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EP-4741800-A1 - FLOW CYTOMETER HAVING A VARIABLE DEPTH SAMPLE LINE INJECTION TUBE, AND METHODS OF USING THE SAME

EP4741800A1EP 4741800 A1EP4741800 A1EP 4741800A1EP-4741800-A1

Abstract

Aspects of the present disclosure include flow cytometers having variable depth sample line injection tubes. Flow cytometers according to certain embodiments include a flow cell; and a sample injection tube (SIT) assembly comprising a sample line operably coupled to the flow cell; wherein the SIT assembly is configured to provide for variable sample line depth in a sample container. Methods of cytometrically processing samples, e.g., in analysis and/or sorting applications, are also provided.

Inventors

  • Dembski, Kyle Thomas
  • Lankila, Henry J.
  • Grand Fong, Richard

Assignees

  • BECTON, DICKINSON AND COMPANY

Dates

Publication Date
20260513
Application Date
20251110

Claims (15)

  1. A flow cytometer comprising: a flow cell; a sample container receiving region; a sample injection tube (SIT) assembly comprising a sample line fluidically coupled to the flow cell; and an actuator coupled to the SIT assembly configured to move the SIT assembly; wherein the SIT assembly is configured to provide for variable sample line depth in a sample container present in the sample container receiving region.
  2. The flow cytometer according to Claim 1, wherein the SIT assembly comprises: an arm having a first proximal end coupled to the actuator and a second distal end comprising a through-hole; and a sample line subassembly seated in the through-hole; wherein the sample line subassembly is reversibly movable in the z-direction relative to the x-y plane of the second end.
  3. The flow cytometer according to Claim 2, wherein the sample line subassembly is reversibly movable in the z-direction relative to the x-y plane of the second end by a predetermined displacement distance.
  4. The flow cytometer according to Claim 3, wherein the predetermined displacement distance ranges from 1 mm to 2 mm.
  5. The flow cytometer according to Claim 4, wherein the predetermined displacement distance is 1.5 mm.
  6. The flow cytometer according to any of Claims 2 to 5, wherein the sample line subassembly comprises: a threaded bushing at least a portion of which is present in the through-hole, wherein the threaded bushing comprises a threaded hole and a sample line hole therethrough; and a sample line screw coupled to the threaded hole and comprising an inner bore receiving the sample line.
  7. The flow cytometer according to Claim 6, wherein the sample line subassembly further comprises a ferrule in the threaded hole, wherein the sample line passes through the ferrule.
  8. The flow cytometer according to any of Claims 2 to 7, wherein the SIT assembly further comprises a spring configured to compress the sample line subassembly onto the arm.
  9. The flow cytometer according to Claim 8, wherein the spring comprises a return flexure.
  10. The flow cytometer according to Claim 9, wherein the return flexure comprises an opening at a first end through which the sample line screw of the sample line subassembly passes.
  11. The flow cytometer according to Claim 10, wherein the return flexure is coupled to the arm.
  12. The flow cytometer according to any of the Claims 2 to 11, wherein the SIT assembly further comprises a restraining member configured to limit movement of the sample line subassembly in the z-direction relative to the x-y plane of the second end.
  13. The flow cytometer according to Claim 12, wherein the flow cytometer further comprises a sensor that measures displacement of the return flexure.
  14. The flow cytometer according to Claim 13, wherein the flow cytometer is configured to provide an assessment of sample line positioning in a sample container based on output from the sensor.
  15. A method of analyzing a sample, the method comprising: (a) introducing a particulate sample from a sample container into a flow cytometer with a sample injection tube (SIT) assembly comprising a sample line operably coupled to a flow cell, wherein the SIT assembly is configured to provide for variable sample line depth in the sample container; 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. 63/718,451 filed November 8, 2024, the disclosure of which application is incorporated herein by reference in its entirety. 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 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 sleave 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 sleave 20, such that the distal end 25a of the sample line 25 may be moved into the outer sleave 20 for washing and out of the outer sleave 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 the currently employed sample injection tube designs, such as illustrated in FIG. 1A, do not account for varied sample container bottom configurations. As such, currently employed sample injection tube configurations result in a varied amount of dead volume (i.e., sample in a container which is not drawn into the flow cytomer), which dead volume differs among the many disparate sample container types and manufacturers. In a given workflow, the dead volume may vary, ranging in some instances from 5-30mL depending on the type and height of the sample container device bo