to introduce a fixed amount of liquid sample from a sample container, such as a tube or well (such as a well in a multiwell plate), into a sample fluid line connected to a flow cell of a flow cytometer. The SIT assemblies of embodiments of the present application are configured to draw a sample from a sample container into the lumen of the sample line of the SIT assembly, from which the drawn sample can be transported by a flow cytometer to the flow cell of the flow cytometer.

[0026] As summarized above, the SIT assemblies of embodiments of this disclosure are SIT assemblies with variable sample line depth. Therefore, they exhibit variable line depth with respect to the sample line of the SIT assembly; that is, the SIT assembly is configured to provide a variable sample line depth depending on a given sample container from which the sample line draws fluid. Variable sample line depth means that the SIT assembly can adapt to a given container to provide, if any, very little dead volume without movement by (one or more) actuators that control the z-movement of the arm and / or the SIT assembly as a whole. Thus, movement of the sample line of the SIT assembly to adapt to a given sample container is achieved without movement of (one or more) z-actuators that may be coupled to the proximal end of the SIT assembly, e.g., the SIT arm and / or the proximal end of the entire SIT assembly. Because the SIT assembly is a SIT assembly with variable sample line depth, it offers significant advantages with respect to sample utilization. Specifically, by using the SIT assembly of the embodiments of this disclosure, the occurrence of sample dead volume, i.e., sample volume present in a given sample container that is inaccessible by the flow cytometer (i.e., cannot be drawn into the flow cytometer by the sample injection tube), is substantially reduced, if not eliminated. Thus, embodiments of the SIT assembly can provide sample dead volume including less than 1 ml, e.g., less than 0.5 ml, and less than 0.1 ml, e.g., less than 0.01 ml, and in some cases, no sample may remain in the sample container, i.e., no sample dead volume can be provided. Therefore, the SIT assemblies disclosed herein are advantageous in applications such as those where analysis of the entire sample is desired, or where avoidance of sample waste is desired.

[0027] Figure 1B shows a diagram of a SIT assembly according to one embodiment of the present disclosure. As shown in Figure 1B, the SIT assembly 100 includes an arm 110 having a first proximal end 145 coupled to an actuator (not shown) and a second distal end 140 including a through hole 130. Also shown is a sample line subassembly 120 (in the illustrated embodiment, collectively consisting of a sample line screw 122, a sliding bush 124, a ferrule 126, and a sample line 128) installed in the through hole. As shown, the sample line subassembly is reversibly movable in the z direction by a displacement distance (d) relative to the xy plane of the second end of the arm 110. When loading a sample from a sample container into a flow cytometer using the illustrated SIT assembly, the open end 128a of the sample line 128 is initially positioned within a sample container (not shown) having a bottom 150 by moving the arm 110 with an actuator (not shown) such that the arm 110 moves downward as indicated by arrow Z1 and the bottom 150 of the sample container moves upward toward the open end 128a of the sample line 128 as indicated by arrow Z2. Optionally, the displacement distance (d) may be predetermined or variable. By predetermined, the displacement distance is within a set range such that the sample line subassembly cannot move outside this range. In some cases, the predetermined displacement distance is in the range of 0.05 to 5 mm, e.g., 1 to 3 mm, e.g., 1 to 2 mm, and in some cases, 1.5 mm.

[0028] In embodiments, the sample line subassembly may be of any kind, as long as it is configured to hold the sample line at the second end of the sample arm and to provide a variable sample line depth within the sample container, as described, for example. Thus, the sample line subassembly of embodiments of the present disclosure is a subassembly coupled to an arm and movable in the z direction by a displacement distance relative to the xy plane of the arm. Depending on the particular configuration, the sample line subassembly may move the z-displacement distance using any convenient method, such as a mechanical method (e.g., using a spring), a motor-driven method, or a pneumatic method.

[0029] Figure 1B shows one embodiment of a sample line subassembly that uses spring force to vary the sample line depth. In Figure 1B, the sample line subassembly 120 includes a sample line thread 122, a sliding bush 124, a ferrule 126, and a sample line 128. As shown, the sliding bush 124 is installed in a through hole 130 of the arm 110. The sliding bush includes an upper wall having threads and a bottom having a central hole sized to allow the sample line to pass through. The sample line thread 122 is screwed into the sliding bush. Furthermore, the sample line 128 passes through the central axis of the sample line thread and the central hole of the sliding bush 124. The ferrule 126 is positioned between the sample line thread 122 and the bottom of the sliding bush 124, and the sample line 128 passes through the ferrule. The length of the sample line 128 extending below the bottom of the sliding bush 124 may be varied as desired.

[0030] As shown in Figure 1B, the SIT assembly 100 further includes a spring configured to compress the sample line subassembly onto the arm. In the embodiment shown in Figure 1B, the spring is configured as a return bend 150. The return bend 150 contacts the upper flange 160 of the sliding bush 124 and applies a compressive force so that the spring biases the sliding bush downward in the z direction. The magnitude of the compressive force applied by the spring is such that it accommodates the upward movement of the sample line in the z direction when the sample line reaches the bottom of the sample container without deforming the sample line. The compressive force applied by the return bend 150 on the flange 160 of the sliding bush 124 may vary, and in some cases may be in the range of 0.5 to 2.5 Newtons, for example, 0.75 to 1.25 Newtons, or for example, 1 Newton. Figures 1C and 1D show a top view and a bottom view, respectively, of the SIT assembly 100 shown in Figure 1B. As shown in the figure, the return bend is an elongated, flat strip having an opening 155 at a first end through which the sample line thread 122 of the sample line subassembly passes. The return bend 150 is also connected to the arm at a second end by a wing nut 170.

[0031] In embodiments including a return bend as shown in Figures 1B to 1D, the SIT assembly may further include a restraint member configured to restrict the movement of the sample line subassembly in the z direction relative to the xy plane at the second end. Specifically, the restraint member may be included to prevent upward movement in the z direction by a distance greater than desired, for example, beyond a predetermined displacement distance. The restraint member, if present, may have any convenient configuration. One embodiment of a suitable restraint member is shown in Figure 1E. As shown, the restraint member 180 is an extension of the arm 110 and is configured to restrict the upward movement of the sample line subassembly in the z direction by extending over the end of the return bend 150. Also shown is an actuator 190 that moves the arm of the SIT assembly up and down in the z direction relative to the outer sleeve 20. As shown, the proximal end of the arm 110 of the SIT assembly is coupled to the actuator 190 that provides the up and down movement of the SIT arm relative to the outer sleeve 20. Any convenient actuator may be used. Figure 1E shows a diagram of an actuator that may be used to provide z-direction movement of the SIT arm in embodiments of the present disclosure. As shown in Figure 1E, actuator 190 is a dual guide rod cylinder actuator. Also shown is a second actuator 195 for moving the entire SIT assembly up and down in the z-direction. Actuator 195 may be any convenient actuator, such as a motor-driven actuator. In some embodiments, actuator 195 is a pneumatically driven actuator, such as the one described in concurrently pending application No. 60 / __________ (Agent reference number BECT-388PRV), filed on the same date as this application, whose disclosure is incorporated herein by reference.

