Flow cytometer with pneumatically driven automatic sample loader and method of use thereof

Abstract

Aspects of this disclosure include flow cytometers having a pneumatically driven automatic sample loader. A flow cytometer according to a particular embodiment includes a flow cell and a pneumatically driven automatic sample loader configured to automatically acquire a sample from a sample container and transport the sample to the flow cell. For example, a method for cytologically processing a sample for analytical and / or sorting applications is also provided. The pneumatically driven automatic sample loader also includes a sample container receiving area, a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving area, and a loader door configured to regulate access to the sample container receiving area, wherein the operation of the SIT assembly and the loader door is pneumatically driven by a pneumatic assembly.

Description

[Background technology]

[0001] Cross-reference of related applications In accordance with 35 U.S.C. § 119(e), this application claims priority to the filing dates of U.S. Provisional Patent Application No. 63 / 813,208, filed on 28 May 2025, and U.S. Provisional Patent Application No. 63 / 718,455, filed on 8 November 2024, the disclosures thereof incorporated herein by reference.

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

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

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

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

[0006] The inventors have recognized that a motor-driven actuator, such as the one shown in Figure 1A, requires a motor and gears, in addition to an additional printed circuit board (PCB) and firmware, to operate the motor. Therefore, these devices have a high level of complexity, which leads to increased manufacturing and maintenance costs. Furthermore, these components increase the factors for failure, and thus increase potential repair costs. For example, these components require multiple sources of supply for acquisition, additional software updates, lubrication, and additional space within the flow cytometer housing. Moreover, a failure in any of these points could result in the sample injection tube 10 failing to reach the sample for analysis. This problem is exacerbated by the presence of additional moving parts, each requiring its own motor, gears, PCB, and firmware.

[0007] The embodiments described herein solve this complexity caused by multiple motors, gears, and PCBs. In the embodiments, a pneumatic system including a pump and pneumatic cylinders is used to actuate the sample inlet tube (i.e., SIT). Some embodiments include a second pneumatic cylinder for actinguator doors. Additional embodiments include a third pneumatic cylinder for actinguating sample line subassemblies. In the embodiments, a single pump is coupled to two or more pneumatic cylinders. Such devices reduce complexity and the space occupied within the housing, as such embodiments have fewer moving parts and other components (e.g., PCBs). These embodiments reduce costs (including maintenance costs and labor).

[0008] A flow cytometer according to a particular embodiment includes a flow cell and a pneumatically driven automatic sample loader configured to automatically acquire a sample from a sample container and transport the sample to the flow cell. Methods for cytologically processing a sample for analytical and / or sorting applications are also provided.

[0009] A flow cytometer is provided. An embodiment of the flow cytometer provided includes a flow cell and a pneumatically driven automatic sample loader configured to automatically acquire a sample from a sample container and transport the sample to the flow cell. In some cases, the pneumatically driven automatic sample loader includes a sample container receiving area, a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving area, and a loader door configured to regulate access to the sample container receiving area, the operation of the SIT assembly and the loader door being pneumatically driven by a pneumatic assembly. In some cases, the pneumatic assembly includes a pneumatic pump that provides positive pressure to a first pneumatic line, a first switch configured to fluidly communicate with the pneumatic pump via the first pneumatic line and to guide the positive pressure to the second and third lines so that the third pneumatic line is not pressurized when positive pressure is applied to the second pneumatic line, and so that the second pneumatic line is not pressurized when positive pressure is applied to the third pneumatic line, and a second switch configured to fluidly communicate with the second and third pneumatic lines, such that pressurization of the second pneumatic line moves the SIT assembly to the sampling position and pressurization of the third pneumatic line moves the SIT assembly to the resting position. The assembly includes a first pneumatic cylinder mechanically connected to the IT assembly; a second switch configured to fluidly communicate with a pneumatic pump via a first pneumatic line and to direct positive pressure to the fourth and fifth lines so that the fifth pneumatic line is not pressurized when positive pressure is applied to the fourth pneumatic line, and so that the fourth pneumatic line is not pressurized when positive pressure is applied to the fifth pneumatic line; and a second pneumatic cylinder mechanically connected to the loader door and fluidly communicating with the fourth and fifth pneumatic lines such that pressurization of the fourth pneumatic line moves the loader door to the closed position and pressurization of the fifth pneumatic line moves the loader door to the open position.In certain cases, the pneumatic assembly further includes a third switch configured to fluidly communicate with a pneumatic pump via a first pneumatic line and to direct positive pressure to the sixth and seventh lines so that the seventh pneumatic line is not pressurized when positive pressure is applied to the sixth pneumatic line, and so that the sixth pneumatic line is not pressurized when positive pressure is applied to the seventh pneumatic line; and a third pneumatic cylinder mechanically connected to a sample line subassembly having a sample line that fluidly communicates with the sixth and seventh pneumatic lines and fluidly communicates with a flow cell such that pressurization of the sixth pneumatic line moves the sample line subassembly to a loading position and inserts the sample line into a sample container, and pressurization of the seventh pneumatic line moves the sample line subassembly to a retracted position.

[0010] In some cases, the operation of the SIT assembly, loader door, and / or sample line subassembly is not driven by a stepping motor and worm gear. In certain cases, the operation of the SIT assembly, loader door, and / or sample line subassembly does not require a separate circuit board. In some cases, the pneumatic pump, the first, second, and third pneumatic cylinders, and the first, second, and third switches operate from a single circuit board. In certain cases, the pneumatic pump, the first, second, and third pneumatic cylinders, and the first, second, and third switches do not require firmware.

[0011] In certain embodiments, one or more of the first, second, and third switches include a flow regulator for adjusting pressure equalization in an unpressurized 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 of the first, second, and third switches, the pneumatic pump pressurizing the pressure reservoir, and the pressure reservoir providing positive pressure to the first pneumatic line. In certain embodiments, the pneumatic assembly includes connectors on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines so that the pneumatic lines can be disconnected and reconnected. In various embodiments, the pneumatic assembly further includes a mounting bracket on which the pneumatic pump, pressure reservoir, switches, and connectors are mounted. In many embodiments, the pneumatic assembly further includes a pressure gauge for measuring the pressure of the pressure reservoir and 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 falls below a threshold pressure, the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines are repressurized. In certain cases, the threshold pressure is in the range of 25 psi to 50 psi.

[0013] In some embodiments, the loader door protects the sample from ambient light by reducing the transmission of ambient light. In various embodiments, the loader door is translucent. In some cases, the SIT assembly resides on an XY movable stage. Some embodiments include a calibration plate, chassis, and manual tube port, the calibration plate and chassis configured to calibrate the height of the manual tube port to minimize dead volume of the sample when transitioning between acquiring a sample from a sample container in the pneumatically driven automatic sample loader and acquiring a sample from the manual tube loading position. In some cases, the pneumatically driven automatic sample loader further includes a platform configured to hold one or more samples. In certain cases, the platform is configured to mix, heat, and / or cool one or more samples.

[0014] Various examples include one or more light sources configured to irradiate a flow cell at interrogation points and a detector configured to collect particle-modulated light from the flow cell.

[0015] In various embodiments, the flow cytometer is a particle analyzer, a particle sorter, and / or an imaging flow cytometer.

[0016] Aspects of the present disclosure include a method for analyzing a sample, the method including introducing a sample container containing the sample into a pneumatically driven sample loader of a flow cytometer, the pneumatically driven sample loader being configured to automatically acquire the sample from the sample container such that the sample is conveyed to a flow cell of the flow cytometer, and analyzing the sample by flow cytometry.

Brief Description of the Drawings

[0017] The present disclosure can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures. [Figure 1A] Shows a diagram of a sample injection tube (SIT) assembly used in a currently used flow cytometer. [Figure 1B] Shows various diagrams of a pneumatically driven sample loader according to a particular embodiment. [Figure 1C] Shows various diagrams of a pneumatically driven sample loader according to a particular embodiment. [Figure 1D] Shows various diagrams of a pneumatically driven sample loader according to a particular embodiment. [Figure 1E] Shows various diagrams of a pneumatically driven sample loader according to a particular embodiment. [Figure 2] Shows a flow cytometry system according to a particular embodiment. [Figure 3-1] Shows an image-corresponding particle sorter according to a particular embodiment. [Figure 3-2] Shows an image-corresponding particle sorter according to a particular embodiment. [Figure 4]A functional block diagram of a particle analysis system according to a specific embodiment is shown. [Figure 5] A functional block diagram of an example of a control system according to a specific embodiment is shown. [Figure 6A] A schematic diagram of a particle sorting machine system according to a specific embodiment is shown. [Figure 6B] A schematic diagram of a particle sorting machine system according to a specific embodiment is shown. [Figure 7] This shows an embodiment of a computer-controlled system according to a specific example. [Figure 8] This shows a diagram of the BD FACSDICOVER (trademark) A8 Plate Loader setup user interface. [Figure 9A] This shows the effect of cell concentration on mixing efficiency. [Figure 9B] This shows the effect of well volume on mixing efficiency. [Figure 9C] This shows the effect of stirring intensity on mixing efficiency. [Figure 9D] This shows the effect of stirring frequency on mixing efficiency. [Figure 9E] This shows the effect of the default stirring settings on survival rate. [Figure 9F] This shows the effect of the default stirring settings on fluorescence. [Figure 10A] This indicates an A8 carryover for PBMC. [Figure 10B] This shows A8 carryover in HT-29 cells. [Figure 10C] This shows the throughput of the FACSDiscover(trademark) A8 Loader. [Modes for carrying out the invention]

[0018] Aspects of the present disclosure include flow cytometers having a pneumatically driven automatic sample loader. A flow cytometer according to a particular embodiment includes a flow cell and a pneumatically driven automatic sample loader configured to automatically acquire a sample from a sample container and transport the sample to the flow cell. Methods for cytologically processing samples for analytical and / or sorting applications are also provided, for example.

[0019] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and can therefore naturally vary. It should also be understood that the scope of this disclosure is limited only by the appended claims, and that the terms used herein are intended solely to describe and not to limit the specific embodiments.

[0020] Where a range of values ​​is presented, it should be understood that each value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless otherwise explicitly stated in the context, and any other stated or intermediate values ​​within that stated range, are included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, except for any specifically excluded limits within the stated range, and are also included in this disclosure. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0021] In this specification, a particular range is presented with the term “approximately” preceding a number. The term “approximately” is used herein to provide literal support for the exact number preceding the term, as well as for any number that is close to or approximates the number preceding the term. In determining whether a number is close to or approximates a specifically enumerated number, any close or approximate number that is not enumerated may be a number that, in the context in which the number is presented, presents a substantial equivalent of the specifically enumerated number.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but representative exemplary methods and materials are described here.

[0023] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and explain the methods and / or materials to which the publications are relatedly cited. Any reference to a publication is for the purpose of disclosing a publication prior to the filing date, and this disclosure should not be construed as an acknowledgment that such publication has no prior rights by prior disclosure. Furthermore, the publication dates presented may differ from the actual publication dates, and the actual publication dates may need to be verified independently.

[0024] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly stated in the context. Furthermore, it should be noted that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “exclusively” and “only” in relation to the enumeration of elements of the claims or the use of “negative” limitations.

[0025] As will be obvious to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any method of description may be carried out in the order of the described events or in any other logically possible order.

