Patent ID: 12228494

It will be recognized that some, or all, of the Figures are for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown. The Figures are provided for the purpose of illustrating one or more embodiments with the explicit understanding that they will not be used to limit the scope or the meaning of the claims.

DETAILED DESCRIPTION

In the following detailed description of the embodiments, numerous specific details are set forth. However, it will be obvious to one skilled in the art that the embodiments may be practiced without these specific details. In other instances, well known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the embodiments. The various sections of this description are provided for organizational purposes. However, many details and advantages apply across multiple sections.

Referring now toFIG.1A, to put the disclosed embodiments in context, a basic conceptual diagram of a cell sorter system90and a flow cytometer system90is shown. Five major subsystems of the system90include an excitation optics system102, a fluidics system104, an emission optics system106, an acquisition system108, and an analysis system110. In the case of a cell sorter system, the system further includes a cell sorting system109. Generally, a “system” includes (electrical, mechanical, and electro-mechanical) hardware devices, software devices, or a combination thereof.

The excitation optics system102includes, for example, a laser device112, an optical element114, an optical element116, and an optical element,118. Example optical elements include an optical prism and an optical lens. The excitation optics system102illuminates an optical interrogation region120. The fluidics system104carries fluid samples122through the optical interrogation region120. The emission optics system106includes, for example, an optical element130and various optical detectors including a side scatter (SSC) channel detector, fluorescent wavelength range one (FL1) detector, fluorescent wavelength range two (FL2) detector, fluorescent wavelength range three (FL3) detector, fluorescent wavelength range four (FL4) detector, and fluorescent wavelength range five (FL5) detector. The emission optics system106gathers photons emitted or scattered from passing particles. The emission optics system106focuses these photons onto the optical detectors SSC, FL1, FL2, FL3, FL4, and FL5. Optical detector SSC is a side scatter channel. Optical detectors FL1, FL2, FL3, FL4, and FL5are fluorescent detectors may include band-pass, or long-pass, filters to detect a particular and differing fluorescence wavelength ranges. Each optical detector converts photons into electrical pulses and sends the electrical pulses to the acquisition (electronics) system108. The acquisition system108, including one or more analog to digital converters and digital storage devices, processes and prepares these signals for analysis in the analysis system110.

The disclosed embodiments are employed for the most part in the fluidics system104of a cell sorter system and a flow cytometer system. U.S. patent application Ser. No. 15/817,277 and U.S. patent application Ser. No. 15/942,430 disclose exemplary flow cytometer systems that are incorporated by reference. U.S. Pat. No. 9,934,511 discloses a cell sorter system that is incorporated herein by reference.

Fluidics System Overview

Referring now toFIG.1B, a schematic diagram of a central source pressure-based cytometer fluidics system100. A legend150is provided to help identity some components of the system100and to help keep the figures uncluttered. The system100may include, without limitation, one or more of each of the following components: a sheath tank102, a manifold assembly104, one or more isolation valves115(e.g., valves V1-V5, and valve P2), one or more ports (e.g., ports p1-p1), a linear valve106, a waste tank108, a rotary valve110, a sample probe112, a sample loop114(e.g., 1-ml sample loop), one or more pinch valves116(e.g., valves V6and V7), a check valve118, a pump120, and a flow cell122, a flow meter124, a transducer126, an 0/1 sensor128(e.g., sensors S1-S8), a degasser130(e.g., degasser vacuum switch S18), a flow switch132(e.g., waste switch S8), a filter134, a pressure regulator136, a junction138, a sheath line140, a waste line142, an air/gas line144, and a sample line146. Lines are identified as lines1-23. A line is a conduit or tube through which fluid (e.g., gas or liquid) flows. The components may be coupled as shown to form the system100.

