Flow cytometer with pneumatically driven autosampler and method of use thereof

Abstract

Aspects of the present disclosure include a flow cytometer with a pneumatically driven autoinjector. According to certain embodiments, a flow cytometer comprises: a flow cell; and a pneumatically driven autoinjector configured to automatically retrieve a sample from the sample container and deliver the sample to the flow chamber. The present disclosure also provides methods of flow cytometry of a sample, for example in analysis and / or sorting applications.

Description

Cross-reference to related applications

[0001] Pursuant to Section 119(e) of Chapter 35 of the United States Code, this application claims priority over the filing dates of U.S. Provisional Application No. 63 / 813,208, filed May 28, 2025, and U.S. Provisional Application No. 63 / 718,455, filed November 8, 2024; the disclosures of both patent applications are incorporated herein by reference. Background Technology

[0002] Characterization of analytes in biofluids has become an important component of biological research, medical diagnostics, and the assessment of overall patient health and well-being. Detection of analytes in biofluids, such as human blood or blood-derived products, can provide results that can play a role in determining treatment plans for patients with multiple disease states.

[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 blood, and a sheath fluid reservoir containing sheath fluid. The flow cytometer delivers particles (including cells) from the fluid sample as a cell stream to the flow chamber, while simultaneously directing the sheath fluid into the flow chamber. To characterize the components of the flow stream, the flow stream is illuminated. Changes in the material within the flow stream, such as the presence of morphological or fluorescent labels, can cause changes in the observed light, and these changes can be used for characterization and separation. To characterize the components in the flow stream, light must be illuminating and collected from the flow stream. The light source of a flow cytometer can vary and may include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the irradiated particles is collected and quantified.

[0004] Biological particles can be separated by adding sorting or collection capabilities to flow cytometers. In the separated stream, particles with one or more desired characteristics are separated from the sample stream by mechanical or electrical removal. A commonly used flow cytometry sorting technique employs droplet sorting, where the stream containing linearly separated particles is broken into droplets. Droplets containing the particles of interest are charged and deflected into the collection tube as they pass through an electric field. Typically, linearly separated 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 to reach the droplet breakup point and separate from the stream into droplets can be predicted. Ideally, a brief charge is applied to the stream just before the droplet containing the selected particles is about to detach from the fluid stream, and then the stream is grounded immediately after detachment. The droplet to be sorted remains charged upon detachment from the fluid stream, while all other droplets remain uncharged.

[0005] Samples analyzed in a flow cytometer may initially be contained in sample containers, such as test tubes or wells of multi-well plates. Sample injection tubes are used to introduce the sample from the sample container into the flow chamber of the flow cytometer. Figure 1A It provides sample injection tubes commonly used in current flow cytometers. For example... Figure 1A As shown, the sample injection tube assembly 10 includes a sample tubing 25, which is secured to the first end 40 of a support arm 30 by a sample tubing bolt 15. Because the sample tubing bolt secures the sample tubing to the end 40 of the support arm 30, the sample tubing 25 does not move relative to the end 40 of the support arm 30 in the z-direction. An outer tube 20, as part of a droplet containment system, is also shown in the figure. One end 45 of the support arm 30 is connected to a first actuator 35, which moves the support arm relative to the outer tube 20 in the z-direction, allowing the distal end 25a of the sample tubing 25 to be moved into the outer tube 20 for cleaning or to extend from the outer tube 20 to aspirate a sample from a sample container. A second actuator 50, a motor-driven actuator, is also shown in the figure, allowing the entire assembly to move up and down in the z-direction. Summary of the Invention

[0006] The inventors realized that, Figure 1AThe motor-driven actuators shown require additional printed circuit boards (PCBs) and firmware to operate the motor, in addition to the motor and gears. Therefore, these devices are highly complex, leading to increased manufacturing and maintenance costs. Furthermore, these components add points of failure, thus increasing potential repair costs. For example, these components require supplies from multiple sources, additional software updates, lubrication, and occupy extra space within the flow cytometer housing. Additionally, any point of failure could prevent the sample injection tube 10 from extending to extract samples for analysis. This problem is exacerbated by adding additional moving parts, as each moving part requires its own motor, gears, PCB, and firmware.

[0007] The embodiments described herein address the complexity introduced by multiple motors, gears, and PCBs. In these embodiments, a pneumatic system comprising a pump and cylinders is used to actuate the sample injection tube (i.e., SIT). Some embodiments include a second cylinder for actuating the injection gate. Other embodiments include a third cylinder for actuating the sample tubing subassemblies. In some embodiments, a single pump is connected to more than one cylinder. Such devices reduce complexity and decrease the space occupied within the housing because these embodiments have fewer moving parts and other components (e.g., PCBs). These embodiments also reduce costs, including maintenance and labor costs.

[0008] According to some embodiments, a flow cytometer includes a flow chamber and a pneumatically driven autosampler configured to automatically acquire samples from a sample container and deliver the samples to the flow chamber. Furthermore, methods for cytometry of samples, for example, in analytical and / or sorting applications, are also provided.

[0009] A flow cytometer is provided. Aspects of the provided flow cytometer include: a flow chamber; and a pneumatically driven autosampler configured to automatically acquire samples from sample containers and deliver them to the flow chamber. In some cases, the pneumatically driven autosampler includes: a sample container receiving area; a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container present in the sample container receiving area; and an injection gate configured to regulate access to the sample container receiving area; wherein actuation of the SIT assembly and the injection gate is pneumatically driven by a pneumatic assembly. In some cases, the pneumatic assembly includes: a pneumatic pump for providing positive pressure to a first pneumatic line; a first switch, fluidly connected to the pneumatic pump via the first pneumatic line and configured to direct positive pressure to a second and a third line such that when positive pressure is applied to the second pneumatic line, the third pneumatic line is not pressurized, and vice versa; and a first cylinder, fluidly connected to the second and third pneumatic lines and mechanically connected to the SIT assembly, such that when the second pneumatic line is pressurized, the SIT assembly moves to a sampling position, while when the third pneumatic line is pressurized, the SIT assembly moves to a sampling position. When the pneumatic tubing is pressurized, the SIT assembly moves to the rest position; the second switch, which is fluidly connected to the pneumatic pump via the first pneumatic tubing and is configured to direct positive pressure to the fourth and fifth tubings, such that when positive pressure is applied to the fourth pneumatic tubing, the fifth pneumatic tubing is not pressurized, and vice versa; and the second cylinder, which is fluidly connected to the fourth and fifth pneumatic tubings and mechanically connected to the injection gate, such that when the fourth pneumatic tubing is pressurized, the injection gate moves to the closed position, and when the third pneumatic tubing is pressurized, the injection gate moves to the open position. In some cases, the pneumatic assembly further includes: a third switch, which is in fluid communication with the pneumatic pump via a first pneumatic line and configured to direct positive pressure to the sixth and seventh lines such that when positive pressure is applied to the sixth pneumatic line, the seventh pneumatic line is not pressurized, and vice versa; and a third cylinder, which is in fluid communication with the sixth and seventh pneumatic lines and mechanically connected to a sampling line subassembly, the sampling line subassembly including a sampling line in fluid communication with a flow chamber such that when the sixth pneumatic line is pressurized, the sampling line subassembly moves to a loading position to insert the sampling line into the sample container, and when the seventh pneumatic line is pressurized, the sampling line subassembly moves to a retracted position.

[0010] In some cases, the actuation of the SIT assembly, injection gate, and / or sample tubing subassemblies is not driven by stepper motors and worm gears. In some cases, the actuation of the SIT assembly, injection gate, and / or sample tubing subassemblies does not require a separate circuit board. In some cases, the pneumatic pump, first cylinder, second cylinder, and third cylinder, as well as the first switch, second switch, and third switch, are operated from a single circuit board. In some cases, the pneumatic pump, first cylinder, second cylinder, and third cylinder, as well as the first switch, second switch, and third switch, do not require firmware.

[0011] In some implementations, one or more of the first switch, second switch, and third switch include a flow regulator for regulating the pressure balance in the non-pressurized pneumatic line.

[0012] In some embodiments, the pneumatic assembly further includes a pressure reservoir in fluid communication with a pneumatic pump and one or more first, second, and third switches, wherein the pneumatic pump pressurizes the pressure reservoir, and the pressure reservoir provides positive pressure to a first pneumatic line. In some embodiments, the pneumatic assembly includes connectors located on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines, allowing the pneumatic lines to be disconnected and reconnected. In various embodiments, the pneumatic assembly also includes a mounting bracket on which the pneumatic pump, pressure reservoir, switches, and connectors are mounted. In many embodiments, the pneumatic assembly also includes a pressure gauge for measuring the pressure in the pressure reservoir and in one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines. In some cases, when the pressure measured by the pressure gauge is below the threshold pressure, the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines will be repressurized. In some cases, the threshold pressure range is 25 psi to 50 psi.

[0013] In some implementations, the sample gate protects the sample from ambient light by reducing its transmission. In various implementations, the sample gate is semi-transparent. In some cases, the SIT assembly is located on an XY movable stage. Some implementations also include a calibration plate, a chassis, and a manual test tube port, wherein the calibration plate and chassis are configured to calibrate the height of the manual test tube port to minimize sample dead volume during the transition from sample acquisition from the sample container in the pneumatically driven autosampler to sample acquisition from the manual test tube loading position. In some cases, the pneumatically driven autosampler also includes a platform for accommodating one or more samples. In some cases, this platform is configured to mix, heat, and / or cool one or more samples.

[0014] Various examples include one or more of a light source configured to illuminate the flow chamber at the detection point and a detector for collecting particle-modulated light from the flow chamber.

[0015] In various implementations, the flow cytometer can be a particle analyzer, a particle sorter, and / or an imaging flow cytometer.

[0016] This disclosure also includes a method for analyzing samples, wherein the method comprises: introducing a sample container containing the sample into a pneumatically driven autosampler of a flow cytometer, wherein the pneumatically driven autosampler is configured to automatically acquire the sample from the sample container such that the sample is delivered to the flow chamber of the flow cytometer; and performing flow cytometry analysis on the sample. Attached Figure Description

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

[0018] Figure 1A This provides a view of the Sample Injection Tube (SIT) assembly used in current flow cytometers.

[0019] Figures 1B to 1E Various views of a pneumatically driven autosampler according to certain implementation schemes are provided.

[0020] Figure 2 A flow cytometry system according to certain implementation schemes is demonstrated.

[0021] Figure 3 A particle sorting instrument with image functionality according to certain embodiments is described.

[0022] Figure 4 A functional block diagram of a particle analysis system according to certain implementation schemes is depicted.

[0023] Figure 5 A functional block diagram of an example control system according to certain implementation schemes is depicted.

[0024] Figures 6A to 6B A schematic diagram of a particle sorting system according to certain implementation schemes is depicted.

[0025] Figure 7 Various aspects of a computer control system according to certain implementation schemes are described.

[0026] Figure 8 The user interface for configuring the BD FACSDICOVER™ A8 plate sampler is shown.

[0027] Figure 9A The effect of cell concentration on mixing efficiency was described; Figure 9B The effect of the volume inside the pores on the mixing efficiency was described; Figure 9C The effect of mixing intensity on mixing efficiency is described; Figure 9D The effect of mixing frequency on mixing efficiency was described; Figure 9E The effect of default mixing settings on cell viability was depicted. Figure 9F The effect of the default mixing settings on fluorescence was described.

[0028] Figure 10A and Figure 10B The illustration shows A8 remnants: peripheral blood mononuclear cells (PBMCs, Fig. 10A) and HT-29 cells ( Figure 10B ); Figure 10C The diagram illustrates the throughput of the FACSDiscover™ A8 injector. Detailed Implementation

[0029] This disclosure includes aspects of flow cytometers having a pneumatically driven autosampler. A flow cytometer according to certain embodiments includes: a flow chamber; and a pneumatically driven autosampler configured to automatically acquire a sample from a sample container and deliver the sample to the flow chamber. Methods for cytometry of samples, for example, in analytical and / or sorting applications, are also provided.

[0030] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, as they can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure is limited by the appended claims.