[0032] If desired, the return bend SIT assembly, for example, as shown in Figures 1B to 1E, may include a sensor for measuring the displacement of the bend arm. Such a sensor may be used to determine the position of the sample line, such as the opening of the sample line relative to the sample container, such as at the bottom of the sample container. In such embodiments, the flow cytometer may be configured to provide an evaluation of the sample line positioning within the sample container based on the output from the sensor. For example, there may be a processor that uses the output of the sensor to provide an evaluation of the sample line positioning within the sample container. The sensor may be any convenient position sensor, if present, and examples of position sensors include, but are not limited to, capacitive displacement sensors, eddy current sensors, Hall effect sensors, induction sensors, laser Doppler vibrometers (optical), linear variable differential transformers (LVDTs), photodiode arrays, piezoelectric transducers (piezoelectric), position encoders, absolute encoders, incremental encoders, linear encoders, rotary encoders, potentiometers, proximity sensors (optical), string potentiometers, ultrasonic sensors, and the like.

[0033] As described above, the SIT assembly of the embodiment is configured to obtain a sample from a sample container, such as a tube or a well of a multiwell plate, by drawing the sample into the sample line of the SIT assembly and then transporting the drawn sample to a flow cell, either directly or via an additional (one or more) fluid line. The flow cell of interest includes a cuvette configured to transport particles in a flow stream. As used herein, “flow cell” is described in its conventional sense, referring to an element that includes a channel for a liquid flow stream for transporting particles in a sheath fluid. The cuvette of interest has a passage (i.e., a channel) through which it passes. The flow stream through which the channel is formed may include a liquid sample injected from a sample tube. In certain cases, the flow cell includes a light-accessible channel. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed from silica, such as fused silica. In some cases, the flow cell is configured to be illuminated from a light source at one or more interrogation points. As discussed herein, “interrogation point” refers to a region within a flow cell where particles are irradiated by light from a light source, for example, for analysis. The size of the interrogation point may vary as desired. For example, if 0 μm represents the optical axis of the light emitted by the light source, the interrogation point may be in the range of -15 μm to 30 μm, e.g., -25 μm to 40 μm. Depending on specific considerations (e.g., the number and arrangement of lasers), multiple irradiation points may exist within the flow cell.

[0034] In some embodiments, the flow cell includes, or is configured to be used with, a sample injection port configured to provide the sample to the flow cell, for example, by a SIT assembly and, if present, any intervening fluid lines. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the internal chamber (i.e., flow path) of the flow cell. Depending on the desired characteristics of the flowstream, the flow rate of the sample delivered to the flow cell chamber by the sample injection port is 1 μL / min or more, e.g., 2 μL / min or more, e.g., 3 μL / min or more, e.g., 5 μL / min or more, e.g., 10 μL / min or more, e.g., 15 μL / min or more, e.g., 25 μL / min or more, e.g., 50 μL / min or more, and may include 100 μL / min or more. In some cases, the flow rate of the sample delivered to the flow cell chamber by the sample injection port is 1 μL / second or more, e.g., 2 μL / second or more, e.g., 3 μL / second or more, e.g., 5 μL / second or more, e.g., 10 μL / second or more, e.g., 15 μL / second or more, e.g., 25 μL / second or more, e.g., 50 μL / second or more, and may include 100 μL / second or more.

[0035] The sample injection port may be an orifice positioned in the wall of the internal chamber, or a conduit positioned at the proximal end of the internal chamber. If the sample injection port is an orifice positioned in the wall of the internal chamber, the sample injection port orifice may be any suitable shape, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc., curved cross-sectional shapes such as circles, ellipses, etc., and irregular shapes such as parabolic bottoms coupled to flat tops. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm.

[0036] In certain cases, the sample injection port is a conduit positioned at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned so that the orifice of the sample injection port is aligned with the flow cell orifice. If the sample injection port is a conduit positioned in line with the flow cell orifice, the cross-sectional shape of the sample injection tube may be any suitable shape, including, but not limited to, straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as parabolic bottoms joined to flat tops. The orifice of the conduit may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a slanted tip having a 5° inclination angle in the range of 1° to 10°, for example 2° to 9°, for example 3° to 8°, for example 4° to 7°.

[0037] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to provide a flow of sheath fluid into the internal chamber of the flow cell, for example, together with the sample, to create a layered flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of sheath fluid delivered to the flow cell chamber may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, and may include, for example, 2500 μL / sec or more.

[0038] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the internal chamber. The sheath fluid injection port orifice may have any suitable cross-sectional shape of interest, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a flat top. The size of the sheath fluid injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm.

[0039] The flow cytometers of this disclosure include light sources configured to irradiate particles in the flow stream at an interrogation point in the flow cell. The number of light sources in the flow cytometer can vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, the flow cytometer may include multiple light sources in some cases. In some such cases, the number of light sources ranges from 2 to 10, for example, 2 to 5, or for example, 2 to 4. Any convenient light source may be used as a light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous-wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, this flow cytometer includes dye lasers such as stilbene lasers, coumarin lasers, or rhodamine lasers. In yet other cases, the laser of interest includes 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, and combinations thereof.In other examples, this flow cytometer includes solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium 2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0040] A laser light source according to a particular embodiment may also include one or more optical tuning components. In a particular embodiment, the optical tuning component may include any device located between the light source and the flow cell that can change the spatial width of the irradiation, or any other characteristics of the irradiation from the light source, such as the direction of irradiation, wavelength, beam width, beam intensity, and focus. The optical tuning protocol may include, but is not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof, any convenient device for tuning one or more characteristics of the light source. In a particular embodiment, the flow cytometer of interest includes one or more focusing lenses. The focusing lenses may, in one example, be reduction lenses. In yet another embodiment, the flow cytometer of interest includes optical fibers.

[0041] The light source may be positioned at any suitable distance from the flow cell, for example, the light source and the flow cell are separated by 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 25 mm or more, and include, for example, a distance of 100 mm or more. Furthermore, the light source may be positioned at any suitable angle with respect to the flow cell, for example, within an angle range including angles of 10 to 90 degrees, e.g., 15 to 85 degrees, e.g., 20 to 80 degrees, e.g., 25 to 75 degrees, e.g., 30 to 60 degrees, e.g., 90 degrees.

[0042] In some embodiments, the light source of interest includes a plurality of lasers configured to provide laser light for discrete irradiation of a flowstream, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, and e.g., fifteen or more lasers configured to provide laser light for discrete irradiation of a flowstream. Depending on the desired wavelength of light for irradiating the flowstream, each laser may have a specific wavelength, e.g., 400 nm to 800 nm, varying from 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm. In certain embodiments, the laser of interest may include one or more of the following: a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0043] In certain embodiments, the light source is a light beam generator configured to generate two or more frequency-shifted light beams. In some cases, the light beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optical device to generate two or more angle-deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. For example, the laser in the light beam generator of interest may include the above.

