Pipeline volume calibration system and method

Abstract

Systems and methods for pipeline volume calibration and measurement of fluid samples delivered to a point of interrogation are provided. In various embodiments, a known fluid volume comprising a sample pipeline fluid and a secondary fluid is delivered to a fluid boundary sensor. The fluid boundary sensor helps determine the positioning of boundaries between various fluids, and the positioning of these boundaries is used to determine the sample pipeline fluid volume.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to U.S. Patent Application No. 62 / 858,507, “Pipeline Volume Calibration System and Method” (filed June 7, 2019), the entire contents of which are incorporated herein by reference for any and all purposes. Technical Field

[0003] This disclosure generally relates to the measurement of pipeline volume of fluid samples. Background Technology

[0004] When analyzing very small sample volumes, such as at the microliter scale, fluid analysis instruments must be extremely precise in measuring sample volume and collecting data. For example, flow cytometry can analyze individual cells suspended in a fluid as it passes through a highly focused laser beam, obtaining information about the cells based on scattered light. In such experiments, the precise volume of the fluid sample (i.e., the pipeline volume) must be known to accurately correlate the timing of scattered light from the cells (e.g., forward scattered light, side scattered light) and also the fluorescence from fluorescent labels associated with the cells in the fluid sample. Similarly, the precise timing of sample entry into and exit from the interrogation region is crucial for determining the start / stop times for data collection and analysis. Therefore, accurately determining the fluid pipeline volume between the sample initiation point and the interrogation point is critical to system performance.

[0005] Current approaches to addressing these challenges include delaying the start of analysis of the sample fluid and ending the analysis before the sample actually begins and ends. Analysis at the front and rear ends is cut off when the sample fluid passes the interrogation point, ensuring that what is being analyzed is the sample fluid, not air or other non-sample fluids in the tubing.

[0006] However, a drawback of this method is that a portion of the sample fluid is wasted and not analyzed. This can be particularly problematic during experiments attempting to detect and analyze rare events (e.g., cells expressing rare proteins in the sample fluid), as rare event analytes present only in dilution may be missed if some sample fluid containing such analytes is discarded or not analyzed. Furthermore, some events of interest may ultimately not be analyzed because they are located at the beginning or end of the fluid sample.

[0007] Careful sample tubing fabrication methods aim to provide consistent and reliable sample tubing volumes because such methods attempt to ensure that the sample tubing volume falls within a certain tolerance or range. However, this approach typically results in higher costs for both sample tubing fabrication and testing. Furthermore, sample arrival times must be calculated with a significant margin of error during analysis to allow for variations in tubing volume. This, in turn, leads to the waste of potentially valuable samples, and portions at both ends must be discarded during analysis (and with this discarding, the potential loss of valuable information that may be critical to research objectives and clinical purposes associated with patient diagnosis).

[0008] Therefore, there has long been a need in the art for improved methods and systems for pipeline volume calibration and sample measurement. Summary of the Invention

[0009] In addressing the described challenges, this disclosure first provides systems and methods for measuring, estimating, and / or determining sample volume. One method may include: placing a calibration volume of a first fluid into the sample line such that the calibration volume completely fills the sample line, the completely filled sample line defining a volume SL therein; transferring the calibration volume of the first fluid and a displacement volume of a second fluid into a sample region, the calibration volume of the first fluid and the displacement volume of the second fluid defining a total volume; transferring the total volume from the sample region to a sensor configured to identify a boundary between the first fluid and the second fluid; and estimating the volume SL of the sample line based on the boundary.

[0010] This disclosure also provides a system and method comprising: a sample line enclosing a volume SL therein; a sample region configured to receive a first fluid from the sample line; and a fluid delivery column configured to: (a) deliver a volume of the first fluid into the sample line; (b) deliver a calibration volume CV of the first fluid completely filling the sample line from the sample line into the sample region; and (c) deliver a displacement volume D of a second fluid into the sample region such that the calibration volume CV of the first fluid and the displacement volume D of the second fluid are such that... Define a total volume TV; a sensor region configured to receive the first fluid and the second fluid from the sample region and detect the boundary between the first fluid and the second fluid; a shunt column configured to: (a) place the sample line in fluid communication with the sample region; (b) place the sample region in fluid communication with the sensor; or both (a) and (b); and optionally a processor configured to determine the volume surrounded by the first sample line based at least on the difference between the volume D of the displacement volume of the second fluid and the total volume TV.

[0011] Additionally, a system and method for estimating the volume of a sample fluid are provided, the method comprising: delivering a quantity of a first fluid into a catheter; delivering a quantity of a second fluid into the catheter to displace the first fluid within the catheter; estimating the total volume of the first fluid and the second fluid in the catheter; delivering the first fluid and the second fluid from the catheter to a sensor capable of determining a boundary between the first fluid and the second fluid; determining the volume of the second fluid in the total volume; and estimating the volume of the sample fluid based at least on the volume of the first fluid and the estimated total volume of the sample fluid and the first fluid.