[0031] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, and any other specified value or intermediate value within the specified range, is included in this disclosure, wherein, unless the context expressly specifies otherwise, the precision of the intermediate value is one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this disclosure, but are subject to any explicitly excluded limits within the specified range. When a specified range contains one or two limits, the range excluding one or both of the included limits is also included in this disclosure.

[0032] Certain ranges shown herein use the term “about” before the numerical value. As used herein, the term “about” provides written support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately the specifically stated number, the unstated number that is close to or approximately the number may be a number that is substantially equivalent to the specifically stated number when shown.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure, those described herein are representative and illustrative.

[0034] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually designated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publications. Any reference to a publication is for its disclosure prior to the filing date and should not be construed as an admission that this disclosure has no right to precede that publication by virtue of a prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

[0035] It should be noted that, unless the context clearly indicates otherwise, as used herein and in the appended claims, the absence of a quantifier before an element includes both singular and plural forms. It should also be noted that the claims may exclude any optional element. Therefore, this statement is intended to serve as a preliminary basis for the use of exclusive terms such as “solely” or “only” or for the use of negative limitations when relating to the elements of a claim.

[0036] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has independent components and features, which can be readily separated from or combined with features of any other several embodiments without departing from the scope or spirit of this disclosure. Any described method may be performed in the order of the described events or in any other logically possible order.

[0037] Although the system and method have been or will be described and their functions explained for grammatical fluency, it should be clearly understood that, unless expressly provided for in Chapter 35 of the United States Code, a claim shall not in any case be construed as necessarily being limited to “method” or “step”, but shall conform to the judicial principles of equivalence and the meaning and full scope of the equivalent as defined in the claim. When a claim is explicitly drafted in accordance with Section 112 of Chapter 35 of the United States Code, the claim shall be in full conformity with the legal equivalents in Section 112 of Chapter 35 of the United States Code.

[0038] Flow cytometer including pneumatically driven autosamplers

[0039] As described above, aspects of this disclosure include a pneumatically driven autosampler. In various aspects, the pneumatically driven autosampler includes: a sample container receiving area; a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located within the sample container receiving area; and an injection gate configured to regulate access to the sample container receiving area, wherein actuation of both the SIT assembly and the injection gate is pneumatically driven by the pneumatic assembly. The SIT assembly disclosed herein is used to introduce a quantity of liquid sample from a sample container—e.g., a tube or orifice (such as the orifice of a multi-well plate)—into a sample fluid line leading to the flow chamber of a flow cytometer. The SIT assembly of an embodiment of this application is configured to aspirate a sample from a sample container into the lumen of the sample line of the SIT assembly, which can then be delivered by the flow cytometer to the flow chamber of the flow cytometer.

[0040] As described above, the autosampler of the embodiments of this disclosure is pneumatically driven. In some cases, the autosampler of these embodiments includes a sample receiving area, a sample injection tube (SIT) assembly configured to introduce sample tubing into a sample container located within a sample container area, and an injection gate configured to regulate access to the sample container receiving area, wherein actuation of the SIT assembly and the injection gate is pneumatically driven by the pneumatic assembly. In some cases, the pneumatic assembly includes a pneumatic pump for providing positive pressure to one or more cylinders. Positive pressure, as opposed to negative pressure, refers to pressure above ambient pressure, while negative pressure refers to pressure below ambient pressure. Ambient pressure at sea level (1 atmosphere) is approximately equal to 14.7 pounds per square inch (psi). However, ambient pressure can vary with altitude, environmental conditions (high-pressure or low-pressure systems), and / or pressure control in laboratory or clinical environments. For example, clinical wards typically maintain higher ambient pressures to avoid contamination, while laboratory environments maintain lower ambient pressures to avoid contamination of corridors or other transitional spaces. Such cylinders can be mechanically connected to components such as SIT assemblies, injection gates, and / or sampling tubing subassemblies. By applying positive pressure to the cylinder, a component can be actuated to a desired position. These components can be actuated between two positions (e.g., sampling position and rest position, closed position and open position, and / or loaded position and retracted position) using one or more switches. Using a pneumatic drive system, embodiments can actuate one or more of the SIT assembly, injection gate, and / or sample tubing subassemblies without stepper motors and worm gears. A worm gear, also known as a drive screw, is a linear component with a grooved outer surface whose radial rotation moves a device along its longitudinal axis. Because there is no stepper motor, embodiments do not require separate circuit boards (e.g., printed circuit boards or PCBs) to actuate each of the SIT assembly, injection gate, and / or sample tubing. In embodiments, actuation of the SIT assembly, injection gate, and / or sample tubing is accomplished by a single circuit board. Furthermore, since there are no stepper motors and circuit boards, these implementations may operate without firmware—or rather, the pneumatic pump, the first cylinder, the second cylinder, and the third cylinder, as well as the first switch, the second switch, and the third switch, may also be firmware-free.

[0041] Figure 1B A pneumatically driven autosampler according to one embodiment of this disclosure is provided. Figure 1B In this design, a sample container receiving area 102 is provided. This area 102 can be a platform, support, notch, or other location for inserting a support, tray, or plate (e.g., a perforated plate). Additionally, Figure 1B Also shown is a SIT assembly 104, configured to introduce a sample line 105 into a sample container located in the sample container area—further details about the SIT assembly 104 will be provided below. Although Figure 1BThe sample inlet door is not shown (to avoid obscuring certain details), but the sample inlet door mounting assembly 106 is shown. The sample inlet door can be actuated between an open position and a closed position, wherein the open position allows the sample to be loaded into the sample container receiving area 102, and the closed position allows the sample inlet door to protect the sample from ambient light and to protect the user from injury by moving parts.

[0042] When using, such as Figure 1B When the apparatus is shown, the sample inlet gate can be opened to allow a user to access the sample container receiving area 102. Samples (or multiple samples) can be placed on the sample container receiving area 102. These samples can be placed in test tubes and test tube racks, or in multi-well plates (e.g., 96-well plates, 384-well plates, 1536-well plates, etc.). The sample container receiving area 102 may include a mixing mechanism to allow mixing, resuspension, or otherwise homogenizing of the samples. According to some embodiments, the sample container receiving area 102 may also include heating and / or cooling mechanisms, for example, for maintaining the samples at a desired temperature (e.g., refrigerated storage, reaction temperature, etc.). Heating and cooling mechanisms known in the art include heat pumps, reverse heat pumps, Peltier devices, water circulators, heat pipes, condensers, and / or any other related mechanisms. After the samples are loaded (or possibly for storage or protection of internal components of the apparatus), the sample inlet gate can be actuated to the closed position.

[0043] Figure 1B An XY movable stage 108 is also shown, on which the SIT assembly 104 is located or mounted. The movable stage 108 allows the SIT assembly to move in the XY plane, thereby moving sample tubing between test tubes on a sample holder or between wells on a multiwell plate. Movement in the XY direction can be achieved using motors, gears, rails, magnetic tracks, and / or any other suitable method to precisely move the platform. The movable stage 108 allows the SIT assembly 104 to be positioned above a specific test tube or well, thereby enabling the SIT assembly to reach the injection position for sample acquisition or collection by actuating the injection position.

[0044] Figure 1C The illustration shows the SIT component 104 in the injection position according to various embodiments of the present disclosure. Figure 1C In this embodiment, the SIT assembly 104 includes a first cylinder 110 for actuating the SIT assembly 104 between an injection position and a rest position. To maintain stability, the SIT assembly 104 can move along a stabilizing track 112 to avoid jamming or stopping due to torque acting on the arm length of the SIT assembly 104. Some embodiments may include a balancing device or other system to limit or avoid torque acting on the SIT assembly 104.

[0045] The sample tubing subassembly 114 is illustrated as a parallel arm on the SIT assembly 104. A third cylinder 116 is also shown for actuating the sample tubing subassembly 114 between the injection position and the retracted position. As shown, the SIT assembly 104 is in the sampling position, while the sample tubing subassembly 114 is in the retracted position. In this position, the flow cytometer is in a position before or after sample acquisition. Figure 1C The SIT assembly shown includes a return flexible element 118 that enables variable sample tubing depth. Such SIT assemblies are further described in U.S. Patent Application No. 60 / ___,____ (Attorney's File No.: P-30353.US01PRO / BECT-386PRV), filed on the same date as this application, the disclosure of which is incorporated herein by reference.

[0046] Figure 1D Another view of the pneumatic assembly according to an embodiment is shown. In this illustration, a mounting bracket 150 is shown attached to a pneumatic pump 152. A first pneumatic line 156 is in fluid communication with the pneumatic pump 152, such that the pneumatic pump 152 provides positive pressure to an optional pressure reservoir 154, which is further connected to the first pneumatic line 156. As shown, the first pneumatic line 156 is attached to a manifold 158, which directs pressure to switches 160a to 160c. Switches 160a to 160c can be used to distribute positive pressure between pneumatic lines 162a to 162f. For example, the first switch 160a can distribute positive pressure between a second pneumatic line 162a and a third pneumatic line 162b, wherein the second pneumatic line 162a and the third pneumatic line 162b are connected to a first cylinder ( Figure 1C , 110) fluid communication. When positive pressure is directed to the second pneumatic line 162a, the first cylinder will bring the SIT assembly ( Figure 1C , 104) is actuated to the down or sampling position. Similarly, when positive pressure is directed to the third pneumatic line 162b, the first cylinder will actuate the SIT assembly ( Figure 1C ,104) Actuate to the upward or resting position.

[0047] Similarly, switches 160b to 160c are connected to pneumatic lines 162c to 162f to direct positive pressure to the second and third cylinders, thereby actuating the sample inlet gate between the closed and open positions and switching the sample tubing subassembly between the sampling and retracted positions. Figure 1C (114). Additionally, connector 164 is shown in the figure, which allows disconnection and reconnection of the pneumatic lines 162a to 162f, thereby enabling the replacement of one or more components without interfering with the operation of any other components.

[0048] Returning to switches 160a to 160c, these switches allow unpressurized lines to vent to the atmosphere or environment, for example, to allow actuation or movement of the associated cylinder. In some embodiments, switches 160a to 160c include a flow regulator 164 for regulating the pressure balance in the unpressurized pneumatic line. This flow regulation can achieve smoother or slower actuation to prevent damage that may result from unrestrained or restricted movement of components.

[0049] Implementations include pressure gauges or other pressure gauges that can be used to assess the pressure at one or more points in the illustrated configuration. In some implementations, the pressure gauge can measure the pressure in a pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines. In such implementations, when the pressure measured by the pressure gauge is below a threshold pressure, the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines will be repressurized. This repressurization can be achieved by activating a pump to add additional pressure to the system, opening a switch to increase pressure, and maintaining the position of the cylinder. In various implementations, the threshold pressure can range from approximately 5 psi to approximately 100 psi, including subranges thereof. These subranges include (but are not limited to) 10 psi to 90 psi, 15 psi to 80 psi, 20 psi to 70 psi, 25 psi to 60 psi, 30 psi to 50 psi, 35 psi to 40 psi, etc. In some embodiments, the threshold pressure range is 25 psi to 50 psi. In some embodiments, repressurization occurs when the measured pressure is below 100 psi, 90 psi, 80 psi, 70 psi, 60 psi, 50 psi, 40 psi, 30 psi, 20 psi, or below 10 psi. In some cases, repressurization terminates when the measured pressure reaches approximately 30 psi, approximately 40 psi, approximately 50 psi, approximately 60 psi, approximately 70 psi, approximately 80 psi, approximately 90 psi, or approximately 100 psi.

[0050] Figure 1E A photograph of a flow cytometer according to an embodiment is provided. In the photograph, a portion of the housing has been removed to allow viewing of the mounting bracket 150 (as well as the pneumatic pump 152, reservoir 154, and switches 160a to 160c, which are not individually labeled). In this figure, the sample inlet gate 175 is shown in the closed position, thereby protecting the sample container receiving area 102 from ambient light and / or interference from external sources. In some embodiments, the sample inlet gate 175 protects the sample from ambient light by reducing the transmission of ambient light. In some embodiments, the sample inlet gate 175 is translucent.