A goal of the system100is to push sheath fluid and sample fluid to the flow cell122in the most stable means possible. The pressure regulator136applies pressure to the sheath tank102. That pressure pushes sheath fluid and sample fluid throughout the system100. Pressurized sheath fluid and sample fluid can flow to the flow cell122and through the flow cell122. The flow cell122counts events, for example, counts sample particles of interest passing through the flow cell122. The pressurized fluid (e.g., sample and sheath) streams out of a nozzle at the bottom of the flow cell122. The system100breaks up the pressurized fluid that streams out of the nozzle into droplets. The system100can then selectively impart an electric charge on particularly identified droplets emanating from the nozzle. The electric charge on the droplets enables the system100to deflect each charged droplet off center, away from a normal center stream. The deflection enables the system100to sort the fluid (droplets) emanating from the nozzle. The system100can thereby collect charged droplets (sorted fluid) in one or more separate vessels from those that were uncharged (unsorted fluid). Unsorted fluid, usually in a center stream, is typically waste. Side streams are typically the charged droplets (sorted fluid) of interest. The system100sorts the fluids and sends unsorted fluids to waste via an aspirator (not shown) through valve V7. Accordingly, the aspirator aspirates air/gas, the waste fluid, and sheath fluid into the manifold assembly104. The manifold assembly104has a pump that pumps the waste fluids into the waste tank108.

In more detail, sheath fluid enters the system100via line1and flows through a valving network. The sheath line from the sheath tank splits into two branches. In a first branch, sheath fluid (100% pure sheath) flows to the flow cell122via line6. In a co-flowing second branch (e.g., parallel second branch), sample fluid (e.g., blood sample) flows through the sample loop114, which runs through the rotary valve110and into the flow cell122. A rotary valve110determines the components to which the sample loop114is coupled. For example, when the system100is aspirating sample fluid, the rotary valve110couples the sample loop114to the sample probe112. When the system100is running the rotary valve110couples the sample loop114to the flow cell122. The system100opens valve V5. Sample fluid and sheath fluid flow into the flow cell122. Both the sample fluid and sheath fluid are driven by the same pressure but are flowing through paths of different resistances (e.g., different restrictions).

The restriction of the sample loop114is substantially higher than the restriction of the sheath line140. The restrictions ratio (sample restriction/fluid restriction) governs the ratio of sample fluid to sheath fluid. The linear valve106modulates the restriction in the sheath line140to obtain different sample fluid flow rates through the sample loop114. The linear valve106can modulate the restriction by varying the length of the sheath line140. Differences in path lengths and diameters also contribute to differences in restrictions.

In a traditional system (not shown), a first pressure regulator pressurizes a sheath tank, while a second pressure regulator pressurizes a sample tube. A pressure difference between the sheath fluid pressure and the sample fluid pressure governs a flow rate of the sample fluid that is pushed into a flow cell. So, there is a separate sample tank (like the sheath tank) that has a second pressure regulator that is different than the first pressure regulator for the sheath tank. Unfortunately, such a traditional system is problematic. The multiple pressure regulators might not be perfectly in sync, but the multiple pressure regulators must be perfectly in sync. Destructive back filling might occur on the sample fluid, thereby destroying the sample fluid and introducing undesirable bubbles into the traditional system. Also, having multiple pressure regulators is pricey.

Advantageously, the present system100solves the problems that are present in the traditional system by using one pressure regulation system for both the sheath fluid (e.g., sheath line140) and the sample fluid (e.g., sample loop114). The pressure regulation system includes the single pressure regulator136and the transducer126. The system100has no other regulated pressure source than the pressure regulator136. Accordingly, the system100does not experience sync issues with pressurization. Also, a system100with a single pressure regulator136is less expensive, simpler, easier to manufacture, and has lower maintenance costs than a traditional system that has multiple pressure regulators.

For the system100to analyze a sample fluid, there needs to be a mechanism for getting the sample fluid into the sample114. In this case, the linear valve106(e.g., piston pump, syringe drive) splits duties. Most of the time, the linear valve106is governing restriction. When it is time to pull sample fluid into the sample loop114, the linear valve106switches duties and acts like a syringe drive. The rotary valve110then switches position, while the linear valve106draws sample into the sample loop114. The rotary valve110then switches to a normal rotary valve position, while the linear valve106returns to the restrictive position. A valve is opened to allow both sheath fluid and sample fluid to enter the flow cell122at substantially the same time.

Electrical Analog

FIG.2is a schematic diagram showing an electrical analog204to a simplified model202of the system100. The simplified model202includes, without limitation, sheath fluid210(e.g., sheath-in), sample fluid212, waste fluid214(e.g., waste-out), a shear valve216, the linear valve106, the sample loop114, the pressure transducer126, the flow cell122, and the valves V1, V2, V4-V7, and P2.