[0012] A system for automatically determining the volume of a sample is also provided, the system comprising: a sensor region; a fluid delivery column configured to deliver a second fluid and a first fluid, respectively, to the sensor region, the sensor region being configured to measure a signal passing through the sensor region, the signal being different based on the presence of the first fluid or the second fluid in the sensor region; and a processor configured to determine the volume of the sample based on the signal measured when the first fluid and the second fluid are delivered through the sensor region. Attached Figure Description

[0013] The invention and the following detailed description will be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of the disclosed subject matter are shown in the drawings for the purpose of illustrating the subject matter; however, the disclosed subject matter is not limited to the specific methods, compositions, and apparatuses disclosed. Furthermore, the drawings are not necessarily drawn to scale. In the drawings:

[0014] Figure 1A-1I A description of exemplary systems and methods according to this disclosure is provided;

[0015] Figure 2 A description of exemplary embodiments according to this disclosure is provided; and

[0016] Figure 3 A flowchart of an embodiment of the disclosed technology is provided. Detailed Implementation

[0017] This disclosure can be more readily understood by referring to the following detailed description of embodiments, which are incorporated in conjunction with the accompanying drawings and examples that form a part of this disclosure. It should be understood that this disclosure is not limited to the specific apparatus, methods, applications, conditions, or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed subject matter.

[0018] Furthermore, as used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and unless the context clearly specifies otherwise, a reference to a particular numerical value includes at least that particular value. As used herein, the term “a plurality” means more than one. When expressing a series of values, another embodiment includes from one particular value and / or to other particular values. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the particular value forms another embodiment. All scopes are inclusive and composable. It should be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0019] It should be understood that, for clarity, certain features of the disclosed subject matter described herein in the context of a single embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed subject matter described in the context of a single embodiment may also be provided individually or in any sub-combination. Furthermore, any reference to values ​​described in the scope includes every value within the scope. Any references to any document herein are incorporated herein by reference in their entirety for any and all purposes.

[0020] On one hand, this disclosure provides systems and methods for measuring, estimating, and determining sample volume. Various embodiments relate to instruments that use fluid systems to deliver samples for analysis to an inquiry point. In such systems, accurately determining the fluid line volume between the sample initiation point and the inquiry point is critical to system performance. Without accurate measurement, the time it takes for the sample to reach the inquiry point is uncertain and may lead to potential data loss or corruption, as discussed herein.

[0021] Therefore, this invention offers many unique advantages over conventional systems and methods. For example, regarding manufacturing, variations during manufacturing are compensated for by determining the pipeline volume after the system is completed. More precise measurements of the pipeline volume allow for analysis using a larger percentage of the sample. This is especially important when using small sample sizes. Furthermore, if components of the system change at the customer's site, the pipeline volume can be easily determined again to accommodate any possible variations.

[0022] In various embodiments, the pipeline volume determination and sample measurement systems and methods can be installed and implemented in one or more existing systems. That is, the disclosed embodiments are not limited to the depicted illustrations and descriptions, and can be applied to current analytical systems, such as flow cytometers, to improve analysis and data collection.

[0023] Figure 1A-1I Example systems and methods for determining the volume enclosed within a sample pipeline are illustrated. In embodiments, as discussed herein, the sample pipeline may contain sample fluid for analysis in one or more instruments.

[0024] Graphic element legend

[0025] For convenience, the following is a combination Figure 1A-1I List of elements used:

[0026] 102: Fluid (e.g., focused fluid) tank

[0027] 104: First fluid (e.g., focusing fluid)

[0028] 106: Pump (configured to connect to fluid 104 from fluid tank 102)

[0029] 108: Flow divider (e.g., valve)

[0030] 110: Fluid boundary sensor (configured to detect signals related to the boundary between fluids, such as focusing on the boundary between fluid and air)

[0031] 112: Pump (configured to draw fluid into fluid boundary sensor 110)

[0032] 114: Pump (configured to draw fluid into sample area 116, or configured to discharge fluid from sample area 116)

[0033] 116: Sample area – The sample area may be located, for example, within a flow cytometer analyzer (with acoustic and / or hydrodynamic focusing of cells and other particles), an imager, a flow cytometer unit, and other particle sorters / separators.

[0034] 116a: The fluid volume filling the entire sample line 118 to the end of the sample line tip 122.

[0035] 116b: After the sample line volume 116a is connected to the sample region 116, a second fluid (e.g., an air bubble) is connected to the sample region 116.

[0036] 118: Sample line (shown as dashed line for easy reference)

[0037] 120: Overflow container (configured to receive fluid exiting the sample line head 122 of the sample line 118)

[0038] 122: Sample tubing tip (shown as dashed line for easy reference)

[0039] 124: Pump (e.g., configured to draw fluid from an overflow container and connect the fluid to tank 126)

[0040] 126: Jar

[0041] Figure 1A The initial state of a system comprising a sample line having a determined sample line volume is shown. A fluid reservoir (e.g., a focusing fluid reservoir) 102 may contain a first fluid (e.g., a focusing fluid) 104. As shown, portions of the focusing fluid can be drawn from the fluid reservoir 102 into the sample line 118. In various embodiments, the sample line and other conduits through which the fluid flows may be tubes comprising one or more of various materials.