[0051] The figure also shows a manual tube inlet 176. In embodiments, the manual tube inlet allows for the handling of single samples without the need for plates or tube racks, such as when a small amount of sample needs to be placed into a flow cytometer. Various embodiments with a manual tube inlet include calibration plates and chassis configured to calibrate the height of the manual tube inlet to minimize sample dead volume during transitions between sample loading from a sample container in a pneumatically driven autosampler and from a manual tube loading position. Further details regarding strategies for avoiding dead space within SIT components, including the pneumatically driven autosampler and the manual tube loading position, can be found in co-pending application number 63 / 718,451 (Attorney General's Case No. P-30353.US01PRO / BECT-386PRV), filed on the same date as this application, the disclosure of which is incorporated herein by reference.

[0052] As described above, the SIT assembly of the pneumatically driven autosampler in the embodiments is configured to obtain a sample from a sample container (e.g., a test tube or a well plate) by drawing the sample into the sample tubing of the SIT assembly and then delivering the drawn sample directly or via additional fluid tubing to the flow chamber. The flow chamber of interest includes a cuvette configured to deliver particles in a flow stream. As described herein, a “flow cell” generally refers to a component containing a liquid flow channel for delivering particles in a sheath fluid. The cuvette of interest has a passageway (i.e., a flow channel) extending therethrough. The flow stream contained in this flow channel may include a liquid sample injected from a sample test tube. In some cases, the flow chamber includes a light-transmitting flow channel. The cuvette may be made of materials such as quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is made of silica (e.g., fused silica). In some cases, the flow cell is configured to receive light source illumination at one or more detection points. A “detection point” discussed herein refers to an area within the flow cell where particles are illuminated by a light source, for example, for analysis. The size of the detection point may vary as needed. For example, if 0 μm represents the optical axis of the light emitted by the light source, the range of detection points can be from -50 μm to 50 μm, such as from -25 μm to 40 μm, including from -15 μm to 30 μm. Depending on certain factors (such as the number and arrangement of lasers), there may be multiple irradiation points within the flow cell.

[0053] In some embodiments, the flow chamber includes or is configured to be used in conjunction with a sample injection port, which is configured to provide a sample to the flow chamber, for example, a sample delivered via the SIT assembly and any intermediate fluid conduit (if present). In embodiments, the sample injection system is configured to provide a suitable flow rate of sample to the inner chamber of the flow chamber (i.e., the flow channel). Depending on the desired flow characteristics, the rate at which the sample is delivered through the sample injection port to the flow chamber can be 1 μL / min or higher, for example, 2 μL / min or higher, for example, 3 μL / min or higher, for example, 5 μL / min or higher, for example, 10 μL / min or higher, for example, 15 μL / min or higher, for example, 25 μL / min or higher, for example, ... Such as 50 μL / min or higher, including 100 μL / min or higher, wherein, in some cases, the sample is delivered to the flow chamber via the sample injection port at a rate of 1 μL / s or higher, such as 2 μL / s or higher, such as 3 μL / s or higher, such as 5 μL / s or higher, such as 10 μL / s or higher, such as 15 μL / s or higher, such as 25 μL / s or higher, such as 50 μL / s or higher, including 100 μL / s or higher.

[0054] In some cases, a flow cytometer is an analytical flow cytometer with multiple acquisition speeds. The term "analytical flow cytometer" refers to a flow cytometer configured for analyzing particles (including cells) suspended in a fluid stream. The fluid stream flowing through an analytical flow cytometer is typically connected to a waste container to dispose of analyzed particles as waste, rather than sorting and collecting them for further use or analysis. In an analytical flow cytometer, the fluid stream can be driven by a vacuum pump connected to a waste line, which can be coupled to the outlet fluid of the flow chamber, allowing fluid to be drawn from the flow chamber into the waste line under vacuum conditions. As used herein, "multiple acquisition speeds" refers to the analytical flow cytometer's ability to capture or acquire data at more than one fluid speed within its flow chamber. Multiple acquisition speeds add functionality to an analytical flow cytometer, allowing it to be used in both high-speed analysis modes with faster fluid speeds and low-speed imaging modes with slower fluid speeds. In some implementations, multiple acquisition speeds can be achieved through an adjustable hydrodynamic system. Multiple velocities are achieved by adjusting the fluid resistance within the waste line of the flow cytometer. In analyzing some embodiments of the flow cytometer, the fluid flow through the flow chamber is driven by a vacuum pump fluidly coupled to the waste line. By increasing the fluid resistance within the waste line, the fluid velocity within the flow chamber decreases. In some embodiments, the reduced fluid velocity is sufficient for particle imaging. Similarly, by decreasing the fluid resistance within the waste line, the fluid velocity within the flow chamber increases. In some embodiments, the increased fluid velocity is sufficient for particle analysis. For further details on such flow cytometers, see U.S. Patent Application No. 19 / 076,246, filed March 11, 2025; the disclosure of which is incorporated herein by reference.

[0055] The sample injection port can be an orifice located on the inner wall of the cavity or a conduit located proximal to the inner chamber. When the sample injection port is an orifice located on the inner wall of the cavity, the orifice can be of any suitable shape, wherein the cross-sectional shape of interest includes, but is not limited to, straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ovals, etc.; and irregular shapes, such as the bottom of a parabola connected to the top of a plane. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on its shape, and in some cases, its opening is 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0056] In some cases, the sample injection port is a conduit located proximal to the flow chamber cavity. For example, the sample injection port can be a conduit positioned such that its orifice aligns with the orifice of the flow chamber. When the sample injection port is positioned aligned with the flow chamber orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes, such as the base of a parabola connected to the top of a plane. The orifice of the conduit can vary depending on its shape, and in some cases, its opening is 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm. The tip shape of the sample injection port can 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 beveled tip with a bevel angle ranging from 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, including a bevel angle of 5°.

[0057] In some embodiments, the flow chamber further includes a sheath fluid injection port configured to supply sheath fluid to the flow chamber. In some embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid into the flow chamber cavity, for example, together with a sample, to form a laminar flow of sheath fluid surrounding a sample flow. Depending on the desired flow characteristics, the sheath fluid flow rate delivered to the flow chamber cavity can be 25 μL / s or higher, for example 50 μL / s or higher, for example 75 μL / s or higher, for example 100 μL / s or higher, for example 250 μL / s or higher, for example 500 μL / s or higher, for example 750 μL / s or higher, for example 1000 μL / s or higher, including 2500 μL / s or higher.

[0058] In some embodiments, the sheath fluid injection port is an orifice located in the inner wall of the cavity. The sheath fluid injection port orifice can be of any suitable shape, with cross-sectional shapes of interest including, but not limited to: straight cross-sectional shapes, such as squares, rectangles, irregular quadrilaterals, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ovals; and irregular shapes, such as the bottom of a parabola connected to the top of a plane. The size of the sheath fluid injection port orifice can vary depending on the shape, and in some cases, its opening is 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0059] The flow cytometer disclosed herein includes a light source configured to illuminate particles in a flow stream at a detection point within a flow chamber. The number of light sources in the flow cytometer can vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, the flow cytometer may include multiple light sources in some cases. In some such cases, the number of light sources ranges from 2 to 10, for example, 2 to 5, including 2 to 4. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser can be any convenient laser, such as a continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer includes dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers. In still other cases, lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other cases, the flow cytometer includes a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:YCa4O(BO3)3 laser, an Nd:YCOB laser, a titanite laser, a thulium YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, or combinations thereof.

[0060] According to some embodiments, the laser source may also include one or more optical adjustment elements. In some embodiments, the optical adjustment element is positioned between the source and the flow chamber, and it may include any device capable of altering the spatial width of illumination from the source or certain other characteristics of the illumination, such as illumination direction, wavelength, beam width, beam intensity, and focal spot. The optical adjustment scheme may include any convenient device for adjusting one or more characteristics of the source, including but not limited to lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation schemes, and combinations thereof. In some embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens may be a reducing lens. In other embodiments, the flow cytometer of interest includes optical fibers.

[0061] The light source can be positioned at any suitable distance from the flow chamber, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, including 100 mm or more. Furthermore, the light source can be positioned at any suitable angle relative to the flow chamber, such as angles from 10 to 90 degrees, from 15 to 85 degrees, from 20 to 80 degrees, from 25 to 75 degrees, including 30 to 60 degrees, for example, an angle of 90 degrees.

[0062] In some embodiments, the light source of interest includes multiple lasers configured to provide laser light for discretely irradiating the flowing stream. For example, two or more lasers, three or more lasers, four or more lasers, five or more lasers, ten or more lasers, or even fifteen or more lasers are configured to provide laser light for discretely irradiating the flowing stream. Depending on the wavelength of the light desired for irradiating the flowing stream, the specific wavelength of each laser can vary from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, from 300 nm to 1000 nm, from 350 nm to 900 nm, or even from 400 nm to 800 nm. In some embodiments, the laser of interest may include one or more 405 nm, 488 nm, 561 nm, and 635 nm lasers.

[0063] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some cases, the beam generator includes a laser and a radio frequency (RF) generator, the RF generator being configured to apply an RF drive signal to the acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, lasers in the beam generator of interest include those listed above.

[0064] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. In this system, the acousto-optic device is configured to generate an angle-deflected laser beam using light emitted from a laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device from any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0065] In some implementations, the controller is configured to apply radio frequency (RF) drive signals to the acousto-optic device to generate a desired number of angle-deflected laser beams in the output laser beam. For example, it is configured to apply 3 or more RF drive signals, such as 4 or more RF drive signals, such as 5 or more RF drive signals, such as 6 or more RF drive signals, such as 7 or more RF drive signals, such as 8 or more RF drive signals, such as 9 or more RF drive signals, such as 10 or more RF drive signals, such as 15 or more RF drive signals, such as 25 or more RF drive signals, such as 50 or more RF drive signals, and is configured to apply 100 or more RF drive signals.

[0066] In some cases, in order to generate an intensity distribution of an angle-deflected laser beam in the output laser beam, the controller is configured to apply an RF drive signal with varying amplitude, for example from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to 50V, for example from about 2V to 40V, for example from 3V to about 30V, including from about 5V to about 25V. In some embodiments, the frequency of each applied radio frequency drive signal is from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, including from about 5 MHz to about 50 MHz.

[0067] In some embodiments, the controller has a processor with a memory operatively connected to the processor such that the memory contains instructions stored therein, which, when executed by the processor, cause the processor to generate an output laser beam with angled deflections of a desired intensity distribution. For example, the memory may contain instructions for generating two or more (e.g., three or more, four or more, five or more, ten or more, twenty-five or more, fifty or more) angled laser beams of equal intensity; the memory may contain instructions for generating one hundred or more angled laser beams of equal intensity. In other embodiments, the memory may include instructions for generating two or more (e.g., three or more, four or more, five or more, ten or more, twenty or more, fifty or more) angle-deflected laser beams with different intensities, and the memory may contain instructions for generating 100 or more angle-deflected laser beams with different intensities.

[0068] In some embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity gradually increases from the center to the edge along a horizontal axis. In these cases, the intensity range of the angle-deflected laser beam at the center of the output beam can be from 0.1% to about 99%, for example 0.5% to about 95%, for example 1% to about 90%, for example 2% to about 85%, for example 3% to about 80%, for example 4% to about 75%, for example 5% to about 70%, for example 6% to about 65%, for example 7% to about 60%, for example 8% to about 55%, including about 10% to about 50% of the intensity of the angle-deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity gradually increases from its edge along the horizontal axis to its center. In these cases, the range of angular deflection laser beam intensity at the edge of the output beam can be from 0.1% to 99% of the intensity of the deflection laser beam at the center of the output laser beam along the horizontal axis, for example, 0.5% to 95%, 1% to 90%, 2% to 85%, 3% to 80%, 4% to 75%, 5% to 70%, 6% to 65%, 7% to 60%, 8% to 55%, including 10% to 50% of the intensity of the deflection laser beam at the center of the output laser beam along the horizontal axis. In some other embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a Gaussian intensity profile along a horizontal axis. In still other embodiments, the controller has a processor with a memory operatively connected to the processor, such that the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a flat-topped intensity profile along a horizontal axis.