[0026] While systems and methods are described for grammatical fluidity with functional descriptions, claims should not necessarily be interpreted as being limited by constructing a limitation of “means” or “steps” unless explicitly formulated under 35 U.S. SC § 112, and should be given the meaning of the definitions and the full scope of equivalents provided by the claims under the doctrine of equivalents, and if the claims are explicitly formulated under 35 U.S. SC § 112, it should be clearly understood that a full set of statutory equivalents should be given under 35 U.S. SC § 112.

[0027] Flow cytometer equipped with a pneumatically driven automatic sample loader As summarized above, aspects of the present disclosure include pneumatically driven automatic sample loaders. In various embodiments, the pneumatically driven automatic sample loader includes a sample container receiving area, a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving area, and a loader door configured to regulate access to the sample container receiving area, the operation of the SIT assembly and the loader door being pneumatically driven by a pneumatic assembly. The SIT assemblies disclosed herein are used to introduce a certain amount of liquid sample from a sample container, e.g., a tube or well (such as a well in a multiwell plate), into a sample fluid line connected to a flow cell of a flow cytometer. The SIT assemblies of embodiments of the present application are configured to draw a sample from the sample container into the lumen of the sample line of the SIT assembly, from which the drawn sample can be transported by the flow cytometer to the flow cell of the flow cytometer.

[0028] As summarized above, the automatic loaders of embodiments of this disclosure are pneumatically driven. In an example, the pneumatically driven automatic sample loader includes a sample receiving area, a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving area, and a loader door configured to regulate access to the sample container receiving area, the operation of the SIT assembly and the loader door being pneumatically driven by a pneumatic assembly. In an example, the pneumatic assembly includes a pneumatic pump for providing positive pressure to one or more pneumatic cylinders. Positive pressure refers to a pressure higher than ambient pressure, as opposed to negative pressure, which refers to a pressure lower than 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 by elevation or altitude, environmental conditions (high-pressure or low-pressure systems), and / or air control within a laboratory or clinical environment. For example, a clinical room may typically be maintained at a higher ambient pressure to avoid contamination, while a laboratory environment may be at a lower ambient pressure to avoid contamination of corridors or other boundary spaces. Such pneumatic cylinders may be mechanically coupled to components such as SIT assemblies, loader doors, and / or sample line subassemblies. By providing positive pressure to the pneumatic cylinders, the components can be actuated to desired positions. By using one or more switches, these components can be actuated between two positions (e.g., sampling position versus rest position, closed position versus open position, and / or loading position versus retracted position). By using a pneumatic drive system, embodiments actuate one or more of the SIT assemblies, loader doors, and / or sample line subassemblies that are not driven by stepping motors and worm gears. A worm gear, also called a drive screw, refers to a linear component having a grooved profile so that radial rotation allows the device to move along the longitudinal axis of the linear component. In the absence of a stepping motor, embodiments do not require separate circuit boards (e.g., printed circuit boards or PCBs) to actuate each of the SIT assemblies, loader doors, and / or sample lines.In this embodiment, the operation of the SIT assembly, loader door, and / or sample line is controlled by a single circuit board. Furthermore, the absence of stepping motors and circuit boards allows for operation without the need for firmware, or the pneumatic pump, the first, second, and third pneumatic cylinders, and the first, second, and third switches do not require firmware.

[0029] Figure 1B shows a diagram of a pneumatically driven automatic sample loader according to one embodiment of the present disclosure. In Figure 1B, a sample container receiving area 102 is provided. Such an area 102 may be a platform, rack, recess, or other location for inserting a rack, tray, or plate (e.g., a multiwell plate). Furthermore, Figure 1B shows a SIT assembly 104 configured to introduce a sample line 105 into a sample container located in the sample container receiving area. Further details regarding the SIT assembly 104 are given below. The loading door is not shown in Figure 1B (to avoid obscuring some details), but a loading door mounting assembly 106 is shown. The loading door can be operated between an open position and a closed position, the open position allowing the sample to be loaded into the sample container receiving area 102, and the closed position allowing the loading door to protect the sample from ambient light and the user from moving parts.

[0030] When using a device such as the one shown in Figure 1B, the loading door can be operated to the open position to allow the user to access the sample container receiving area 102. A sample (or more samples) can be placed in the sample container receiving area 102. Such (one or more) samples can be housed in tubes and tube racks or multi-well plates (e.g., 96-well, 384-well, 1,536-well plates, etc.). The sample container receiving area 102 may include a shaking mechanism to allow mixing, resuspension, or other agitation of the sample. In some embodiments, the sample container receiving area 102 may also include a heating and / or cooling mechanism to maintain the sample at a desired temperature (e.g., refrigeration temperature for storage, reaction temperature, etc.). Such heating and cooling mechanisms are known in the art, including heat pumps, inverse heat pumps, Peltier elements, water circulators, heat pipes, condensers, and / or any other related mechanisms. Once the sample is loaded (or can be stored or protected inside the device), the loading door can be operated to the closed position.

[0031] Furthermore, Figure 1B shows an XY movable stage 108 on which the SIT assembly 104 is present or mounted. The movable stage 108 can enable movement of the SIT assembly in the XY plane to move a sample line between tubes in a rack or between wells in a multiwell plate. XY movement can be achieved using motors, gears, tracks, magnetic tracks, and / or any other applicable method for precisely moving the platform. The movable stage 108 allows the SIT assembly 104 to be positioned on a particular tube or well so that it can be placed in a loaded position for sample acquisition or retrieval by actuation to a loading position.

[0032] Figure 1C shows a more detailed view of the SIT assembly 104 in the loaded position according to various embodiments of the present disclosure. In Figure 1C, the SIT assembly 104 includes a first pneumatic cylinder 110 for operating the SIT assembly 104 between the loaded position and the resting position. For stability, the SIT assembly 104 may be moved along a stabilization track 112 to avoid seizing or stopping due to torque over the arm length of the SIT assembly 104. Certain embodiments may include a counterbalance or other system to limit or avoid torque on the SIT assembly 104.

[0033] The sample line subassembly 114 is shown as a parallel arm of the SIT assembly 104. A third pneumatic cylinder 116 is shown to actuate the sample line subassembly 114 between a loaded position and a retracted position. As shown, the SIT assembly 104 is in the sampling position and the sample line subassembly 114 is in the retracted position. In this position, the flow cytometer is either in the position immediately before or immediately after acquiring the sample. In Figure 1C, the illustrated SIT assembly includes a return flexure 118 that provides a variable sample line depth. Such SIT assemblies are further described in U.S. Patent Application No. 60 / 718,451 (Agent Reference Number P-30353.US01PRO / BECT-386PRV), filed on the same day as this specification, and its disclosure is incorporated herein by reference.

[0034] Figure 1D shows another diagram of a pneumatic assembly according to an embodiment. This diagram shows a mounting bracket 150 attached to a pneumatic pump 152. A first pneumatic line 156 is in fluid communication with the pneumatic pump 152 so that the pneumatic pump 152 provides positive pressure to an optional pressure reservoir 154 further connected to the first pneumatic line 156. As shown, the first pneumatic line 156 is mounted on a manifold 158, which directs pressure to switches 160a-c. Using switches 160a-c, positive pressure can be directed between pneumatic lines 162a-f. For example, the first switch 160a can direct positive pressure between the second pneumatic line 162a and the third pneumatic line 162b, which are in fluid communication with the first pneumatic cylinder (Figure 1C, 110). When positive pressure is directed to the second pneumatic line 162a, the first pneumatic cylinder acts on the SIT assembly (Figure 1C, 104) to the lower or sampling position. Similarly, when positive pressure is directed to the third pneumatic line 162b, the first pneumatic cylinder acts on the SIT assembly (Figure 1C, 104) to the upper or resting position.

[0035] Similarly, switches 160b-c are directed to pneumatic lines 162c-f to guide positive pressure to the second and third pneumatic cylinders in order to operate the loader door between the closed and open positions and the sample line subassembly (Figure 1C, 114) between the sampling and retracted positions. Furthermore, connectors 164 are shown that allow each of the pneumatic lines 162a-f to be separated and reconnected, for example, to allow the replacement of one or more components without interfering with the operation of any other components.

[0036] Returning to switches 160a-c, these switches can allow the unpressurized line to vent to the atmosphere or environment, for example, to enable the operation or movement of the associated pneumatic cylinder. In some embodiments, switches 160a-c include flow regulators 164 for regulating pressure equalization within the unpressurized pneumatic line. Such flow regulators can allow for smoother or slower operation to prevent damage that may occur due to the unrestrained or unrestricted movement of components.

[0037] The embodiments include a pressure gauge or other pressure gauge that can be used to evaluate the pressure at one or more points in the illustrated configuration. In some embodiments, the pressure gauge can measure the pressure in the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines. In such embodiments, when the pressure measured by the pressure gauge falls below a threshold pressure, the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines are repressurized. Such repressurization can be performed by operating a pump to add pressure to the system, opening a switch to apply pressure, and maintaining the position of the pneumatic cylinder. In various embodiments, the threshold pressure may be in the range of about 5 psi to about 100 psi, including its partial range. Such partial ranges 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 is in the range of 25 psi to 50 psi. In certain embodiments, repressurization occurs when the measured pressure drops to less than 100 psi, less than 90 psi, less than 80 psi, less than 70 psi, less than 60 psi, less than 50 psi, less than 40 psi, less than 30 psi, less than 20 psi, or less than 10 psi. In certain cases, repressurization ends 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.

[0038] Figure 1E is a photograph of a flow cytometer according to an embodiment. In this photograph, a portion of the housing has been removed so that the mounting bracket 150 is visible (along with the pneumatic pump 152, reservoir 154, and switches 160a-c, although these are not separately labeled). In this image, the loader door 175 is shown in the closed position, thus protecting the sample container receiving area 102 from ambient light and / or interference from external sources. In some embodiments, the loader door 175 can protect the sample from ambient light by reducing the transmission of ambient light. In certain embodiments, the loader door 175 is translucent.

[0039] A manual tube port 176 is also shown. In embodiments, the manual tube port allows for the processing of a single sample without the need for a plate or tube rack, for example, when a limited number of samples are placed in the flow cytometer. Various embodiments having a manual tube port include a calibration plate and chassis, which are configured to calibrate the height of the manual tube port to minimize dead volume of the sample when transitioning between acquiring a sample from a sample container in a pneumatically driven automatic sample loader and acquiring a sample from the manual tube loading position. Further details regarding strategies for avoiding dead space in the SIT assembly, including dead space located in the pneumatically driven automatic sample loader and the manual tube loading position, are described in concurrent application 63 / 718,451 (Agent reference number P-30353.US01PRO / BECT-386PRV), filed on the same date as this specification, and its disclosure is incorporated herein by reference.