[0042] like Figure 1B As shown, pump 106 draws first fluid 104 from fluid tank 102 and directs it toward distributor 108 (which may be, for example, a valve or rotary valve) and sample line 118. (For ease of reference, the presence of first fluid 104 is indicated by a thick black line.)

[0043] The splitter 108 can be in a state where fluid is delivered from tank 102 to sample line 118 via one or more fluid lines (unlabeled); as an example, the splitter 108 can be in a state where sample line 118 is in fluid communication with tank 102 but not with sample area 116. It should be understood that the splitter 108 can be a single unit (e.g., a single valve capable of having multiple states). In some embodiments, the splitter 108 may include multiple units (e.g., multiple valves).

[0044] The sample line 118 contains the total fluid volume to be determined. In some embodiments, the focusing fluid volume serves as the calibration volume. The sample line 118 may further include a sample line tip 122, which in some embodiments is contained in (or emptied into) an overflow container 120 configured to receive excess fluid present at the end of the sample line 118. It should be understood that the total volume of the sample line 118 includes the volume contained within the filled sample line tip 122, which is also the end of the sample line 118.

[0045] When the focusing fluid is drawn into sample line 118, such as Figure 1C As seen, excess focusing fluid is aspirated and deposited into overflow container 120. In embodiments, this may occur because the total volume of the sample line is initially unknown, thus excess fluid is pumped through to ensure that sample line 118 is completely filled. In some embodiments, the operating precision of pump 106 may be varied to aspirate excess focusing fluid 104 into the sample line. It is contemplated that operation is particularly suitable to ensure sufficient fluid communication to sample line 118 such that sample line 118, including sample head 122, is completely filled. Excess fluid will exit sample line head 122 into overflow container 120, which may be in fluid communication with the end of the sample line. In some embodiments, a fluid delivery column may be used to remove fluid from overflow container 120 while fluid is retained within sample line 118 and sample head 122.

[0046] In some embodiments, the amount of excess fluid in the sample line tip 122 and the overflow container 120 is minimized. One or more sensors in the sample line tip 122 and the overflow container 120 may provide feedback to one or more components of the depicted system (including, but not limited to, pump 106) to indicate when the sample line tip is filled and / or when excess fluid has been realized in the overflow container 120, thereby minimizing the amount of wasted focusing fluid. In this way, the system can operate in an automated manner such that, for example, when excess fluid leaves the sample line tip 122 and is detected, the system stops supplying additional fluid to the sample line 118 and removes excess fluid that has left the sample line tip 122 and accumulated in the container 120.

[0047] As shown, Figure 1D It is shown that any excess fluid in the top container can be connected to the waste tank 126 via fluid delivery column 124 (e.g., a pump). Therefore, column 124 is configured to draw fluid from overflow container 120 and connect the fluid to waste tank 126 or other waste areas. It should be understood that column 124 can be configured not to remove any focusing fluid from sample line 118. Therefore, sample line 118 remains fully filled with fluid 104 throughout its passage through the end of the sample line at sample line head 122; the volume of fluid filling sample line 118 to the end of the line is shown by 116a.

[0048] After the sample line 118 has been fully filled, the pump 114 can be operated to connect the focusing fluid contained in the sample line to the sample zone 116, which may be a sample loop. (It should be understood that although 114 is referred to as a pump for convenience, 114 may include one or more pumps, valves, etc.)

[0049] Figure 1E The volume of sample line fluid 116a is shown after fluid 116a has been connected to sample region 116. In addition to the fluid 116a from the sample line, pump 114 can also pump a second fluid, such as an air bubble, "after" the sample line fluid volume 116a. The second fluid is shown as air bubble 116b within sample region 116. Therefore, the total volume pumped into sample region 116 by pump 114 is the sample line fluid volume 116a plus the volume of air bubble 116b.

[0050] In some embodiments, pump 114 may be a pump configurable to draw upward a predetermined and / or user-specified volume of fluid, such as 450 μl (e.g., a syringe pump or other precise method). In other words, pump 114 can draw a known amount of fluid (including both the sample line fluid volume 116a and the air bubble 116b) into sample region 116. Therefore, the amount of fluid 116c drawn into sample region 116 is known because this amount 116c is the amount of fluid that pump 114 has already drawn into sample region 116.

[0051] Figure 1F The pump 112 is shown drawing fluid (including sample line fluid volume 116a and air bubble 116b) from sample region 116 toward fluid boundary sensor 110 via distributor 108 (e.g., rotary valve). Figure 1FAs shown, bubble 116b is about to enter sensor 110, with sample volume 116a following bubble 116b. A fluid boundary sensor is configured to detect a signal associated with the boundary between fluids. In some embodiments, the fluid is immiscible. That is, a sensor capable of detecting signals (such as optical, electrical, acoustic, or any combination thereof) can determine a first signal associated with a first fluid (e.g., air, bubble 116b, etc.) or calibration fluid, and then the signal obtained by the boundary sensor changes as the first fluid passes through because the properties and quality of the focusing fluid differ from the measurements of the initial calibration fluid.

