Flow and vacuum controlled fluid management system for a flow particle analyzer

Abstract

To provide a flow and vacuum control type fluid management system for a flow type particle analyzer such as a flow cytometer.SOLUTION: A fluid management system includes a pump modulation type sheath fluid subsystem and a vacuum modulation type effluent subsystem. In addition, for example, a method for using a flow type particle analyzer having the fluid management system of the present invention, for particle analysis is also provided.SELECTED DRAWING: Figure 2

Description

[Background technology]

[0001] Flow particle analyzers, such as flow cytometers, are analytical tools that allow for the characterization of particles in a fluid stream based on optical parameters such as light scattering and fluorescence. The fluid stream may contain particles such as molecules, analyte-bound beads, or individual cells in fluid suspension. The particles are typically exposed to excitation light from one or more lasers and passed through one or more detectors where the light scattering and fluorescence properties of the particles are measured.

[0002] Each particle, or its subcomponents, can be labeled with a variety of spectrally distinct fluorescent dyes. Detection or characterization is performed using multiple photodetectors, typically one for each different dye to be detected. Analysis is performed while the fluid stream passes through a channel in an optical cuvette, as typically used in analytical flow cytometers.

[0003] In a typical flow cytometer, as the particle-laden sample fluid passes through the detection region, it is surrounded by a particle-free sheath fluid that forms a coaxial annular flow with the sample fluid, thereby producing a hydrodynamically focused flow of the particle-laden sample fluid at the center of the fluid stream surrounded by the particle-free sheath fluid. Typically, the ratio of sheath fluid to sample fluid is high, with the sample fluid forming only a small portion of the total fluid flow passing through the detection region. Summary of the Invention

[0004] A flow and vacuum controlled fluid management system for a flow particle analyzer, such as a flow cytometer, is provided. Aspects of the fluid management system include a pump-modulated sheath fluid subsystem and a vacuum-modulated waste subsystem. Methods of using a flow particle analyzer having the fluid management system of the present invention, for example, in particle analysis applications, are also provided. [Brief explanation of the drawings]

[0005] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0006] [Figure 1] 1 is a schematic diagram of the operating principle of a fluid management system according to one embodiment of the present invention; [Figure 2] FIG. 1 is a schematic diagram of a fluid management system of a flow cytometer, in accordance with one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0007] A flow and vacuum controlled fluid management system for a flow particle analyzer, such as a flow cytometer, is provided. Aspects of the fluid management system include a pump-modulated sheath fluid subsystem and a vacuum-modulated waste subsystem. Methods of using a flow particle analyzer having the fluid management system of the present invention, for example, in particle analysis applications, are also provided.

[0008] Before the present invention is described in detail, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0009] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within this stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0010] In this specification, certain ranges are presented with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically recited number, the close or approximately unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0012] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications and patents are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0013] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0014] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0015] Although apparatus and methods have been or will be described with functional descriptions for the sake of grammatical fluidity, it is expressly understood that unless expressly recited under 35 U.S.C. § 112, the claims should not necessarily be construed as limited by construction of "means" or "step" limitations, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of legal equivalents, and that if the claims are expressly recited under 35 U.S.C. § 112, they should be accorded the full legal equivalents under 35 U.S.C. § 112.

[0016] In further describing various aspects of the present invention, first, flow and vacuum controlled fluid management systems and flow particle analyzers, e.g., flow cytometers, including same, are reviewed in more detail. Then, a review of methods of using flow particle analyzers is provided.

[0017] Flow and vacuum controlled fluid management systems and flow particle analyzers including same As summarized above, a flow and vacuum controlled fluid management system for a flow particle analyzer, such as a flow cytometer, is provided. The fluid management system of the present invention is configured to manage the flow of fluids, such as sheath fluid, sample fluid, and waste fluid, within the flow particle analyzer, for example, as described below. Thus, the fluid management system can be used to control how fluids, such as sheath fluid, sample fluid, and waste fluid, flow through the fluid flow paths of the flow particle analyzer. Specifically, the fluid management system of the present invention can be configured to control the flow rates of sample fluid, sheath fluid, and waste fluid through the fluid system of the flow particle analyzer.

[0018] Flow particle analyzers, such as flow cytometers, typically include a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow particle analyzer transports particles (e.g., cells) in the fluid sample as a particulate stream to a flow cell, while directing a seal fluid into the flow cell via a flow cell input. Within the flow cell, a liquid sheath forms around the particulate stream, imparting a substantially uniform velocity to the particulate stream. The flow cell hydrodynamically focuses particles, e.g., cells, within the stream so that they pass through the center of a light source within the examination region of the flow cell. Light from the examination region, e.g., in the form of scattered light (e.g., side-scattered or forward-scattered light) as sufficiently emitted fluorescent light, is then detected by a suitable optical detection system for use, e.g., in subsequent particle analysis. Fluid exiting the flow cell output enters a waste management system, which conveys the fluid from the flow cell output to a waste reservoir.

[0019] The fluid management system of the present invention is a flow- and vacuum-controlled fluid management system. By "flow- and vacuum-controlled," we mean that the fluid management system is configured to control the sample flow rate within the system through the combined action of a pump-modulated sheath fluid subsystem and a vacuum-modulated waste subsystem, and includes both a sheath fluid flow-modulating pump and a vacuum-applying device to control fluid flow through the fluid system of a flow particle analyzer. In some cases, sample flow is controlled solely by the combined action of the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem, such that sample flow through the system is not directly controlled.

[0020] In some cases, the fluid management system is configured to have a constant fluid resistance during operation. Thus, during use, the fluid resistance within the system does not change. Because the fluid management system is configured to have a constant fluid resistance during operation, during use, the fluid management system is a closed system. Because the fluid management system is a closed system during use, the fluid lines and reservoirs or other components do not have internal areas exposed to the external environment. A closed system may be characterized in that no internal space or location of the system is exposed to the system's external environment. Thus, changes in pressure in one portion of the system affect the flow of fluid through another portion of the system.