[0044] The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in this system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal can be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital combiner (DDS), arbitrary waveform generator (AWG), or electric pulse generator.

[0045] In embodiments, the controller is configured to apply high-frequency drive signals to an acousto-optical device to generate a desired number of angularly deflected laser beams within the output laser beam, and includes being configured to apply, for example, three or more high-frequency drive signals, for example, four or more high-frequency drive signals, for example, five or more high-frequency drive signals, for example, six or more high-frequency drive signals, for example, seven or more high-frequency drive signals, for example, eight or more high-frequency drive signals, for example, nine or more high-frequency drive signals, for example, ten or more high-frequency drive signals, for example, fifteen or more high-frequency drive signals, for example, twenty-five or more high-frequency drive signals, for example, fifty or more high-frequency drive signals, and being configured to apply one hundred or more high-frequency drive signals.

[0046] In some cases, to create an intensity profile of an angularly deflected laser beam within the output laser beam, the controller is configured to apply a high-frequency drive signal having an amplitude including, for example, about 5V to about 25V, which varies, for example, from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V. Each applied high-frequency drive signal has a frequency range of approximately 5 MHz to approximately 50 MHz, for example, approximately 0.001 MHz to approximately 500 MHz, for example, approximately 0.005 MHz to approximately 400 MHz, for example, approximately 0.01 MHz to approximately 300 MHz, for example, approximately 0.05 MHz to approximately 200 MHz, for example, approximately 0.1 MHz to approximately 100 MHz, for example, approximately 0.5 MHz to approximately 90 MHz, for example, approximately 1 MHz to approximately 75 MHz, for example, approximately 2 MHz to approximately 70 MHz, for example, approximately 3 MHz to approximately 65 MHz, for example, approximately 4 MHz to approximately 60 MHz.

[0047] In certain embodiments, the controller has a processor having memory operably coupled to the processor such that the memory contains stored instructions for generating an output laser beam having an angle-deflected laser beam having a desired intensity profile when executed by the processor. For example, the memory may contain instructions for generating two or more angle-deflected laser beams having the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may contain instructions for generating 100 or more angle-deflected laser beams having the same intensity. In other embodiments, the memory may contain instructions for generating two or more angle-deflected laser beams having different intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and the memory may contain instructions for generating 100 or more angle-deflected laser beams having different intensities.

[0048] In certain embodiments, the controller has a processor having memory operably coupled to the processor such that the memory contains stored instructions, when executed by the processor, for generating an output laser beam having an intensity that increases from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, and may also include a range of about 10% to about 50%, such as 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%, etc. In other embodiments, the controller has a processor having memory operably coupled to the processor such that the memory contains stored instructions, when executed by the processor, for generating an output laser beam having an intensity that increases from the edge to the center of the output laser beam along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in the range of 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, and may also include a range of about 10% to about 50%, such as 0.5% to about 95%, 1% to about 90%, 2% to about 85%, 3% to about 80%, 4% to about 75%, 5% to about 70%, 6% to about 65%, 7% to about 60%, 8% to about 55%, etc. In yet another embodiment, the controller has a processor having a memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis.In yet another embodiment, the controller has a processor having a memory operably coupled to the processor such that the memory contains stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis.

[0049] In some embodiments, the light beam generator of interest may be configured to generate spatially separated, angularly deflected laser beams in the output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams may be spaced apart by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, and may include spacing of 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams in the output laser beam that overlap with adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) may be an overlap of 0.001 μm or more, for example, an overlap of 0.005 μm or more, for example, an overlap of 0.01 μm or more, for example, an overlap of 0.05 μm or more, for example, an overlap of 0.1 μm or more, for example, an overlap of 0.5 μm or more, for example, an overlap of 1 μm or more, for example, an overlap of 5 μm or more, for example, an overlap of 10 μm or more, and may include overlaps of 100 μm or more.

[0050] In certain cases, a light beam generator configured to generate two or more frequency-shifted light beams is subject to 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. This includes laser excitation modules described in 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.

[0051] Furthermore, the flow cytometer includes a detector configured to collect light emitted by the irradiated particles. The photodetector is configured to detect particle-modulated light carried by an optical fiber focusing element and to generate a signal based on the characteristics of the light (e.g., intensity). For example, one or more particle-modulated photodetectors may include one or more side-scatter photodetectors for detecting the side-scatter wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particles). In some embodiments, the flow cytometer includes a single side-scatter photodetector. In other embodiments, the flow cytometer includes a plurality of side-scatter photodetectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more.

[0052] Any convenient detector for detecting the collected light can be used in the side-scattered light detector described herein. Among detectors of interest, particularly optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof, but not limited to these. In certain embodiments, the collected light is measured by a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor or an N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is 0.01 cm 2 ~10 cm 2 , for example 0.05 cm 2 ~9 cm 2 , for example 0.1 cm 2 ~8 cm 2 , for example 0.5 cm 2 ~7 cm 2 of, 1 cm 2 ~5 cm 2 is a photomultiplier tube such as a photomultiplier tube having an active detection surface area for each region in the range including.

[0053] In embodiments, the flow cytometer also includes a fluorescence detector configured to detect one or more fluorescence wavelengths of light. In other embodiments, the flow cytometer includes a plurality of, for example two or more, for example three or more, for example four or more, five or more, ten or more, fifteen or more, including twenty or more fluorescence detectors.

[0054] Any convenient detector for detecting the collected light may be used in the fluorescence detector described herein. Detectors of interest include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the collected light is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is 0.01 cm 2 ~10cm 2 For example, 0.05 cm 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 1cm 2 ~5cm 2 This is a photomultiplier tube, such as a photomultiplier tube, having an activity detection surface area in each region within the range including [specific region].

[0055] If the flow cytometer includes multiple fluorescence detectors, each fluorescence detector may be the same, or the array of fluorescence detectors may be a combination of different types of detectors. For example, if the flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector (or imaging sensor) is a CMOS type device. In other embodiments, both the first and second fluorescence detectors are CCD type devices. In yet another embodiment, both the first and second fluorescence detectors are CMOS type devices. In yet another embodiment, the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In yet another embodiment, the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment, both the first and second fluorescence detectors are photomultiplier tubes.

[0056] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure the collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths, and includes measuring the light emitted by the sample in the flow stream at 400 or more different wavelengths. In some embodiments, two or more detectors in the module described herein are configured to measure the same or overlapping wavelengths of the collected light.

[0057] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light over one or more wavelengths in the 200 nm to 1000 nm range. In yet another embodiment, the detector of interest is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a module may include one or more detectors configured to measure light at one or more of the following wavelengths: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm and any combination thereof. In certain embodiments, one or more detectors may be configured to pair with specific fluorophores, such as those used with a sample in a fluorescence assay.