[0052] In some embodiments, the fluid boundary sensor is a bubble sensor. In some embodiments, the fluid passage between the sample region 116 and the fluid boundary sensor 110 is pre-filled with a pre-filled fluid, such as the same fluid as the first fluid 104. The pre-filled fluid may be a fluid that is immiscible with the air bubble 116b.

[0053] When pump 112 has completely drawn bubble 116b into fluid boundary sensor 110, as Figure 1G As shown, sensor 110 can determine the start and end points of each of the bubble 116b and the sample line fluid volume 116a. (e.g.) Figure 1G As shown, bubble 116b has entered sensor 110, and sample line volume 116a is about to enter sensor 110; the sensor is configured to detect the “start” and “end” of bubble 116b.

[0054] As pump 112 continues to draw fluid volume 116a from the sample line to fluid boundary sensor 110, Figure 1H In the sample line fluid volume, the start and end points of the sample line fluid volume 116a may be included, as well as any additional air bubbles (if any). In various embodiments, once the sample line fluid is first identified, the presence of additional air bubbles may indicate an air leak and / or a problem with one or more components of the system (e.g., orifices in one or more pipes) that allow air bubbles to be introduced.

[0055] Therefore, the use of a fluid boundary sensor provides the start and end times of each of the various fluids initially contained in the sample region. Based on the fluid boundary information and the known volume (116c) aspirated into the sample region 116, Figure 1E As shown), the user and / or system can determine the bubble volume 116b, and thereby determine the sample line fluid volume 116a by subtracting the bubble volume 116b from the total aspirated volume 116c, i.e., 116c = 116a + 116b.

[0056] In various embodiments, readings from a fluid boundary sensor can be analyzed at a computing device including a processor, which determines the bubble volume 116b and the sample line fluid volume 116a from the boundary readings in the manner provided above. For example, certain aspects can be performed by a machine such as a computer via program code. The program code (i.e., instructions) can be embodied in a tangible storage medium or a memory medium implemented as a storage device, such as a magnetic or optical medium, a volatile or non-volatile medium. One or more programs incorporating the processes described herein can be implemented or utilized, for example, by using an application programming interface (API), reusable controls, etc., and one or more operations disclosed herein can be automated.

[0057] Figure 1I The diagram illustrates pump 112 aspirating all sample line fluid volume 116a through fluid boundary sensor 110. After passing through boundary sensor 110, the sample line fluid volume should be known, and the sample fluid can be delivered to one or more conduits for use. Sample line volume 116a can be aspirated through sensor 110 (after bubble 116b has been aspirated through sensor 110), allowing sensor 110 to detect the presence of any other bubbles (or other adsorptions) in sample line fluid 116a. Without being bound by any particular theory, the presence of one or more bubbles in sample line fluid 116a can indicate a leak (e.g., an air leak) somewhere in the system, and the identification of bubbles in sample line fluid 116a can therefore be used for system diagnostics, thereby informing the user of the presence (or absence) of a leak in the system.

[0058] It should be understood that the disclosed systems and methods are not limited to Figure 1A-1I The figures are depicted in detail. Furthermore, the figures are not necessarily drawn to scale, and the text labels on various parts of the figures are illustrative only and do not limit the disclosed technology.

[0059] In various embodiments, the pump described herein can be any of a variety of pumps, including, but not limited to, syringe pumps and diaphragm pumps, for drawing fluid to its intended location. Additionally, any of a variety of pumps can be used to load a user-specified or predetermined amount of fluid that can be implemented. For each pump referenced in the figures, one or more pumps may be used according to the embodiments described herein. It should be understood that the examples depicted are not limited to the embodiments provided in this disclosure.

[0060] Figure 2 A depiction of another exemplary system 200 according to this disclosure is provided.

[0061] In the depicted embodiment, there is an automated sampler 206 and an instrument 220. The instrument may be a flow cytometer analyzer, such as Attune.TM The NXT flow cytometer analyzer (Thermo Fisher Scientific Inc.), other flow cytometer analyzers, flow cytometer sorters / splitters, or other fluid instruments. An autosampler 206 and instrument 220 are used according to the disclosed system and method. As shown, sample fluid, such as focusing fluid, stored in storage volume 204 (e.g., a bottle or other container), is pumped upwards by pump 210 (which may be, for example, a syringe pump) through tubing (212 / 212a / 212b) across tubing 208 to sample tubing (214, 214a, 214b) spanning the autosampler 206 and instrument 220 (e.g., a cell counter). As shown, the autosampler 206 and instrument 220 may be positioned at a distance from each other, said distance may be crossed by line 212 / 212a. Pump 218 (which may be, for example, a diaphragm pump) can induce sample fluid into sample lines 214 / 214a / 214b. Pump 218 may include one or more check valves to ensure that fluid flows in only one direction. Pump 218 may also be configured to completely fill the sample lines. Container 230 can receive sample fluid from line 214. Line 216 (if present) can connect container 230 to pump 218. Instrument 220 may include a sample injection port and / or one or more sample holders; sample holders can accommodate tubes or other sample containers.