The electrical analog204is a circuit that includes, without limitation, a sample resistor Rsa, a sheath resistor Rsh, a flow cell resistor Rfc, and a nozzle resistor Rn. The electrical analogy164also shows the context of the sheath fluid210, the waste fluid214, the linear valve106, the sample loop114, the pressure transducer126, the flow cell122, and a nozzle218of the flow cell122.

The sample resistor Rsais analogous to the resistance (e.g., restriction) in the sample fluid path at the sample loop114. The variable sheath resistor Rshis analogous to the resistance (e.g., restriction) in the sheath fluid path at the linear valve linear valve106. The flow cell resistor Rfcis analogous to the resistance (e.g., restriction) at the flow cell122. The nozzle resistor Rnis analogous to the resistance (e.g., restriction) at the nozzle218of the flow cell122.

A total current Qtotis analogous to the flow rate of the total fluid or sheath fluid210. A sample current Qsais analogous to the flow rate of the sample fluid212at the sample loop114. A sheath current Qshis analogous to the flow rate of the sheath fluid210at the linear valve106.

The sample resistor Rsaand the variable sheath resistor Rshare coupled in parallel and share a first node and a second node. The flow cell resistor Rfcis coupled to the second node. The flow cell resistor Rfcis coupled to the nozzle resistor Rnat a third node. The total current Qtotenters the electrical analog204at the first node and is split between the sample current Qsaand the sheath current Qsh. The sample current Qsapasses through the sample resistor Rsa. The sheath current Qshpasses through the variable sheath resistor Rsh. The sample current Qsaand the sheath current Qshcombine to form the total current Qtot. So, the sample current Qsaplus the sheath current Qshequals the total current Qtot. The total current Qtotpasses through the flow cell resistor Rfcand the nozzle resistor Rn. The sample current Qsacan be described by using the following equation:

Qsa=Qtot⁢11+RsaRshEq.1

Accordingly, the flow rate of the sample fluid212can be described by using the following equation:

Flow⁢Ratesa=Flow⁢Ratetot⁢11+RestrictionsaRestrictionshEq.2

Restrictionsais the resistance (e.g., restriction) in the sample fluid path at the sample loop114. Restrictionshis the restriction (e.g., restriction) in the sheath fluid path at the linear valve linear valve106. Flow Ratetotis the flow rate of the total fluid or sheath fluid210. Flow Ratesais the flow rate of the sample fluid212at the sample loop114.

Fluidics Bucket

FIG.13is a conceptual drawing of a fluidics bucket1300for the central source pressure-based cytometer fluidics system100.FIG.13provides a more realistic depiction of some components of the fluidics system100. The fluidics bucket1300includes, without limitation, the manifold assembly104, the linear valve106, the check valve118, the aspirator pump120, the degasser element130, the degasser pump133, the degasser switch S18, the sheath filter134, and the sheath regulator136.

Start Stream

FIG.3is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a start stream path302of sheath fluid. In one embodiment, the start stream path302includes a simple path (or simplest path), and a low restriction path (or lowest restriction path), for sheath fluid to flow to the flow sell122. The sheath fluid flows from the sheath tank to102through line1, line2, degasser130, valve V4, valve V1, line6, and the flow cell122. An example total flow rate of fluids in the start stream path302is about 6 milliliters per minute (ml/min). Because sample fluid is not running in this startup operation, the flow rate of the sheath fluid in the start stream path302is equal to the total flow rate (e.g., 6 ml/min). The pressure regulator136maintains the total flow rate in the system100.

The pressure regulator136and the transducer126are coupled via a feedback loop (e.g., electronics feedback circuit). The transducer126is in line with the fluid that is at, under, or after valve V4. The transducer126may include, for example, a piezoelectric transducer that measures pressure at valve V4. The transducer126converts pressure-to-voltage or pressure-to-current (e.g., the opposite of what the pressure regulator136does).

