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EP-4741799-A1 - FLOW CYTOMETER HAVING A PNEUMATICALLY DRIVEN SAMPLE AUTOLOADER, AND METHODS OF USING THE SAME

EP4741799A1EP 4741799 A1EP4741799 A1EP 4741799A1EP-4741799-A1

Abstract

Aspects of the present disclosure include flow cytometers having a pneumatically driven sample autoloader. Flow cytometers according to certain embodiments include a flow cell; and a pneumatically driven sample autoloader configured to automatically obtain a sample from a sample container and convey the sample to the flow cell. Methods of cytometrically processing samples, e.g., in analysis and/or sorting applications, are also provided.

Inventors

  • Lankila, Henry J
  • MANZARRAGA, JORGE
  • FUNDERBURK, JEANNINE K.

Assignees

  • BECTON, DICKINSON AND COMPANY

Dates

Publication Date
20260513
Application Date
20251110

Claims (15)

  1. A flow cytometer comprising: a flow cell; and a pneumatically driven sample autoloader configured to automatically obtain a sample from a sample container and convey the sample to the flow cell.
  2. The flow cytometer according to Claim 1, wherein the pneumatically driven sample autoloader comprises: a sample container receiving region; a sample injection tube (SIT) assembly configured to introduce a sample line into a sample container present in the sample container region; and a loader door configured to modulate access to the sample container receiving region; wherein actuation of the SIT assembly and the loader door is pneumatically driven by a pneumatic assembly.
  3. The flow cytometer according to Claim 2, wherein the pneumatic assembly comprises: a pneumatic pump to provide a positive pressure to a first pneumatic line; a first switch in fluid communication with the pneumatic pump via the first pneumatic line and configured to direct the positive pressure to a second line and a third line, such that when the positive pressure is applied to the second pneumatic line, the third pneumatic line is not pressurized, and vice versa; a first pneumatic cylinder in fluid communication with the second and third pneumatic lines and mechanically linked to the SIT assembly, such that pressurization to the second pneumatic line moves the SIT assembly to a sampling position and pressurization to the third pneumatic line moves the SIT assembly to a rest position; a second switch in fluid communication with the pneumatic pump via the first pneumatic line and configured to direct the positive pressure to a fourth line and a fifth line, such that when the positive pressure is applied to the fourth pneumatic line, the fifth pneumatic line is not pressurized, and vice versa; and a second pneumatic cylinder in fluid communication with the fourth and fifth pneumatic lines and mechanically linked to the loader door, such that pressurization to the fourth pneumatic line moves the loader door to a closed position and pressurization to the third pneumatic line moves the loader door to an open position.
  4. The flow cytometer according to Claim 3, wherein the pneumatic assembly further comprises: a third switch in fluid communication with the pneumatic pump via the first pneumatic line and configured to direct the positive pressure to a sixth line and a seventh line, such that when the positive pressure is applied to the sixty pneumatic line, the seventh pneumatic line is not pressurized, and vice versa; and a third pneumatic cylinder in fluid communication with the sixth and seventh pneumatic lines and mechanically linked to a sample line subassembly comprising a sample line in fluid communication to the flow cell, such that pressurization to the sixth pneumatic line moves the sample line subassembly into a loading position such that sample line is inserted into a sample container and pressurization to the seventh pneumatic line moves the sample line subassembly to a retracted position.
  5. The flow cytometer according to Claim 3 or 4, wherein the actuation of the SIT assembly, the loader door, and/or sample line subassembly are not driven by a stepper motor and worm gear.
  6. The flow cytometer according to any of Claims 3-5, wherein the actuation of the SIT assembly, the loader door, and/or sample line subassembly do not require separate circuit boards.
  7. The flow cytometer according to any of Claims 3-6, wherein the pneumatic pump, first, second, and third pneumatic cylinders, and first, second, and third switches operate from a single circuit board.
  8. The flow cytometer according to any of Claims 3-7, wherein the pneumatic pump, first, second, and third pneumatic cylinders, and first, second, and third switches do not require firmware.
  9. The flow cytometer according to any of Claims 3-8, wherein one or more of the first, second, and third switches comprise a flow regulator to modulate pressure equalization in a non-pressurized pneumatic line.
  10. The flow cytometer according to any of Claims 3-9, 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, where 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 of Claims 3-10, wherein the pneumatic assembly comprises a connector on one or more of the first, second, third, fourth, fifth, sixth, and seventh pneumatic lines, such that the pneumatic line may be decoupled and recoupled.
  12. The flow cytometer according to any of Claims 3-11, wherein the pneumatic assembly further comprises a mounting bracket upon which the pneumatic pump, the pressure reservoir, the switches, and the connector are mounted.
  13. The flow cytometer according to any of Claims 3-12, the pneumatic assembly further comprises an manometer to measure pressure within one or more of the pressure reservoir and the second, third, fourth, fifth, sixth, and seventh pneumatic lines.
  14. The flow cytometer according to any of Claims 2-13, wherein the loader door protects a sample from ambient light by reducing transmission of the ambient light.
  15. A method of flow cytometrically analyzing a sample with a flow cytometer, the method comprising: (a) introducing a sample container comprising the sample into a pneumatically driven sample autoloader of the flow cytometer, wherein the pneumatically driven sample autoloader is configured to automatically obtain a sample from the sample container such that the sample is conveyed to a flow cell of the flow cytometer; and (b) flow cytometrically analyzing the sample.