[0058] The flow cytometer may include any suitable (one or more) mechanisms for supplying the sample solution and sheath solution to the sample solution input coupler and sheath solution input coupler. For example, the sample solution input coupler may be fluidly connected to a sample solution line (e.g., a tube) fluidly connected to a sample solution reservoir. Similarly, the sheath solution input coupler may be fluidly connected to a sheath solution line fluidly connected to a sheath solution reservoir. Similarly, the flow cytometer may include any suitable (one or more) mechanisms for managing waste from the flow stream. A fluid discharge coupler may be fluidly connected to a waste line fluidly connected to a waste reservoir. A fluid management system that may be adapted for use with this flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0059] Appropriate flow cytometry systems include: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (ed.), 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 Examples include, but are not limited to, those described in Syst.24(3):203-255, which are incorporated herein by reference.In specific cases, the flow cytometry systems of interest include BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Accuri® C6 Plus flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortessa® X-20 flow cytometer, BD Biosciences FACSPresto® flow cytometer, BD Biosciences FACSVia® flow cytometer, and BD Biosciences FACSCalibur® cell sorter, BD Biosciences FACSCount® cell sorter, and BD Biosciences This includes FACSLyric™ cell sorters, BD Biosciences Via™ cell sorters, BD Biosciences Influx™ cell sorters, BD Biosciences Jazz™ cell sorters, BD Biosciences Aria™ cell sorters, BD Biosciences FACSAria™ II cell sorters, BD Biosciences FACSAria™ III cell sorters, BD Biosciences FACSAria™ Fusion cell sorters, and BD Biosciences FACSMelody™ cell sorters, BD Biosciences FACSymphony™ S6 cell sorters, BD Biosciences FACSDiscover™ cell sorters, and others.

[0060] In some embodiments, this system is based on U.S. Patents No. 10,663,476, No. 10,620,111, No. 10,613,017, No. 10,605,713, No. 10,585,031, No. 10,578,542, No. 10,578,469, No. 10,481,074, and No. 10,302,545. , No. 10,145,793, No. 10,113,967, No. 10,006,852, No. 9,952,076, No. 9,933,341, No. 9, No. 726,527, No. 9,453,789, No. 9,200,334, No. 9,097,640, 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, No. 875, No. 7,129,505, No. 6,821,740, No. 6,813,017, No. 6,809,804, No. 6,372,506, No. 5, Flow cytometry systems such as those described in Patent Nos. 700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766 (these disclosures are incorporated herein by reference in their entirety).

[0061] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in certain cases, this system is based on Diebold, et al. Nature Photonics. Vol.7(10), 806-810(2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, and Flow cytometry systems configured to image particles in a flow stream by fluorescence imaging using high-frequency tagged emission (FIRE), such as those described in Patent Nos. 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851, are incorporated herein by reference. In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in U.S. Patent Nos. 10,324,019, 10,620,111, 11,105,728, and 11,774,343, and U.S. Patent Applications Nos. 18 / 537,103, 18 / 657,618, 18,657,623, and 18 / 657,633, the disclosures of which are incorporated herein by reference in their entirety.

[0062] Figure 2 shows a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. Although the example in Figure 2 shows a single laser, it will be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to a focusing lens 202, which focuses the beam onto the portion of the fluid stream where the particles 211 of the sample in the flow cell 210 are located. The flow cell 210 is part of a fluid system that guides particles in the stream to the focused laser beam, typically one at a time, for interrogation. Alternatively, a nozzle top may be used if the flow cytometer is a stream-in-air cytometer.

[0063] As shown in Figure 2, the flow cell 210 is fluidically connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a conduit (i.e., sheath fluid line) 207. In addition, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce particles 211 into the flow cell 210. The particles 211 are hydrodynamically focused through the sheath fluid entering from the sheath fluid injection port 208 so that a flowstream 214 is formed downstream of the tapered portion 212 of the flow cell 210. Particles released at the distal end of the flow cell 210 can be disposed of and / or collected via any suitable protocol. For example, depending on the type of flow cytometry performed, the particles may be collected at the distal end of the flow cell 210, for example, via a waste line. Alternatively, the particles may be sorted.

[0064] Light from (one or more) laser beams interacts with particles 211 in the sample by diffraction, refraction, reflection, scattering, and absorption, with re-emission at various different wavelengths, depending on the particle's characteristics, such as particle 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 sent to one or more detectors. In particular, forward scatter (FSC) is sent to a forward scatter detector 223. The forward scatter detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect the diffracted light, i.e., the excitation light that travels mainly forward through or around the particles. The intensity of the light detected by the forward scatter detector 223 depends on the overall size of the particles. The forward scatter detector may include, for example, a photodiode. An optical filter 221a and a scattering bar 222 are positioned between the forward scatter detector 223 and the beam. The optical filter 221a may be configured to remove non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scatter light detector 223.

[0065] Furthermore, side-scattered light (SSC) is detected by a side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the complexity of the particle structure increases. In the example in Figure 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 and allow non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scattered light detector 224. Fluorescence detectors 225a-225c, each configured to detect fluorescence of different wavelengths, are also shown. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a and allow light of other wavelengths to pass through. The optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to a first wavelength (or wavelength range) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b and to allow light of a third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. The optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to a second wavelength (or wavelength range) from being detected by the fluorescence detector 225b. Furthermore, the optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to a third wavelength (or wavelength range) from being detected by the fluorescence detector 225c.

[0066] Those skilled in the art will recognize that the flow cytometer according to the embodiments of the present disclosure is not limited to the flow cytometer shown in Figure 2, but may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations. For example, the embodiment in Figure 2 shows three fluorescence detectors for illustrative purposes, but it will be understood that any suitable number of fluorescence detectors may be used.

[0067] During operation, the cytometer's operation is controlled by the controller / processor 290, and measurement data from the detector is stored in memory 295 and can be processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detector to receive output signals from the detector and may also be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function 297 may also be provided within the system. The memory 295, controller / processor 290, and I / O 297 may be provided as a single integrated part of the flow cytometer. In such embodiments, a display may also form part of the I / O function 297 for presenting experimental data to the user of the cytometer 200. Alternatively, some or all of the memory 295, controller / processor 290, and I / O functions may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and controller / processor 290 can communicate with the cytometer 200 wirelessly or via a wired connection. The controller / processor 290 can be configured to work in conjunction with the memory 295 and I / O 297 to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0068] Different fluorescent molecules in a fluorescent dye panel used in a flow cytometer experiment emit light in their own characteristic wavelength bands. Specific fluorescent labels used in the experiment, and their associated fluorescence emission bands, may be selected to substantially match the detector's filter window. I / O297 can be configured to receive data for flow cytometer experiments with a panel of fluorescent labels, and for multiple cell populations having multiple markers, with each cell population having a subset of multiple markers. I / O297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 295. The controller / processor 290 can be configured to evaluate the assignment of one or more labels to the markers.

[0069] In some embodiments, the system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, whose disclosure is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference.