[0040] As described above, the SIT assembly of the pneumatically driven automatic loader of the embodiment is configured to acquire a sample from a sample container, such as a tube or a well of a multiwell plate, by drawing the sample into the sample line of the SIT assembly and then transporting the drawn sample to a flow cell, either directly or via an additional (one or more) fluid line. The flow cell of interest includes a cuvette configured to transport particles in a flow stream. As used herein, “flow cell” is described in its conventional sense, referring to a component that includes a channel for a liquid flow stream for transporting particles in a sheath fluid. The cuvette of interest has a passage (i.e., a channel) through which the channel passes. The flow stream, through which the channel is formed, may include a liquid sample injected from a sample tube. In certain cases, the flow cell includes a channel to which light can reach. The cuvette may be made of, for example, quartz, glass, transparent plastic, etc. In some embodiments, the cuvette is formed from silica, such as fused silica. In some cases, the flow cell is configured to be illuminated from a light source at one or more interrogation points. As discussed herein, an "interrogation point" refers to a region (investigation area) within a flow cell where particles are irradiated by light from a light source, for example, for analysis. The size of the interrogation point can vary as desired. For example, if 0 μm represents the optical axis of the light emitted by the light source, the interrogation point may be in the range of -50 μm to 50 μm, e.g., -25 μm to 40 μm, and e.g., -15 μm to 30 μm. Depending on specific considerations (e.g., the number and arrangement of lasers), multiple irradiation points may exist within the flow cell.

[0041] In some embodiments, the flow cell includes, or is configured to be used with, a sample injection port configured to provide the sample to the flow cell, for example, by a SIT assembly and any intervening fluid lines, if present. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the internal chamber (i.e., flow path) of the flow cell. Depending on the desired characteristics of the flowstream, the flow rate of the sample delivered to the flow cell chamber by the sample injection port may be 1 μL / min or more, e.g., 2 μL / min or more, e.g., 3 μL / min or more, e.g., 5 μL / min or more, e.g., 10 μL / min or more, e.g., 15 μL / min or more, e.g., 25 μL / min or more, e.g., 50 μL / min or more, and e.g., 100 μL / min or more. In some cases, the flow rate of the sample delivered to the flow cell chamber by the sample injection port may be 1 μL / second or more, e.g., 2 μL / second or more, e.g., 3 μL / second or more, e.g., 5 μL / second or more, e.g., 10 μL / second or more, e.g., 15 μL / second or more, e.g., 25 μL / second or more, e.g., 50 μL / second or more, and e.g., 100 μL / second or more.

[0042] In some cases, a flow cytometer is an analytical flow cytometer having multiple acquisition rates. “Analytical flow cytometer” refers to a flow cytometer configured to analyze particles (including cells) suspended in a fluid stream. The fluid flow through the analytical flow cytometer is typically connected to a waste container, and the analyzed particles are disposed of as waste rather than being sorted and collected for further use or analysis. In an analytical flow cytometer, the fluid flow may be driven by a vacuum pump connected to a waste line, and the waste line may be fluidically coupled to the outlet of the flow cell so that the fluid can be drawn from the flow cell into the waste line under vacuum. As used herein, “multiple acquisition rates” refers to the ability of an analytical flow cytometer to capture or acquire data at two or more fluid rates within the flow cell. Multiple acquisition rates add functionality to an analytical flow cytometer, allowing a single flow cytometer to be used in a high-speed analysis mode at faster fluid rates and a low-speed imaging mode at slower fluid rates. In some embodiments, multiple acquisition rates are enabled by an adjustable fluid system. Multiple rates are achieved by adjusting the fluid resistance in the waste line of the flow cytometer. In some embodiments of analytical flow cytometers, the fluid flow through the flow cell is driven by a vacuum pump fluidically coupled to a waste line. By increasing the fluid resistance in the waste line, the fluid velocity in the flow cell is reduced. In some embodiments, the reduced fluid velocity is sufficient for particle imaging. Similarly, by reducing the fluid resistance in the waste line, the fluid velocity in the flow cell is increased. In some embodiments, the increased fluid velocity is sufficient for particle analysis. Further details relating to such flow cytometers can be found in U.S. Patent Application No. 19 / 076,246, filed March 11, 2025, the disclosures of which are incorporated herein by reference.

[0043] The sample injection port may be an orifice located in the wall of the internal chamber, or a conduit located at the proximal end of the internal chamber. If the sample injection port is an orifice located in the wall of the internal chamber, the sample injection port orifice may be any suitable shape, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc., curved cross-sectional shapes such as circles, ellipses, etc., and irregular shapes such as parabolic bottoms joined to a flat top. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm.

[0044] In certain cases, the sample injection port is a conduit located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned so that the orifice of the sample injection port is aligned with the flow cell orifice. If the sample injection port is a conduit positioned in line with the flow cell orifice, the cross-sectional shape of the sample injection tube may be any suitable shape, including, but not limited to, straight cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a flat top. The orifice of the conduit may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, and e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a slanted tip having an inclination angle in the range of 1° to 10°, for example 2° to 9°, for example 3° to 8°, for example 4° to 7°, and for example 5°.

[0045] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid to the internal chamber of the flow cell, for example, together with the sample, to create a layered flow stream in which the sheath fluid surrounds the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of sheath fluid delivered to the flow cell chamber may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, and e.g., 2500 μL / sec or more.

[0046] In some embodiments, the sheath fluid injection port is an orifice located in the wall of the internal chamber. The sheath fluid injection port orifice may have any suitable cross-sectional shape of interest, including, but not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved cross-sectional shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a flat top. The size of the sheath fluid injection port orifice may vary depending on the shape, and in certain cases, it may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, and e.g., 1.25 mm to 1.75 mm, e.g., an opening of 1.5 mm.

[0047] The flow cytometers of this disclosure include light sources configured to irradiate particles in the flow stream at an interrogation point in the flow cell. The number of light sources in the flow cytometer can vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, the flow cytometer may include multiple light sources in some cases. In some such cases, the number of light sources is in the range of 2 to 10, e.g., 2 to 5, and e.g., 2 to 4. Any convenient light source may be used as a light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous-wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, this flow cytometer includes dye lasers such as stilbene lasers, coumarin lasers, or rhodamine lasers. In yet other cases, the laser of interest includes metal vapor lasers such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof.In other examples, this flow cytometer includes solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium 2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0048] A laser light source according to a particular embodiment may also include one or more optical adjustment components. In a particular embodiment, the optical adjustment component may include any device located between the light source and the flow cell that can change the spatial width of the irradiation, or any other characteristics of the irradiation from the light source, such as the direction of irradiation, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol may include, but is not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof, any convenient device for adjusting one or more characteristics of the light source. In a particular embodiment, the flow cytometer of interest includes one or more focusing lenses. The focusing lenses may, in one example, be reduction lenses. In yet another embodiment, the flow cytometer of interest includes optical fibers.

[0049] The light source can be positioned at any appropriate distance from the flow cell, for example, the light source and the flow cell may be separated by 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, e.g., 25 mm or more, and e.g., 100 mm or more. Furthermore, the light source can be positioned at any appropriate angle with respect to the flow cell, for example, at angles in the range of 10 to 90 degrees, e.g., 15 to 85 degrees, e.g., 20 to 80 degrees, e.g., 25 to 75 degrees, and e.g., 30 to 60 degrees, e.g., at an angle of 90 degrees.

[0050] In some embodiments, the light source of interest includes multiple lasers configured to provide laser light for discrete irradiation of a flowstream, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, and e.g., fifteen or more lasers. Depending on the desired wavelength of light for irradiating the flowstream, each laser may have a specific wavelength such as 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and e.g., 400 nm to 800 nm. In certain embodiments, the laser of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0051] In certain embodiments, the light source is a light beam generator configured to generate two or more frequency-shifted light beams. In some cases, the light beam generator includes a laser and a high-frequency generator configured to apply a high-frequency drive signal to an acousto-optical device to generate two or more angle-deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous-wave laser. For example, the laser in the light beam generator of interest includes those described above.

[0052] An acousto-optic device can be any convenient acousto-optic protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in this system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal can be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital combiner (DDS), arbitrary waveform generator (AWG), or electric pulse generator.

[0053] In one embodiment, the controller is configured to apply high-frequency drive signals to an acousto-optical device to generate a desired number of angle-deflected laser beams within the output laser beam, for example, by applying three or more high-frequency drive signals, four or more high-frequency drive signals, five or more high-frequency drive signals, six or more high-frequency drive signals, seven or more high-frequency drive signals, eight or more high-frequency drive signals, nine or more high-frequency drive signals, ten or more high-frequency drive signals, fifteen or more high-frequency drive signals, twenty-five or more high-frequency drive signals, fifty or more high-frequency drive signals, and one hundred or more high-frequency drive signals.

[0054] In some cases, to generate an intensity profile of the angularly deflected laser beam within the output laser beam, the controller is configured to apply a high-frequency drive signal having amplitudes such as, for example, about 0.001V to about 500V, about 0.005V to about 400V, about 0.01V to about 300V, about 0.05V to about 200V, about 0.1V to about 100V, about 0.5V to about 75V, about 1V to 50V, about 2V to 40V, about 3V to about 30V, and about 5V to about 25V. Each applied high-frequency drive signal has a frequency of approximately 0.001 MHz to approximately 500 MHz, for example approximately 0.005 MHz to approximately 400 MHz, for example approximately 0.01 MHz to approximately 300 MHz, for example approximately 0.05 MHz to approximately 200 MHz, for example approximately 0.1 MHz to approximately 100 MHz, for example approximately 0.5 MHz to approximately 90 MHz, for example approximately 1 MHz to approximately 75 MHz, for example approximately 2 MHz to approximately 70 MHz, for example approximately 3 MHz to approximately 65 MHz, for example approximately 4 MHz to approximately 60 MHz, and for example approximately 5 MHz to approximately 50 MHz.

[0055] In certain embodiments, the controller is a processor having memory operably coupled to the processor, wherein the memory contains stored instructions, and when the instructions are executed by the processor, the processor causes the processor to generate an output laser beam having an angle-deflected laser beam having a desired intensity profile. For example, the memory may contain instructions for generating two or more angle-deflected laser beams having the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and e.g., 100 or more. In other embodiments, the memory may contain instructions for generating two or more angle-deflected laser beams having different intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., 25 or more, e.g., 50 or more, and e.g., 100 or more.

[0056] In certain embodiments, the controller is a processor having memory operably coupled to the processor, wherein the memory contains stored instructions, and when the instructions are executed by the processor, the processor causes the processor to generate an output laser beam whose intensity increases from the edge to the center along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., In other embodiments, the controller is a processor having memory operably coupled to a processor, wherein the memory contains stored instructions, and when an instruction is executed by the processor, the processor causes the processor to generate an output laser beam whose intensity increases from the edge to the center along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., In yet another embodiment, the controller is a processor having memory operably coupled to a processor, wherein the memory contains stored instructions, and when an instruction is executed by the processor, the processor causes the processor to generate an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis.In yet another embodiment, the controller is a processor having memory operably coupled to the processor, wherein the memory contains stored instructions, and when the instructions are executed by the processor, the processor causes the processor to generate an output laser beam having a top-hat intensity profile along the horizontal axis.

[0057] In some embodiments, the light beam generator of interest may be configured to generate spatially separated, angularly deflected laser beams within the output laser beam. Depending on the applied high-frequency drive signal and the desired irradiation profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, e.g., 100 μm or more, e.g., 500 μm or more, e.g., 1000 μm or more, e.g., 5000 μm or more, e.g., 15000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams within the output laser beam that overlap with adjacent angularly deflected laser beams along the horizontal axis of the output laser beam, etc. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be of 0.001 μm or more, e.g., 0.005 μm or more, e.g., 0.01 μm or more, e.g., 0.05 μm or more, e.g., 0.1 μm or more, e.g., 0.5 μm or more, e.g., 1 μm or more, e.g., 5 μm or more, e.g., 10 μm or more, and e.g., 100 μm or more.