[0069] In some embodiments, the beam generator of interest can be configured to generate spatially separated, angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired output laser beam illumination profile, the spacing between the angle-deflected laser beams can be 0.001 μm or higher, for example, 0.005 μm or higher, for example, 0.01 μm or higher, for example, 0.05 μm or higher, for example, 0.1 μm or higher, for example, 0.5 μm or greater, for example, 1 μm or higher, for example, 5 μm or higher, for example, 10 μm or higher, for example, 100 μm or higher, for example, 500 μm or higher, for example, 1000 μm or higher, including 5000 μm or higher. In some embodiments, the system is configured to generate angle-deflected laser beams within the output laser beam that overlap, for example, with angle-deflected laser beams adjacent along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., spot overlap) can be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, including 100 μm or more.

[0070] In some cases, beam generators configured to produce two or more frequency-shifted beams include those described in U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and 10,684,211. Laser excitation modules as described in U.S. Patent Nos. 10,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369, and 11,946,851; the disclosures of these U.S. Patents are incorporated herein by reference.

[0071] In addition, the flow cytometer includes detectors configured to collect light emitted by irradiated particles. These photodetectors are configured to detect particle-modulated light transmitted by the fiber optic light collection element and generate a signal based on the characteristics of the light, such as intensity. For example, one or more particle-modulated photodetectors may include one or more side-scatter light detectors for detecting wavelengths of side-scattered light (i.e., light refracted and reflected from the surface and internal structure of the particle). In some embodiments, the flow cytometer includes a single side-scatter light detector. In other embodiments, the flow cytometer includes multiple side-scatter light detectors, such as two or more, three or more, four or more, including five or more.

[0072] The side-scattering light detector described herein can be any convenient detector used to detect the collected light. Detectors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-connected devices (CCDs), enhancement-mode charge-connected devices (ICCDs), light-emitting diodes, photon counters, radiometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or combinations thereof, and other detectors. In some embodiments, the collected light is measured using a charge-connected device (CCD), semiconductor charge-connected device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, for example, with an effective detection surface area of ​​0.01 cm² per region. 2 Up to 10cm 2 For example, 0.05cm 2 Up to 9cm 2 For example, 0.1cm 2 Up to 8cm 2 For example, 0.5cm 2 Up to 7cm 2 And including 1cm 2 up to 5cm 2 Photomultiplier tubes.

[0073] In one embodiment, the flow cytometer further includes a fluorescence detector configured to detect light of one or more fluorescence wavelengths. In other embodiments, the flow cytometer includes multiple fluorescence detectors, such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, including twenty or more.

[0074] The fluorescence detector described herein can be any convenient detector used to detect the collected light. Detectors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-connected devices (CCDs), enhancement-mode charge-connected devices (ICCDs), light-emitting diodes, photon counters, radiometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or combinations thereof, and other detectors. In some embodiments, the collected light is measured using a charge-connected device (CCD), semiconductor charge-connected device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the detector is a photomultiplier tube, for example, with an effective detection surface area of ​​0.01 cm² per region. 2 Up to 10cm 2 For example, 0.05cm 2 Up to 9cm 2 For example, 0.1cm 2 Up to 8cm 2 For example, 0.5cm 2 Up to 7cm 2 And including 1cm 2 up to 5cm 2 Photomultiplier tubes.

[0075] When the flow cytometer includes multiple fluorescence detectors, each fluorescence detector can be identical, or the collection of fluorescence detectors can be a combination of detectors of different types. For example, when the flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector (or imaging sensor) is a CMOS-type device. In other embodiments, both the first and second fluorescence detectors are CCD-type devices. In other embodiments, both the first and second fluorescence detectors are CMOS-type devices. In other embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector is a photomultiplier tube (PMT). In other embodiments, the first fluorescence detector is a CMOS-type device and the second fluorescence detector is a photomultiplier tube. In other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0076] In embodiments of this disclosure, the fluorescence detector of interest is configured to measure one or more wavelengths of light collected, such as two or more wavelengths, five or more different wavelengths, ten or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths, and includes measuring 400 or more different wavelengths of light emitted by a sample in a flowing stream. In some embodiments, two or more detectors in the module described herein are configured to measure collected light of the same or overlapping wavelengths.

[0077] In some embodiments, the detector of interest is configured to measure collected light within a wavelength range (e.g., 200 nm to 1000 nm). In some embodiments, the detector of interest is configured to collect the spectrum of light within a wavelength range. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light within a wavelength range of 200 nm to 1000 nm. In other embodiments, the detector of interest is configured to measure light emitted by a sample in the flow stream at one or more specific wavelengths. For example, the module may include one or more detectors configured to measure light at one or more of the following wavelengths: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some implementations, one or more detectors may be configured to be paired with a specific fluorophore—such as a fluorophore used with the sample in a fluorescence assay.

[0078] Flow cytometers may include any suitable mechanisms for supplying sheath fluid and sample fluid to sample fluid input connectors and sheath fluid input connectors. For example, the sample fluid input connector may be fluidly connected to a sample fluid line (e.g., a conduit) that is in turn fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input connector may be fluidly connected to a sheath fluid line that is in turn fluidly connected to a sheath fluid reservoir. Similarly, flow cytometers may include any suitable mechanisms for handling waste fluid from the flowing stream. Fluid output connectors may be fluidly connected to a waste fluid line that is in turn fluidly connected to a waste fluid reservoir. A fluid management system suitable for the flow cytometer is provided in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0079] Suitable flow cytometry systems may include, but are not limited to, those described in the following literature: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49 (pt 1): 17-28; Linden et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison et al., J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; its publication details are incorporated herein by reference. In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto. TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BDBiosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BDBiosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BDBiosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TMFlow cytometer and BD Biosciences FACSCalibur TM Cell sorting instrument, BDBiosciences FACSCount TM Cell sorting instrument, BD Biosciences FACSLyric TM Cell sorting instrument, BDBiosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorting instrument, BD Biosciences FACSAria TM II Cell Sorter, BD Biosciences FACSAria TM III Cell Sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorting instrument, BDBiosciences FACSymphony TM S6 cell sorter, BD Biosciences FACSDiscover™ cell sorter, etc.

[0080] In some embodiments, the system is a flow cytometry system, as described in U.S. Patent Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,585,031, 10,578,542, 10,578,469, and 10,481,0 No. 74; No. 10,302,545; No. 10,145,793; No. 10,113,967; No. 10,006,852; No. 10,006,852; No. 9,952,076; No. 9,933,341; No. 9,726,527; 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. 7,201,875; No. 7,129,505; No. 6,821,740; No. 6,813,017; No. 6,809 The flow cytometry systems described in Nos. 804, 6,372,506, 5,700,692, 5,643,796, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766, the disclosures of which are incorporated herein by reference in their entirety.

[0081] In some implementations, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the system is a flow cytometry system configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE), as described in Diebold et al., Nature Photonics. As described in Vol. 7 (10); 806-810 (2013) and in U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,036,699; 10,078,045; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,451,538; 10,620,111; and 10,684,211 U.S. Patent Nos. 10,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369, and 11,946,851, the disclosures of which are incorporated herein by reference. In some embodiments, when the flow cytometer is a particle sorter, the particle sorter is an image-assisted particle sorter. Image-assisted particle sorting devices are described in U.S. Patent Nos. 10,324,019, 10,620,111, 11,105,728, 11,774,343, and U.S. Patent Application Nos. 18 / 537,103, 18 / 657,618, 18,657,623, and 18 / 657,633; the entire contents of these patents and patent applications are incorporated herein by reference.

[0082] Figure 2 A flow cytometry system 200 according to one illustrative embodiment of the present disclosure is shown. System 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at a detection point 215 within a flow chamber 210. Although Figure 2The example shows a single laser, but it should be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to focusing lens 202, which focuses the beam onto the fluid flow portion containing sample particles 211 within flow chamber 210. Flow chamber 210 is part of a hydrodynamic system that directs particles (typically one at a time) in a flow to the focused laser beam for detection. Alternatively, if the flow cytometer is an air flow cytometer, a nozzle can be used.

[0083] like Figure 2 As shown, the flow chamber 210 is fluidly connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid inlet 208 via a conduit (i.e., a sheath fluid line) 207. Furthermore, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., a sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample syringe 213 (e.g., a sample injection needle) configured to introduce particles 211 into the interior of the flow chamber body 210. The particles 211 are hydrodynamically focused via the sheath fluid entering from the sheath fluid injection port 208, thereby forming a flow stream 214 downstream of the conical portion 212 of the flow chamber 210. Particles exiting from the distal end of the flow chamber 210 can be disposed of and / or collected using any suitable method. For example, depending on the type of flow cytometry performed, particles can be collected at the distal end of the flow chamber 210, for example, via a waste line. Alternatively, the particles can be sorted.

[0084] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at several different wavelengths depending on the characteristics of the particles, such as size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be directed to one or more detectors. Specifically, forward scattered light (FSC) is directed to a forward scattered light detector 223. The forward scattered light detector 223 is positioned slightly off-center from the axis of the direct beam passing through the flow chamber 210 and is configured to detect diffracted light, i.e., excitation light that passes through or around the particles primarily in the forward direction. The intensity of the light detected by the forward scattered light detector 223 depends on the overall size of the particles. The forward scattered light detector may include, for example, a photodiode. An optical filter 221a and a scattering blocker 222 are arranged between the forward scattered light detectors 223. The optical filter 221a can be configured to filter out non-FSC light of at least one wavelength, while the scattering blocker 222 can be configured to prevent the incident beam (i.e. non-scattered light) from the laser 201 from being detected by the forward scattering detector 223.

[0085] Furthermore, side-scattered light (SSC) is detected by side-scattered light detector 224. In other words, side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of particle 211, the intensity of which tends to increase with increasing particle structure complexity. Figure 2 In the example, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to a side-scatter light detector 224 while allowing non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to block non-SSC light of at least one wavelength from being detected by the side-scatter light detector 224. Fluorescence detectors 225a-225c are also shown in the figure, each configured to detect fluorescence of different wavelengths. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a while allowing light of other wavelengths to pass through. The optical filter 221c may be configured to block light of at least one wavelength—that does not correspond to the first wavelength (or wavelength range)—from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect fluorescence corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b, while allowing light of a third wavelength (or wavelength range) to be detected by the fluorescence detector 225c. The optical filter 221d is configured to block light of at least one wavelength—that does not correspond to the second wavelength (or wavelength range)—from being detected by the fluorescence detector 225b. Furthermore, the optical filter 221e is configured to block light of at least one wavelength—that does not correspond to the third wavelength (or wavelength range)—from being detected by the fluorescence detector 225c.

[0086] Those skilled in the art will recognize that the flow cytometer according to one embodiment of this disclosure is not limited to... Figure 2 The flow cytometer shown may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors, which may have different wavelengths and different configurations. For example, although Figure 2 The illustrated embodiment shows three fluorescence detectors for ease of explanation, but it should be understood that any suitable number of fluorescence detectors can be used.

[0087] During operation, the cytometer is controlled by a controller / processor 290, and measurement data from the detector can be stored in memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is connected to the detector to receive signals output by it, and can be connected to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. Input / output (I / O) capabilities 297 may also be provided in the system. Memory 295, controller / processor 290, and I / O 297 may be provided as an integral part of the flow cytometer. In such embodiments, a display for showing experimental data to the user of the cytometer 200 may also constitute part of the I / O capabilities 297. Alternatively, some or all of the memory 295 and controller / processor 290, as well as the I / O capabilities, may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and controller / processor 290 may be in wireless or wired communication with the cytometer 210. The controller / processor 290, combined with memory 295 and I / O 297, can be configured to perform a variety of functions related to the preparation and analysis of flow cytometry experiments.

[0088] Different fluorescent molecules in a luciferin pairing scheme used in flow cytometry experiments emit light at their respective characteristic wavelengths. Specific fluorescent labels used in the experiment and their associated fluorescence emission wavelengths can be selected to approximately match the detector's filter window. I / O 297 can be configured to receive data about a flow cytometry experiment having a set of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O 297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and flow cytometry configuration data. Flow cytometry experiment data, such as label spectral characteristics and flow cytometry configuration data, can also be stored in memory 295. Controller / processor 290 can be configured to evaluate the assignment of a label to one or more markers.