[0021] Figure 1 shows a schematic diagram of the operating principle of a fluid management system according to one embodiment of the present invention. In Figure 1, the sheath and sample paths are modeled as two parallel resistors, which combine to pass through the flow cell, cuvette, and waste paths, which collectively represent a third fluidic resistor. The operation of the system is based on fluidic circuit principles, where the pressure drop (delta P) across a closed fluidic path is assumed to be equal to the product of the liquid flow rate (Q) and the fluid resistance (R). The sheath and sample paths can be modeled as two parallel resistors, which combine to pass through the flow cell, cuvette, and waste paths, which collectively represent a third fluidic resistor.

[0022] Control of the sheath flow rate (Q_sheath) and vacuum pressure (P_vacuum) can be used to control the sample flow rate. With a constant sample line resistance (R_sample) and waste pressure drop (delta P), the vacuum pressure (P_vacuum) is used to set the sample flow rate (Q_sample). The sheath pumps are simultaneously controlled to maintain a constant overall pressure drop through the flow cell, cuvette, and waste path. To change the sheath flow rate, and therefore the particle velocity in the cuvette, the sheath supply pump can be controlled to change the pressure drop to a new value, and the vacuum pump control is adjusted accordingly.

[0023] As discussed above, aspects of the fluid management system include a pump-modulated sheath fluid subsystem and a vacuum-modulated waste subsystem, each of which is discussed in more detail below.

[0024] Pump-Modulated Sheath Fluid Subsystem Aspects of the fluid management system include a pump-modulated sheath fluid subsystem for supplying sheath fluid to the flow cell and providing a flow rate of sheath fluid through the flow cell. The pump-modulated sheath fluid subsystem may be configured to generate the flow rate of sheath fluid by pumping sheath fluid from a sheath fluid source to an input of the flow cell. The pump-modulated sheath fluid subsystem may include a sheath fluid source, a pump (e.g., a positive displacement pump), a degassing device, a pulsation damper, and a sheath supply valve. Pressure within the pump-modulated sheath fluid subsystem may be measured by a sheath fluid subsystem pressure transducer.

[0025] The pump-modulated sheath fluid subsystem of the fluid management system includes a pump that modulates the flow of liquid through the sheath fluid portion of the fluid system of the flow particle analyzer. The sheath fluid pump may pump sheath fluid from a sheath fluid source to the input of the flow cell. The flow rate of the sheath fluid provided by the sheath fluid pump may vary, possibly ranging from 1 to 30 ml / min, such as 2 to 20 ml / min. In the broadest sense, a pump may be any device that moves liquid through mechanical action. The pump-mediated sheath fluid subsystem may include any suitable pump, such as a positive displacement pump. As used herein, a "positive displacement pump" refers to a pump that moves fluid by capturing a volume and expelling (displacing) that captured volume from the device; such pumps may operate in a series of actuation cycles, each cycle capturing a volume of fluid and mechanically moving the fluid through the pump and into the fluid system. Positive displacement pumps that may be used include, but are not limited to, rotary positive displacement pumps such as peristaltic pumps, internal gear pumps, screw pumps, shuttle block pumps, flexible or sliding vane pumps, circumferential piston pumps, flexible impeller pumps, helical twisted roots pumps, or liquid ring pumps; reciprocating positive displacement pumps such as piston pumps, plunger pumps, or diaphragm pumps; and linear positive displacement pumps such as rope pumps and chain pumps. In some cases, the sheath fluid subsystem pump is modulated by a peristaltic pump.

[0026] In addition to the sheath fluid pump, the pump-modulated sheath fluid subsystem may further include a pulsation damper positioned downstream of the pump, i.e., between the pump output and the flow cell input. As used herein, a "pulsation damper" refers to a device configured to damp fluid pulsations in the pump-mediated sheath fluid subsystem. A given pulsation damper may function to dampen pulsations in the sheath fluid subsystem, for example, by temporarily expanding / contracting, thereby storing / releasing sheath fluid to dampen pulsations in the sheath fluid. The pulsation damper may include a fluid channel, a first fluidic device adapted to dampen pulsations, and a second fluidic device adapted to dampen pulsations. The first fluidic device may include a first fluidic resistor and a first fluidic capacitor, and the second fluidic device may include a second fluidic resistor and a second fluidic capacitor. The first fluidic resistor and the second fluidic resistor may be resistive channels. The first fluidic capacitor and the second fluidic capacitor may include membranes that expand to accumulate fluid and then contract to reintroduce the accumulated fluid into the fluidic channel. In some cases, the pulsation damper may include a fluidic channel, a first fluidic device adapted to attenuate pulsations with a shallow roll-off slope, and a second fluidic device adapted to attenuate pulsations with a shallow roll-off slope. The first fluidic device and the second fluidic device may be connected to the fluidic channel such that they cooperatively attenuate pulsations with a steep roll-off slope. The first fluidic device may include a first fluidic resistor and a first fluidic capacitor, and the second fluidic device may include a second fluidic resistor and a second fluidic capacitor. In certain embodiments, the pulsation damper is arranged in the following order, similar to a second-order low-pass filter: (1) a first fluidic resistor, (2) a first fluidic capacitor, (3) a second fluidic resistor, and (4) a second fluidic capacitor. Any convenient pulsation damper may be used, such as, but not limited to, those described in U.S. Pat. Nos. 7,328,722, 7,857,005, 8,017,402, and 8,715,573, the disclosures of which are incorporated herein by reference.

[0027] In certain embodiments, the pump-modulated sheath fluid subsystem includes a degassing device. As used herein, "degassing device" refers to a device for removing air bubbles from a fluid stream. The degassing device may be positioned in the flow line at a location downstream of the pump and may receive sheath fluid from the pump. In some cases, the degassing device is between the output of the pump and the input of the pulsation damper. Degassing devices of interest that may be incorporated into a pump-mediated sheath fluid subsystem include, but are not limited to, air bubble filters, and the like.