[0058] In certain cases, a light beam generator configured to produce two or more frequency-shifted light beams is subject to U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and 10,684,211. This includes laser excitation modules as described in Patent Nos. 10,845,295, 10,935,482, 10,935,485, 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851, the disclosures of which are incorporated herein by reference.

[0059] Furthermore, the flow cytometer includes a detector configured to collect light emitted by the irradiated particles. The photodetector is configured to detect particle-modulated light carried by an optical fiber focusing element and to generate a signal based on the characteristics of the light (e.g., intensity). For example, one or more particle-modulated photodetectors may include one or more side-scatter photodetectors for detecting the side-scatter wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particles). In some embodiments, the flow cytometer includes a single side-scatter photodetector. In other embodiments, the flow cytometer includes multiple side-scatter photodetectors, e.g., two or more, e.g., three or more, e.g., four or more, and e.g., five or more.

[0060] Any convenient detector for detecting the collected light can be used in the side-scattered light detector described herein. Among detectors of interest, optical sensors or detectors such as, but not limited to, active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors or photodiodes and combinations thereof can be mentioned. In certain embodiments, the collected light is measured by a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor or an N-type metal-oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector has an active detection surface area in each region in the range of 0.01 cm 2 ~10 cm 2 、 for example 0.05 cm 2 ~9 cm 2 、 for example 0.1 cm 2 ~8 cm 2 、 for example 0.5 cm 2 ~7 cm 2 、 and for example 1 cm 2 ~5 cm 2 such as a photomultiplier tube.

[0061] In embodiments, the flow cytometer also includes a fluorescence detector configured to detect one or more fluorescence wavelengths of the light. In other embodiments, the flow cytometer includes multiple, for example two or more, for example three or more, for example four or more, five or more, ten or more, fifteen or more, and for example twenty or more fluorescence detectors.

[0062] Any convenient detector for detecting the collected light may be used in the fluorescence detector described herein. Detectors of interest include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In certain embodiments, the collected light is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is 0.01 cm 2 ~10cm 2 For example, 0.05 cm 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 , and for example, 1 cm 2 ~5cm 2 These are photomultiplier tubes that have an activity detection surface area in each region within the specified range.

[0063] If the flow cytometer includes multiple fluorescence detectors, each fluorescence detector may be the same, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, if the flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector (or imaging sensor) is a CMOS type device. In other embodiments, both the first and second fluorescence detectors are CCD type devices. In yet another embodiment, both the first and second fluorescence detectors are CMOS type devices. In yet another embodiment, the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In yet another embodiment, the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. In yet another embodiment, both the first and second fluorescence detectors are photomultiplier tubes.

[0064] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure the collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths, and to measure the light emitted by the sample in the flow stream at e.g., 400 or more different wavelengths. In some embodiments, two or more detectors in the module described herein are configured to measure the same or overlapping wavelengths of the collected light.

[0065] In some embodiments, the fluorescence detector of interest is configured to measure collected light over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect the spectrum of light over one or more wavelengths within the 200 nm to 1000 nm range. In yet another embodiment, the detector of interest is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a module may include one or more detectors configured to measure light at one or more of the following wavelengths: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm and any combination thereof. In certain embodiments, one or more detectors may be configured to pair with specific fluorophores, such as those used with a sample in a fluorescence assay.

[0066] The flow cytometer may include any suitable (one or more) mechanisms for supplying the sample liquid and sheath liquid to the sample liquid inlet coupler and sheath liquid inlet coupler. For example, the sample liquid inlet coupler may be fluidly connected to a sample liquid line (e.g., a tube) fluidly connected to a sample liquid reservoir. Similarly, the sheath liquid inlet coupler may be fluidly connected to a sheath liquid line fluidly connected to a sheath liquid reservoir. Likewise, the flow cytometer may include any suitable (one or more) mechanisms for managing waste from the flow stream. A fluid discharge coupler may be fluidly connected to a waste line fluidly connected to a waste reservoir. A fluid management system that may be adapted for use with this flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the disclosure of which is incorporated herein by reference in its entirety.

[0067] Appropriate flow cytometry systems include: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (ed.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49 (pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Examples of disclosures included, but not limited to, those described in Syst.24(3):203-255, are incorporated herein by reference.In specific cases, the flow cytometry systems of interest include BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Accuri® C6 Plus flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortessa® X-20 flow cytometer, BD Biosciences FACSPresto® flow cytometer, BD Biosciences FACSVia® flow cytometer, and BD Biosciences FACSCalibur® cell sorter, BD Biosciences FACSCount® cell sorter, and BD Biosciences This includes FACSLyric™ cell sorters, BD Biosciences Via™ cell sorters, BD Biosciences Influx™ cell sorters, BD Biosciences Jazz™ cell sorters, BD Biosciences Aria™ cell sorters, BD Biosciences FACSAria™ II cell sorters, BD Biosciences FACSAria™ III cell sorters, BD Biosciences FACSAria™ Fusion cell sorters, and BD Biosciences FACSMelody™ cell sorters, BD Biosciences FACSymphony™ S6 cell sorters, BD Biosciences FACSDiscover™ cell sorters, and others.

[0068] In some embodiments, this system is provided for in accordance with U.S. Patents No. 10,663,476, No. 10,620,111, No. 10,613,017, No. 10,605,713, No. 10,585,031, No. 10,578,542, No. 10,578,469, No. 10,481,074, and No. 10,302,545. No. 10,145,793, No. 10,113,967, No. 10,006,852, No. 9,952,076, No. 9,933,341, No. 9 ,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034 No. 8,975,595, No. 8,753,573, No. 8,233,146, No. 8,140,300, No. 7,544,326, No. 7,20 1,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, Flow cytometry systems such as those described in Patent Nos. 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766 are incorporated herein by reference.

[0069] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in certain cases, this system is based on Diebold, et al. Nature Photonics. Vol.7(10);806-810(2013), and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, and Flow cytometry systems configured to image particles in a flow stream by fluorescence imaging using high-frequency tagged emission (FIRE), such as those described in Patent Nos. 11,105,728, 11,280,718, 11,327,016, 11,366,052, 11,371,937, 11,692,926, 11,630,053, 11,774,343, 11,940,369 and 11,946,851, are incorporated herein by reference. In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in U.S. Patent Nos. 10,324,019, 10,620,111, 11,105,728 and 11,774,343, and U.S. Patent Applications Nos. 18 / 537,103, 18 / 657,618, 18,657,623 and 18 / 657,633, the disclosures of which are incorporated herein by reference in their entirety.

[0070] Figure 2 shows a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to irradiate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. Although the example in Figure 2 shows a single laser, it will be understood that multiple lasers can also be used. The laser beam from laser 201 is directed to a focusing lens 202, which focuses the beam onto the portion of the fluid stream where the particles 211 of the sample in the flow cell 210 are located. The flow cell 210 is part of a fluid system that guides particles in the stream to the focused laser beam, typically one at a time, for interrogation. Alternatively, a nozzle top may be used if the flow cytometer is a stream-in-air cytometer.

[0071] As shown in Figure 2, the flow cell 210 is fluidically connected to a sheath fluid reservoir 203 containing sheath fluid and a sample fluid reservoir 204 containing sample fluid. Sheath fluid from the sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via a conduit (i.e., sheath fluid line) 207. In addition, sample fluid containing particles 211 from the sample fluid reservoir 204 is supplied to a sample injection port 206 via a conduit (i.e., sample fluid line) 205. The sample injection port 206 is fluidly connected to a sample injector 213 (e.g., a sample injection needle) configured to introduce particles 211 into the flow cell 210. The particles 211 are hydrodynamically focused through the sheath fluid entering from the sheath fluid injection port 208 so that a flowstream 214 is formed downstream of the tapered portion 212 of the flow cell 210. Particles released at the distal end of the flow cell 210 can be disposed of and / or collected via any suitable protocol. For example, depending on the type of flow cytometry performed, the particles may be collected at the distal end of the flow cell 210, for example, via a waste line. Alternatively, the particles may be sorted.

[0072] Light from (one or more) laser beams interacts with particles 211 in the sample by absorption with diffraction, refraction, reflection, scattering, and re-emission at various different wavelengths, depending on the particle's characteristics, such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles. The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be sent to one or more detectors. In particular, forward scatter (FSC) is sent to a forward scatter detector 223. The forward scatter detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect the diffracted light, i.e., the excitation light that travels mainly forward through or around the particles. The intensity of the light detected by the forward scatter detector 223 depends on the overall size of the particles. The forward scatter detector can include, for example, a photodiode. An optical filter 221a and a scattering bar 222 are positioned between the forward scatter detector 223 and the beam. The optical filter 221a may be configured to remove non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scatter light detector 223.

[0073] Furthermore, side-scattered light (SSC) is detected by a side-scattered light detector 224. In other words, the side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structure of the particle 211, which tends to increase as the complexity of the particle structure increases. In the example in Figure 2, the flow cytometer 200 includes a dichroic mirror 220a configured to reflect SSC light to the side-scattered light detector 224 and allow non-SSC light (e.g., fluorescence) to pass through. An optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by the side-scattered light detector 224. Fluorescence detectors 225a-225c, each configured to detect fluorescence of different wavelengths, are also shown. For example, the dichroic mirror 220b may be configured to reflect fluorescence (FL) corresponding to a first wavelength (or wavelength range) to the fluorescence detector 225a and allow light of other wavelengths to pass through. The optical filter 221c may be configured to prevent at least one wavelength of light that does not correspond to a first wavelength (or wavelength range) from being detected by the fluorescence detector 225a. Similarly, the dichroic mirror 220c is configured to reflect FL light corresponding to a second wavelength (or wavelength range) to the fluorescence detector 225b and to allow light of a third wavelength (or wavelength range) to pass through for detection by the fluorescence detector 225c. The optical filter 221d is configured to prevent at least one wavelength of light that does not correspond to a second wavelength (or wavelength range) from being detected by the fluorescence detector 225b. Furthermore, the optical filter 221e is configured to prevent at least one wavelength of light that does not correspond to a third wavelength (or wavelength range) from being detected by the fluorescence detector 225c.

[0074] Those skilled in the art will recognize that the flow cytometer according to the embodiments of the present disclosure is not limited to the flow cytometer shown in Figure 2, but may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations. For example, the embodiment in Figure 2 shows three fluorescence detectors for illustrative purposes, but it will be understood that any suitable number of fluorescence detectors may be used.

[0075] During operation, the cytometer's operation is controlled by the controller / processor 290, and measurement data from the detector is stored in memory 295 and can be processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detector to receive output signals from the detector and may also be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function 297 may also be provided within the system. The memory 295, controller / processor 290, and I / O 297 may be provided as a single integrated part of the flow cytometer. In such embodiments, a display may also form part of the I / O function 297 for presenting experimental data to the user of the cytometer 200. Alternatively, some or all of the memory 295, controller / processor 290, and I / O functions may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 295 and controller / processor 290 can communicate with the cytometer 200 wirelessly or via a wired connection. The controller / processor 290 can be configured to work in conjunction with the memory 295 and I / O 297 to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0076] Different fluorescent molecules in a fluorescent dye panel used in a flow cytometer experiment emit light in their own characteristic wavelength bands. Specific fluorescent labels used in the experiment, and their associated fluorescence emission bands, may be selected to substantially match the detector's filter window. I / O297 can be configured to receive data relating to a panel of fluorescent labels and a flow cytometer experiment having multiple markers and multiple cell populations, each having a subset of multiple markers. I / O297 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 295. The controller / processor 290 can be configured to evaluate the assignment of one or more labels to the markers.