[0089] In some embodiments, the system is a particle sorting system configured to sort particles using a closed particle sorting module, such as the particle sorting system described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as the sorting decision module described in U.S. Patent Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, the system for sorting sample components includes a particle sorting module with deflection plates, such as the one described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0090] In some implementations, the system is a fluorescence imaging particle sorter that utilizes radio frequency tag emission imaging technology, such as... Figure 3 As shown, the particle sorter 300 includes an illumination assembly 300a containing a light source 301 (e.g., a 488nm laser). The light source 301 generates an output beam 301a, which is split into beams 302a and 302b by a beam splitter 302. Beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a, which includes beams deflected at one or more angles. In some cases, the output beam 303a generated by the acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a, which includes beams deflected at one or more angles. In some cases, the output beam 304a generated by the acousto-optic device 304 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 303a and 304a, generated by acousto-optic devices 303 and 304 respectively, are combined by beam splitter 305 to generate output beam 305a, which is transmitted via optical element 306 (e.g., objective lens) to illuminate particles in flow chamber 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into multiple beams, each with a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam and then overlaps it with the beam array at beam combiner 305. In some embodiments, the light illumination system having a light source and acousto-optic devices may also include the systems described in Schraivogel et al. (“High-Speed ​​Fluorescence Imaging Cell Sorting,” Science (2022), 375(6578):315-320) and U.S. Patent Publication No. 2021 / 0404943, the disclosure of which is incorporated herein by reference.

[0091] Output beam 305a irradiates sample particles 308 flowing through flow chamber 307 (e.g., together with sheath fluid 309) in irradiation region 310. As shown, in irradiation region 310, multiple beams (e.g., angle-deflected radio frequency offset beams represented by dots in irradiation region 310) overlap with the reference local oscillator beam (represented by shaded lines in irradiation region 310). Due to their different optical frequencies, the overlapping beams exhibit beat characteristics, resulting in each beam operating at a different frequency f. 1-n Carrying sinusoidal modulation.

[0092] Light from the irradiated sample is transmitted to a light detection system 300b, which includes multiple photodetectors. The light detection system 300b includes a forward-scattering photodetector 311 for generating a forward-scattering image 311a and a side-scattering photodetector 312 for generating a side-scattering image 312a. The light detection system 300b also includes a bright-field photodetector 313 for generating a light loss image 313a. In some embodiments, the forward-scattering detector 311 and the side-scattering detector 312 are photodiodes (e.g., avalanche photodiodes, APDs). In some cases, the bright-field photodetector 313 is a photomultiplier tube (PMT). Fluorescence from the irradiated sample is also detected by fluorescence photodetectors 314-317. In some cases, photodetectors 314-317 are photomultiplier tubes. The light beam from the irradiated sample is directed by a beam splitter 320 to the side-scattering detection channel 312 and the fluorescence detection channels 314-317. The optical detection system 300b includes bandpass optics 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optics 321 has a 534 nm / 40 nm bandpass. In some cases, optics 322 has a 586 nm / 42 nm bandpass. In some cases, optics 323 has a 700 nm / 54 nm bandpass. In some cases, optics 324 has a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm to each side of the center of the spectral band, or from 500 nm to 520 nm.

[0093] 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 digitally processed in real time by processors 350 and 351. Based on the data signals generated in processors 350 and 351, images 311a-317a can be generated in each light detection channel. Image-assisted sorting is triggered by a sorting signal generated in sorting trigger 352. Sorting assembly 300c includes deflection plate 331 for deflecting particles into sample container 332 or waste stream 333. In some cases, sorting assembly 300c is configured to sort particles using a closed particle sorting module, such as the module described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0094] In some implementations, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4 It can be used to analyze and characterize particles, and to physically sort particles into collection containers or not sort them into collection containers. Figure 4 A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a hydrodynamic system. Particle analysis system 401 includes hydrodynamic system 402. Hydrodynamic system 402 may include a sample tube 405 and a moving fluid column located within the sample tube, or connected to the sample tube 405 and the moving fluid column within the sample tube, in which particles 403 (e.g., cells) in the sample move along a common sample path 409.

[0095] The particle analysis system 401 includes a detection system 404 configured to collect signals generated from each particle as it passes through one or more detection stations along a common sample path. Detection station 408 typically refers to a monitored area 407 along the common sample path. In some embodiments, detection may include detecting light or one or more other properties as particle 403 passes through monitored area 407. Figure 4 A detection station 408 and a monitored area 407 are shown. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor more than one area.

[0096] Each signal is assigned a signal value, thus forming a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multi-dimensional data point, containing values ​​of various attributes measured for the particle. The detection system 404 is configured to continuously acquire such data points within a first time interval.

[0097] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system shown may be operationally associated with the hydrodynamic system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the number of data points collected by the detection system 404 within the first time interval. The control system 406 is also configured to generate an experimental signal frequency based on the number of data points for a portion of the first time interval. Furthermore, the control system 406 may compare the experimental signal frequency with the calculated signal frequency or a predetermined signal frequency.

[0098] Figure 5 A functional block diagram of a particle analyzer control system is shown, including, for example, 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.

[0099] The particle analysis or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel may be included between the particle analysis or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via this data communication channel.

[0100] Analysis controller 500 may be configured to receive biological event data from particle analysis or sorting system 502. The biological event data received from particle analysis or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display—including a first graph of the biological event data—to display device 506. For example, analysis controller 500 may also be configured to gate a region of interest around a population of biological event data displayed on display device 506 and overlay it onto the first graph. In some embodiments, gating may be a logical combination of one or more graphical regions of interest plotted on a single-parameter histogram or bivariate graph. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0101] The analysis controller 500 can also be configured to display the gated biological event data on the display device 506 in a manner different from other events in the gated biological event data. For example, the analysis controller 500 can be configured to distinguish the color of the gated biological event data from the color of the gated biological event data. The display device 506 can be implemented in the form of a monitor, tablet computer, smartphone, or other electronic device configured to display a graphical interface.

[0102] The analysis controller 500 can be configured to receive a gating selection signal from a first input device that identifies a gating. For example, the first input device can be implemented as a mouse 510. The mouse 510 can generate a gating selection signal for the analysis controller 500 that identifies a gating to be displayed on or operated via the display device 506 (e.g., by clicking when the cursor is positioned on or within the desired gating). In some embodiments, the first device can be implemented as a keyboard 508 or other means of providing input signals to the analysis controller 500 (such as a touchscreen, stylus, optical detector, or voice recognition system). Some input devices may include multiple input functions. In such embodiments, each input function can be considered an input device. For example, such as... Figure 5 As shown, the mouse 510 can include a right mouse button and a left mouse button, and each button can generate a trigger event.

[0103] Triggering events can cause the analysis controller 500 to change the way the data is displayed, change the portion of the data actually displayed on the display device 506, and / or provide input for further processing (such as selecting groups of interest for particle sorting).

[0104] In some implementations, the analysis controller 500 may be configured to detect when the mouse 510 initiates gating selection. The analysis controller 500 may also be configured to automatically modify the plotting visualization to facilitate the gating process. Modifications may be based on the specific distribution of the biological event data received by the analysis controller 500.

[0105] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0106] Display device 506 can be configured to receive display data from analysis controller 500. The display data may include graphs of biological event data and gating of outlines circling portions of the graphs. Display device 506 can also be configured to change the displayed information based on input received from analysis controller 500 and input from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0107] In some implementations, the analysis controller 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include controls for receiving sample events or sample images. Sample events, images, or sample gating may be provided before collecting event data from the sample, or based on an initial set of events from a portion of the sample.

[0108] Figure 6A This is a schematic diagram of a particle sorting system 600 (e.g., particle analysis or sorting system 502) according to some embodiments shown herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6A As shown, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is connected to a fluid conduit 601, which may be connected to, include, or be a nozzle 603. Within the fluid conduit 601, sheath fluid 604 is hydrodynamically focused into a sample fluid 606 containing particles 609 in a moving fluid column 608 (e.g., a flow). Within the moving fluid column 608, the particles 609 (e.g., cells) align in a single file to pass through a monitoring region 611 (e.g., a laser-flow intersection) and are irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to break into multiple droplets 610, some of which contain particles 609.

[0109] In operation, a detection station 614 (e.g., an event detector) identifies a particle (or cell) of interest as it crosses a monitoring area 611. The detection station 614 inputs to a timing circuit 628, which in turn inputs to a flash charging circuit 630. At the droplet break point, a timing droplet delay (Δt) is used to flash charge the moving fluid column 608 to charge the droplet of interest. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplet can then be sorted by deflecting it into a container, such as a collection tube, a porous sample plate, or a microporous sample plate, through an activated deflector plate (not shown), where the pores or micropores can be associated with the specific droplet of interest. Figure 6A As shown, the droplets can be collected into the drain container 638.

[0110] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest passes through a monitoring region 611. Exemplary droplet boundary detectors are described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be input to an amplitude signal 620 and / or a phase signal 618, which are then input (via amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0111] In some embodiments, sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be connected to a memory configured to store detected events and sorting decisions based thereon. The event data of the particles may include sorting decisions. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or as a communication connection, such that one of the detection system 616 or detection station 614 collects event measurements and provides them to non-collecting elements.

[0112] Figure 6B This is a schematic diagram of a particle sorting system based on some of the embodiments shown in this article. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via a current-charged wire in the barbs. This forms a droplet stream 610 containing particles 609 for analysis. The particles can be irradiated with one or more light sources (e.g., a laser) to generate light scattering and fluorescence information. The particle information is analyzed by sorting electronics or other detection systems. Figure 6B (Not shown in the image). Deflecting plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby guiding the droplets toward a target collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to guide particles along a first path 662 toward container 674 or along a second path 668 toward container 678. If a particle is not of interest (e.g., does not exhibit scattering or irradiation information within a specified sorting range), the deflector plates can allow the particle to continue along flow path 664. Such uncharged droplets can enter a waste container, for example, via a suction device 670.

[0113] Sorting electronics can be included to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to induce particle sorting. Figure 6B Example implementations of the illustrated scheme include BDFACS Aria provided by Becton, Dickinson and Company (Franklin Lakes, NJ). TM A series of flow cytometers.

[0114] method

[0115] In some cases, the samples analyzed in this method are biological samples. The term "biological sample," used in its conventional sense, refers to a whole organism, plant, fungus, or a subset of animal tissue, animal cells, or animal components, which in some cases can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Therefore, "biological sample" refers to a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory sections, gastrointestinal sections, cardiovascular sections, and urogenital sections, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be tissues of any type of organism, including healthy tissues and diseased tissues (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc. In some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger puncture (wherein the blood may be combined with any reagent or not before testing, such as preservatives, anticoagulants, etc.).

[0116] In some embodiments, the source of the sample is "mammal" or "milk," terms that are widely used to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the object is a human. The methods can be applied to samples obtained from human objects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, teenagers, and adults), wherein in some embodiments, the human object is an adolescent, teenager, or adult. Although this disclosure can be applied to samples from human objects, it is to be understood that these methods can also be applied to samples from other animal objects (i.e., "non-human objects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0117] Cells of interest can be directed / targeted for characterization based on a variety of parameters, such as phenotypic features identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect the analyzed droplet (which is determined to contain the target cell). The method can be used to characterize a variety of cells. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells with readily available cell surface markers or antigens that can be captured or labeled by their readily available affinity agents or conjugates. For example, target cells may contain cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialiacoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells derived from whole blood, bone marrow, or umbilical cord blood, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+).

[0118] In practicing the method, a certain amount of initial fluid sample is injected into the flow cytometer. The amount of sample injected into the particle sorting module can vary, for example, from 0.001 mL to 1000 mL, such as 0.005 mL to 900 mL, such as 0.01 mL to 800 mL, such as 0.05 mL to 700 mL, such as 0.1 mL to 600 mL, such as 0.5 mL to 500 mL, such as 1 mL to 400 mL, such as 2 mL to 300 mL, and including samples from 5 mL to 100 mL.