[0070] In certain embodiments, the system is fluorescence imaging using a high-frequency tagged emission image-enabled particle sorter, as shown in Figures 3-1 and 3-2. The particle sorter 300 includes an optical illumination component 300a, which includes a light source 301 (e.g., a 488 nm laser) that generates an output optical beam 301a, which is split into beam 302a and beam 302b using a beam splitter 302. The optical beam 302a is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a having one or more angularly deflected optical beams. In some cases, the output beam 303a generated from the acousto-optical device 303 includes a local oscillator beam and multiple high-frequency comb beams. The optical beam 302b is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a having one or more angularly deflected optical beams. In some cases, the output beam 304a generated from the acousto-optic device 304 includes a local oscillator beam and multiple high-frequency comb beams. The output beams 303a and 304a generated from the acousto-optic devices 303 and 304, respectively, are combined with a beam splitter 305 to generate an output beam 305a, which is then transported through an optical component 306 (e.g., an objective lens) to irradiate particles in the flow cell 307. In certain embodiments, the acousto-optic device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in a beam combiner 305. In certain embodiments, the light irradiation system having a light source and an acousto-optical device may also include those described in Schraivogel, et al. ("High-speed fluorescence image-enabled cell sorting," Science (2022), 375(6578):315-320) and U.S. Patent Application Publication No. 2021 / 0404943, which are incorporated herein by reference.

[0071] The output beam 305a irradiates sample particles 308 propagating through the flow cell 307 (e.g., together with the sheath fluid 309) in the irradiation area 310. As shown in the irradiation area 310, multiple beams (e.g., angle-deflected high-frequency shifted light beams shown as dots across the irradiation area 310) are superimposed on the reference local oscillator beam (indicated by diagonal lines across the irradiation area 310). Due to their different optical frequencies, the overlapping beams exhibit pulsating behavior, thereby giving each beamlet a distinct frequency f 1-n This is used to carry a sine wave modulation signal.

[0072] Light from the irradiated sample is delivered to a photodetection system 300b, which includes multiple photodetectors. The photodetection system 300b includes a forward scatter photodetector 311 for generating a forward scatter image 311a and a side scatter photodetector 312 for generating a side scatter image 312a. The photodetection system 300b also includes a bright-field photodetector 313 for generating an optical loss image 313a. In some embodiments, the forward scatter detector 311 and the side scatter detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the irradiated sample is also detected by fluorescence detectors 314-317. In some cases, the photodetectors 314-317 are photomultiplier tubes. Light from the irradiated sample is directed through a beam splitter 320 to the side scatter detection channel 312 and the fluorescence detection channels 314-317. The photodetector system 300b includes bandpass optical components 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optical component 321 is 534 nm / 40 nm bandpass. In some cases, optical component 322 is 586 nm / 42 nm bandpass. In some cases, optical component 323 is 700 nm / 54 nm bandpass. In some cases, optical component 324 is 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number indicates the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on both sides of the center of the spectral band, i.e., from 500 nm to 520 nm.

[0073] Data signals generated in response to light detected in scattered light detection channels 311 and 312, bright-field light detection channel 313, and fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-responsive sorting is performed in response to sorting signals generated by sorting trigger 352. The sorting component 300c includes deflection plates 331 for deflecting particles into the sample container 332 or into the waste stream 333. In some cases, the sorting component 300c is configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0074] In some embodiments, the system is a particle analyzer and can analyze and characterize particles using the particle analysis system 401 (Figure 4), whether or not the particles are physically sorted into a collection container. Figure 4 shows a functional block diagram of the particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid system 402. The fluid system 402 includes or can include a sample tube 405 and a moving fluid column in the sample tube through which sample particles 403 (e.g., cells) move along a common sample path 409.

[0075] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting light or one or more other properties of a particle 403 as it passes through the monitoring area 407. Figure 4 shows one detection station 408 with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas.

[0076] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be called event data. The data points can be multidimensional data points containing the values ​​of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0077] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuits and / or frequency control circuits. The illustrated control system can be operably associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during a first time interval. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may further compare the experimental signal frequency with that of a calculated signal frequency or a predetermined signal frequency.

[0078] Figure 5 shows a functional block diagram of an example of a particle analyzer control system, such as 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.

[0079] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0080] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. The analysis controller 500 can be configured to provide a display device 506 with a graphic display including a first plot of the biological event data. The analysis controller 500 can be further configured to render a region of interest overlaid on the first plot, for example, as a gate around the collection of biological event data shown by the display device 506. In some embodiments, the gate may be a logical combination of one or more graphic regions of interest depicted in a histogram or bivariate plot of a single parameter. In some embodiments, a display may be used to display particle parameters or saturation detector data.

[0081] The analysis controller 500 can be further configured to display biological event data on the display device 506 within the gate in a different way from other events in the biological event data outside the gate. For example, the analysis controller 500 can be configured to render the colors of the biological event data contained within the gate differently from the colors of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0082] The analysis controller 500 can be configured to receive gate selection signals from a first input device that identify gates. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 that identify gates to be displayed on the display device 506 or operated via the display device (for example, by clicking on a desired gate when the cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 508, or as other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, photodetector, or speech recognition system. Some input devices can include multiple input functions. In such implementations, each of those input functions can be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0083] The trigger event can cause the analysis controller 500 to change how the data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting a population of interest for particle sorting.

[0084] In some embodiments, the analysis controller 500 can be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification may be based on a specific distribution of biological event data received by the analysis controller 500.

[0085] The analysis controller 500 can be connected to the storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to enable the analysis controller 500 to acquire biological event data, such as flow cytometry event data.

[0086] The display device 506 can be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that outline sections of the plots. The display device 506 can be further configured to modify the information presented according to the input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the memory device 504, the keyboard 508, and / or the mouse 510.

[0087] In some implementations, the analysis controller 500 can generate a user interface for receiving exemplary events for selection. For example, the user interface may include controls for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates may be provided before the collection of event data for the sample, or based on an initial set of events for a portion of the sample.

[0088] Figure 6A is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to a nozzle 603, may include a nozzle 603, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample solution 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a line across a monitoring area 611 (e.g., where laser streams intersect) irradiated by an irradiation source 612 (e.g., a laser). The vibration of the droplet-forming transducer 602 divides the moving fluid column 608 into multiple droplets 610, some of which contain particles 609.

[0089] During operation, a detection station 614 (e.g., an event detector) identifies a particle (or cell) of interest as it crosses the monitoring area 611. The detection station 614 supplies power to a timing circuit 628, which supplies power to a flash charge circuit 630. A flash charge can be applied to the moving fluid column 608 so that the droplet of interest becomes charged at the droplet departure point, which is indicated by a timed drop delay (Δt). 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 the droplet into a container such as a collection tube or a multi-well or microwell sample plate, and the well or microwell can be associated with a specific droplet of interest. As shown in Figure 6A, the droplets can be collected in a drain receptacle 638.