The pressure regulator136maintains pressure at a substantially fixed level and regulates the total flow rate in the liquid path302by regulating air. The pressure regulator136may include, for example, a piezoelectric transducer that converts voltage-to-pressure or current-to-pressure (e.g., the opposite of what the transducer126does). The pressure regulator136attempts to maintain the liquid pressure that the transducer126senses at valve V4regardless of what is running through the start stream path302. Accordingly, the behavior of the pressure regulator136does not depend on whether sheath fluid or sample fluid is running through lines.

In contrast, a traditional system (not shown) typically includes a transducer in the sheath tank to measure the pressure of the air/gas above the fluid in the sheath tank. The traditional system attempts to regulate sheath fluid pressure via the air/gas in the sheath tank. Such a setup can be problematic because whenever the fluid level in the sheath tank changes, the air/gas gap above the fluid changes, and the effective pressure of the sheath fluid changes. The traditional system must compensate for such changes. Pressure instabilities often occur.

Advantageously, the present system100eliminates such problems. For example, the transducer126senses a measured pressure in the start stream path302passing through valve V4, as opposed to a pressure regulator trying to regulate the air pressure within the sheath tank102. The transducer126converts and/or translates that liquid pressure into a voltage and/or current. The transducer126communicates that voltage and/or current to the pressure regulator136via the electrical feedback loop. The transducer126measures pressure independently of the sheath tank102. By regulating pressure via the single pressure regulator136, the system can substantially eliminate undesirable head effects (e.g., frothy foam) in the sheath tank102.

The transducer126changes the level of an electrical signal (e.g., voltage and/or current) applied to the pressure regulator136via the feedback loop. For example, the transducer126sends, and the pressure regulator136receives, an electrical signal between a lower voltage (e.g., 0 Volts) and an upper voltage (e.g., 10 Volts). The pressure regulator136translates that electrical signal into a regulated pressure. Accordingly, the transducer126modulates the pressure regulator136. The pressure regulator136, in turn, regulates air pressure such that the liquid pressure, and liquid flow velocity, downstream from valve V4remains substantially constant.

The degasser130helps minimize the amount of air/gas bubbles in the system100. The degasser130is situated in line between line1and line2and actively pulls air/gas molecules (e.g., nitrogen and oxygen) out of the sheath fluid path. Consider, for example, a nozzle on the flow cell122having a diameter of 70 micrometers running at 70 psi, taken to a nozzle having a diameter of 100-micrometer nozzle running at 18 psi. When the pressure is dropped in such a manner, air/gas can come out of out of solution in the sheath tank, which leads to air/gas bubbles at the flow cell. The bubbles are typically microscopic and may not cause problems at the transducer126. However, the microscopic bubbles tend to show up at the flow cell as an unacceptable amount of background noise. The degasser130helps minimize such microscopic bubbles and background noise.

Purge Flow Cell

FIG.4is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a flow cell purge path302. A purpose of the flow cell purge path302is to purge air/gas from the start stream. In one embodiment, the flow cell purge path402includes, without limitation, line12, port p6, and valve V2of the manifold assembly104.

On a first path, the sheath essentially flows like fluid in the start stream path302that is discussed with reference toFIG.3. For example, the sheath fluid flows into the manifold assembly104via port p1, flows through valve V4, flows through valve V1, flows through line6, and enters the flow cell122. Ideally, the system100ejects a continuous liquid jet through a nozzle at the terminus of the flow cell122and into an aspirator.

However, at system start up, there may be air/gas in the lines and components that causes the sheath stream not to form a straight and stable liquid jet. For example, the sheath fluid that is flowing out of the nozzle of the flow cell122into an aspirator initially may not be flowing out of the nozzle straight and predictably, due to air/gas in the system100. The diameter of the opening of the nozzle of the flow cell122may be, for example, 70 micrometers. Unfortunately, unwanted air/gas in the flow cell122causes energy to be wasted on compressing the unwanted air/gas, as opposed to producing accurately sample droplets from the nozzle of the flow cell122. Unwanted air/gas bubbles in the system100act like shock absorbers, which is undesirable.