[0077] In some embodiments, the system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, particles of a sample (e.g., cells) are sorted using a sorting decision module having multiple sorting decision units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, whose disclosure is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference.

[0078] In certain embodiments, the system is fluorescence imaging using a radiofrequency tagged emission image-enabled particle sorter, as shown in Figure 3. The particle sorter 300 includes an optical illumination component 300a, which includes a light source 301 (e.g., a 488 nm laser) that generates an output optical beam 301a, which is split into beam 302a and beam 302b using a beam splitter 302. The optical beam 302a is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 303 to generate an output beam 303a having one or more angularly deflected optical beams. In some cases, the output beam 303a generated from the acousto-optical device 303 includes a local oscillator beam and multiple radiofrequency comb beams. The optical beam 302b is propagated through an acousto-optical device (e.g., an acousto-optic deflector, AOD) 304 to generate an output beam 304a having one or more angularly deflected optical beams. In some cases, the output beam 304a generated from the acousto-optic device 304 includes a local oscillator beam and multiple high-frequency comb beams. The output beams 303a and 304a generated from the acousto-optic devices 303 and 304, respectively, are combined using a beam combiner 305 to generate an output beam 305a, which is then transported through an optical component 306 (e.g., an objective lens) to irradiate particles in the flow cell 307. In certain embodiments, the acousto-optic device 303 (AOD) splits a single laser beam into an array of beamlets, each having a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in the beam combiner 305. In certain embodiments, the light irradiation system having a light source and an acousto-optical device may also include those described in Schraivogel, et al. ("High-speed fluorescence image-enabled cell sorting," Science (2022), 375(6578):315-320) and U.S. Patent Application Publication No. 2021 / 0404943, which are incorporated herein by reference.

[0079] The output beam 305a irradiates sample particles 308 propagating through the flow cell 307 (e.g., together with the sheath fluid 309) in the irradiation area 310. As shown in the irradiation area 310, multiple beams (e.g., angle-deflected high-frequency shifted light beams shown as dots across the irradiation area 310) overlap with the reference local oscillator beam (indicated by diagonal lines across the irradiation area 310). Due to their different optical frequencies, the overlapping beams exhibit beat behavior, thereby giving each beamlet a distinct frequency f 1-n This is used to carry a sine wave modulation signal.

[0080] Light from the irradiated sample is delivered to a photodetector system 300b, which includes multiple photodetectors. The photodetector system 300b includes a forward-scattered light photodetector 311 for generating a forward-scattered image 311a and a side-scattered light photodetector 312 for generating a side-scattered image 312a. The photodetector system 300b also includes a bright-field photodetector 313 for generating an optical loss image 313a. In some embodiments, the forward-scattered detector 311 and the side-scattered 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. Light from the irradiated sample is directed through a beam splitter 320 to the side-scattered detection channel 312 and the fluorescence detection channels 314-317. The photodetector system 300b includes bandpass optical components 321, 322, 323, and 324 (e.g., dichroic mirrors) for propagating light of a predetermined wavelength to photodetectors 314-317. In some cases, optical component 321 is a 534 nm / 40 nm bandpass. In some cases, optical component 322 is a 586 nm / 42 nm bandpass. In some cases, optical component 323 is a 700 nm / 54 nm bandpass. In some cases, optical component 324 is a 783 nm / 56 nm bandpass. The first digit represents the center of the spectral band. The second digit indicates the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on both sides of the center of the spectral band, i.e., from 500 nm to 520 nm.

[0081] Data signals generated in response to light detected by scattered light detection channels 311 and 312, bright-field light detection channel 313, and fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-responsive sorting is performed in response to sorting signals generated by sorting trigger 352. The sorting component 300c includes deflection plates 331 for deflecting particles into the sample container 332 or into the waste stream 333. In some cases, the sorting component 300c is configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, whose disclosure is incorporated herein by reference. In certain embodiments, the sorting component 300c includes a sorting decision module having multiple sorting decision units, such as those described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0082] In some embodiments, the system is a particle analyzer and can analyze and characterize particles using the particle analysis system 401 (Figure 4), whether or not the particles are physically sorted into a collection container. Figure 4 shows a functional block diagram of the particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 includes a fluid system 402. The fluid system 402 includes or can include a sample tube 405 and a moving fluid column in the sample tube in which sample particles 403 (e.g., cells) move along a common sample path 409.

[0083] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting light or one or more other properties of a particle 403 as it passes through the monitoring area 407. Figure 4 shows one detection station 408 with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor two or more areas.

[0084] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be called event data. The data points can be multidimensional data points containing values ​​for each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0085] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuits and / or frequency control circuits. The illustrated control system can be operably associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of a first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during a first time interval. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may further compare the experimental signal frequency with that of a calculated signal frequency or a predetermined signal frequency.

[0086] Figure 5 shows a functional block diagram of an example of a particle analyzer control system, such as an analysis controller (i.e., processor) 500, for analyzing and displaying biological events. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0087] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0088] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. The analysis controller 500 can be configured to provide a display device 506 with a graphical display including a first plot of the biological event data. The analysis controller 500 can be further configured to render regions of interest overlaid on the first plot, for example, as a gate around the collection of biological event data shown by the display device 506. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest depicted in a histogram or bivariate plot of a single parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0089] The analysis controller 500 can be further configured to display biological event data within the gate on the display device 506 in a different manner from other events in the biological event data outside the gate. For example, the analysis controller 500 can be configured to render the colors of the biological event data contained within the gate differently from the colors of the biological event data outside the gate. The display device 506 can be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0090] The analysis controller 500 can be configured to receive gate selection signals from a first input device that identify gates. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate gate selection signals to the analysis controller 500 that identify gates to be displayed on or operated via the display device 506 (for example, by clicking when the cursor is positioned on or inside a desired gate). In some implementations, the first device can be implemented as a keyboard 508, or as other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, photodetector, or voice recognition system. Some input devices can include multiple input functions. In such implementations, each of these input functions can be considered an input device. For example, as shown in Figure 5, the mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0091] The trigger event can cause the analysis controller 500 to change how the data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting a population of interest for particle sorting.

[0092] In some embodiments, the analysis controller 500 can be configured to detect when gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a specific distribution of biological event data received by the analysis controller 500.

[0093] The analysis controller 500 can be connected to the storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to enable the analysis controller 500 to acquire biological event data, such as flow cytometry event data.

[0094] The display device 506 can be configured to receive display data from the analysis controller 500. The display data may include plots of biological event data and gates that outline sections of the plots. The display device 506 can be further configured to change the information presented according to the input received from the analysis controller 500, in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0095] In some implementations, the analysis controller 500 can generate a user interface for receiving exemplary events for sorting. For example, the user interface may include a controller for receiving exemplary events or exemplary images. The exemplary events or images or exemplary gates may be provided before the collection of event data for the sample, or based on an initial set of events for a portion of the sample.

[0096] Figure 6A is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in Figure 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to a nozzle 603, contain a nozzle 603, or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample solution 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are arranged in a line across a monitoring area 611 (e.g., where the laser and the stream intersect) which is irradiated by an irradiation source 612 (e.g., a laser). The vibration of the droplet-forming transducer 602 divides the moving fluid column 608 into multiple droplets 610, some of which contain particles 609.

[0097] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring area 611. The detection station 614 inputs to a timing circuit 628, which then inputs to a flash charge circuit 630. A flash charge can be applied to the moving fluid column 608 so that the droplet of interest becomes charged at the droplet departure point, which is indicated by a timed droplet delay (Δt). The droplet of interest may contain one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a container such as a collection tube or a multi-well or microwell sample plate, where the wells or microwells can be associated with specific droplets of interest. As shown in Figure 6A, the droplets can be collected in a drain receptacle 638.

[0098] The detection system 616 (e.g., a droplet boundary detector) plays a role in automatically determining the phase of the droplet driving signal as particles of interest pass through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 enables the instrument to accurately calculate the location of each detected particle in the droplet. The detection system 616 may supply an amplitude signal 620 and / or a phase signal 618, which are supplied (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 control the droplet formation transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0099] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit, or they can be communicatively coupled so that either the detection system 616 or the detection station 614 can collect event measurements and provide them to non-collecting elements.

[0100] Figure 6B is a schematic diagram of a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in Figure 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream-charging wire in a barb. This creates a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed by sorting electronics or other detection systems (not shown in Figure 6B). The deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, guiding the droplets toward a destination collection receptacle (e.g., one of 672, 674, 676, or 678). As shown in Figure 6B, deflection plates 652 and 654 can be controlled to guide particles toward receptacle 674 along the first path 662 or toward receptacle 678 along the second path 668. If the particles are not of interest (e.g., do not exhibit scattering or illumination information within a specified sorting range), the deflection plates may allow the particles to continue along the flow path 664. Such uncharged droplets may enter the waste receptacle via the aspirator 670 or the like.

[0101] Sorting electronics may be included to initiate measurement data collection, receive fluorescence signals from particles, and determine how to adjust the deflection plates to sort the particles. An exemplary implementation of the embodiment shown in Figure 6B includes the BD FACSAria® line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0102] method In some cases, the samples analyzed in this method are biological samples. The term “biological sample” is used in its conventional sense to refer to an entire organism, an entire plant, an entire fungus, or, in specific cases, a subset of animal tissues, cells, or components that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to both a naturally occurring organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin, respiratory, gastrointestinal, cardiovascular and urogenital tract sections, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of living tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some cases, the sample is a blood sample containing whole blood, such as blood obtained from a venipuncture or fingertip puncture (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0103] In certain embodiments, the source of the sample is “mammal” or “mammalian,” and these terms are broadly used to describe organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs and rats), and Primates (e.g., humans, chimpanzees and monkeys). In some cases, the subject is human. The method may also be applied to samples obtained from human subjects of both sexes and any developmental stage (i.e., neonatal, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is juvenile, adolescent, or adult. While this disclosure may be applied to samples derived from human subjects, it should be understood that the method may also be performed on samples from other animal subjects (i.e., “non-human subjects”), such as, for example, birds, mice, rats, dogs, cats, livestock, and horses, without limitation.

[0104] Cells of interest can be targeted for characterization based on various parameters, such as phenotypic features identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analyzed droplets determined to contain target cells. Various cells can be characterized using this method. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells having favorable cell surface markers or antigens that can be captured or labeled by favorable affinity factors or their conjugates. For example, target cells may contain cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0105] When performing this method, a certain amount of initial fluid sample is injected into a flow cytometer. The amount of sample injected into the particle sorting module may vary, for example, samples in the range of 0.001 mL to 1000 mL, 0.005 mL to 900 mL, 0.01 mL to 800 mL, 0.05 mL to 700 mL, 0.1 mL to 600 mL, 0.5 mL to 500 mL, 1 mL to 400 mL, 2 mL to 300 mL, and 5 mL to 100 mL.