[0090] The detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet driving signal as particles of interest pass through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 enables the instrument to accurately calculate the location of each detected particle in the droplet. The detection system 616 can supply amplitude signals 620 and / or phase signals 618, which supply amplitude signals and / or phase signals to amplitude control circuits 626 and / or frequency control circuits 624 (via amplifier 622). The amplitude control circuits 626 and / or frequency control circuits 624 control the droplet formation transducer 602. The amplitude control circuits 626 and / or frequency control circuits 624 may be included in a control system.

[0091] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit, or they can be communicatively coupled so that either the detection system 616 or the detection station 614 can collect event measurements and provide them to non-collecting elements.

[0092] Figure 6B is a schematic diagram of a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in Figure 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream-charging wire in a barb. This creates a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronics or other detection systems (not shown in Figure 6B). The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, guiding the droplets toward a target collection receptacle (e.g., one of 672, 674, 676, or 678). As shown in Figure 6B, deflection plates 652 and 654 can be controlled to direct particles toward receptacle 674 along the first path 662 or toward receptacle 678 along the second path 668. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within a specified sorting range), the deflection plates may allow the particles to continue along the flow path 664. Such uncharged droplets may enter the waste receptacle via an aspirator 670 or the like.

[0093] Sorting electronics may be included to initiate measurement data collection, receive fluorescence signals from particles, and determine how to adjust the deflection plates to sort the particles. An exemplary implementation of the embodiment shown in Figure 6B includes the BD FACSAria® line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0094] method In some cases, the samples analyzed in this method are biological samples. The term “biological sample” is used in its conventional sense to refer to an entire organism, an entire plant, an entire fungus, or, in specific cases, a subset of animal tissues, cells, or components that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to, but is not limited to, both a naturally occurring organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, for example, plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, gastrointestinal tract, cardiovascular and urogenital tract sections, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of living tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some cases, the sample is a blood sample containing whole blood, such as blood obtained from a venipuncture or fingertip puncture (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0095] In certain embodiments, the source of the sample is “mammal” or “mammalian,” and these terms are used broadly to describe organisms belonging to the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In some cases, the subject is human. The method may also be applied to samples obtained from human subjects of both sexes and any developmental stage (i.e., neonatal, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is juvenile, adolescent, or adult. While this disclosure may be applied to samples derived from human subjects, it should be understood that the method may also be applied to samples from other animal subjects (i.e., “non-human subjects”), such as birds, mice, rats, dogs, cats, livestock, and horses, but is not limited to these.

[0096] Cells of interest can be targeted for characterization by various 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 analyzed droplets determined to contain target cells. Various cells can be characterized using this method. 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 having favorable cell surface markers or antigens that can be captured or labeled by favorable affinity factors or their 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 alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0097] When performing this method, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module may vary, for example, 0.001 mL to 1000 mL, 0.005 mL to 900 mL, 0.01 mL to 800 mL, 0.05 mL to 700 mL, 0.1 mL to 600 mL, 0.5 mL to 500 mL, 1 mL to 400 mL, 2 mL to 300 mL, or any other amount including 5 mL to 100 mL.

[0098] The method according to embodiments of the present disclosure includes counting labeled particles (e.g., target cells) in a sample and selectively sorting them. When carrying out the method, a fluid sample containing particles is first introduced into the system's flow nozzle. Exiting the flow nozzle, the particles pass through the sample interrogation region substantially one at a time, where each particle is irradiated with a light source, and light scattering parameters and, in some cases, desired fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded separately for each particle. Depending on the characteristics of the interrogated flowstream, the light may be irradiated to a length of 0.001 mm or more of the flowstream, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more of the flowstream. In certain embodiments, the method includes irradiating a planar cross section of the flowstream within the sample interrogation region with a laser (as described above). In other embodiments, the method includes irradiating a sample interrogation region with a predetermined length of flowstream, for example, a length corresponding to the irradiation profile of a diffuse laser beam or lamp.

[0099] In certain embodiments, the method includes irradiating a flowstream at or near the nozzle orifice of the flow cell. For example, the method may include irradiating a flowstream at a position including 1 mm or more, for example, 0.001 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, from the nozzle orifice. In certain embodiments, the method includes irradiating a flowstream immediately adjacent to the nozzle orifice of the flow cell.

[0100] In embodiments of the method, a detector such as a photomultiplier tube (PMT) is used to record the light passing through each particle (called forward scattering in certain cases), the light reflected perpendicular to the direction of particle flow through the detection region (called orthogonal or side scattering in some cases), and, if the particles are labeled with a fluorescent marker, the fluorescence emitted from the particles as they pass through the detection region and are illuminated by an energy source. Each of forward scattering (FSC), side scattering (SSC), and fluorescence emission involves distinct parameters for each particle (or each “event”). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle can, if desired, be analyzed in real time or stored in data storage and analysis means such as a computer.

[0101] In certain embodiments, particles are detected and uniquely identified, as desired, by exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted in the detection channels used to identify the particles and the associated binding complexes may be measured after excitation by a single light source or separately after excitation by individual light sources. If separate excitation light sources are used to excite particle labels, the labels may be selected so that all labels are excitable by each of the excitation light sources used.

[0102] The method, in certain embodiments, also includes data acquisition, analysis, and recording using a computer or the like, with multiple data channels recording data from each detector about the light scattering and fluorescence emitted by each particle as it passes through the sample interrogation area of ​​the particle sorting module. In these embodiments, the analysis includes sorting and counting the particles so that each particle is presented as a set of digitized parameter values. The system may be set up with a trigger on a selected parameter to distinguish the particle of interest from background and noise. A “trigger” refers to a preset threshold for detecting the parameter and may be used as a means to detect the passage of a particle through a light source. Detection of an event exceeding the threshold of the selected parameter triggers the acquisition of light scattering and fluorescence data for the particle. For particles or other components in the assayed medium that cause a response below the threshold, no data is acquired. The trigger parameter may be the detection of forward scattered light caused by the passage of a particle through a light beam. The flow cytometer then detects and collects the light scattering and fluorescence data for the particle.

[0103] Next, a specific subpopulation of interest is further analyzed by “gating” based on data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This procedure can be performed by plotting forward light scattering (FSC) versus side (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles (i.e., their cells in the gate) is selected, and particles not in the gate are excluded. If desired, the gate may also be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Then, only those particles in the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to yield a count of the particles of interest in the sample.

[0104] The methods of interest may further include the use of particles in research, laboratory testing, or therapy. In some embodiments, the methods include obtaining individual cells prepared from biological samples of a target fluid or tissue. For example, the methods include obtaining cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods include obtaining cells from fluid or tissue samples used in therapy. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissues, is prepared and can be introduced into a subject as a therapeutic procedure. Conditions that can be treated by administration of samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, and organ damage.

[0105] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification, culture and in vitro growth, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with the collection of viable cells and tissues from the subject's source tissues to generate cell and / or tissue samples. Samples may be collected by any appropriate procedure, including, for example, administering a cell recruiter to the subject, drawing blood from the subject, or removing bone marrow from the subject. After sample collection, cell enrichment may be performed by several methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, and fluorescence-activated cell sorting (FACS). In some cases, enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and grown in vitro. In some cases, cells are preserved, for example, by cryopreservation, and stored for future use, where they can be thawed and administered to a patient, for example, by injection.