A co-flowing second path (e.g., parallel flow cell purge path402) couples the flow cell to the manifold assembly104. To purge air/gas bubbles, the system100cycles several times between the operations of the flow cell purge path402and the start stream path302. Operations of the flow cell purge path302include forcing air/gas bubbles in the flow cell122to get swept out through line12and purged into the bottom of the manifold assembly104. The system then cycles to operations of the start stream path302, as discussed with reference toFIG.3. The cycling can occur, for example, about 3-5 times in rapid succession. The cycling breaks up air/gas in the flow cell122into many smaller bubbles and purges that air into the bottom of the manifold assembly104.

Advantageously, the flow cell purge path302improves the effectiveness of a drop drive, which is the ability of the flow cell122to produce consistent and predictable droplets from the nozzle. The flow cell purge path302includes a droplet driving transducer in the flow cell assembly to work more accurately and efficiently. The drop drive transducer is a vibrating piezo element. For example, the transducer may be vibrating at a frequency of between 20,000 Hz and 80,000 Hz. When the stream exits the nozzle of the flow cell122, the transducer vibration frequency (e.g., 20,000 Hz-80,000 Hz) should produce droplets at the same rate (e.g., 20,000-80,000 droplets per second). Without unwanted air/gas in the flow cell122, energy is not wasted on compressing the unwanted air/gas. Instead, the system100can focus energy from the transducer on producing droplets accurately and effectively.

The nozzle at the end of the flow cell122is a convectively dominated component. A pressure drop at the nozzle of the flow cell122is primarily due to Bernoulli's principle, which states that the speed of a fluid increases linearly as the decrease of the square root of the static pressure. The nozzle of the flow cell122is accelerating the flow of fluid from one diameter to another. That acceleration is substantially independent of viscosity. If the pressure drop can be held constant across the nozzle, then the flow cell122is effectively a constant volume device. Importantly, a constant volume device that performs convective flow across the nozzle is substantially independent of temperature. So, if the system100can regulate the line pressure in the system, then the flow cell122can have a volumetric flow rate that is, in some embodiments, accurate to within +/−2% across a broad operating temperature range. Stability is enhanced by regulating on feedback from the liquid line pressure provided by the Pressure Transducer126instead of air pressure over the sheath tank. Doing so eliminates the sheath tank's gravity head from the regulation process and helps maintain a constant pressure drop across the nozzle. This, ensures that droplets produced by the jet emanating from the nozzle at the end of the flow cell122can be formed in a substantially identical manner.

Aspirate Sample Fluid

FIG.5is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for sample aspiration path502. A purpose of the sample aspiration path502is to prepare the sample fluid for running in the system. On a first path, the sheath flows like fluid in the start stream path302that is discussed with reference toFIG.3. For example, the sheath fluid flows into the manifold assembly104via port p1, flows through valve V4, flows through valve V1, flows through line6, and enters the flow cell122. The system100ejects a continuous sheath stream through a nozzle and an aspirator of the flow cell122.

On a co-flowing second path (e.g., parallel aspirate sample path502), the rotary valve couples the sample loop114to the sample probe112. The linear valve106starts at a top position (e.g., piston in bore is at a top position). The linear valve106acts like a highly accurate syringe by increasing volume in the linear valve. For example, the piston in the bore moves downward and/or moves to increase vacuum suction in the linear valve. The linear valve106thereby pulls sample fluid into the sample loop114from the sample probe112. In one embodiment, the sample loop114includes line9, the rotary valve110, the flow meter124, line7, the manifold assembly at ports p2and port p3, and line4.

However, the flow meter124is typically calibrated for sheath fluid (e.g., an aqueous solution). Sample fluid (e.g., blood sample) running through the flow meter124could cause the flow meter124to be off accuracy by as much as 5%. So, the sample fluid typically goes only partially through the sample loop114and does not reach the flow meter124. The flow meter124typically only has sheath fluid (e.g., an aqueous solution) passing therethrough and is not contaminated with sample fluid. For example, the linear valve106may aspirate 0.5 ml of sample fluid, while the volume of the sample loop114is 1.0 ml. Such a volume of sample fluid would not reach the flow124if the system100is aspirated properly.

Advantageously, the linear valve106has a variable volume that can, for example, accurately aspirate 480 ml of sample fluid and leave 20 ml of air/gas right up near the rotary valve110in line9. The system100wastes a minimal amount of sample fluid in the sample loop114. The rotary valve110can then switch over to line8without introducing unwanted air/gas into the system100. The rotary valve110enables substantial freedom on the placement of the sample probe112with respect to other components of the system100. In one embodiment, sample probe112is near the flow cell122. However, the sample probe112can be placed almost anywhere.