[0028] In certain embodiments, the pump-modulated sheath fluid subsystem includes at least one valve. The at least one valve may be a sheath supply valve that facilitates control of the flow of sheath fluid. In some cases, the sheath supply valve restricts fluid flow within the pump-mediated sheath fluid subsystem, allowing for a variable flow rate of sheath fluid. The sheath supply valve may be positioned between the pump and the flow cell. In some cases, the sheath supply valve may be positioned between the pulsation damper and the input of the flow cell. In some cases, the volumetric pumped sheath fluid subsystem includes multiple valves. Suitable valves for use in volumetric pumped sheath fluid subsystems include, but are not limited to, check valves, etc.

[0029] The pump-modulated sheath fluid subsystem may further include a sheath fluid subsystem pressure transducer. The sheath fluid subsystem pressure transducer may be any device configured to measure the pressure within the sheath fluid subsystem at any suitable location along the flow line through which the sheath fluid is pumped. In some cases, the sheath fluid subsystem pressure transducer may be positioned immediately prior to the flow cell and configured to measure and provide output data representative of the sheath fluid pressure immediately prior to the flow cell. The sheath fluid subsystem pressure transducer may further be connected to a controller that adjusts the flow rate of the sheath fluid or sample fluid based on the pressure measured by the sheath fluid subsystem pressure transducer. The sheath fluid subsystem pressure transducer may be any suitable pressure monitoring device, i.e., a pressure sensor, such as, but not limited to, force collector-type pressure sensors, such as piezoresistive strain gauges, capacitive sensors, electromagnetic sensors, piezoelectric sensors, strain gauge sensors, optical sensors, and potentiometer sensors, as well as other types of pressure sensors, such as resonant sensors, thermal sensors, and ionization sensors. In some cases, the pump-mediated sheath fluid subsystem includes multiple sheath fluid subsystem pressure transducers coupled to flow lines through which the sheath fluid is pumped, and each sheath fluid subsystem pressure transducer can be connected to a controller for adjusting the flow rate of the sheath fluid or sample fluid based on the measured pressure.

[0030] In certain embodiments, the pump-modulated sheath fluid subsystem may be fluidly coupled to a sheath fluid source such that the sheath fluid source supplies sheath fluid to the input of the pump-mediated sheath fluid subsystem. The sheath source may be any suitable reservoir or container for holding sheath fluid.

[0031] Vacuum Modulated Waste Subsystem Aspects of the fluid management system include a vacuum-modulated waste subsystem that can be configured to generate a flow rate of waste fluid by drawing waste fluid from an output of a flow cell to a waste reservoir via application of a vacuum. The waste fluid can include sample fluid and sheath fluid that pass through the flow cell of the flow particle analyzer and exit the output of the flow cell.

[0032] The vacuum-modulated waste subsystem includes a vacuum application device positioned along a waste line between the flow cell output and a waste storage. The vacuum application device may vary as needed and may include a vacuum pump operably coupled to a vacuum accumulator, which may be positioned along the waste subsystem between the pump and the flow cell. The vacuum pump components of the vacuum application device may vary as needed. In some cases, the vacuum pump is a positive displacement pump. Positive displacement vacuum pumps that may be used include, but are not limited to, rotary vane pumps, diaphragm pumps, piston pumps, scroll pumps, screw pumps, gear pumps, peristaltic pumps, and the like. In some cases, the vacuum pump is a diaphragm pump. In some cases, the vacuum pump is not the same as the pump of the pump-modulated sheath fluid subsystem. In some cases, the vacuum pump is a diaphragm pump and the pump of the sheath fluid subsystem is a peristaltic pump.

[0033] Operatively coupled to the pump is a vacuum accumulator. As used herein, "vacuum accumulator" refers to a sealed container having an internal volume that is twice the stroke volume of the pump, and in some cases, the internal volume of the container exceeds the internal volume of the stroke volume by 25% or more, e.g., 10-100%, such as 20-100%, including 50% or more, by 10% or more. The vacuum accumulator can maintain the vacuum level generated by the vacuum pump. In some cases, the absolute vacuum pressure within the vacuum accumulator determines the flow rate of sample fluid from the sample source through the sample input line and through the flow cell. In some cases, the pump and vacuum accumulator of the flow cytometer are the vacuum source and vacuum accumulator described in U.S. Patent No. 8,528,427, the disclosure of which is incorporated herein by reference.

[0034] In certain embodiments, the vacuum-modulated waste subsystem includes at least one valve. The at least one valve may be a waste valve that can facilitate control of the flow of waste. In some cases, the waste valve restricts fluid flow within the vacuum-modulated waste subsystem, allowing for a variable flow rate of waste. The waste valve may be positioned on a flow line of the vacuum-pumped waste subsystem between the flow cell and the pump. In some cases, the waste valve is positioned on a flow line between the output of the flow cell and the vacuum accumulator. In some cases, the vacuum-pumped waste subsystem includes multiple valves. Suitable valves for use in the vacuum-pumped waste subsystem may vary and may include, but are not limited to, adjustable valves, etc.

[0035] The vacuum-modulated effluent subsystem may further include a effluent subsystem pressure transducer. The effluent subsystem pressure transducer may measure the vacuum pressure in the effluent subsystem. In some cases, the effluent subsystem pressure transducer is configured to measure the vacuum pressure in the vacuum accumulator. The effluent subsystem pressure transducer may be connected to a controller that adjusts the flow rate of the effluent based on the measured vacuum pressure. The effluent subsystem pressure transducer may be any suitable pressure monitoring device, for example, a pressure sensor, such as, but not limited to, force collector type pressure sensors, such as piezoresistive strain gauges, capacitive sensors, electromagnetic sensors, piezoelectric sensors, strain gauge sensors, optical sensors, potentiometer sensors, and other types of pressure sensors, such as, for example, resonant sensors, thermal sensors, ionization sensors, and the like. In some cases, the vacuum-modulated sheath fluid subsystem includes multiple effluent subsystem pressure transducers. In some cases, the vacuum pumped sheath fluid subsystem includes multiple effluent subsystem pressure transducers coupled to flow lines through which the effluent is pumped, and each effluent subsystem pressure transducer can be connected to a controller for adjusting the flow rate of the effluent based on the measured pressure.