Description

CROSS-REFERENCE TO RELATED APPLICATION Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63/813,208 filed May 28, 2025 and to United States Provisional Application Serial No. 63/718,455 filed on November 8, 2024; the disclosures of which are herein incorporated by reference. INTRODUCTION The characterization of analytes in biological fluids has become an important part of biological research, medical diagnoses and assessments of overall health and wellness of a patient. Detecting analytes in biological fluids, such as human blood or blood derived products, can provide results that may play a role in determining a treatment protocol of a patient having a variety of disease conditions. Flow cytometry is a technique used to characterize and often times sort biological material, such as cells of a blood sample or particles of interest in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transports the particles (including cells) in the fluid sample as a cell stream to a flow cell, while also directing the sheath fluid to the flow cell. To characterize the components of the flow stream, the flow stream is irradiated with light. Variations in the materials in the flow stream, such as morphologies or the presence of fluorescent labels, may cause variations in the observed light and these variations allow for characterization and separation. To characterize the components in the flow stream, light must impinge on the flow stream and be collected. Light sources in flow cytometers can vary and may include one or more broad spectrum lamps, light emitting diodes as well as single wavelength lasers. The light source is aligned with the flow stream and an optical response from the illuminated particles is collected and quantified. Isolation of biological particles has been achieved by adding a sorting or collection capability to flow cytometers. Particles in a segregated stream, detected as having one or more desired characteristics, are individually isolated from the sample stream by mechanical or electrical removal. A common flow sorting technique utilizes drop sorting in which a fluid stream containing linearly segregated particles is broken into drops. The drops containing particles of interest are electrically charged and deflected into a collection tube by passage through an electric field. Typically, the linearly segregated particles in the stream are characterized as they pass through an observation point situated just below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, the time at which it will reach the drop break-off point and break from the stream in a drop can be predicted. Ideally, a brief charge is applied to the fluid stream just before the drop containing the selected particle breaks from the stream and then grounded immediately after the drop breaks off. The drop to be sorted maintains an electrical charge as it breaks off from the fluid stream, and all other drops are left un-charged. Samples that are analyzed in flow cytometers may initially be provided in a sample container, such as a tube or well of a multi-well plate. For introducing samples from a sample container into the flow cytometer flow cell, sample injection tubes may be employed. FIG. 1A provides a view of a sample injection tube found in currently employed flow cytometers. As illustrated in FIG. 1A, sample injection tube assembly 10 includes a sample line 25 fixed to a first end 40 of support arm 30 by sample line screw 15. Because the sample line screw fixes the sample line at end 40 of support arm 30, sample line 25 does not move in the z-direction relative to end 40 of the support arm 30. Also shown is outer sleeve 20 which serves as part of a droplet containment system. Support arm 30 is coupled at end 45 to a first actuator 35, which moves the arm in the z-direction relative to the outer sleeve 20, such that the distal end 25a of the sample line 25 may be moved into the outer sleeve 20 for washing and out of the outer sleeve for drawing in sample from a sample container. Also shown is second actuator 50, which is a motor-driven actuator and moves the entire assembly up and down in the z direction. SUMMARY The inventors have realized that motor-driven actuators, such as illustrated in FIG. 1A, require motors and gears in addition to additional printed circuit boards (PCBs) and firmware to operate the motors. As such, these devices have high levels of complexity leading to increased manufacturing and maintenance costs. Furthermore, these parts increase points of failure, thus increasing potential repair costs. For example, these parts require multiple sources for acquisition, added software updates, lubrication, and additional space