[0106] Computer control system 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 in which instructions for performing the steps of this method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, as well as input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of several other processors that are available or will be available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically works with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0107] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as permanent hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or floppy disk drives. Such types of memory storage devices typically read from and / or write to program storage media such as compact disks (not shown). Any of these program storage media, or others currently in use or to be developed in the future, may be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in program storage devices used in conjunction with system memory and / or memory storage devices.

[0108] In some embodiments, a computer program product is described that includes a computer-usable medium on which control logic (a computer software program including program code) is stored. When the control logic is executed by the computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of a hardware state machine for performing the functions described herein will be obvious to those skilled in the art.

[0109] Memory may be any suitable device on which the processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including magnetic or optical disks, or tapes or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium having the necessary program code. The programming may be supplied to the processor remotely via a communication channel, or it may be pre-stored in a computer program product such as memory or some other portable or fixed computer-readable storage medium using any of the memory-related devices. For example, a magnetic or optical disk may have a program which can be read by a disk writer / reader. The system of this disclosure also includes programming in the form of a computer program product, algorithms for use in carrying out the methods described above. The programming according to this disclosure may be recorded on a computer-readable medium, for example, any medium which 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 hybrids of these categories such as magnetic / optical storage media.

[0110] The processor can also access communication channels to communicate with users in remote locations. Remote locations mean that the user is not in direct contact with the system, but rather relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0111] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication such as code division multiple access (CDMA) and global system for mobile communications (GSM).

[0112] In one embodiment, the communication interface is configured to include one or more physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port that enables data communication between the system and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0113] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol that enables the system to communicate with other devices such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0114] In one embodiment, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP), Short Message Service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0115] In one embodiment, the system is configured to communicate wirelessly with a server device via a communication interface using common standards such as 802.11, Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or electrical appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD), as well as input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0116] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device using one or more of the communication protocols and / or mechanisms described above, for example, data stored in the system, for example, an optional data storage unit.

[0117] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a remote user, for example, via the internet, telephone or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML or XML documents, email or other files, or data in other formats. The data may also include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present in this system may be any type of known computer platform or a type to be developed in the future, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, whether networked or not. They may be located in the same place or physically separated. Depending perhaps on the type and / or manufacturer of the selected computer platform, various operating systems may be used on any of the computer platforms.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.

[0118] Figure 7 shows a general architecture of an exemplary computing device 700 according to a particular embodiment. The general architecture of the computing device 700 shown in Figure 7 includes the configuration of computer hardware and software components. However, not all of these generally conventional elements need to be illustrated in order to provide an implementable disclosure. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 may provide a connection to one or more networks or computing systems. Thus, the processing unit 710 can receive information and instructions from other computing systems or services via the network. The processing unit 710 also communicates with memory 770 and may further provide output information to an optional display 750 via the input / output device interface 740. For example, analytical software (e.g., data analysis software or program such as FlowJo®) stored as executable instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 740 may also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input devices.

[0119] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Data may be stored in a data storage device 790. Memory 770 may further include computer program instructions and other information for implementing embodiments of the present disclosure.

[0120] kit In addition to the components described above, the kit may further include instructions (in some embodiments). These instructions may be present in the kit in various forms, and one or more of them may be present in the kit. One possible form of these instructions is information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the kit's packaging, or accompanying documents. Yet another form of these instructions is a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), or portable flash drive. Yet another possible form of these instructions is a website address that can be used via the internet to access the information at a remote site.

[0121] usefulness Embodiments of this disclosure are used in applications where cells prepared from biological samples may be desired for use in research, laboratory testing, or therapeutic settings. In some embodiments, the methods and devices may facilitate obtaining and / or analyzing individual cells prepared from biological samples of target fluids or tissues. For example, the methods and systems may facilitate obtaining cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods and systems may facilitate obtaining cells from fluid or tissue samples used in therapeutic settings.

[0122] Notwithstanding the attached claims, this disclosure is also defined by the following clauses:

[0123] 1. Flow cell and, Sample container housing area, A sample injection tube (SIT) assembly having a sample line fluidly coupled to a flow cell, An actuator coupled to the SIT assembly, configured to move the SIT assembly A flow cytometer comprising, A flow cytometer in which the SIT assembly is configured to provide a variable sample line depth within the sample container, located in the sample container housing region.

[0124] 2. The SIT assembly is, An arm having a first proximal end connected to an actuator and a second distal end having a through hole, A sample line subassembly installed inside the through-hole and Equipped with, The flow cytometer according to Clause 1, wherein the sample line subassembly is reversibly movable in the z direction with respect to the xy plane of the second end.

[0125] 3. The flow cytometer according to Clause 2, wherein the sample line subassembly is reversibly movable in the z direction by a predetermined displacement distance relative to the xy plane of the second end.

[0126] 4. A flow cytometer as described in Clause 3, wherein the specified displacement distance is in the range of 1 mm to 2 mm.

[0127] 5. A flow cytometer as described in Clause 4, wherein the specified displacement distance is 1.5 mm.

[0128] 6. The sample line subassembly is A threaded bush having at least a portion located within a through hole, and comprising a threaded hole and a sample line hole through which the threaded bush passes; A sample line thread, which has an inner bore for receiving the sample line, is coupled to a screw hole. A flow cytometer comprising any one of clauses 2 to 5.

[0129] 7. The sample line subassembly further comprises a ferrule in a threaded hole, and the sample line passes through the ferrule, as described in Clause 6 of the flow cytometer.

[0130] 8. The flow cytometer according to any one of clauses 2 to 7, wherein the SIT assembly further comprises a spring configured to compress the sample line subassembly onto an arm.

[0131] 9. The flow cytometer described in Clause 8, wherein the spring has a return bend.

[0132] 10. The flow cytometer according to Clause 9, wherein the return bend section has an opening at the first end through which the sample line thread of the sample line subassembly passes.

[0133] 11. The return bend is connected to the arm, as described in Clause 10 of the flow cytometer.

[0134] 12. The flow cytometer according to any one of clauses 2 to 11, wherein the SIT assembly further comprises a restraining member configured to restrict the movement of the sample line subassembly in the z direction relative to the xy plane of the second end.

[0135] 13. The flow cytometer as described in Clause 12, further comprising a sensor for measuring the displacement of the return bend.

[0136] 14. The flow cytometer according to Clause 13, configured to provide an evaluation of the sample line positioning within a sample container based on the output from a sensor.

[0137] 15. The actuator comprises a dual guide rod cylinder actuator, as described in any one of Clauses 2 to 14.

[0138] 16. A flow cytometer according to any one of clauses 1 to 15, further comprising a light source configured to irradiate a flow cell at an interrogation point.

[0139] 17. A flow cytometer according to any one of the clauses 1 to 16, further comprising a detector configured to collect particle-modulated light from a flow cell.