[0106] Methods according to embodiments of the present disclosure include counting labeled particles (e.g., target cells) in a sample and selectively sorting them. When performing the method, a fluid sample containing particles is first introduced into a flow nozzle of the system. Exiting the flow nozzle, the particles pass through the sample interrogation region substantially one at a time, where each particle is irradiated with a light source, and light scattering parameters and, in some cases, fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded separately for each particle. Depending on the characteristics of the interrogated flowstream, the light may be irradiated to a length of 0.001 mm or more, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, and e.g., 1 mm or more of the flowstream. In certain embodiments, the method includes irradiating a planar cross section of the flowstream within the sample interrogation region with a laser (as described above), etc. In other embodiments, the method includes irradiating a sample interrogation region with a predetermined length of flowstream, for example, a length corresponding to the irradiation profile of a diffuse laser beam or lamp.

[0107] In certain embodiments, the method includes irradiating a flow stream at or near the nozzle orifice of the flow cell. For example, the method may include irradiating a flow stream at a position of about 0.001 mm or more from the nozzle orifice, e.g., 0.005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, and e.g., 1 mm or more. In certain embodiments, the method includes irradiating a flow stream immediately adjacent to the nozzle orifice of the flow cell.

[0108] In embodiments of this method, a detector such as a photomultiplier tube (PMT) is used to record the light passing through each particle (called forward scattering in certain cases), the light reflected perpendicular to the direction of particle flow through the detection region (called orthogonal or side scattering in some cases), and, if the particles are labeled with a fluorescent marker, the fluorescence emitted from the particles as they pass through the detection region and are illuminated by an energy source. Each of forward scattering (FSC), side scattering (SSC), and fluorescence emission involves distinct parameters for each particle (or each “event”). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle can, if desired, be analyzed in real time or stored in data storage and analysis means such as a computer.

[0109] In certain embodiments, particles are detected and uniquely identified, as desired, by exposing the particles to excitation light and measuring the fluorescence of each particle in one or more detection channels. The fluorescence emitted in the detection channels used to identify the particles and the associated binding complexes may be measured after excitation by a single light source or separately after excitation by individual light sources. If separate excitation light sources are used to excite particle labels, the labels may be selected so that all labels are excitable by each of the excitation light sources used.

[0110] The method, in certain embodiments, also includes data acquisition, analysis, and recording using a computer or the like, with multiple data channels recording data from each detector about the light scattering and fluorescence emitted by each particle as it passes through the sample interrogation area of ​​the particle sorting module. In these embodiments, the analysis includes sorting and counting the particles so that each particle is presented as a set of digitized parameter values. The system may be set up as a trigger for a selected parameter to distinguish the particle of interest from background and noise. A “trigger” refers to a preset threshold for detecting the parameter and may be used as a means to detect the passage of a particle through a light source. Detection of an event exceeding the threshold of the selected parameter triggers the acquisition of light scattering and fluorescence data for the particle. For particles or other components in the assayed medium that cause a response below the threshold, no data is acquired. The trigger parameter may be the detection of forward scattered light caused by the passage of a particle through a light beam. The flow cytometer then detects and collects the light scattering and fluorescence data for the particle.

[0111] Next, a specific subpopulation of interest is further analyzed by “gating” based on data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible subpopulation separation. This procedure can be performed by plotting forward light scattering (FSC) versus side (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles (i.e., their cells in the gate) is selected, and particles not in the gate are excluded. If desired, the gate may also be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Then, only those particles in the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to yield a count of the particles of interest in the sample.

[0112] The methods of interest may further include the use of particles in research, laboratory testing, or therapy. In some embodiments, the methods include obtaining individual cells prepared from a biological sample of a target fluid or tissue. For example, the methods include obtaining cells from a fluid or tissue sample used as a research or diagnostic specimen for a disease such as cancer. Similarly, the methods include obtaining cells from a fluid or tissue sample used in therapy. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissues, is prepared and can be introduced into a subject as a therapeutic procedure. Conditions that can be treated by administration of flow cytometry-sorted samples include, but are not limited to, blood disorders, immune system disorders, and organ damage.

[0113] A typical cell therapy protocol may include the following steps: sample collection, cell isolation, genetic modification, culture and in vitro growth, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of cells into the subject. The protocol may begin with collecting viable cells and tissues from the subject's source tissue to generate cell and / or tissue samples. Samples may be collected by any appropriate procedure, including, for example, administering a cell recruiter to the subject, drawing blood from the subject, or extracting bone marrow from the subject. After sample collection, cell enrichment may be performed by several methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, and fluorescence-activated cell sorting (FACS). In some cases, enriched cells may be genetically modified by any convenient method, for example, nuclease-mediated gene editing. Genetically modified cells can be cultured, activated, and grown in vitro. In some cases, cells are preserved, for example, by cryopreservation, and stored for future use, where they can be thawed and administered to a patient, for example, by injection.

[0114] Computer control system The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access memory in which instructions for performing the steps of the method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, as well as input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or one of other processors that are available or will be available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages ​​known in the art, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof. The operating system typically works with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0115] System memory may be any of the various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as permanent hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices may be any of the various known or future devices, including compact disk drives, tape drives, or diskette drives. Such types of memory storage devices typically read from and / or write to program storage media such as compact disks (not shown). Any of these program storage media, or others currently in use or to be developed in the future, may be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in program storage devices used in conjunction with system memory and / or memory storage devices.

[0116] In some embodiments, a computer program product is described that includes a computer-usable medium on which control logic (a computer software program including program code) is stored. When the control logic is executed by the computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of a hardware state machine for performing the functions described herein will be obvious to those skilled in the art.

[0117] Memory may be any suitable device on which the processor can store and retrieve data, such as a magnetic storage device, an optical storage device, or a solid-state storage device (including magnetic or optical disks, or tapes or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium having the necessary program code. The programming may be provided to the processor remotely via a communication channel, or it may be pre-stored in a computer program product, such as any other portable or fixed computer-readable storage medium using either memory or a device connected to memory. For example, a magnetic or optical disk may have programming which can be read by a disk writer / reader. The system of this disclosure also includes programming in the form of a computer program product, algorithms for use in carrying out the methods described above. The programming according to this disclosure may be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0118] The processor can also access communication channels to communicate with users in remote locations. Remote locations mean that the user is not in direct contact with the system, but rather relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0119] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0120] In one embodiment, the communication interface is configured to include one or more physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port that enables data communication between the system and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0121] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol that enables the system to communicate with other devices such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0122] In one embodiment, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP), Short Message Service (SMS), wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0123] In one embodiment, the system is configured to communicate wirelessly with a server device via a communication interface using common standards such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or electrical appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD), as well as input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0124] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device using one or more of the communication protocols and / or mechanisms described above, for example, data stored in the system, for example, an optional data storage unit.

[0125] The output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a remote user, for example, via the internet, telephone or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML or XML documents, email or other files, or data in other formats. The data may also include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present in this system may be any type of known computer platform or a type to be developed in the future, but they are typically computers of a class commonly referred to as servers. However, they may also be mainframe computers, workstations, or other types of computers. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, and may or may not be networked. They may be located in the same place or physically separated. Various operating systems may be used on any computer platform, depending on the type and / or manufacturer of the computer platform selected.Suitable operating systems include Windows® NT®, Windows® XP, Windows® 7, Windows® 8, Windows® 10, iOS®, macOS®, Linux®, Ubuntu®, Fedora®, OS / 400®, i5 / OS®, IBM i®, Android®, SGI IRIX®, Oracle Solaris®, and others.

[0126] Figure 7 shows a general architecture of an exemplary computing device 700 according to a particular embodiment. The general architecture of the computing device 700 shown in Figure 7 includes the arrangement of computer hardware and software components. However, not all of these generally conventional elements need to be illustrated in order to provide a usable disclosure. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via the network. The processing unit 710 may also communicate with memory 770 and further provide output information to an optional display 750 via the input / output device interface 740. For example, analysis software (such as data analysis software or programs like FlowJo®) stored as executable instructions in the non-temporary memory of the analysis system can display flow cytometry event data to the user. The input / output device interface 740 may also accept input from an optional input device 760, such as a keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, speech recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0127] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-temporary computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Data may be stored in a data storage device 790. Memory 770 may further include computer program instructions and other information for implementing embodiments of the present disclosure.

[0128] usefulness Embodiments of this disclosure are used in applications where cells prepared from biological samples may be desired for use in research, laboratory testing, or therapeutic settings. In some embodiments, the methods and devices may facilitate obtaining and / or analyzing individual cells prepared from biological samples of a target fluid or tissue. For example, the methods and systems may facilitate obtaining cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods and systems may facilitate obtaining cells from fluid or tissue samples used in therapeutic settings.

[0129] The following are provided as examples, not as limitations.

[0130] experiment Loader performance and optimal settings for high-throughput data generation using BD FACSDiscover® A8 Cell Analyzer A. Summary The variables presented during data acquisition using automated loaders can be complex and, despite their good design, can disrupt experiments. Factors such as cell concentration, resuspension volume, acquisition criteria, mixing frequency, and duration can all play a significant role in determining the relative success of an experiment. Using a BD FACSDiscover® A8 Cell Analyzer incorporating an automated pneumatic loader (e.g., one described above) and a dual-speed function (e.g., as described in U.S. Patent Application No. 19 / 076,246 filed March 11, 2025, the disclosure of which is incorporated herein by reference), the inventors present a systematic approach to optimizing loader performance to achieve consistent, high-quality data. Mixing efficiency is shown as a function of carrier type, cell concentration, sample volume, agitation, and agitation frequency. The output metric for mixing efficiency is based on the number of events acquired in a given volume. Careful tracking of these metrics facilitates the establishment of setting ranges for optimizing loader performance. Furthermore, the inventors present data for evaluating the effects of carrier agitation on cell viability and fluorescence stability. Other confounding issues, such as acquisition criteria for the minimum number of events required to achieve statistically robust results in the context of immunophenotyping applications, were investigated. Based on these data, the inventors present a set of guidelines for achieving maximum throughput without sacrificing data quality. Furthermore, strategies for evaluating throughput, carryover, and dead volume are presented.

[0131] B. Method A series of experiments designed to evaluate the loader integrated into the FACSDiscover® A8 Cell Analyzer were performed. These experiments were designed to identify the performance limits of the loader in the context of mixing efficiency. Mixing efficiency experiments were performed using both cell lines (Jurkat T cells) and peripheral blood mononuclear cells (PBMCs). The consistency of the number of cells obtained in a constant acquisition volume from a sample with a constant cell concentration was used as a measure of relative mixing efficiency. Deviations from the default agitation settings were investigated to determine the effectiveness of these settings and the impact of deviations. Carryover and throughput experiments were performed with default settings in both imaging mode and fast mode (carryover data for fast mode are not shown). Similar experimental sets were performed using other supported (corresponding) carriers, such as 40-tube racks and deep-bottom 96-well plate formats. The setup is shown in Figure 8.

[0132] C. Results 1. Mixing efficiency a. Effect of cell concentration on mixing efficiency: The number of cells obtained in a 25 mL acquisition volume was plotted for each well in a 96-well plate. Cell concentrations ranging from 500,000 cells / mL to 10,000,000 cells / mL were tested. In all cases, the default loader settings were used, and orbital mixing occurring every 4 wells was employed at 1400 rpm for 5 seconds. The coefficient of variation (%CV) was calculated for each condition. Data points shown in red are those outside the range defined as 2 × standard deviation above or below the mean. The results are shown in Figure 9A.