[0036] In certain embodiments, the vacuum-modulated waste subsystem can be fluidly coupled to a waste reservoir, such as a container configured to store waste fluid, such that waste fluid flows from the output of the pump into the waste reservoir. The waste reservoir can be any suitable container for holding sheath fluid.

[0037] Sample Source In some cases, the flow particle analyzer includes a sample source. The sample source may be any suitable reservoir or container for holding a sample fluid. The sample source may be fluidly coupled to a sample input line leading to the flow cell and may supply the sample fluid to the sample input line.

[0038] controller Aspects of the fluid management system further include a controller for modulating the flow rates of fluids within the flow cytometer. In some cases, the controller modulates the flow rate of sheath fluid from a sheath fluid source to an input of the flow cell. In certain embodiments, the controller modulates the flow rate of sample fluid from a sample source to an input of the flow cell. In certain embodiments, the controller modulates the flow rate of waste fluid from an output of the flow cell to a waste reservoir. The controller may adjust the flow rates of the sheath fluid, sample fluid, and waste fluid based on pressure measured by a system pressure transducer. In some cases, the controller adjusts the flow rates of the sheath fluid, sample fluid, and waste fluid based on pressure measured immediately before the flow cell by a sheath fluid subsystem pressure transducer. In some cases, the controller adjusts the flow rates of the sheath fluid, sample fluid, and waste fluid based on vacuum pressure measured in a vacuum accumulator by a waste subsystem pressure transducer.

[0039] The controller may provide any suitable sample fluid flow rate through the flow cell. In some cases, the controller modulates the sample fluid flow rate based on fluid pressure measured by a sheath fluid subsystem pressure transducer. In some cases, the controller modulates the sample fluid flow rate based on vacuum pressure measured by a waste subsystem pressure transducer. In some cases, the controller modulates the sample fluid flow rate based on a pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem, as measured by a sheath fluid subsystem transducer and a waste subsystem transducer. In some cases, the controller may be configured to provide a sample fluid flow rate in the range of 1 to 1000 μl / min, e.g., 5 to 200 μl / min.

[0040] In some cases, the controller modulates the flow rate of the sample fluid through joint modulation or joint control of the sheath fluid flow rate and the vacuum pressure of the waste subsystem. The controller may modulate the flow rate of the sheath fluid based on a measured pressure difference between the positive displacement pumped sheath fluid subsystem and the vacuum pumped waste subsystem, as measured by the sheath fluid subsystem transducer and the waste subsystem transducer. The controller may be configured to provide a sheath fluid flow rate in the range of 1 to 30 ml / min, e.g., 2 to 20 ml / min.

[0041] In some cases, the controller is configured to control a control feedback circuit for regulating the flow rate of fluids within the flow cytometer. In certain embodiments, the controller may be configured to control a sheath fluid subsystem control feedback circuit for regulating the pump-modulated sheath fluid subsystem based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem. In some cases, the controller controls a waste subsystem control feedback circuit for regulating the vacuum-modulated waste subsystem based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-pumped waste subsystem.

[0042] Flow Particle Analyzer The fluid management systems described herein may be used in a variety of different flow particle analyzers. Suitable flow cytometry systems in which the subject fluid management systems may be used include, but are not limited to, those described in U.S. Patent Nos. 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, and 8,1 40,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, and 5,602,039, the disclosures of which are incorporated herein by reference in their entireties. In some cases, flow cytometry systems of interest include a BD Biosciences FACSCanto™ II flow cytometer, a BD Accuri™ flow cytometer, a BD Biosciences FACSCelesta™ flow cytometer, a BD Biosciences FACSLyric™ flow cytometer, a BD Biosciences FACSVerse™ flow cytometer, a BD Biosciences FACSymphony™ flow cytometer, a BD Biosciences LSRFortessa™ flow cytometer, a BD Biosciences LSRFortess™ X-20 flow cytometer, or the like.

[0043] In certain embodiments, the subject system is a flow cytometry system having an excitation module that generates multiple beams of frequency-shifted light using radio frequency multiplexing excitation. In these embodiments, the laser light generator may include multiple lasers and one or more acousto-optical components (e.g., acousto-optical polarizers, acousto-optical frequency shifters) for generating multiple frequency-shifted comb beams. One or more of the frequency-shifted comb beams and the local oscillator beam may be configured to be received by a beam shaping component as described herein to generate one or more beams of frequency-shifted light having a substantially constant intensity profile. In some cases, the subject system is a flow cytometry system having a laser excitation module as described in U.S. Patent Nos. 9,423,353, 9,784,661, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0044] Specific Embodiments FIG. 2 shows a schematic diagram of a fluid management system of a flow cytometer according to one embodiment of the present invention. In the sheath fluid subsystem of the fluid management system, sheath fluid is pumped from a sheath fluid source to a hydrodynamic focusing channel. The sheath fluid is combined with a sample fluid to be optically analyzed in the imaging flow channel. A sheath fluid supply tank 10 containing sheath fluid is coupled to a flow line that supplies sheath fluid to a sheath supply peristaltic pump 11. Using the pump to control the sheath flow rate allows for head-pressure-independent flow control and continuous variation of the sheath fluid and sample flow rates. The sheath fluid passes through the flow line of the sheath fluid subsystem and flows through a degassing device 12 (e.g., a bubble filter) to remove bubbles from the fluid stream and a pulsation damper 13 to dampen pulsations in the fluid subsystem. The fluid line includes a sheath supply valve 14 for restricting fluid flow, positioned before a flow cell containing a hydrodynamic focusing channel 16 and an imaging flow channel 18. The pressure within the flow cytometer in front of the flow cell is measured by the sheath fluid subsystem pressure transducer 15 .