[0140] 18. A flow cytometer is a particle analyzer, as defined in any one of clauses 1 to 17.

[0141] 19. A flow cytometer is a particle separator, as described in any one of clauses 1 to 18.

[0142] 20. A flow cytometer is an imaging flow cytometer, as described in any one of clauses 1 to 19.

[0143] 21. A method for analyzing a sample, the method being: (a) Introducing a particulate sample from a sample container into a flow cytometer using a sample injection tube (SIT) assembly having a sample line operably coupled to a flow cell, wherein the SIT assembly is configured to provide a variable sample line depth within the sample container, (b) Analyze the sample by flow cytometry and Methods that include...

[0144] 22. The SIT assembly is An arm having a first proximal end connected to an actuator and a second distal end having a through hole, A sample line subassembly installed inside the through-hole and Equipped with, The method according to clause 21, wherein the sample line subassembly is reversibly movable in the z direction with respect to the xy plane of the second end.

[0145] 23. The method according to clause 22, wherein the sample line subassembly is reversibly movable in the z direction by a predetermined displacement distance with respect to the xy plane of the second end.

[0146] 24. The method according to clause 23, wherein the specified displacement distance is in the range of 1 mm to 2 mm.

[0147] 25. The method according to Clause 24, wherein the specified displacement distance is 1.5 mm.

[0148] 26. The sample line subassembly is A threaded bush having at least a portion located within a through hole, and comprising a threaded hole and a sample line hole through which the threaded bush passes; A sample line thread, which has an inner bore for receiving the sample line, is coupled to a screw hole. The method according to any one of the clauses 22 to 25, comprising:

[0149] 27. The method according to Clause 26, wherein the sample line subassembly further comprises a ferrule in a threaded hole, and the sample line passes through the ferrule.

[0150] 28. The method according to any one of the clauses 22 to 27, wherein the SIT assembly further comprises a spring configured to compress a sample line subassembly onto an arm.

[0151] 29. The method according to Clause 28, wherein the spring has a return bend portion.

[0152] 30. The method according to Clause 29, wherein the return bend portion has an opening at the first end through which the sample line thread of the sample line subassembly passes.

[0153] 31. The method according to clause 30, wherein the return bend is connected to the arm.

[0154] 32. The method according to any one of the claims 22 to 31, wherein the SIT assembly further comprises a restraining member configured to restrict the movement of the sample line subassembly in the z direction relative to the xy plane of the second end.

[0155] 33. The method according to any one of the clauses 29 to 32, wherein the flow cytometer further comprises a sensor for measuring the displacement of the return bend.

[0156] 34. The method according to clause 33, further comprising obtaining an evaluation of the sample line positioning in the sample container based on the output from the sensor.

[0157] 35. The actuator comprises a dual guide rod cylinder actuator, as described in any one of the clauses 22 to 34.

[0158] 36. The method according to any one of the clauses 21 to 35, further comprising a light source configured to irradiate a flow cell at an interrogation point.

[0159] 37. The method according to any one of the clauses 21 to 36, wherein the flow cytometer further comprises a detector configured to collect particle-modulated light from a flow cell.

[0160] 38. A flow cytometer is a particle analyzer as described in any one of the clauses 21 to 37.

[0161] 39. A flow cytometer is a particle separator as described in any one of the clauses 21 to 38.

[0162] 40. The flow cytometer is an imaging flow cytometer, as described in any one of the clauses 21 to 39.

[0163] Therefore, the foregoing is merely illustrative of the principles of the present disclosure. Those skilled in the art will understand that various configurations embodying the principles of the present disclosure and that fall within its spirit and scope can be devised, although not expressly described or shown herein. Furthermore, all examples and conditional statements described herein are primarily intended to help the reader understand the principles of the present disclosure and the concepts to which the inventors have contributed to advancing the art, and should be interpreted as not being limited to such specifically described examples and conditions. Furthermore, all descriptions herein listing the principles, aspects, and embodiments of the present disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any developed elements that perform the same function regardless of their structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether such disclosure is expressly described in the claims or not.

[0164] Accordingly, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) are expressly defined as applying to the limitation in the claims only if the exact phrase “means” or the exact phrase “step” is stated at the beginning of such limitation in the claims, and if such exact phrase is not used in the limitation of the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) does not apply.

Claims

1. Flow cell and, Sample container housing area, A sample injection tube (SIT) assembly comprising a sample line fluidly coupled to the flow cell, An actuator coupled to the SIT assembly is configured to move the SIT assembly. A flow cytometer comprising, A flow cytometer in which the SIT assembly is configured to provide a variable sample line depth within the sample container located in the sample container housing region.

2. The aforementioned SIT assembly is An arm having a first proximal end connected to the actuator and a second distal end having a through hole, A sample line subassembly installed in the through hole and Equipped with, The flow cytometer according to claim 1, wherein the sample line subassembly is reversibly movable in the z direction with respect to the x-y plane of the second end.

3. The flow cytometer according to claim 2, wherein the sample line subassembly is reversibly movable in the z direction by a predetermined displacement distance with respect to the x-y plane of the second end.

4. The flow cytometer according to claim 3, wherein the predetermined displacement distance is in the range of 1 mm to 2 mm.

5. The flow cytometer according to claim 4, wherein the predetermined displacement distance is 1.5 mm.

6. The aforementioned sample line subassembly is A threaded bush, at least a portion of which is located within the through hole, comprising a threaded hole and a sample line hole through which the threaded bush passes, A sample line screw having an inner bore for receiving the sample line is coupled to the screw hole. A flow cytometer according to any one of claims 2 to 5, comprising:

7. The flow cytometer according to claim 6, wherein the sample line subassembly further comprises a ferrule in the screw hole, and the sample line passes through the ferrule.

8. The flow cytometer according to any one of claims 2 to 7, wherein the SIT assembly further comprises a spring configured to compress the sample line subassembly onto the arm.

9. The flow cytometer according to claim 8, wherein the spring has a return bend portion.

10. The flow cytometer according to claim 9, wherein the return bend portion has an opening at the first end through which the sample line thread of the sample line subassembly passes.

11. The flow cytometer according to claim 10, wherein the return bend portion is connected to the arm.

12. The flow cytometer according to any one of claims 2 to 11, wherein the SIT assembly further comprises a restraining member configured to restrict the movement of the sample line subassembly in the z direction with respect to the x-y plane at the second end.

13. The flow cytometer according to claim 12, further comprising a sensor for measuring the displacement of the return bend portion.

14. The flow cytometer according to claim 13, wherein the flow cytometer is configured to provide an evaluation of the positioning of the sample line in the sample container based on the output from the sensor.

15. A method for analyzing a sample, wherein the method is (a) Introducing a particulate sample from a sample container into a flow cytometer using a sample injection tube (SIT) assembly having a sample line operably coupled to a flow cell, wherein the SIT assembly is configured to provide a variable sample line depth within the sample container, (b) Analyze the sample by flow cytometry. Methods that include...

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