[0133] b. Effect of well volume on mixing efficiency: The number of cells obtained in a 25 mL acquisition volume was plotted for each well in a 96-well plate. Well volumes ranging from 50 mL to 200 mL were tested. In all cases, the default loader settings were used, and orbital mixing occurring every 4 wells was employed at 1400 rpm for 5 seconds. The coefficient of variation (%CV) was calculated for each condition. Data points shown in red are those outside the range defined as 2 × standard deviation above or below the mean. The results are shown in Figure 9B.

[0134] c. Effect of stirring intensity on mixing efficiency: The number of cells obtained in a 25 mL acquisition volume was plotted for each well in a 96-well plate. Stirring intensities ranging from 500 RPM to 1400 RPM were tested. In all cases, the default loader settings were used, and orbital mixing occurring every 4 wells in 5 seconds was employed. The coefficient of variation (%CV) was calculated for each condition. The results are shown in Figure 9C.

[0135] d. Effect of stirring frequency on mixing efficiency: The number of cells obtained in a 25 mL acquisition volume was plotted for each well in a 96-well plate. Stirring frequencies ranging from every 2 wells to every 12 wells were tested. In all cases, the default loader settings were used, and orbital mixing occurring over 5 seconds at 1400 rpm was employed. The coefficient of variation (%CV) was calculated for each condition. The results are shown in Figure 9D.

[0136] e. Effect of default agitation settings on viability: Using a well volume of 200 mL and default agitation settings (1400 RPM, 5 seconds every 4 wells), the viability of both Jurkat (upper plot) and PBMC (lower plot) was measured for each of the 96 wells throughout the plate acquisition process. Cell viability was measured by 7-AAD staining. For reference, an aliquot of Jurkat or PBMC stored at 4°C during plate acquisition was measured as a no-agitation control. The viability of the no-agitation control was tested before and after plate processing. The results are shown in Figure 9E.

[0137] f. Effect of default stirring settings on fluorescence: Using a 200 mL well volume and default stirring settings (1400 RPM, 5 seconds every 4 wells), the effect of stirring on measured fluorescence was investigated using a 7-color TBNK panel. The upper plot shows the median fluorescence intensity (MFI) of several key markers in the panel, and the lower plot shows the relative frequency of populations that can be quantified using the panel. The results are shown in Figure 9F.

[0138] 2. Carryover and Throughput a. A8 ​​Carryover: PBMC (2A) and HT-29 cells (2B). Carryover was tested using a standard protocol with 10 million cells / mL loaded into one well of a 96-well plate (left plot is 2APBMC, right plot is 2B-HT-29, an extremely clumpy cell line). Following the highly concentrated well were four blank wells loaded with PBS only. The number of cells remaining in the system in the blank wells was then measured as a “carryover” event. This was done in both fast mode (data not shown) and imaging mode, with similar results. To establish background noise in PBS, the PBS blank wells were acquired before the first well. The bar graph in the lower left of each figure plots the mean carryover events in 10 replicates (5 wells total) of a series of carryovers. The results are shown in Figures 10A and 10B.

[0139] b. Throughput of FACSDiscover® A8 Loader: Plate processing time was measured for each fluid mode using a 200 μL well volume and a cell concentration of 1 million cells / mL at the default agitation rate and frequency. The high-speed mode (102 ml / min) throughput time is shown for a standard 96-well plate (green plot) with and without SIT flush (right plot) (left plot). The acquisition processing time for 2 mL, 5 mL, 10 mL, 25 mL and 50 mL is shown. The same measurements were performed in imaging mode (30 mL / min) (blue bar graph on the right). The results are shown in Figure 10C.

[0140] conclusion The loader integrated into the BD FACSDiscover® A8 Cell Analyzer features customizable characteristics, including agitation frequency, duration, and intensity (RPM), that allow users to achieve maximum sample mixing efficiency during plate acquisition. These settings should be optimized based on user needs. However, the instrument comes with default settings that have been tested to yield optimal results. The purpose of this study was to demonstrate how these default values ​​were determined and to provide some guidance on the potential impact of deviations from these settings on the user workflow. The inventors found that the default settings produced the most reliable data, and in most cases, a significant deviation was required to affect the results. Furthermore, the inventors investigated loader performance in the context of carryover and throughput in both imaging and high-speed modes.

[0141] Notwithstanding the attached claims, this disclosure is also defined by the following clauses:

[0142] 1. Flow cell and, A pneumatically driven automatic sample loader configured to automatically acquire a sample from a sample container and transport the sample to a flow cell, A flow cytometer equipped with the following features.

[0143] 2. The pneumatically driven automatic sample loader, Sample container receiving region, A sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving region, A loader door configured to regulate access to the sample container receiving area and Equipped with, A flow cytometer as described in Clause 1, wherein the operation of the SIT assembly and the loader door is pneumatically driven by a pneumatic assembly.

[0144] 3. The pneumatic assembly is A pneumatic pump that provides positive pressure to the first air pressure line, A first switch is configured to fluidly communicate with a pneumatic pump via a first pneumatic line, to guide positive pressure to the second and third lines so that the third pneumatic line is not pressurized when positive pressure is applied to the second pneumatic line, and to guide the positive pressure to the second and third lines so that the second pneumatic line is not pressurized when positive pressure is applied to the third pneumatic line. A first pneumatic cylinder is mechanically connected to the SIT assembly, having fluid communication with second and third pneumatic lines, such that pressurization of the second pneumatic line moves the SIT assembly to the sampling position and pressurization of the third pneumatic line moves the SIT assembly to the resting position. A second switch is configured to fluidly communicate with a pneumatic pump via a first pneumatic line, to guide positive pressure to the fourth and fifth lines so that the fifth pneumatic line is not pressurized when positive pressure is applied to the fourth pneumatic line, and to guide the positive pressure to the fourth and fifth lines so that the fourth pneumatic line is not pressurized when positive pressure is applied to the fifth pneumatic line. A second pneumatic cylinder is mechanically connected to the loader door, and is in fluid communication with the fourth and fifth pneumatic lines, such that pressurization of the fourth pneumatic line moves the loader door to the closed position and pressurization of the fifth pneumatic line moves the loader door to the open position. A flow cytometer as described in Clause 2, comprising the features described in Clause 2.

[0145] 4. The pneumatic assembly, A third switch is configured to fluidly communicate with a pneumatic pump via a first pneumatic line, to guide positive pressure to the sixth and seventh lines so that the seventh pneumatic line is not pressurized when positive pressure is applied to the sixth pneumatic line, and to guide the positive pressure to the sixth and seventh lines so that the sixth pneumatic line is not pressurized when positive pressure is applied to the seventh pneumatic line. A third pneumatic cylinder is mechanically connected to a sample line subassembly that has fluid communication with the sixth and seventh pneumatic lines, and the sample line has fluid communication with the flow cell such that pressurization of the sixth pneumatic line moves the sample line subassembly to the loading position and inserts the sample line into the sample container, and pressurization of the seventh pneumatic line moves the sample line subassembly to the retracted position. A flow cytometer as described in Clause 3, comprising the features described in Clause 3.

[0146] 5. A flow cytometer as described in Clause 3 or 4, wherein the operation of the SIT assembly, loader door and / or sample line subassembly is not driven by a stepping motor and worm gear.

[0147] 6. A flow cytometer as described in any one of Clauses 3 to 5, wherein the operation of the SIT assembly, loader door, and / or sample line subassembly does not require a separate circuit board.

[0148] 7. A flow cytometer according to any one of clauses 3 to 6, wherein the pneumatic pump, the first, second and third pneumatic cylinders, and the first, second and third switches are operated on a single circuit board.

[0149] 8. A flow cytometer as described in any one of clauses 3 to 7, comprising a pneumatic pump, first, second and third pneumatic cylinders, and first, second and third switches, which does not require firmware.

[0150] 9. A flow cytometer according to any one of the first, second, and third switches, wherein one or more switches are equipped with a flow regulator for adjusting pressure equalization in an unpressurized pneumatic line.

[0151] 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 a pneumatic pump and one or more of the first, second, and third switches, the pneumatic pump pressurizes the pressure reservoir, and the pressure reservoir provides positive pressure to a first pneumatic line.

[0152] 11. A flow cytometer according to any one of clauses 3 to 10, wherein the pneumatic assembly is provided with connectors on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines so that the pneumatic lines can be separated and reconnected.

[0153] 12. A flow cytometer as described in any one of Clauses 3 to 11, wherein the pneumatic assembly further comprises a mounting bracket on which a pneumatic pump, a pressure reservoir, a switch, and a connector are mounted.

[0154] 13. A flow cytometer according to any one of clauses 3 to 12, wherein the pneumatic assembly further comprises a pressure reservoir and a pressure gauge for measuring the pressure of one or more of the second, third, fourth, fifth, sixth and seventh pneumatic lines.

[0155] 14. A flow cytometer according to Clause 13, wherein when the pressure measured by the pressure gauge falls below a threshold pressure, the pressure reservoir and one or more of the second, third, fourth, fifth, sixth, and seventh pneumatic lines are repressurized.

[0156] 15. A flow cytometer as described in Clause 14, wherein the threshold pressure is in the range of 25 psi to 50 psi.

[0157] 16. A flow cytometer as described in any one of clauses 2 to 15, wherein the loader door protects the sample from ambient light by reducing the transmission of ambient light.

[0158] 17. A flow cytometer as described in Clause 16, wherein the loader door is translucent.

[0159] 18. A flow cytometer as described in any one of clauses 2 to 17, wherein the SIT assembly is located on an XY movable stage.

[0160] 19. A flow cytometer according to any one of Clauses 1 to 18, further comprising a calibration plate, a chassis, and a manual tube port, wherein the calibration plate and chassis are configured to calibrate the height of the manual tube port to minimize dead volume of the sample when transitioning between acquiring a sample from a sample container in a pneumatically driven automatic sample loader and acquiring a sample from a manual tube loading position.

[0161] 20. A flow cytometer according to any one of the clauses 1 to 19, comprising a platform having a sample container receiving area configured to hold one or more samples.

[0162] 21. A flow cytometer as described in Clause 20, wherein the platform is configured to mix, heat and / or cool one or more samples.

[0163] 22. A flow cytometer according to any one of the clauses 1 to 21, further comprising a light source configured to irradiate a flow cell at an interrogation point.

[0164] 23. A flow cytometer according to any one of clauses 1 to 22, further comprising a detector configured to collect particle-modulated light from a flow cell.

[0165] 24. A flow cytometer as described in any one of clauses 1 to 23, wherein the flow cytometer is a particle analyzer.

[0166] 25. A flow cytometer as described in any one of clauses 1 to 24, wherein the flow cytometer is a particle separator.

[0167] 26. A flow cytometer as described in any one of clauses 1 to 25, wherein the flow cytometer is an imaging flow cytometer.

[0168] 27. A method for flow cytometry analysis of a sample using a flow cytometer, (a) A step of introducing a sample container containing a sample into a pneumatically driven automatic sample loader of a flow cytometer, wherein the pneumatically driven automatic sample loader is configured to automatically retrieve the sample from the sample container so that the sample is transported to the flow cell of the flow cytometer. (b) Steps to perform flow cytometry analysis on the sample and Methods that include...

[0169] 28. The pneumatically driven automatic sample loader, Sample container receiving region, A sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving region, A loader door configured to regulate access to the sample container receiving area and Equipped with, The method according to Clause 27, wherein the operation of the SIT assembly and the loader door is pneumatically driven by a pneumatic assembly.

