US-20260126373-A1 - FLOW CYTOMETER SAMPLE INJECTION NEEDLE

US20260126373A1US 20260126373 A1US20260126373 A1US 20260126373A1US-20260126373-A1

Abstract

Flow cells including sample injection needles for operatively coupling a sample injection line to a body of a flow cell are provided. The subject sample injection needles include: a sample injection needle adaptor having a sample tube adapter fixed to a needle for delivering sample fluid from a sample injection line to a flow cell body; and a clamp for operatively coupling the sample injection needle to the flow cell body. Also provided are kits including the subject sample injection needles and flow cytometers having the subject flow cells as well as methods of use and assembly thereof.

Inventors

Henry J. Lankila
Jorge Manzarraga
Kyle Dembski
Christopher Ghazi

Assignees

BECTON, DICKINSON AND COMPANY

Dates

Publication Date: 20260507
Application Date: 20241106

Claims (20)

1 . A flow cytometer comprising: a flow cell comprising: a flow cell body for transporting particles in a core stream of a flow stream from a proximal end to a distal end, wherein the flow cell body comprises a flow cell cone at the proximal end; and a sample injection needle having a passage therethrough for delivering sample fluid from a sample injection line at a proximal end to the flow cell body at a distal end to generate the core stream, wherein the sample injection needle comprises: a sample injection needle adaptor comprising a sample tube adapter attached to a needle; and a clamp operatively coupling the sample injection needle to the flow cell body; wherein in an unclamped configuration of the flow cell the sample injection needle adaptor rotates freely with respect to the flow cell cone and the clamp, and wherein in a clamped configuration of the flow cell the sample injection needle adaptor is fixed with respect to the flow cell cone and the clamp; a light source configured to irradiate the particles in the flow stream at a sample interrogation region within the flow cell; and a detector configured to collect light emitted by the irradiated particles.
2 . The flow cytometer according to claim 1 , wherein the needle of the sample injection needle adaptor comprises a proximal end attached to the sample tube adapter and a distal end positioned within the flow cell cone.
3 . The flow cytometer according to claim 2 , wherein in the clamped configuration the sample injection needle adaptor is fixed with respect to the flow cell cone and the clamp by compression of the clamp.
4 . The flow cytometer according claim 2 , wherein the clamp is compressed by one or more fastening members.
5 . The flow cytometer according to claim 4 , wherein the clamp is compressed by a plurality of screws.
6 . The flow cytometer according to claim 5 , wherein the clamp comprises a set of holes receiving each of the plurality of screws.
7 . The flow cytometer according to claim 6 , wherein the flow cell body comprises a set of holes aligned with the set of clamp holes and receiving each of the plurality of screws.
8 . The flow cell according to claim 5 , wherein the clamp is configured such that the tilt of the sample injection needle with respect to the flow cell body is adjustable by manipulating the torque of at least one of the plurality of screws.
9 . The flow cytometer according to claim 2 , wherein the clamp comprises a distal end in contact with the sample tube adapter and a proximal end fluidically connecting the sample injection line to the sample injection needle.
10 . The flow cytometer according to claim 9 , wherein the distal end of the clamp comprises a recess in which at least a portion of the sample tube adapter is positioned and a surface in contact with the proximal end of the flow cell body.
11 . The flow cytometer according to claim 9 , wherein the proximal end of the clamp comprises a connector configured to minimize a dead volume of sample fluid when sample fluid is flowing from the sample injection line to the sample injection needle.
12 . The flow cytometer according to claim 2 , wherein the sample tube adapter comprises a proximal end positioned in a recess of the clamp and a distal end in contact with the proximal end of the flow cell body.
13 . The flow cytometer according to claim 11 , wherein the sample tube adapter comprises a flange in contact with the proximal end of the flow cell body.
14 . The flow cytometer according to claim 11 , wherein the distal end of the sample tube adapter is pressed against the proximal end of the flow cell body by the clamp such that the distal end of the needle of the sample injection needle adaptor is at a fixed position within the flow cell cone.
15 . The flow cytometer according to claim 2 , wherein the needle of the sample injection needle adaptor comprises a taper at the distal end.
16 . The flow cytometer according to claim 2 , wherein the distal end of the needle of the sample injection needle adaptor is positioned within the flow cell cone in a manner that enables an intact core stream to be maintained under flow conditions that vary by a magnitude or more.
17 . The flow cytometer according to claim 2 , wherein the flow cell body comprises a sheath fluid introduction port for delivering sheath fluid to the flow cell cone.
18 . The flow cytometer according to claim 17 , wherein the distal end of the needle of the sample injection needle adaptor is separated from the sheath fluid introduction port by a longitudinal distance ranging from 17 mm to 26 mm.
19 . The flow cytometer according to claim 2 , wherein the distal end of the flow cell body comprises a cuvette for transporting particles in the core stream past the sample interrogation region, wherein the cuvette is positioned at the distal end of the flow cell body by a clamp fixed to the flow cell body.
20 . The flow cytometer according to claim 19 , wherein the cuvette is positioned by the flow cell body clamp such that the sample interrogation region is optimally aligned with the cuvette for optical detection of particles in the core stream.

Description

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. Some flow cytometer systems use pressure-driven fluidics to supply both the sample fluid and the sheath fluid to a flow cell. In these systems, the sample and sheath fluids are delivered to the flow cell, which contains an interrogation region (i.e., wherein particles are illuminated by a light source), under greater than ambient pressures. Changes in the flow rate through the flow cell of a pressure-driven fluidics system are achieved by varying the pressure in the sample tube and/or the sheath fluid reservoir that feed into the flow cell. The ratio of sample fluid to sheath fluid flowing through the flow cell is governed both by the pressure levels in the sample tube and sheath fluid reservoir, and by the ratio of the resistances of the sample fluid and sheath fluid paths. Alternatively, flow cytometer systems have been implemented using vacuum-driven fluidics in which a vacuum pump draws a vacuum downstream of the flow cell, and the sample and sheath fluids are held at ambient pressure. For vacuum-driven fluidic systems, changes in the flow rate through the flow cell are achieved by varying the vacuum drawn by the vacuum pump, and the ratio of sample fluid to sheath fluid flowing through the flow cell is governed by the ratio of the resistances of sample fluid and sheath fluid paths. In order to allow for characterization and isolation of biological material at the individual particle level, some flow cytometers include an injection needle for introducing sample fluid to a flow cell. Using a sample injection needle, sample fluid may be combined with sheath fluid in the flow cell in a manner sufficient to create a focused core stream of particle containing sample fluid. The core stream may then transport particles through a detection region and/or sorting means in a single-file manner. This technique is known as hydrodynamic focusing. SUMMARY The present inventors have realized that imaging particles using a flow cytometer requires precise control over the speed and position at which sample fluid is combined with sheath fluid within a flow cell. In particular, it was discovered