[0045] The system further comprises a sample input 23 containing a sample fluid. The sample input delivers the sample fluid to a fluid line leading to a flow cell containing a hydrodynamic focusing channel 16 and an imaging flow channel 18. The sample fluid combines with sheath fluid pumped by a sheath-feed peristaltic pump 11, and the two fluids flow through the input of the flow cell.

[0046] In the waste subsystem of the fluidic system, waste fluid, including sample fluid and sheath fluid, is drawn from the flow cell output to a waste storage tank. Given proper control of the sheath flow rate by the sheath feed peristaltic pump 11, the sample fluid is drawn through the flow cell by a vacuum created by the diaphragm pump 21 drawing the waste fluid. After the waste fluid passes through the flow cell output, the diaphragm pump 21 draws the waste fluid through the flow line to a waste storage tank 22. The diaphragm pump also generates a vacuum in a vacuum accumulator 20 to apply a vacuum to the waste line of the waste subsystem. The vacuum accumulator may be a small, sealed plastic bottle. A second pressure transducer 19 is connected to the vacuum accumulator 20 to measure the pressure in the vacuum accumulator. The waste subsystem further includes a waste valve positioned in the flow line between the output of the imaging flow channel 18 and the vacuum accumulator 20. For a given sample flow rate, the vacuum pressure in the associated vacuum accumulator is controlled to match the fluid resistance of the sample line via vacuum pump 21. The pressure drop across the flow cell, defined as the difference in pressure measured from the first and second transducers, is then used as a feedback control for the sheath supply pump 11 for the appropriate sheath flow rate.

[0047] method Also provided are methods of using a flow particle analyzer including a fluid management system of the present invention, e.g., as described above. The method may include flowing a sample fluid through a flow cytometer including a flow cell having an input and an output, a pump-modulated sheath fluid subsystem for fluidly coupling a sheath fluid source to the input, and a vacuum-modulated waste subsystem for fluidly coupling a waste reservoir to the output, e.g., as detailed above.

[0048] In some embodiments, the sample fluid contains an initial sample that is a biological sample. The term "biological sample" is used in its conventional sense to refer to a whole organism, plant, fungus, or, in some cases, a subset of animal tissues, cells, or components that can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, and urinary tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample can be any type of biological 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 a derivative thereof, e.g., plasma, tears, urine, semen, etc., and in some cases the sample is a blood sample, including whole blood, such as blood obtained from a venipuncture or fingerstick (which may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0049] In certain embodiments, the source of the sample is a "mammal" or "mammalian," terms used broadly to refer to organisms within the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodents (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is human. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., newborn, infant, juvenile, adolescent, adult); in certain embodiments, the human subject is a juvenile, adolescent, or adult. It should be understood that while the present invention may be applied to samples from human subjects, it may also be practiced on samples from other animal subjects (i.e., within "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0050] When performing the subject methods, a volume of sample fluid is pumped into a flow cytometer. The volume of sample fluid pumped into the flow cytometer can vary from 0.001 mL to 1000 mL, including samples from, e.g., 0.005 mL to 900 mL, e.g., 0.01 mL to 800 mL, e.g., 0.05 mL to 700 mL, e.g., 0.1 mL to 600 mL, e.g., 0.5 mL to 500 mL, e.g., 1 mL to 400 mL, e.g., 2 mL to 300 mL, and 5 mL to 100 mL.

[0051] The method may further include modulating the flow rate of the sample fluid based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem, measured by, for example, the sheath fluid subsystem transducer and the waste subsystem transducer, as detailed above. The flow rate of the sample fluid may be regulated by a controller connected to the sheath fluid subsystem transducer and the waste subsystem transducer. The controller may receive signals from each of the transducers that provide pressure within the fluidic subsystems of the flow cytometer before and after the flow cell. The controller may be in communication with the pumps of the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem, and may control the operation of the pumps to control the flow rate of the sample fluid.

[0052] Computer Control System Aspects of the present disclosure further include computer control systems for carrying out the subject methods, the systems further comprising one or more computers for full or partial automation of the systems for carrying out the methods described herein. In some embodiments, the systems comprise a computer having a computer-readable storage medium having stored thereon a computer program, the computer program, when loaded into the computer, comprising instructions for operating a fluid management system, e.g., as described above.

[0053] 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 accesses a memory having stored instructions to perform the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a 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 or become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, such as Java, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the 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, all in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0054] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write compact disk, a flash memory device, or other memory storage device. The memory storage device can be any of a variety of known or future devices, including a compact disk drive, a tape drive, a removable hard disk drive, or a disk drive. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as flash memory, an SD card, a solid-state hard drive, or other forms of optical or magnetic memory devices, respectively. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or in program storage devices used in conjunction with the memory storage devices.

[0055] In some embodiments, a computer program product is described comprising a computer-usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor of a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementation of hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art.

[0056] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, or tape, or RAM, or any other suitable device, either fixed or portable). The processor may include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communications channel, or may be pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, using any of these devices together with the memory. For example, a magnetic or optical disk may carry the programming and be readable by a disk writer / reader. The system of the present invention also includes programming in the form of a computer program product, e.g., algorithms for use in implementing the above-described methods. The programming according to the present invention may be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media, hard disk storage media, optical storage media such as DVD, Blu-ray, and CD-ROM, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories, such as magnetic / optical storage media.

[0057] The processor may also have access to a communication channel for communicating with a user at a remote location, meaning that the user does not have direct contact with the system but 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 smartphone.

[0058] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications (e.g., radio frequency identification (RFID), WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocols, and cellular communications such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0059] In one embodiment, the communications interface is configured to include one or more communications ports, e.g., a physical port or interface such as a USB port, a Lightning port, a USB-C port, or any other suitable electrical connection port, to enable data communications between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communications.