Boost Sample Fluid

FIG.6is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a boost sample path602. On a first path, the sheath essentially flows like fluid in the start stream path302that is discussed with reference toFIG.3. For example, the sheath fluid flows into the manifold assembly104via port p1, flows through valve V4, flows through valve V1, flows through line6, and enters the flow cell122. The system100ejects a continuous sheath stream through a nozzle and an aspirator of the flow cell122.

On a co-flowing second path (e.g., parallel boost sample path602), the rotary valve110is switched over to line8without introducing unwanted air/gas into the system100. The linear valve106can then perform a boost sample operation. The linear valve106moves to decrease volume in the linear valve106. For example, the piston in the bore moves upward and/or moves to decrease vacuum suction in the linear valve. The linear valve106can drive an accurate amount of sample fluid such that the sample fluid abuts (or is substantially near) components of the flow cell124. In that way, when the system later begins to run the sample fluid, the sample fluid is available at the flow cell124immediately or within a minimal amount of time. In one embodiment, the volume in line8between the rotary valve110and the flow cell124is about 25 ml. The system is then ready to run the sample fluid.

Run Sample Fluid

FIG.7is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a run sample path704. The sample fluid is already in the sample loop114. By turning valve V4off and then turning valves V5and P2on, the system100enters a co-flowing path scenario (e.g., parallel path scenario). The sheath pressurizes line2at port p1. That pressure then causes fluid to flow through valve V5and exit ports p2and p3, simultaneously.

On a first path (e.g., run sample path704), sample fluid exits the manifold assembly104at port p2, flows through line7, flows through the rotary valve110, flows through line8, and enters the flow cell122. Advantageously, this first path is relatively simple and facilitates cleaning of sample fluid in the system100. Sample fluid can be difficult to clean because sample fluid tends to get stuck in any crevices or tight spots in the sample fluid path.

On a co-flowing second path (e.g., parallel sheath path702), sheath fluid exits port p3, flows through line3, flows through line8, enters the linear valve106at a piston. The sheath fluid exits the linear valve106at valve P2, flows through line5, enters the manifold assembly104at port p5, flows through valve V1, exits the manifold assembly at port p4, flows through line6, and enters the flow cell122.

The linear valve106acts like a variable restrictor (or a variable resistor in an electronics analogy) on the sheath fluid. The linear valve106includes a piston and a bore. The linear valve modulates the amount of restriction on the sheath fluid by varying the length of the gap between the piston and the bore. When the piston is lowered in the bore, the sheath fluid must go through a shorter gap path in the linear valve106and thereby undergoes less restriction. When the piston is raised in the bore, the sheath fluid must go through a longer gap path in the linear valve106and thereby undergoes more restriction.

Accordingly, the linear valve106enables control of flow rates in the system100. To get a higher sample flow rate in the system100, the piston in the linear valve106is raised, which causes sheath to be more restricted. However, the system100maintains a balance between the sheath fluid flow rate and the sample fluid flow rate, such that the total flow rate in the system100remains substantially constant. So, the system100responds to the decrease in the flow rate of sheath fluid by automatically and instantaneously increasing the flow rate of the sample fluid through the system100. More sample fluid thereby gets pushed through the flow cell122.

The pressure transducer126, which is coupled below valves V1and V4, is in liquid communication with both co-flowing paths (e.g., parallel sheath fluid path and parallel sample fluid path). The transducer126is also discussed with reference toFIG.3.

Recover Sample Fluid

FIG.8is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a recover sample path802. A purpose of the recover sample path802is to recover unused sample fluid back into the sample probe112.

On a first path, the sheath essentially flows like fluid in the start stream path302, which is discussed with reference toFIG.3. For example, the sheath fluid flows into the manifold assembly104via port p1, flows through valve V4, flows through valve V1, flows through line6, and enters the flow cell122. The system100ejects a continuous sheath stream through a nozzle and an aspirator of the flow cell122.