[0062] Excess focusing fluid at the end of sample line 214 can be collected and delivered to waste container 202. For example, a diaphragm pump can draw excess fluid from the head of sample line 214 into waste container 202. At this point, the exact volume of the sample line may or may not be known.

[0063] Valve 224 can be configured to allow sample fluid in lines 214 / 214a / 214b to enter sample ring 226, but not to allow sample fluid to enter lines 212b or 228. Sample fluid can be propelled into sample ring 226 by a pump, which can be a pump that delivers a known amount of fluid to the sample ring. The known amount of fluid can be, for example, all sample fluid residing in lines 214 / 214a / 214b plus an additional amount of another fluid, such as air or other gas, or other fluid. The other fluid can be a fluid immiscible with the sample fluid.

[0064] For example, after filling sample lines 214 / 214a / 214b, a pump connected to valve 224 (which can put sample ring 226 and sample lines 214 / 214a / 214b in fluid communication with each other) draws sample fluid, along with air bubbles, upward from sample lines 214 / 214a / 214b into sample ring 226; the total volume of fluid (i.e., the volume of sample line fluid plus air bubbles) is known.

[0065] A rotary valve can be actuated to fluidly communicate the sample loop and the bubble sensor with each other, and an air bubble and sample fluid can then be drawn from the sample loop through the bubble sensor using a pump. Measurements from the bubble sensor, which identifies the boundary positioning of the air bubble and sample fluid, along with a known amount drawn upwards into the sample loop, can be used to determine the volume of the air bubble and the volume of the sample fluid. When the sample line is fully filled, the volume of the sample fluid can then correspond to the volume in the sample line.

[0066] For example, the pump can be configured to draw, for example, 450 μl of fluid into the sample loop. (The total amount drawn into the sample loop, i.e., sample fluid plus air, will be greater than the total amount contained in the sample line to ensure that air bubbles are present within the sample loop.) In some cases, the amounts of focusing fluid and air can be estimated based on one or more known factors about the system (e.g., 370 μl of fluid, 80 μl of air), and in other cases, the amount of each fluid is unknown.

[0067] The valve is then adjusted (e.g., a rotary valve) to direct fluid from the sample loop 226 toward the bubble sensor 222. As the air bubble and fluid pass through the bubble sensor 222, the start and end points of each of the bubble and focusing fluid are recorded. Based on this information and the known amounts of air and focusing fluid contained in the sample loop, the bubble size can be determined. For example, if the air bubble volume is determined to be 83 μl based on the bubble sensor reading, the actual sample line fluid volume would be 367 μl (i.e., 450 μl – 83 μl = 367 μl).

[0068] It should be understood that although the figures in the examples of this invention are in the range of hundreds of microliters, the disclosed systems and methods are not limited to the microliter range. For example, based on the system and the volume to be determined, the disclosed technical concepts can be applied to determine hundreds of milliliters or more.

[0069] Furthermore, the size of the bubble is not critical and ultimately depends on the type of bubble sensor (or other fluid boundary sensor), its sensitivity, and the intended use. For example, using an ultrasonic bubble sensor, such as for flow cytometry, might lead to attempts to ensure the bubble is larger than 10 μL. However, smaller bubbles can be used. Users may want the bubble to be larger than the sensor's minimum size and be able to ensure the entire bubble is detected.

[0070] Valve 224 can then be adjusted to allow sample fluid (and other fluids) from sample ring 226 to enter line 228 (but not line 212b). The sample fluid is then transferred to sensor 222 (which may be, for example, a bubble sensor). As described elsewhere herein, the amount of fluid in sample ring 226 is known, and therefore the amount of fluid transferred from sample ring 226 to sensor 222 is also known.

[0071] Sensor 222 then detects the interface between the sample fluid and other fluids in communication with the sensor. By detecting the interface, the system can then determine the volume of fluid initially contained in sample lines 214 / 214a / 214b. In this way, the user—as described elsewhere in this document—can conduct experiments with less waste and higher throughput, because the user will know how much sample fluid is delivered to the sample loop for each experimental run performed in the sample loop.

[0072] In some embodiments, the disclosed systems and processes may be performed infrequently, such as during installation or when the system changes (e.g., after maintenance or servicing, or after replacing one or more system components). Additionally and / or alternatively, the disclosed systems and methods may be used as part of regular maintenance activities (e.g., every few months or on another recurring schedule).

[0073] Figure 3 An exemplary flowchart of method 300 according to the disclosed technology is provided. As shown, method 300 may include step 302 of (completely) filling a sample line with a first fluid. Such fluid may be, for example, a buffer solution, a sheath fluid (e.g., for acoustic and / or hydrodynamic focusing).