[0060] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or any other communication device that a user may use in conjunction with.

[0061] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing the Internet Protocol (IP) via a cellular network, short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0062] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communications interface using common standards such as, for example, 802.11 or Bluetooth® RF protocols, or the IrDA infrared protocol. The server device may be another portable device, such as a smartphone, tablet computer, or notebook computer, or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device includes a display, such as a liquid crystal display (LCD) or light-emitting diode display (LED), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0063] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored in the subject system, e.g., any data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0064] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When 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 a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The 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 user at a remote location, for example, via the Internet, telephone, or satellite network, in accordance with known techniques. Presentation of data by the output manager may be performed in accordance with various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject system are typically of a class of computers commonly referred to as servers, but may be any type of known or future-developed computer platform. Alternatively, they may be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cabling or other communication system, including networked or non-networked wireless systems. They may be co-located or physically separated.In some cases, various operating systems may be used on any of the computer platforms, depending on the type and / or configuration of the computer platform selected. Suitable operating systems include Windows 10, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, Zorin OS, etc.

[0065] Utilities The fluidic subsystems and methods described herein are used in a variety of applications where it is desirable to analyze particulate components in a sample in a fluid medium, such as a biological sample. The fluidic subsystems may be incorporated into any suitable analytical flow cytometer system. Embodiments of the present invention allow for the control of continuously varying flow rates of both the sheath and sample within a flow cytometer while maintaining stability of the sheath and sample flow rates. Continuous variation of both the sheath and sample flow rates is provided by the use of a positive displacement pump to pump the sheath fluid and a vacuum pump to generate vacuum pressure within the flow cytometer.

[0066] Flow cytometry systems and methods for analyzing samples in which the subject fluid management systems may be used include, but are not limited to, U.S. Patent Nos. 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, and 8,233,146. Nos. 8,140,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, and 5,602,039, the disclosures of which are incorporated herein by reference in their entireties. In some cases, flow cytometry systems of interest include a BD Biosciences FACSCanto™ II flow cytometer, a BD Accuri™ flow cytometer, a BD Biosciences FACSCelesta™ flow cytometer, a BD Biosciences FACSLyric™ flow cytometer, a BD Biosciences FACSVerse™ flow cytometer, a BD Biosciences FACSymphony™ flow cytometer, a BD Biosciences LSRFortessa™ flow cytometer, a BD Biosciences LSRFortess™ X-20 flow cytometer, or the like.

[0067] In certain embodiments, the subject system is a flow cytometry system having an excitation module that generates multiple beams of frequency-shifted light using radio frequency multiplexing excitation. In these embodiments, the laser light generator may include multiple lasers and one or more acousto-optical components (e.g., acousto-optical polarizers, acousto-optical frequency shifters) for generating multiple frequency-shifted comb beams. One or more of the frequency-shifted comb beams and the local oscillator beam may be configured to be received by a beam shaping component as described herein to generate one or more beams of frequency-shifted light having a substantially constant intensity profile. In some cases, the subject system is a flow cytometry system having a laser excitation module as described in U.S. Patent Nos. 9,423,353, 9,784,661, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0068] Regardless of the scope of the appended claims, the present disclosure is also defined by the following notes.

[0069] 1. A fluid management system for a flow particle analyzer, comprising: a flow cell having an input and an output; a sample input line for fluidly coupling a sample source to said input; a pump-modulated sheath fluid subsystem for fluidly coupling a sheath fluid source to the input; a vacuum modulated waste subsystem for fluidly coupling a waste reservoir to said output; Equipped with configured to have a constant fluid resistance coupling during operation; Fluid management systems. 2. The fluid management system of claim 1, configured to control the flow rate of the sample by the combined action of a pump-modulated sheath fluid subsystem and a vacuum-modulated waste subsystem. 3. The fluid management system of claim 1 or 2, wherein the flow particle analyzer is a flow cytometer. 4. A fluid management system according to any one of claims 1 to 3, wherein the sample input line is fluidly coupled to a sample source, the pump-modulated sheath fluid subsystem is fluidly coupled to a sheath fluid source, and the vacuum-modulated waste subsystem is fluidly coupled to a waste reservoir. 5. The fluid management system of claim 4, wherein the pump-modulated sheath fluid subsystem is configured to generate a flow rate of sheath fluid by pumping sheath fluid from a sheath fluid source to an input of the flow cell.

[0070] 6. The fluid management system of any one of clauses 1-5, wherein the pump-modulated sheath fluid subsystem comprises a positive displacement pump. 7. The fluid management system of claim 6, wherein the positive displacement pump comprises a pump selected from the group consisting of a peristaltic pump, a gear pump, and a diaphragm pump. 8. The fluid management system of claim 7, wherein the positive displacement pump is a peristaltic pump. 9. The fluid management system of any one of clauses 1-8, wherein the pump-modulated sheath fluid subsystem comprises a degassing device. 10. A fluid management system according to any one of claims 1 to 9, wherein the pump-modulated sheath fluid subsystem comprises a pulsation damper configured to damp fluid pulsations within the pump-modulated sheath fluid subsystem.

[0071] 11. The fluid management system of any one of clauses 1-10, wherein the vacuum modulated waste subsystem comprises a vacuum application device. 12. The fluid management system of claim 11, wherein the vacuum application device comprises a vacuum pump operably coupled to the vacuum accumulator. 13. The fluid management system of claim 12, wherein the vacuum pump comprises a positive displacement vacuum pump. 14. The fluid management system of claim 13, wherein the positive displacement vacuum pump is selected from the group consisting of a diaphragm pump, a gear pump, and a peristaltic pump. 15. The fluid management system of claim 14, wherein the positive displacement vacuum pump is a diaphragm pump.