[0119] The method according to embodiments of this disclosure includes counting and optionally sorting labeled particles (e.g., target cells) in a sample. In practicing the method, a fluid sample containing the particles is first introduced into a fluid nozzle of the system. As the sample exits the fluid nozzle, the particles pass substantially one-by-one through a sample detection zone, where each particle is illuminated by a light source, and measurements of light scattering parameters and, in some cases, fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded individually for each particle. Depending on the nature of the detected flow, flows of 0.001 mm or more, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, and flows including 1 mm or more, can be illuminated. In some embodiments, the method includes irradiating a planar cross-section of the flow in the sample detection region, for example, with a laser (as described above). In other embodiments, the method includes irradiating a predetermined length of the flow in the sample detection region, which corresponds, for example, to the irradiation profile of a diffuse laser beam or lamp.

[0120] In some embodiments, the method includes irradiating the flow at or near the flow chamber nozzle orifice. For example, the method may include irradiating the flow at a distance of approximately 0.001 mm or more from the nozzle orifice—such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, and locations including 1 mm or more. In some embodiments, the method includes irradiating the flow immediately adjacent to the flow chamber nozzle orifice.

[0121] In an implementation of the method, as a particle passes through a sensing area and is illuminated by an energy source, a detector—such as a photomultiplier tube (PMT)—records the light passing through each particle (in some cases referred to as forward scattering), the light reflected along a direction orthogonal to the particle's flow through the sensing area (in some cases referred to as orthogonal scattering or side scattering), and, if the particle is labeled with one or more fluorescent markers, the fluorescence emitted by the particle. Each forward scattering (FSC), side scattering (SSC), and fluorescence emission contains independent parameters for each particle (or each "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. If desired, the recorded data for each particle can be analyzed in real time or stored in a data storage and analysis tool (such as a computer).

[0122] In some implementations, if desired, particle detection and unique identification are achieved by exposing particles to excitation light in one or more detection channels and measuring the fluorescence of each particle. In the detection channels, the fluorescence emitted to identify the particles and their associated binding complexes can be measured after excitation with a single light source, or after excitation with different light sources individually. If separate excitation sources are used to excite the particle markers, the markers can be selected such that each excitation source used excites all the markers.

[0123] Some implementations of the method also include data acquisition, analysis, and recording, for example using a computer, where multiple data channels record light scattering and fluorescence data emitted by each particle from each detector as it passes through the sample detection zone of the particle sorting module. In these implementations, the analysis includes classifying and counting the particles, such that each particle is displayed as a set of digitized parameter values. The system can be configured to be triggered by selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for the detection parameter and can be used to detect the particle's passage through the light source. If an event exceeding the selected parameter threshold is detected, the acquisition of light scattering and fluorescence data for the particle is triggered. For particles or other components in the measured medium that elicit a response below the threshold, no data acquisition is performed. The trigger parameter can be the detected value of forward scattered light caused by the particle passing through the light beam. Flow cytometry then detects and collects the particle's light scattering and fluorescence data.

[0124] Subsequently, based on the data collected for the entire population, specific subpopulations of interest are further analyzed using "gating." To select an appropriate gating point, the data is plotted to obtain the best possible subpopulation separation. This process can be performed by plotting a contrast between lateral (i.e., orthogonal) light scattering (SSC) and forward light scattering (FSC) on a two-dimensional scatter plot. Subpopulations of particles (i.e., those cells within the gating point) are then selected, and particles not within the gating point are excluded. If desired, the gating point can be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Those particles within the gating point are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis can be configured to determine the count of particles of interest in the sample.

[0125] Methods of interest may also include the use of particles in research, laboratory testing, or treatment. In some embodiments, the method includes obtaining individual cells prepared from a target fluid biological sample or tissue biological sample. For example, the method includes obtaining cells from a fluid sample or tissue sample for use as a research or diagnostic sample for a disease such as cancer. Similarly, the method includes obtaining cells from a fluid sample or tissue sample for therapeutic purposes. Cell therapy protocols are those in which a living cellular material comprising, for example, cells and tissues is prepared and introduced into the body of a subject as a therapeutic treatment. Conditions that can be treated by applying samples sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, etc.

[0126] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification / genetic engineering, culture, and in vitro expansion, cell collection, sample reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with the collection of live cells and tissues from the subject's source tissue to generate cell and / or tissue samples. Samples can be collected using any suitable procedure, including, for example, administration of cell mobilizing agents to the subject, blood collection from the subject, bone marrow extraction from the subject, etc. After sample collection, cells can be enriched using several methods, including, for example, centrifugation-based methods, filtration-based methods, centrifugation, magnetic separation methods, fluorescence-activated cell sorting (FACS), etc. In some cases, the enriched cells can be genetically modified / genetically engineered using any convenient method, such as nuclease-mediated gene editing. Genetically modified / genetically engineered cells can be cultured, activated, and expanded in vitro. In some cases, cells are preserved, for example, cryopreserved, and stored for future use, thawed, and then administered to the patient, for example, by injecting cells into the patient.

[0127] Computer control system

[0128] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access memory storing instructions for performing the steps of the method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, a memory storage device, and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor, or may be one of other available or soon-to-be-available processors. The processor executes the operating system, which interacts with firmware and hardware in well-known ways and facilitates the processor's coordination and execution of various computer programs written in various programming languages ​​known in the art, such as Java, Perl, C++, Python, other high-level languages ​​or low-level languages, and combinations thereof. The operating system typically works with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file management and data management, memory management, and communication control and related services according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0129] System memory can be any known or future memory storage device. Examples include any generally available random access memory (RAM), magnetic media such as internal hard disks or magnetic tapes, optical media such as those for reading and writing compact optical discs, flash memory devices, or other memory storage devices. Memory storage devices can be any known or unknown device, including compact optical disc drives, magnetic tape drives, or floppy disk drives. These types of memory storage devices typically read and / or write to program storage media (not shown), such as compact optical discs. Any such program storage media or other media in use or that may be developed in the future can be considered computer program products. It will be understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0130] In some embodiments, a computer program product is described, comprising a computer-usable medium containing control logic (computer software program, including program code) stored therein. When the control logic is executed by a computer processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using a hardware state machine. The implementation of the hardware state machine used to perform the functions described herein will be apparent to those skilled in the art.

[0131] The memory can be any suitable device in which a processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including a disk or optical disc or magnetic tape or RAM, or any other suitable fixed or portable device). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel or pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, any of which is connected to the memory for use. For example, a disk or optical disc may carry the programming and can be read by a disk writer / reader. The systems of this disclosure also include programming, for example, existing in the form of a computer program product, i.e., an algorithm for practicing the methods described above. The programming according to this disclosure can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0132] The processor can also access communication channels to communicate with users in remote locations. A remote location refers to a user who does not have direct contact with the system and forwards input information from external devices, such as computers connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones), to the input manager.

[0133] In some embodiments, the system according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., RFID, Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), communication protocols, and cellular communication, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0134] In some implementations, the communication interface is configured to include one or more communication ports, such as physical ports or physical interfaces, such as USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the system and other external devices, such as computer terminals (e.g., computer terminals in a physician's office or hospital environment), which are configured for similar complementary data communication.

[0135] In some implementations, the communication interface is configured for infrared communication, wireless communication, or any other suitable wireless communication protocol to enable the system to communicate with other devices, such as computer terminals and / or networks, mobile phones with communication capabilities, personal digital assistants, or any other communication devices that the user can use in combination.

[0136] In some implementations, the communication interface is configured to provide connectivity for data transmission using the Internet Protocol (IP) via mobile phone networks, short message service (SMS), wireless connection of a personal computer (PC) connected to a local area network (LAN) connected to the Internet, or Wi-Fi connected to the Internet at a Wi-Fi hotspot.

[0137] In some embodiments, the system is configured to wirelessly communicate with a server device via a communication interface, for example using common standards such as 802.11, RF protocols, or IrDA infrared protocols. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0138] In some implementations, the communication interface is configured to communicate automatically or semi-automatically with data stored in the system, such as data stored in an optional data storage unit, using one or more of the above-described communication protocols and / or communication mechanisms.

[0139] The output controller may include controllers for any of the various known display devices used to present information to a user, whether human or machine, locally or remotely. If one of the display devices provides visual information, that information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs used to provide a graphical input and output interface between the system and the user, and to process user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished in alternative implementations using networks or other types of remote communication. The output manager may also provide information generated by the processing module to a user located at a remote location, such as via the Internet, telephone, or satellite networks, according to known technologies. The presentation of data by the output manager may be implemented according to various known technologies. For example, the data may include SQL documents, HTML documents, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses, allowing the user to retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the system may be any type of known computer platform or a type developed in the future, although they typically belong to a class of computers commonly referred to as servers. However, they may also be mainframes, workstations, or other types of computers. They can be connected via any known or future type of cable or other communication system, including wireless systems, whether networked or otherwise. They may be located in the same place or physically separated. Different operating systems can be used on any computer platform, depending on the type and / or model of the computer platform chosen. Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBMi®, Android™, SGI IRIX®, Oracle Solaris®, and others.

[0140] Figure 7 A general architecture of an example computing device 700 according to certain implementation schemes is described. Figure 7The general architecture of the computing device 700 described herein includes the layout of computer hardware components and computer software components. However, it is not necessary to show all these generally conventional components for the purpose of providing a disclosure that can be implemented. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media driver 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 can provide connectivity to one or more networks or computing systems. Therefore, the processing unit 710 can receive information and instructions from other computing systems or computing services via the network. The processing unit 710 can also communicate with and from a memory 770, and can also provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (e.g., data analysis software or programs, such as) stored as executable instructions in the non-transitory memory of an analysis system can display flow cytometry event data to a user. The input / output device interface 740 can also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input device.

[0141] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that the processing unit 710 executes for implementing one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and general operation of the computing device 700. Data may be stored in data storage device 790. Memory 770 may also contain computer program instructions and other information for implementing aspects of this disclosure.

[0142] practicality

[0143] The embodiments of this disclosure are applicable to applications in which cells prepared from biological samples are used for research, laboratory testing, or therapeutic purposes. In some embodiments, the methods and apparatus can facilitate the acquisition and / or analysis of individual cells prepared from a target fluid biological sample or a target tissue biological sample. For example, the methods and systems facilitate the acquisition of cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the methods and systems can facilitate the acquisition of cells from fluid or tissue samples for therapeutic purposes.

[0144] The following content is provided illustratively and is not intended to be restrictive.

[0145] experiment

[0146] Sampler performance and optimal settings for high-throughput data generation using the BD FACSDiscover™ A8 cell analyzer.

[0147] A. Abstract

[0148] When using an autosampler for data acquisition, the influence of various variables can be highly complex and even disrupt a carefully designed experiment. Factors such as cell concentration, resuspension volume, acquisition criteria, mixing frequency, and duration can significantly impact experimental success. Using the BD FACSDiscover™ A8 cell analyzer equipped with a built-in automated pneumatic sampler (e.g., as described above) and dual-speed functionality (e.g., as disclosed in U.S. Patent Application No. 19 / 076,246, filed March 11, 2025, the disclosure of which is incorporated herein by reference), we present a systematic approach to optimize sampler performance for consistently high-quality data. We investigated the relationship between mixing efficiency and carrier type, cell concentration, sample volume, mixing method, and mixing frequency. The output metric for mixing efficiency is based on the number of events acquired within a fixed volume. Careful tracking of these metrics helps establish a set of settings for optimal sampler performance. Furthermore, we provide data to assess the impact of carrier mixing on cell viability and fluorescence stability. We also explored other confounding factors, such as the minimum number of events required to obtain statistically reliable results in the context of immunophenotyping applications. Based on these data, we propose a set of guidelines for achieving maximum throughput without sacrificing data quality. Furthermore, strategies for evaluating throughput, residuals, and dead volumes will be presented.

[0149] B. Method

[0150] To evaluate the integrated sampler of the FACS Discover™ A8 Cell Analyzer, we conducted a series of experiments. These experiments aimed to determine the performance limits of the sampler in terms of mixing efficiency. Mixing efficiency experiments were performed using both cell lines (Jurkat T cells) and peripheral blood mononuclear cells (PBMCs). Consistency in the number of cells collected within a fixed collection volume in samples with fixed cell concentrations was used as a measure of relative mixing efficiency. Deviations from the default mixing settings were also investigated to determine the validity of these settings and the impact of deviations. Residual and throughput experiments were performed at default settings in both imaging and high-speed modes (residual data from high-speed mode are not included). Similar grouping experiments were performed using carriers, for example, supported by 40-well tube racks and deep-well 96-well plate formats. Settings as follows... Figure 8 As shown.