[0074] This can be achieved, for example, by overfilling the sample line, stopping the flow of the first fluid to the sample line, and collecting any overflow fluid from the sample line to make the sample line completely full. The total volume (TV) can then be introduced (step 304) into a holding region consisting of the sample fluid from the fully filled sample line and a certain volume of the second fluid to the holding location. The holding location can be, for example, a sample loop, a container, or other location. The TV of the first and second fluids can be introduced into a sensor region (step 306), for example, a tube, a container, etc. The sensor region can contain a sensor capable of detecting the boundary between two fluids, such as air and water—e.g., a bubble sensor. The bubble sensor can operate by ultrasound, capacitance, or other modes known to those skilled in the art. The total volume (TV) of the first and second fluids can be conveyed through the sensor region, for example, at a known flow rate. The sensor can detect (step 308) the presence of a boundary between the fluids (e.g., between the second fluid and the sample or the first fluid). Based on the boundary detection (step 310), the volume of one (or both) of the first and second fluids in the TV can be determined (manually or automatically).

[0075] As an example, if the volume of fluid (TV) is known to be 500 μL, and TV flows through the sensor region at a rate of 100 μL per minute, and the bubble sensor detects the leading boundary of TV (i.e., the location where the second fluid begins) at t = 0 seconds and the boundary between the first and second fluids at t = 1 minute after the leading boundary, then it can be determined that TV contains 100 μL of the second fluid and 400 μL of the first fluid. This further establishes the volume of the sample line as 400 μL, i.e., the volume of the first fluid in TV. Further analysis (e.g., flow cytometry) can be performed (step 312) because the volume of fluid in the (fully filled) sample line will then be known. In this way, for each analysis performed in the sample region, the precise amount of fluid delivered from the sample line to the sample region (i.e., 400 μL in the aforementioned example) is known.

[0076] Exemplary aspects

[0077] The following aspects are illustrative only and should not be construed as limiting the scope of this specification or the appended claims.

[0078] Aspect 1. A method comprising: placing a calibration volume of a first fluid into a sample line such that the calibration volume completely fills the sample line, the filled sample line defining a volume SL therein; transferring the calibration volume of the first fluid and a displacement volume D of a second fluid into a sample region, the calibration volume of the first fluid and the displacement volume D of the second fluid defining a total volume; transferring the total volume from the sample region to a sensor configured to identify a boundary between the first fluid and the second fluid; and estimating the volume SL of the sample line based on the boundary.

[0079] Fluid lines (e.g., sample lines) can be formed from flexible or rigid materials. Flexible tubing is considered suitable, but not required.

[0080] It should be understood that one or more steps in the disclosed method can be performed automatically. For example, the step of transferring the first fluid and the second fluid into the sample region can be performed automatically.

[0081] like Figure 1A-1I As shown, fluid can be drawn into the sample line, and the valve that positions the sample line in fluid communication with the sample line can be closed, allowing the sample line ( Figure 1C 118) is completely filled. The end of the sample tubing can be immersed in the water collection container (112). Figure 1C In the middle, the water collection container is then emptied, leaving the sample tubing completely filled.

[0082] Aspect 2. The method according to Aspect 1, wherein the sensor detects changes in electrical signals, changes in acoustic signals, changes in optical signals, or any combination thereof. As an example, the sensor may detect changes in optical signals based on a comparison of illuminance received when a liquid is placed within the sensor with illuminance received when air is placed within the sensor. The sensor may also be configured to detect changes (e.g., changes in conductivity, changes in optical signals, changes in acoustic signals) related to the boundary between a first fluid (e.g., air) and a second fluid (e.g., a buffer solution). The first and second fluids may be immiscible with each other.

[0083] Aspect 3. According to either aspect 1 or 2, it further includes transferring a volume of the first fluid exceeding the volume that completely fills the sample line into the sample line and removing the first fluid exceeding the volume that fills the sample line, so that the sample line is completely filled. This can be performed manually or automatically.

[0084] Aspect 4. According to any one of Aspects 1 to 2, it further includes conveying a volume of the first fluid exceeding the volume filling the sample tubing through the sample tubing and then removing the first fluid exceeding the volume filling the sample tubing, so that the sample tubing is filled to the end of the sample tubing. This in Figure 1D and Figure 1E As shown, excess fluid exits sample line 118 and at least partially fills container 120. Figure 1D Then from container 120 ( Figure 1E Remove excess fluid from the sample line 118 to fill the end.

[0085] Aspect 5. According to any one of Aspects 1 to 4, wherein the first fluid and the second fluid are immiscible. As an example, the first fluid may be a buffer solution, growth medium, sheath fluid (also referred to as a focusing fluid in some cases) or other fluid, and the second fluid may be air.

[0086] Aspect 6. According to any one of aspects 1 to 5, wherein the method is performed in an automated manner.

[0087] Aspect 7. According to any one of aspects 1 to 6, wherein the second fluid comprises air.