[0072] 16. The fluid management system of any one of claims 1 to 15, further comprising a controller configured to control the flow rate of the sample through the flow cell by the combined operation of the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem. 17. The fluid management system of claim 16, wherein the controller is configured to provide a flow rate of the sample fluid in the range of 1 to 1000 μl / s. 18. The fluid management system of claim 16, wherein the controller is configured to provide a flow rate of the sample fluid in the range of 5 to 200 μl / s. 19. The fluid management system of claim 17 or 18, wherein the controller modulates the flow rate of the sample fluid through combined modulation of the flow rate of the sheath fluid and the vacuum pressure of the waste subsystem. 20. The fluid management system of claim 19, wherein the controller is configured to provide a flow rate of the sheath fluid in the range of 1 to 30 ml / min.

[0073] 21. A fluid management system according to any one of claims 18 to 20, wherein the controller modulates the flow rate of the sheath fluid based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem. 22. The fluid management system of claim 21, wherein the controller controls a sheath fluid subsystem control feedback circuit to regulate the pump-modulated sheath fluid subsystem based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem. 23. The fluid management system of claim 21 or 22, wherein the pressure in the pump-modulated sheath fluid subsystem is measured by a sheath fluid subsystem pressure transducer. 24. The fluid management system of claim 23, wherein the sheath fluid subsystem pressure transducer is configured to measure the pressure of the sheath fluid immediately before the flow cell. 25. A fluid management system according to any one of claims 18 to 24, wherein the controller controls a waste subsystem control feedback circuit for adjusting the vacuum-modulated waste subsystem based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem.

[0074] 26. The fluid management system of claim 25, wherein the pressure in the vacuum-modulated waste subsystem is measured by a waste subsystem pressure transducer. 27. The fluid management system of claim 26, wherein the waste subsystem pressure transducer is configured to measure the vacuum pressure in the vacuum accumulator.

[0075] 28. A flow particle analyzer comprising a fluid management system according to any one of appendices 1 to 27. 29. The flow particle analyzer of claim 28, wherein the flow particle analyzer is a flow cytometer.

[0076] 30. A method for analyzing particles in a sample, comprising: analyzing particles in a sample using a flow particle analyzer comprising a fluid management system, the fluid management system comprising: a flow cell having an input and an output; a sample input line fluidly coupling a sample source containing a sample to the input; a pump-modulated sheath fluid subsystem fluidly coupling a sheath fluid source to the input; a vacuum modulated waste subsystem fluidly coupling a waste reservoir to said output; Equipped with The fluid management system has a constant fluid resistance coupling. method. 31. The method of claim 30, wherein the fluid management system is configured to control the flow rate of the sample by the combined action of a pump-modulated sheath fluid subsystem and a vacuum-modulated waste subsystem. 32. The method of claim 30 or 31, wherein the flow particle analyzer is a flow cytometer. 33. The method of any one of claims 30 to 32, wherein the pump-modulated sheath fluid subsystem is configured to generate a flow rate of sheath fluid by pumping sheath fluid from a sheath fluid source to an input of the flow cell. 34. The method of any one of claims 30 to 33, wherein the pump-modulated sheath fluid subsystem comprises a positive displacement pump.

[0077] 35. The method of claim 34, wherein the positive displacement pump comprises a pump selected from the group consisting of a peristaltic pump, a gear pump, and a diaphragm pump. 36. The method of claim 35, wherein the positive displacement pump is a peristaltic pump. 37. The method of any one of claims 30 to 36, wherein the pump-modulated sheath fluid subsystem includes a degassing device. 38. The method of any one of claims 30 to 37, wherein the pump-modulated sheath fluid subsystem comprises a pulsation damper configured to damp fluid pulsations within the pump-modulated sheath fluid subsystem. 39. The method of any one of clauses 30-38, wherein the vacuum-modulated waste subsystem comprises a vacuum application device.

[0078] 40. The method of claim 39, wherein the vacuum application device comprises a vacuum pump operably coupled to a vacuum accumulator. 41. The method of claim 40, wherein the vacuum pump comprises a positive displacement vacuum pump. 42. The method of claim 41, wherein the positive displacement vacuum pump is selected from the group consisting of a diaphragm pump, a gear pump, and a peristaltic pump. 43. The method of claim 42, wherein the positive displacement vacuum pump is a diaphragm pump. 44. The method of any one of claims 30 to 43, wherein the fluid management system further comprises a controller configured to control the flow rate of the sample through the flow cell by the combined operation of the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem.

[0079] 45. The method of claim 44, wherein the controller is configured to provide a flow rate of the sample fluid in the range of 1 to 1000 μl / s. 46. ​​The method of claim 45, wherein the controller is configured to provide a flow rate of the sample fluid in the range of 5 to 200 μl / s. 47. The method of claim 45 or 46, wherein the controller modulates the flow rate of the sample fluid through combined modulation of the flow rate of the sheath fluid and the vacuum pressure of the waste subsystem. 48. The method of claim 47, wherein the controller is configured to provide a flow rate of the sheath fluid in the range of 1 to 30 ml / min. 49. The method of any one of claims 46 to 48, wherein the controller modulates the flow rate of the sheath fluid based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem.

[0080] 50. The method of claim 49, wherein the controller controls a sheath fluid subsystem control feedback circuit to adjust the pump-modulated sheath fluid subsystem based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem. 51. The method of claim 49 or 50, wherein the pressure in the pump-modulated sheath fluid subsystem is measured by a sheath fluid subsystem pressure transducer. 52. The method of claim 51, wherein the sheath fluid subsystem pressure transducer is configured to measure the pressure of the sheath fluid immediately before the flow cell. 53. The method of any one of claims 46 to 52, wherein the controller controls a waste subsystem control feedback circuit to regulate the vacuum-modulated waste subsystem based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem. 54. The method of claim 53, wherein the pressure in the vacuum-modulated waste subsystem is measured by a waste subsystem pressure transducer. 55. The method of claim 54, wherein the waste subsystem pressure transducer is configured to measure the vacuum pressure in the vacuum accumulator.