[0151] C. Result

[0152] 1. Mixing efficiency

[0153] a. Effect of Cell Concentration on Mixing Efficiency: In 96-well plates, the number of cells collected in a 25 mL collection volume per well was plotted. Cell concentrations tested ranged from 0.5 million cells / mL to 10 million cells / mL. In all cases, default injector settings were used, with circular mixing at 1400 RPM for 5 seconds every 4 wells. The coefficient of variation (%CV) was calculated for each condition. Data points exceeding two standard deviations above and below the mean are shown in red. Results are as follows: Figure 9A As shown.

[0154] b. Effect of well volume on mixing efficiency: In a 96-well plate, the number of cells collected per well at a collection volume of 25 mL was plotted. The well volumes tested ranged from 50 mL to 200 mL. In all cases, default injector settings were used, with circular mixing at 1400 RPM for 5 seconds every 4 wells. The coefficient of variation (%CV) was calculated for each condition. Data points exceeding two standard deviations above and below the mean are shown in red. Results are as follows: Figure 9B As shown.

[0155] c. Effect of mixing intensity on mixing efficiency: In a 96-well plate, the number of cells collected in each well within a 25 mL collection volume was plotted. The mixing intensity ranged from 500 RPM to 1400 RPM. In all cases, the default injector settings were used, with a 5-second circular mixing motion performed every 4 wells. The coefficient of variation (%CV) was calculated for each condition. Results are shown below. Figure 9C As shown.

[0156] d. Effect of mixing frequency on mixing efficiency: In 96-well plates, the number of cells collected in each well within a 25 mL collection volume was plotted. Different mixing frequencies were tested from every 2 to every 12 wells. In all cases, the default injector settings were used, and circular mixing was performed for 5 seconds at a speed of 1400 RPM. The coefficient of variation (%CV) was calculated for each condition. Results are shown below. Figure 9D As shown.

[0157] e. Effect of default mixing settings on cell viability: Using a well volume of 200 mL and the default mixing settings (1400 RPM, 5 seconds per 4 wells), the viability of Jurkat cells (top) and PBMCs (bottom) was measured in each of 96 wells during a single culture plate run. Cell viability was determined by 7-AAD staining. As a reference, an aliquot of Jurkat cells or PBMCs stored at 4°C during the culture plate run was measured as a control group. The viability of the aliquot of the aliquot of the aliquot of Jurkat cells or PBMCs was tested before and after the culture plate run. Results are shown below. Figure 9E As shown.

[0158] f. Effect of Default Mixing Settings on Fluorescence: Using a well volume of 200 mL and the default mixing settings (1400 RPM, 5 seconds per 4 wells), a 7-color TBNK panel was used to investigate the effect of mixing on the measured fluorescence. The upper figure shows the median fluorescence intensity (MFI) of several key markers in this panel, and the lower figure shows the relative frequencies of each population that can be used for quantitative analysis using this panel. Results are as follows: Figure 9F As shown.

[0159] 2. Residue and flux

[0160] a. A8 ​​Residue: PBMCs (2A) and HT-29 cells (2B). Residue was tested using a standard protocol, i.e., 10 million cells / mL were added to one well of a 96-well plate (2A PBMCs in the left image, 2B HT-29 cells, a highly aggregated cell line, in the right image). Four blank control wells, containing only PBS, followed the high-concentration wells. The number of residual cells in the blank control wells was then measured as a “residue” event. This test was performed in both high-speed mode (data not shown) and imaging mode, with similar results. Data from one PBS blank control well was acquired before the first well to determine the background noise of the PBS. The bar chart in the lower left corner of each image indicates the average number of residual events across 10 replicates (5 wells) in the residue series. Results are as follows: Figure 10A and Figure 10B As shown.

[0161] b. FACSDiscover™ A8 injector throughput: Using an in-well volume of 200 μL and a cell concentration of 1 million cells / mL, plate run times were measured for each fluid mode at the default mixing speed and frequency. The figure shows the throughput time (indicated in green) of a standard 96-well plate in high-speed mode (102 mL / min), corresponding to SIT flushing (right panel) and no SIT flushing (left panel). Acquisition run times are shown for 2, 5, 10, 25, and 50 mL. The same measurements were performed in imaging mode (30 mL / min)—the blue bar graph is shown on the right. Results are as follows: Figure 10C As shown.

[0162] in conclusion

[0163] The integrated sample injector on the FACS Discover™ A8 cell analyzer features customizable characteristics that allow users to maximize sample mixing efficiency during plate acquisition, including mixing frequency, duration, and intensity (rotation speed). These settings should be optimized to meet the user's needs. However, the instrument comes with default settings that have been tested to produce optimal results. This study aims to demonstrate how these default settings were determined and to provide guidance on the potential impact of deviating from them on the user's workflow. We found that the default settings produce the most reliable data, and in most cases, significant deviations are required to affect results. Additionally, we investigated the injector's residual and throughput performance in both imaging and high-speed modes.

[0164] In addition to the appended claims, the invention is also defined by the following terms:

[0165] 1. A flow cytometer, comprising:

[0166] Fluid chamber; and

[0167] A pneumatically driven autosampler is configured to automatically pick up a sample from a sample container and deliver the sample to the flow chamber.

[0168] 2. The flow cytometer according to Clause 1, wherein the pneumatically driven autosampler comprises:

[0169] Sample container receiving area;

[0170] A sample injection tube (SIT) assembly configured to introduce a sample line into the sample container located in the sample container region; and

[0171] A sample inlet gate, configured to regulate access to the sample container receiving area;

[0172] The SIT component and the sample inlet gate are pneumatically driven by a pneumatic component.

[0173] 3. The flow cytometer according to Clause 2, wherein the pneumatic assembly comprises:

[0174] A pneumatic pump is used to provide positive pressure to the first pneumatic line;

[0175] A first switch, which is in fluid communication with the pneumatic pump via the first pneumatic line, is configured to direct positive pressure to the second and third lines such that when positive pressure is applied to the second pneumatic line, the third pneumatic line is not pressurized, and vice versa.

[0176] The first cylinder is in fluid communication with the second and third pneumatic lines and is mechanically connected to the SIT assembly, such that when the second pneumatic line is pressurized, the SIT assembly moves to the sampling position; and when the third pneumatic line is pressurized, the SIT assembly moves to the rest position.

[0177] The second switch is in fluid communication with the pneumatic pump via the first pneumatic line and is configured to direct positive pressure to the fourth and fifth lines such that when positive pressure is applied to the fourth pneumatic line, the fifth pneumatic line is not pressurized, and vice versa.

[0178] The second cylinder is in fluid communication with the fourth and fifth pneumatic lines and is mechanically connected to the sample inlet gate, such that when the fourth pneumatic line is pressurized, the sample inlet gate moves to the closed position, and when the third pneumatic line is pressurized, the sample inlet gate moves to the open position.

[0179] 4. The flow cytometer according to Clause 3, wherein the pneumatic assembly further comprises:

[0180] The third switch, which is in fluid communication with the pneumatic pump via the first pneumatic line and is configured to direct positive pressure to the sixth and seventh lines, such that when positive pressure is applied to the sixth pneumatic line, the seventh pneumatic line will not be pressurized, and vice versa.

[0181] The third cylinder is in fluid communication with the sixth and seventh pneumatic lines and is mechanically connected to the sampling line subassembly, which includes a sample line in fluid communication with the flow chamber. When the sixth pneumatic line is pressurized, the sample line subassembly moves to the injection position, inserting the sample line into the sample container. When the seventh pneumatic line is pressurized, the sample line subassembly moves to the retracted position.

[0182] 5. The flow cytometer according to Clause 3 or Clause 4, wherein the actuation of the SIT assembly, the sample inlet gate and / or the sample tubing subassembly is not driven by a stepper motor and a worm gear.

[0183] 6. A flow cytometer according to any one of Clauses 3 to 5, wherein the actuation of the SIT assembly, the injection gate and / or the sample tubing subassembly does not require a separate circuit board.

[0184] 7. The flow cytometer according to any one of Clauses 3 to 6, wherein the pneumatic pump, the first cylinder, the second cylinder and the third cylinder, and the first switch, the second switch and the third switch are operated from a single circuit board.

[0185] 8. The flow cytometer according to any one of Clauses 3 to 7, wherein the pneumatic pump, the first cylinder, the second cylinder and the third cylinder, and the first switch, the second switch and the third switch do not require firmware.

[0186] 9. The flow cytometer according to any one of Clauses 3 to 8, wherein one or more of the first switch, the second switch and the third switch include a flow regulator for regulating the pressure balance in the non-pressurized pneumatic line.

[0187] 10. A flow cytometer according to any one of Clauses 3 to 9, wherein the pneumatic assembly further comprises a pressure reservoir in fluid communication with the pneumatic pump and one or more of the first switch, the second switch and the third switch, wherein the pneumatic pump pressurizes the pressure reservoir and the pressure reservoir provides positive pressure to the first pneumatic line.

[0188] 11. The flow cytometer according to any one of Clauses 3 to 10, wherein the pneumatic assembly includes a connector located on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines, such that the pneumatic lines can be disconnected and reconnected.

[0189] 12. The flow cytometer according to any one of Clauses 3 to 11, wherein the pneumatic assembly further includes a mounting bracket on which the pneumatic pump, pressure reservoir, switch and connector are mounted.

[0190] 13. The flow cytometer according to any one of Clauses 3 to 12, wherein the pneumatic assembly further comprises a pressure gauge for measuring the pressure in the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines.

[0191] 14. The flow cytometer according to Clause 13, wherein when the pressure measured by the pressure gauge drops below a threshold pressure, one or more of the pressure reservoir and the second, third, fourth, fifth, sixth, and seventh pneumatic lines are repressurized.

[0192] 15. The flow cytometer according to Clause 14, wherein the threshold pressure ranges from 25 psi to 50 psi.

[0193] 16. A flow cytometer according to any one of Clauses 2 to 15, wherein the sample gate protects the sample from ambient light by reducing the transmission of ambient light.

[0194] 17. The flow cytometer according to Clause 16, wherein the sample gate is semi-transparent.

[0195] 18. The flow cytometer according to any one of Clauses 2 to 17, wherein the SIT assembly is located on an XY movable stage.

[0196] 19. The flow cytometer according to any one of Clauses 1 to 18 further includes a calibration plate, a chassis, and a manual tube inlet, wherein the calibration plate and the chassis are configured to calibrate the height of the manual tube inlet to minimize sample dead volume when switching between sample acquisition from a sample container in a pneumatically driven autosampler and sample acquisition from a manual tube inlet position.

[0197] 20. A flow cytometer according to any one of Clauses 1 to 19, wherein the sample container receiving area includes a platform configured to hold one or more samples.

[0198] 21. The flow cytometer according to Clause 20, wherein the platform is configured to mix, heat and / or cool the one or more samples.

[0199] 22. The flow cytometer according to any one of Clauses 1 to 21 further includes a light source configured to illuminate the flow chamber at a detection point.

[0200] 23. The flow cytometer according to any one of Clauses 1 to 22 further includes a detector configured to collect particle-modulated light from the flow chamber.

[0201] 24. The flow cytometer according to any one of Clauses 1 to 23, wherein the flow cytometer is a particle analyzer.

[0202] 25. A flow cytometer according to any one of Clauses 1 to 24, wherein the flow cytometer is a particle sorter.

[0203] 26. The flow cytometer according to any one of Clauses 1 to 25, wherein the flow cytometer is an imaging flow cytometer.

[0204] 27. A method for performing flow cytometry analysis on a sample, the method comprising:

[0205] (a) A sample container containing a sample is introduced into a pneumatically driven autosampler of the flow cytometer, wherein the pneumatically driven autosampler is configured to automatically acquire a sample from the sample container and thereby deliver the sample to the flow chamber of the flow cytometer.

[0206] (b) Perform flow cytometry analysis on the sample.