[0088] Aspect 8. According to any one of Aspects 1 to 7, it further includes operating the sample region to analyze one or more fluid samples, each of the one or more samples having a sample volume of said volume SL. The sample region may contain, for example, a particle concentration series (hydrodynamic concentration, acoustic concentration, or both). The sample region may contain one or more sensors (e.g., an illumination emitter and a detector) configured to interrogate one or more analytes disposed in a sample entering the sample region.

[0089] As described elsewhere in this document, in existing methods, sample arrival time is calculated with a relatively large error margin to allow for variations in tubing volume. This, in turn, leads to the waste of potentially valuable samples, and portions at both ends must be discarded during analysis (and with such discarding, the potential loss of valuable information that may be critical to research objectives and clinical purposes associated with diagnosing patients). By applying the disclosed technique, users can precisely determine the volume of the sample tubing and thus the volume of fluid (e.g., buffer, sheath fluid) sequentially delivered to a sample loop or region, which may contain one or more analytical or processing modules, such as flow cytometers, units, sorters, etc.

[0090] By understanding the amount of fluid delivered to the sample zone, users can therefore operate the sample zone more efficiently without having to discard significant portions of the sample "end" within a given analysis cycle. For example, a user who knows that 30 μL of sample is precisely delivered to the sample zone in each experimental run can then configure the sample zone to handle the middle 29.5 μL of each 30 μL portion of fluid delivered to the sample zone. On the other hand, if a user (e.g., using existing methods) only knows that the sample line contains 25 to 35 μL of sample, the user may be forced to conservatively configure the sample zone, for example, to handle only the middle 20 μL of sample, a significantly reduced volume compared to the volume that can be reliably handled using the disclosed techniques.

[0091] The advantages of the disclosed technique are therefore apparent, as knowing the volume of the sample tubing allows users to analyze a larger proportion of a given sample, since users do not need to establish error margins and discard as many samples as possible. Users can also increase throughput, as they can use fewer samples per experimental run, which in turn increases the number of experimental runs that can be performed per run. Furthermore, since fewer samples can be used per experimental run, samples (such as blood) can be obtained more easily, as less liquid is required per sample. Therefore, the disclosed technique also includes the operation of configuring (whether manually or automatically) the sample area based on the determined volume of the sample tubing. The configuration may include adjusting the sample area to operate with fewer samples, to operate with fewer sample discards (e.g., the sample "end" for a given experimental run), or other adjustments. By using fewer samples per experimental run, users can also perform experiments using fewer reagents.

[0092] Aspect 9. A system comprising: a sample line having a volume SL therein; a sample region configured to receive a first fluid from the sample line; and a fluid delivery column configured to: (a) deliver a volume of the first fluid into the sample line; (b) deliver a calibration volume (CV) of the first fluid completely filling the sample line from the sample line into the sample region; and (c) deliver a displacement volume D of a second fluid into the sample region such that the calibration volume CV of the first fluid and the displacement volume D of the second fluid are such that... D defines the total volume TV; a sensor region configured to receive the first fluid and the second fluid from the sample region and detect the boundary between the first fluid and the second fluid; a shunt configured to: (a) place the sample line in fluid communication with the sample region, (b) place the sample region in fluid communication with the sensor, or both (a) and (b); and optionally a processor configured to determine the volume surrounded by the sample line based at least on the difference between the volume D of the displacement volume of the second fluid and the total volume TV.

[0093] The fluid delivery system may include one or more pumps, valves, distributors, etc. Pumps may be syringe pumps, gear pumps, etc. It should be understood that pumps can be used to discharge fluid, but they can also be used to draw in fluid. As an example (and reference...), Figure 1D , 1E (And 1F), pump 114 can be used to draw sample line fluid 116a together with air bubbles 116b into sample region 116. Pump 114 can then be used to expel fluid 116a and air bubbles 116b from sample region, and to cause fluid 116a and air bubbles 116b to leave sample region 116, through splitter 108, and toward (and even into) sensor 110. Therefore, pump 114 can operate in both forward and reverse modes.

[0094] Reference Figure 1A-1I Pump 112 is optional. (And again refer to...) Figure 1A-1I The location and operation of any of the pumps 106, 124, 114, and 112 can be optional, as one or more of the aforementioned pumps may not be necessary. In other words, any of the pumps 106, 124, 114, and 112 is optional, as the fluid delivery system (and method) can be operated using a single pump and even in a gravity-based manner. The disclosed system and method can be operated with a single pump, but multiple pumps can be used, depending on the arrangement of the valves.

[0095] Aspect 10. The system according to aspect 9, further comprising a container in fluid communication with an end of the sample line, the container being configured to receive fluid delivered through the sample line.

[0096] Aspect 11. The system according to aspect 10, further wherein the fluid delivery column is further configured to remove fluid from the container while the fluid is retained within the sample line, such that the sample line is completely filled.

[0097] Aspect 12. The system according to any one of Aspects 9 to 11, wherein the diverter comprises a single valve.

[0098] Aspect 13. The system according to aspect 12, wherein the single valve is characterized as a rotary valve.

[0099] Aspect 14. The system according to any one of Aspects 9 to 13, wherein the shunt array includes a plurality of valves. As an example, the shunt array may include a valve for regulating flow between the sample line and the sample region, and another valve for regulating flow between the sample region and the sensor region.