[0081] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, provided that such substitution is not technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be within the scope of the subject matter defined by the appended claims.

[0082] It will be understood by those skilled in the art that the terms used herein in general, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including, but not limited to," etc.). It will be further understood by those skilled in the art that where a specific number of introduced claim recitations are intended, such intention will be expressly recited in the claim; in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an," the use of such phrases should not be construed as meaning that introducing a claim enumeration with the indefinite article "a" or "an" limits any particular claim including such an introduced claim enumeration to embodiments including only one such enumeration (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"). The same is true for the use of definite articles used to introduce claim enumerations. Also, even if a specific number of introduced claim enumerations is explicitly recited, those skilled in the art will recognize that such enumeration should be construed to mean at least the recited number (e.g., a bare enumeration of "two enumerations" without any other modifiers means at least two enumerations, or more than two enumerations).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or disjunctive phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0083] Furthermore, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of individual members of the Markush group, or any subgroup of members of the Markush group.

[0084] As will be understood by those skilled in the art, for any and all purposes, including providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations thereof. Any listed range can be readily recognized as fully indicating and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited numbers and can subsequently be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0085] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this invention, certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0086] Thus, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various modifications, not explicitly described or shown herein, which embody the principles of the present invention and are within the spirit and scope of the present invention. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts provided by the inventors to further advance the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended as a public dedication, regardless of whether such disclosure is expressly recited in the claims.

[0087] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) expressly defines a claim limitation as invoking only when the exact phrase "means for" or "step for" is recited at the beginning of the claim limitation. If such exact phrase is not used in the claim limitation, then neither 35 U.S.C. 112(f) nor 35 U.S.C. 112(6) applies.

[0088] CROSS-REFERENCE TO RELATED APPLICATIONS Under 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 764,844, filed August 15, 2018, the disclosure of which is incorporated herein by reference.

Claims

1. 1. A fluid management system for a flow particle analyzer, comprising: a flow cell having an input and an output; a sample input line for fluidly coupling a sample source to said input; a pump-modulated sheath fluid subsystem comprising a positive displacement pump for fluidly coupling a sheath fluid source to the input; a vacuum modulated waste subsystem comprising a vacuum pump operably coupled to a vacuum accumulator for fluidly coupling a waste reservoir to the output, and a waste subsystem pressure transducer configured to measure vacuum pressure within the waste subsystem; Equipped with the fluid management system is configured to have a constant fluid resistance coupling during operation; the fluid management system is configured to control a sample flow rate through the combined operation of the positive displacement pump of the pump-modulated sheath fluid subsystem and the vacuum pump of the vacuum-modulated waste subsystem; the vacuum pump is configured to set a sample flow rate based on the pressure measured at the waste subsystem pressure transducer. Fluid management systems.

2. The fluid management system of claim 1 , wherein the flow particle analyzer is a flow cytometer.

3. 3. The fluid management system of claim 1, wherein the sample input line is fluidly coupled to a sample source, the pump-modulated sheath fluid subsystem is fluidly coupled to a sheath fluid source, and the vacuum-modulated waste subsystem is fluidly coupled to a waste reservoir.

4. 4. The fluid management system of claim 3, wherein the positive displacement pump of the pump-modulated sheath fluid subsystem is configured to generate a flow rate of the sheath fluid by pumping the sheath fluid from the sheath fluid source to the input of the flow cell.

5. The fluid management system of claim 1 or 2, wherein the pump-modulated sheath fluid subsystem comprises a degassing device.

6. The fluid management system of claim 1 or 2, wherein the pump-modulated sheath fluid subsystem comprises a pulsation damper configured to damp fluid pulsations within the pump-modulated sheath fluid subsystem.

7. 3. The fluid management system of claim 1, further comprising a controller configured to control a flow rate of sample through the flow cell by the combined operation of the positive displacement pump of the pump-modulated sheath fluid subsystem and the vacuum pump of the vacuum-modulated waste subsystem.

8. The fluid management system of claim 7 , wherein the controller modulates the flow rate of the sheath fluid based on a measured pressure difference between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem.

9. 9. The fluid management system of claim 8, wherein the controller controls a sheath fluid subsystem control feedback circuit to regulate the pump-modulated sheath fluid subsystem based on a measured pressure differential between the pump-modulated sheath fluid subsystem and the vacuum-modulated waste subsystem.

10. 3. A flow particle analyzer comprising the fluid management system of claim 1 or 2, wherein the flow particle analyzer is a flow cytometer.

11. 1. A method for analyzing particles in a sample, comprising: analyzing the particles in the sample using a flow particle analyzer comprising a fluid management system; The fluid management system comprises: a flow cell having an input and an output; a sample input line fluidly coupling a sample source containing the sample to the input; a pump-modulated sheath fluid subsystem comprising a positive displacement pump fluidly coupling a sheath fluid source to the input; a vacuum modulated waste subsystem comprising a vacuum pump operably coupled to a vacuum accumulator fluidly coupling a waste reservoir to the output, and a waste subsystem pressure transducer configured to measure vacuum pressure within the waste subsystem; Equipped with the fluid management system having a constant fluid resistance coupling; the fluid management system is configured to control a sample flow rate through the combined operation of the positive displacement pump of the pump-modulated sheath fluid subsystem and the vacuum pump of the vacuum-modulated waste subsystem; the vacuum pump is configured to set a sample flow rate based on the pressure measured at the waste subsystem pressure transducer. method.

12. The method of claim 11 , wherein the flow particle analyzer is a flow cytometer.

13. A fluid management system as described in claim 1 or 2, wherein the vacuum accumulator is configured to maintain the vacuum pressure generated by the vacuum pump.

14. A method as described in claim 11 or 12, wherein the vacuum accumulator is configured to maintain the vacuum pressure generated by the vacuum pump.

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