[0207] 28. The method according to Clause 27, wherein the pneumatically driven autosampler comprises:

[0208] Sample container receiving area;

[0209] A sample injection tube (SIT) assembly configured to introduce a sample line into the sample container located in the sample container region; and

[0210] A sample inlet gate, configured to regulate access to the sample container receiving area;

[0211] The SIT component and the sample inlet gate are pneumatically driven by a pneumatic component.

[0212] 29. The method according to Clause 28, wherein the pneumatic assembly comprises:

[0213] A pneumatic pump is used to provide positive pressure to the first pneumatic line;

[0214] A first switch, which is in fluid communication with the pneumatic pump via the first pneumatic line and is configured to direct positive pressure to the second and third lines, such that when positive pressure is applied to the second pneumatic line, the third pneumatic line is not pressurized, and vice versa.

[0215] The first cylinder is in fluid communication with the second and third pneumatic lines and is mechanically connected to the SIT assembly, such that when the second pneumatic line is pressurized, the SIT assembly moves to the sampling position, and when the third pneumatic line is pressurized, the SIT assembly moves to the rest position.

[0216] The second switch, which is in fluid communication with the pneumatic pump via the first pneumatic line and is configured to direct positive pressure to the fourth and fifth lines, such that when positive pressure is applied to the fourth pneumatic line, the fifth pneumatic line will not be pressurized, and vice versa.

[0217] The second cylinder is in fluid communication with the fourth and fifth pneumatic lines and is mechanically connected to the injection gate. When the fourth pneumatic line is pressurized, the injection gate moves to the closed position, and when the third pneumatic line is pressurized, the injection gate moves to the open position.

[0218] 30. The method according to Clause 29, wherein the pneumatic assembly further comprises:

[0219] The third switch, which is in fluid communication with the pneumatic pump via the first pneumatic line and is configured to direct positive pressure to the sixth and seventh lines, such that when positive pressure is applied to the sixth pneumatic line, the seventh pneumatic line will not be pressurized, and vice versa.

[0220] The third cylinder is in fluid communication with the sixth and seventh pneumatic lines and is mechanically connected to the sample tubing assembly. The sample tubing subassembly includes a sample tubing in fluid communication with the flow chamber, such that when the sixth pneumatic line is pressurized, the sample tubing subassembly moves to the injection position, thereby inserting the sample tubing into the sample container; and when the seventh pneumatic line is pressurized, the sample tubing subassembly moves to the retracted position.

[0221] 31. The method according to Clause 29 or Clause 30, wherein the actuation of the SIT assembly, injection gate and / or sample tubing subassembly is not driven by a stepper motor and worm gear.

[0222] 32. The method according to any one of Clauses 29 to 31, wherein the actuation of the SIT assembly, injection gate and / or sample tubing subassembly does not require a separate circuit board.

[0223] 33. The method according to any one of Clauses 29 to 32, wherein the pneumatic pump, the first cylinder, the second cylinder and the third cylinder, and the first switch, the second switch and the third switch are operated by a single circuit board.

[0224] 34. The method according to any one of Clauses 29 to 33, wherein the pneumatic pump, the first cylinder, the second cylinder and the third cylinder, and the first switch, the second switch and the third switch do not require firmware.

[0225] 35. The method according to any one of Clauses 29 to 34, wherein one or more of the first switch, the second switch and the third switch include a flow regulator for regulating the pressure balance in a non-pressurized pneumatic line.

[0226] 36. The method according to any one of Clauses 29 to 35, wherein the pneumatic assembly further comprises a pressure reservoir in fluid communication with the pneumatic pump and one or more of the first switch, the second switch and the third switch, wherein the pneumatic pump pressurizes the pressure reservoir and the pressure reservoir provides positive pressure to the first pneumatic line.

[0227] 37. The method according to any one of Clauses 29 to 36, wherein the pneumatic assembly includes a connector located on one or more of the first pneumatic line, the second pneumatic line, the third pneumatic line, the fourth pneumatic line, the fifth pneumatic line, the sixth pneumatic line, and the seventh pneumatic line, such that the pneumatic line can be disconnected and reconnected.

[0228] 38. The method according to any one of Clauses 29 to 37, wherein the pneumatic assembly further includes a mounting bracket on which the pneumatic pump, pressure reservoir, switch and connector are mounted.

[0229] 39. The method according to any one of Clauses 29 to 38, wherein the pneumatic assembly further comprises a pressure gauge for measuring the pressure in the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines.

[0230] 40. The method according to Clause 39, wherein when the pressure measured by the pressure gauge drops below a threshold pressure, one or more of the pressure reservoir and the second, third, fourth, fifth, sixth, and seventh pneumatic lines are repressurized.

[0231] 41. The method according to Clause 40, wherein the threshold pressure range is 25 psi to 50 psi.

[0232] 42. The method according to any one of Clauses 28 to 41 further includes actuating the opening of the sample inlet gate.

[0233] 43. The method according to any one of Clauses 28 to 42, wherein the sample gate protects the sample from ambient light by reducing the transmission of ambient light.

[0234] 44. The method according to Clause 43, wherein the sample inlet gate is semi-transparent.

[0235] 45. The method according to any one of Clauses 28 to 44, wherein the SIT component is located on an XY movable stage.

[0236] 46. ​​The method according to any one of Clauses 27 to 45 further includes a calibration plate, a chassis, and a manual test tube inlet, wherein the calibration plate and chassis are configured to calibrate the height of the manual test tube inlet to minimize sample dead volume when switching between obtaining a sample from a sample container in the pneumatically driven autosampler and obtaining a sample from the manual test tube inlet position.

[0237] 47. The method according to any one of Clauses 27 to 46, wherein the sample container receiving area includes a platform configured to hold one or more samples.

[0238] 48. The method according to Clause 47, wherein the platform is configured to mix, heat and / or cool the one or more samples.

[0239] 49. The method according to Clause 47 or Clause 48, wherein introducing the particulate sample comprises placing a porous plate or sample tube rack on the platform.

[0240] 50. The method according to any one of Clauses 28 to 49 further includes actuating the closing of the sample inlet gate.

[0241] 51. The method according to any one of Clauses 27 to 50, wherein the flow cytometer further comprises a light source configured to illuminate the flow chamber at a detection point.

[0242] 52. The method according to any one of Clauses 27 to 51, wherein the flow cytometer further includes a detector configured to collect particle-modulated light from the flow chamber.

[0243] 53. The method according to any one of Clauses 27 to 52, wherein the flow cytometer is a particle analyzer.

[0244] 54. The method according to any one of Clauses 27 to 53, wherein the flow cytometer is a particle sorter.

[0245] 55. The method according to any one of Clauses 27 to 54, wherein the flow cytometer is an imaging flow cytometer.

[0246] Therefore, the foregoing merely illustrates the principles of this disclosure. It will be understood that those skilled in the art will be able to devise various arrangements that, while not expressly described or shown herein, embody the principles of this disclosure and are included within its spirit and scope. Furthermore, all instances and conditional language recorded herein are primarily intended to aid the reader in understanding the principles of this disclosure and the concepts contributed by the discloser to advance the art, and should be construed as not being limited to these specifically recorded instances and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of this disclosure, as well as specific examples thereof, are intended to include equivalents of their structure and function. Furthermore, it is intended that such equivalents include both currently known equivalents and future equivalents, i.e., any developed element performing the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly stated in the claims.

[0247] Therefore, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35 U.S.C. 112(f) or 112(6) expressly provides that the foregoing clause is invoked only if a claim begins with the precise expression “meansfor” or “step for”; and if a claim does not use the precise expression, 35 U.S.C. 112(f) or 112(6) is not invoked.

Claims

1. A flow cytometer, comprising: Flow chamber; and A pneumatically driven autosampler is configured to automatically pick up a sample from a sample container and deliver the sample to the flow chamber.

2. The flow cytometer according to claim 1, wherein, The pneumatically driven autosampler includes: Sample container receiving area; A SIT assembly, configured to introduce a sample tubing into the sample container located in the sample container region, wherein SIT refers to a sample injection tube; and A sample inlet gate, configured to regulate access to the sample container receiving area; The SIT component and the sample inlet gate are actuated by a pneumatic component.

3. The flow cytometer according to claim 2, wherein, The pneumatic assembly includes: A pneumatic pump is used to provide positive pressure to the first pneumatic line; A first switch, which is in fluid communication with the pneumatic pump via the first pneumatic line, is configured to direct positive pressure to the second and third pneumatic lines such that when positive pressure is applied to the second pneumatic line, the third pneumatic line is not pressurized, and vice versa. A first cylinder is in fluid communication with the second and third pneumatic lines and mechanically connected to the SIT assembly, such that pressurizing the second pneumatic line moves the SIT assembly to the sampling position; while pressurizing the third pneumatic line moves the SIT assembly to the rest position. The second switch, which is in fluid communication with the pneumatic pump via the first pneumatic line, is configured to direct positive pressure to the fourth and fifth pneumatic lines such that when positive pressure is applied to the fourth pneumatic line, the fifth pneumatic line is not pressurized, and vice versa. The second cylinder is in fluid communication with the fourth and fifth pneumatic lines and is mechanically connected to the injection gate, such that pressurizing the fourth pneumatic line moves the injection gate to the closed position, while pressurizing the third pneumatic line moves the injection gate to the open position.

4. The flow cytometer according to claim 3, wherein, The pneumatic assembly also includes: The third switch is in fluid communication with the pneumatic pump via the first pneumatic line and is configured to direct positive pressure to the sixth and seventh pneumatic lines such that when positive pressure is applied to the sixth pneumatic line, the seventh pneumatic line is not pressurized, and vice versa. The third cylinder is in fluid communication with the sixth and seventh pneumatic lines and is mechanically connected to the sampling line subassembly, which includes a sample line in fluid communication with the flow chamber, such that pressurizing the sixth pneumatic line moves the sample line assembly to the injection position, thereby inserting the sample line into the sample container, while pressurizing the seventh pneumatic line moves the sample line subassembly to the retracted position.

5. The flow cytometer according to claim 3 or 4, wherein, The actuation of the SIT assembly, the injection gate, and / or the sample tubing subassembly is not driven by a stepper motor and a worm gear.

6. The flow cytometer according to any one of claims 3 to 5, wherein, The actuation of the SIT assembly, the injection gate, and / or the sample tubing subassembly does not require a separate circuit board.

7. The flow cytometer according to any one of claims 3 to 6, wherein, The pneumatic pump, the first cylinder, the second cylinder, and the third cylinder, as well as the first switch, the second switch, and the third switch, are operated by a single circuit board.

8. The flow cytometer according to any one of claims 3 to 7, wherein, The pneumatic pump, the first cylinder, the second cylinder, and the third cylinder, as well as the first switch, the second switch, and the third switch, do not require firmware.

9. The flow cytometer according to any one of claims 3 to 8, wherein, One or more of the first switch, the second switch, and the third switch include a flow regulator for regulating the pressure balance in a non-pressurized pneumatic line.

10. The flow cytometer according to any one of claims 3 to 9, wherein, The pneumatic assembly further includes a pressure reservoir in fluid communication with the pneumatic pump and one or more of the first, second, and third switches, wherein the pneumatic pump pressurizes the pressure reservoir and the pressure reservoir provides positive pressure to the first pneumatic line.

11. The flow cytometer according to any one of claims 3 to 10, wherein, The pneumatic assembly includes connectors located on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines, enabling the pneumatic lines to be disconnected and reconnected.

12. The flow cytometer according to any one of claims 3 to 11, wherein, The pneumatic assembly also includes a mounting bracket on which the pneumatic pump, pressure reservoir, switch, and connector are mounted.

13. The flow cytometer according to any one of claims 3 to 12, wherein the pneumatic assembly further comprises a pressure gauge for measuring the pressure in the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines.

14. The flow cytometer according to any one of claims 2 to 13, wherein, The sample inlet gate protects the sample from ambient light by reducing the transmission of ambient light.

15. A method for performing flow cytometry analysis on a sample, the method comprising: (a) A sample container containing a sample is introduced into a pneumatically driven autosampler of the flow cytometer, wherein the pneumatically driven autosampler is configured to automatically acquire a sample from the sample container, thereby delivering the sample to the flow chamber of the flow cytometer. (b) Perform flow cytometry analysis on the sample.

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