In a co-flowing second path (e.g., parallel recover path802), the sample moves in an opposite direction from the aspirated sample fluid path502, which is discussed with reference toFIG.5. In the recover sample path802, the rotary valve110is switched to line9and couples the sample loop114to the sample probe112. The linear valve106starts at a bottom position (e.g., piston in bore is at a bottom position). The linear valve106acts like a highly accurate syringe by moving up (e.g., the piston in the bore moves up) and pushes sample fluid out of the sample loop114and into the sample probe112.

Advantageously, the system100can recover unused sample fluid in a metered and careful way. Sample fluid may cost, for example, $10,000 USD. The system100may have previously pulled in, for example, 500 ml of sample fluid during operations for the aspirate sample path502and may have used only 200 ml of sample fluid during the run sample path704. The linear valve106can then move the piston up a distance to enable the system100to recover most of the 300 ml of unused sample fluid (e.g., the 500 ml of aspirated sample fluid minus the 200 ml of used sample fluid). The system may have to waste a minimal amount of the 300 ml of unused sample fluid (e.g., about 25 ml left over in line8between the rotary valve110and the flow cell122).

In a traditional system (not shown), back filling the sample fluid is a problem. A traditional system separately pressurizes the sample fluid and the sheath fluid by using separate regulators at the same time. If the sample fluid's pressure regulator is malfunctioning, the seal on the sample vessel is not good enough to keep the sample fluid pressurized, and the system starts running the sample fluid, then sheath fluid is pushed right into the sample vessel. The system then backfills, dilutes, and can destroy the sample fluid.

Advantageously, the present system100substantially eliminates the backfilling problem by having one pressure regulator136for the system100, instead of a pressure regulator for sheath fluid and another pressure regulator sample fluid. The system100does not separately pressurize the sample fluid. The system100aspirates sample fluid by using a syringe drive as discussed with reference toFIG.5. The system100boosts sample fluid as discussed with reference toFIG.6. The system100then runs sample fluid as discussed with reference toFIG.7. Accordingly, the system100does not have to rely on a seal to pressurize the sample fluid and push the sample fluid through the system100.

SIT Flush

FIG.9is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a SIT (sample injection tube) flush path904. A purpose of the SIT flush path904is clean the sample loop114to ensure carryover sample fluid is mitigated from sample-to-sample. In one embodiment, the sample loop114to be flushed includes, without limitation, lines1-9. The aspiration line includes, without limitation, line11.

The system100opens the rotary valve110so that the SIT flush path904experiences a full pressure of sheath fluid being pushed through the sample loop114and down through the sample probe112. During this time, the system100opens valve V6and aspirates the droplets coming out of the end of the sample probe112via valve V6before the droplets drip out of the sample probe112. Such aspiration washes the inside and outside of the sample line114simultaneously to eliminate a previous sample fluid, in preparation for a future sample fluid.

Normal Stream Aspiration

FIG.10is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a normal stream aspiration path1004. In one embodiment, the operation for the normal stream aspiration path1004is a background operation that the system100may be performing routinely. In one embodiment, the normal stream aspiration path1004includes, without limitation, line10and port p10of the manifold assembly104.

Valve v7is typically open while the system100is active. If there is any fluid coming out of the nozzle from the flow cell122, then the system100aspirates that fluid away via the normal stream aspiration path1004.

Purge Filter

FIG.11is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a purge filter path1104. A purpose of the purge filter path1104is to remove air/gas from the sheath filter134. In one embodiment, the purge filter path1104includes, without limitation, line20, line21, port p9, and valve V3. The purge filter path1104is important because the sheath fluid must remain wetted. Otherwise, sheath fluid that is not fully wetted affects the resistance of the system in unpredictable ways.

Waste Aspiration

FIG.12is a schematic diagram of the central source pressure-based cytometer fluidics system100during an operation for a waste aspiration path1202. In one embodiment, the waste aspiration path1202includes, without limitation, port p7and lines13-15.

The pump120is couple to a tube/straw that reaches to the bottom of the manifold assembly104. The pump120aspirates away any fluid that is being deposited into the bottom of manifold assembly104. The pump120pumps that fluid into the waste tank108. Such fluid may include waste such as cleaning bleach, contaminated sheath fluid, etc.