[0170] 29. The pneumatic assembly, A pneumatic pump that provides positive pressure to the first air pressure line, A first switch is configured to fluidly communicate with a pneumatic pump via a first pneumatic line, to guide positive pressure to the second and third lines so that the third pneumatic line is not pressurized when positive pressure is applied to the second pneumatic line, and to guide the positive pressure to the second and third lines so that the second pneumatic line is not pressurized when positive pressure is applied to the third pneumatic line. A first pneumatic cylinder is mechanically connected to the SIT assembly, having fluid communication with second and third pneumatic lines, such that pressurization of the second pneumatic line moves the SIT assembly to the sampling position and pressurization of the third pneumatic line moves the SIT assembly to the resting position. A second switch is configured to fluidly communicate with a pneumatic pump via a first pneumatic line, to guide positive pressure to the fourth and fifth lines so that the fifth pneumatic line is not pressurized when positive pressure is applied to the fourth pneumatic line, and to guide the positive pressure to the fourth and fifth lines so that the fourth pneumatic line is not pressurized when positive pressure is applied to the fifth pneumatic line. A second pneumatic cylinder is mechanically connected to the loader door, and is in fluid communication with the fourth and fifth pneumatic lines, such that pressurization of the fourth pneumatic line moves the loader door to the closed position and pressurization of the fifth pneumatic line moves the loader door to the open position. The method described in Clause 28, comprising:

[0171] 30. The pneumatic assembly, A third switch is configured to fluidly communicate with a pneumatic pump via a first pneumatic line, to guide positive pressure to the sixth and seventh lines so that the seventh pneumatic line is not pressurized when positive pressure is applied to the sixth pneumatic line, and to guide the positive pressure to the sixth and seventh lines so that the sixth pneumatic line is not pressurized when positive pressure is applied to the seventh pneumatic line. A third pneumatic cylinder is mechanically connected to a sample line subassembly that has fluid communication with the sixth and seventh pneumatic lines, and the sample line has fluid communication with the flow cell such that pressurization of the sixth pneumatic line moves the sample line subassembly to the loading position and inserts the sample line into the sample container, and pressurization of the seventh pneumatic line moves the sample line subassembly to the retracted position. The method described in Clause 29, comprising:

[0172] 31. The method according to clause 29 or 30, wherein the operation of the SIT assembly, loader door and / or sample line subassembly is not driven by a stepping motor and worm gear.

[0173] 32. The method according to any one of the clauses 29 to 31, wherein the operation of the SIT assembly, loader door and / or sample line subassembly does not require a separate circuit board.

[0174] 33. The method according to any one of the clauses 29 to 32, wherein the pneumatic pump, the first, second and third pneumatic cylinders, and the first, second and third switches are operated from a single circuit board.

[0175] 34. The method described in any one of the clauses 29 to 33, wherein the pneumatic pump, the first, second and third pneumatic cylinders, and the first, second and third switches do not require firmware.

[0176] 35. The method according to any one of the first, second, and third switches, wherein one or more switches are equipped with flow regulators for adjusting pressure equalization in an unpressurized pneumatic line.

[0177] 36. The method according to any one of the clauses 29 to 35, wherein the pneumatic assembly further comprises a pressure reservoir in fluid communication with a pneumatic pump and one or more of the first, second, and third switches, the pneumatic pump pressurizes the pressure reservoir, and the pressure reservoir provides positive pressure to a first pneumatic line.

[0178] 37. The method according to any one of the clauses 29 to 36, wherein the pneumatic assembly is provided with connectors on one or more of the first, second, third, fourth, fifth, sixth and seventh pneumatic lines so that the pneumatic lines can be separated and reconnected.

[0179] 38. The method according to any one of the clauses 29 to 37, wherein the pneumatic assembly further comprises a mounting bracket on which a pneumatic pump, a pressure reservoir, a switch, and a connector are mounted.

[0180] 39. The method according to any one of the clauses 29 to 38, wherein the pneumatic assembly further comprises a pressure reservoir and a pressure gauge for measuring the pressure of one or more of the second, third, fourth, fifth, sixth and seventh pneumatic lines.

[0181] 40. The method according to Clause 39, wherein when the pressure measured by the pressure gauge falls below a threshold pressure, the pressure reservoir and one or more of the second, third, fourth, fifth, sixth and seventh pneumatic lines are repressurized.

[0182] 41. The method according to clause 40, wherein the threshold pressure is in the range of 25 psi to 50 psi.

[0183] 42. The method according to any one of the clauses 28 to 41, further comprising the step of operating the opening of the loader door.

[0184] 43. The method according to any one of the clauses 28 to 42, wherein the loader door protects the sample from ambient light by reducing the transmission of ambient light.

[0185] 44. The method according to Clause 43, wherein the loader door is semi-transparent.

[0186] 45. The method described in any one of the clauses 28 to 44, wherein the SIT assembly is located on an XY movable stage.

[0187] 46. ​​The method according to any one of the clauses 27 to 45, further comprising a calibration plate, a chassis, and a manual tube port, wherein the calibration plate and chassis are configured to calibrate the height of the manual tube port to minimize dead volume of the sample when transitioning between taking a sample from a sample container in a pneumatically driven automatic sample loader and taking a sample from a manual tube loading position.

[0188] 47. The method according to any one of the clauses 27 to 46, wherein the sample container receiving area comprises a platform configured to hold one or more samples.

[0189] 48. The method according to Clause 47, wherein the platform is configured to mix, heat and / or cool one or more samples.

[0190] 49. The method according to clause 47 or 48, wherein the step of introducing a particulate sample includes placing a multiwell plate or a rack of sample tubes on a platform.

[0191] 50. The method according to any one of the clauses 28 to 49, further comprising the step of activating the closing of the loader door.

[0192] 51. The method according to any one of the clauses 27 to 50, wherein the flow cytometer further comprises a light source configured to irradiate a flow cell at an interrogation point.

[0193] 52. The method according to any one of the clauses 27 to 51, wherein the flow cytometer further comprises a detector configured to collect particle-modulated light from a flow cell.

[0194] 53. The method described in any one of clauses 27 to 52, wherein the flow cytometer is a particle analyzer.

[0195] 54. The method according to any one of the clauses 27 to 53, wherein the flow cytometer is a particle separator.

[0196] 55. The method according to any one of the clauses 27 to 54, wherein the flow cytometer is an imaging flow cytometer.

[0197] Therefore, the foregoing is merely illustrative of the principles of the present disclosure. Those skilled in the art will understand that various configurations embodying the principles of the present disclosure and that fall within its spirit and scope can be devised, although not expressly described or illustrated herein. Furthermore, all examples and conditional statements described herein are intended primarily to help the reader understand the principles of the present disclosure and the concepts to which the inventors have contributed to advancing the art, and should be interpreted as not being limited to such specifically described examples and conditions. Furthermore, all descriptions herein listing the principles, aspects and embodiments of the present disclosure, and specific examples thereof, are intended to encompass both structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any developed elements that perform the same function regardless of their structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether such disclosure is expressly described in the claims or not.

[0198] Accordingly, the scope of this disclosure is not intended to be limited to the exemplary embodiments illustrated and described herein. Rather, the scope and spirit of this disclosure are embodied in the appended claims. In the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) are expressly defined as applying to the limitation in the claims only if the exact phrase “means” or the exact phrase “step” is stated at the beginning of such limitation in the claims, and if such exact phrase is not used in the limitation in the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) does not apply.

Claims

1. Flow cell and, A pneumatically driven automatic sample loader configured to automatically acquire a sample from a sample container and transport the sample to the flow cell, A flow cytometer equipped with the following features.

2. The aforementioned pneumatically driven automatic sample loader, Sample container receiving region, A sample injection tube (SIT) assembly configured to introduce a sample line into a sample container located in the sample container receiving region, A loader door configured to regulate access to the sample container receiving area, Equipped with, The flow cytometer according to claim 1, wherein the operation of the SIT assembly and the loader door is pneumatically driven by a pneumatic assembly.

3. The aforementioned pneumatic assembly A pneumatic pump that provides positive pressure to the first air pressure line, A first switch is configured to communicate fluidly with the pneumatic pump via the first pneumatic line, to guide the positive pressure to the second and third lines so that the third pneumatic line is not pressurized when the positive pressure is applied to the second pneumatic line, and to guide the positive pressure to the second and third lines so that the second pneumatic line is not pressurized when the positive pressure is applied to the third pneumatic line, A first pneumatic cylinder is mechanically connected to the SIT assembly, having fluid communication with the second and third pneumatic lines, such that pressurization of the second pneumatic line moves the SIT assembly to the sampling position and pressurization of the third pneumatic line moves the SIT assembly to the resting position. A second switch is configured to fluidly communicate with the pneumatic pump via the first pneumatic line, to guide the positive pressure to the fourth and fifth lines so that the fifth pneumatic line is not pressurized when the positive pressure is applied to the fourth pneumatic line, and to guide the positive pressure to the fourth and fifth lines so that the fourth pneumatic line is not pressurized when the positive pressure is applied to the fifth pneumatic line, A second pneumatic cylinder is mechanically connected to the loader door, and is in fluid communication with the fourth and fifth pneumatic lines, such that pressurization of the fourth pneumatic line moves the loader door to the closed position and pressurization of the fifth pneumatic line moves the loader door to the open position. A flow cytometer according to claim 2, comprising:

4. The aforementioned pneumatic assembly A third switch is configured to fluidly communicate with the pneumatic pump via the first pneumatic line, to guide the positive pressure to the sixth and seventh lines so that the seventh pneumatic line is not pressurized when the positive pressure is applied to the sixth pneumatic line, and to guide the positive pressure to the sixth and seventh lines so that the sixth pneumatic line is not pressurized when the positive pressure is applied to the seventh pneumatic line. A third pneumatic cylinder is mechanically connected to the sample line subassembly, which has fluid communication with the sixth and seventh pneumatic lines, and the sample line is fluid-communicated with the flow cell such that pressurization of the sixth pneumatic line moves the sample line subassembly to the loading position and inserts the sample line into the sample container, and pressurization of the seventh pneumatic line moves the sample line subassembly to the retracted position. A flow cytometer according to claim 3, comprising:

5. The flow cytometer according to claim 3 or 4, wherein the operation of the SIT assembly, the loader door, and / or the sample line subassembly is not driven by a stepping motor and a worm gear.

6. The flow cytometer according to any one of claims 3 to 5, wherein the operation of the SIT assembly, the loader door, and / or the sample line 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, second and third pneumatic cylinders, and the first, second and third switches 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, second and third pneumatic cylinders, and the first, second and third switches do not require firmware.

9. The flow cytometer according to any one of claims 3 to 8, wherein one or more of the first, second, and third switches are equipped with a flow regulator for adjusting the pressure equalization in an unpressurized air line.

10. The flow cytometer according to any one of claims 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, second, and third switches, 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 is provided with connectors on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines so that the pneumatic lines can be separated and reconnected.

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

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 of 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 loading door protects the sample from ambient light by reducing the transmission of ambient light.

15. A method for flow cytometry analysis of a sample using a flow cytometer, (a) A step of introducing a sample container containing the sample into a pneumatically driven automatic sample loader of the flow cytometer, wherein the pneumatically driven automatic sample loader is configured to automatically acquire the sample from the sample container so that the sample is transported to the flow cell of the flow cytometer; (b) The step of performing flow cytometry analysis on the sample Methods that include...

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