Method

FIG.14is a flowchart of a method1400for operating the central source pressure-based cytometer fluidics system100. Other details, discussed with reference to other figures, may also be part of the method1400.

In step1405, the system100starts the sheath fluid flowing in the start stream path302. The system pressurizes sheath fluid via a pressure regulation system, including the single pressure regulator136and the transducer126. The pressure regulator136is not located inside the sheath tank102. The pressure regulator136receives electrical feedback from the transducer126to provide pressure regulation for the system100.

In step1410, the system purges the flow cell122. The system100cycles multiple times between operations for the purge path302and operations for the start stream path302. Like the startup operations, the purging involves the system100using the single pressure regulator136.

In step1415, the system100aspirates the sample fluid. The system100rotates the rotary valve110toward the sample probe112, moves the linear valve106downward, and sucks sample fluid from the sample probe112.

In step1420, the system100boosts the sample fluid. The system100by rotates the rotary valve110toward the flow cell122, moves the linear valve106upward, and forces the sample fluid to abut the flow cell122.

In step1425, the system100runs the sample fluid. The system100pressurizes the sheath fluid via the single pressure regulator136.

In step1430, the system100recovers unused sample fluid. The system100rotates the rotary valve110back toward the sample probe112and moves the linear valve106upward.

In step1435, the system100flushes the SIT. The system100by opens the rotary valve110so the system100experiences sheath fluid at full pressure.

In step1440, the system100aspirates a normal fluid stream. The system100can perform such aspiration as a routine background operation for cleaning and maintenance.

In step1445, the system purges the sheath filter path1104. The purging removes air/gas from the sheath filter path1104.

In step1450, the system100aspirates waste fluid into the bottom of the manifold assembly104.

When implemented in software, the elements of the embodiments of the invention are essentially the program, code segments, or instructions to perform the necessary tasks. The program, code segments, or instructions can be stored in a processor readable medium or storage device that can be read and executed by a processor. The processor readable medium may include any medium that can store information. Examples of the processor readable medium include, without limitation, an electronic circuit, a semiconductor memory device, a read only memory (ROM), a flash memory, an erasable programmable read only memory (EPROM), a floppy diskette, a CD-ROM, an optical disk, and a magnetic disk. The program or code segments may be downloaded via computer networks such as the Internet, Intranet, etc. and stored in the processor readable medium or storage device.

Some portions of the preceding detailed description may have been presented in terms of algorithms and symbolic representations that perform operations on data bits within a computer memory. These algorithmic descriptions and representations are the tools used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art. An algorithm is here, and generally, conceived to be a self-consistent sequence of operations leading to a desired result. The operations are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities may take the form of electrical (e.g., current or voltage) or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, levels, elements, symbols, characters, terms, numbers, or the like.

However, all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. Unless specifically stated otherwise as apparent from the above discussion, it is appreciated that throughout the description, discussions utilizing terms such as “processing” or “computing” or “calculating” or “determining” or “displaying” or the like, refer to the action and processes of a computer system, processing logic, or similar electronic computing device, that automatically or semi-automatically manipulates and transforms data represented as physical (electronic) quantities within the computer system's registers and memories into other data similarly represented as physical quantities within the computer system memories or registers or other such information storage, transmission or display devices.

Additionally, the embodiments of the invention are not described with reference to any particular programming language. It will be appreciated that a variety of programming languages may be used to implement the teachings of the embodiments of the invention as described herein.

This disclosure contemplates other embodiments or purposes. It will be appreciated that the embodiments of the invention can be practiced by other means than that of the described embodiments, which are presented in this description for purposes of illustration and not of limitation. The specification and drawings are not intended to limit the exclusionary scope of this patent document. It is noted that various equivalents for the particular embodiments discussed in this description may be practiced by the claimed invention as well. That is, while specific embodiments of the invention have been described, it is evident that many alternatives, modifications, permutations and variations will become apparent in light of the foregoing description. Accordingly, it is intended that the claimed invention embrace all such alternatives, modifications and variations as fall within the scope of the appended claims. The fact that a product, process, or method exhibits differences from one or more of the described exemplary embodiments does not mean that the product or process is outside the scope (literal scope and/or other legally-recognized scope) of the following claims.