[0100] Aspect 15. The system according to any one of Aspects 9 to 14, wherein the sensor region includes a bubble sensor.

[0101] Aspect 16. The system according to any one of Aspects 9 to 15, further comprising an instrument (e.g., a flow cytometer) configured to analyze a sample disposed in a first fluid received by the instrument from the sample region.

[0102] Aspect 17. The system according to aspect 16, wherein the system is configured to operate the instrument based at least in part on the volume surrounded by the sample tubing.

[0103] Aspect 18. The system according to aspect 17 further includes an automatic sampler.

[0104] Aspect 19. The system according to aspect 18, wherein the automatic sampler is positioned at a distance from the instrument.

[0105] Aspect 20. The system according to Aspect 19, wherein the sample line positions the autosampler in fluid communication with the instrument. In this manner, the system can be configured to determine the volume of the sample line connecting the autosampler to the instrument. This can be done each time the instrument and the autosampler are connected, for example, when the instrument is connected to a new autosampler, such as when the autosampler is replaced. Similarly, this can be done when the instrument is replaced but the autosampler remains in place.

[0106] Aspect 21. The system according to any one of aspects 9 to 20, wherein the system includes a region configured to contact the sample and the first fluid.

[0107] Aspect 22. The system according to aspect 21, wherein the sample comprises cells, cellular components, or both.

[0108] Aspect 23. The system according to any one of Aspects 9 to 22, wherein the volume surrounded by the sample line is in the range of about 30 μL to about 2.5 mL, for example about 50 μL to about 2 mL, about 75 μL to about 1.75 mL, about 100 μL to about 1.5 mL, about 200 μL to about 1.5 mL, about 250 μL to about 1.25 mL, about 350 μL to about 1.1 mL, about 450 μL to about 950 μL, about 550 μL to about 850 μL, or even about 650 μL to about 725 μL.

[0109] Aspect 24. A method for estimating the volume of a first fluid, the method comprising: delivering a quantity of the first fluid into a conduit; delivering a quantity of a second fluid into the conduit to displace the first fluid within the conduit; estimating the total volume of the first fluid and the second fluid in the conduit; delivering the first fluid and the second fluid from the conduit to a sensor capable of determining a boundary between the first fluid and the second fluid; determining the volume of the second fluid in the total volume; and estimating the volume of the first fluid based at least on the volume of the first fluid and the estimated total volume of the first fluid and the second fluid.

[0110] Aspect 25. The method according to aspect 24, wherein the first fluid comprises air.

[0111] Aspect 26. The method according to any one of Aspects 21 to 22, further comprising operating an instrument at least in part based on an estimated volume of the first fluid, the instrument optionally being configured to be in fluid communication with the conduit.

[0112] Aspect 27. The method according to aspect 23, wherein the instrument is a flow cytometer.

[0113] Aspect 28. The method according to any one of Aspects 26 to 27, wherein the first fluid is delivered from the sample line to the instrument.

[0114] Aspect 29. The method according to aspect 28, wherein the volume of the first fluid delivered to the instrument is the volume surrounded by the sample line.

[0115] Aspect 30. The method according to any one of Aspects 28 to 29, wherein the sample line is arranged to place the autosampler in fluid communication with the instrument.

[0116] Aspect 31. The method according to aspect 30, wherein the automatic sampler is positioned at a distance from the instrument.

[0117] Aspect 32. The method according to any one of aspects 21 to 31, wherein the determination of the volume of the second fluid in the total volume is based at least in part on detecting the boundary between the first fluid and the second fluid.

[0118] Aspect 33. A system for automatically determining the volume of a sample, the system comprising: a sensor region; a fluid delivery column configured to deliver a second fluid and a first fluid, respectively, to the sensor region, the sensor region being configured to measure a signal passing through the sensor region, the signal being different based on the presence of the first fluid in the sensor region or the presence of the second fluid in the sensor region; and a processor configured to determine the volume of the sample based on the signal measured when the first fluid and the second fluid are delivered through the sensor region.

[0119] Aspect 34. The system according to aspect 33, wherein the first fluid comprises air.

[0120] Aspect 35. The system according to any one of Aspects 33 to 34, further comprising an instrument and optionally a flow regulator, the system being configured to place the instrument in fluid communication with the sensor region.

[0121] Aspect 36. The system according to aspect 35, wherein the instrument includes a flow cytometer, the flow cytometer optionally being characterized as an acoustic flow cytometer.

[0122] Aspect 37. The system according to any one of Aspects 35 to 36, further comprising a sample line, the system being configured to place the sample line in fluid communication with the instrument.

[0123] Aspect 38. The system according to aspect 37, wherein the instrument is operable based on the estimated volume of the sample pipeline.

[0124] Aspect 39. The system according to any one of Aspects 37 to 38, further comprising a collection column configured to collect excess fluid from the sample line to completely fill the sample line.

[0125] Aspect 40. The system according to any one of Aspects 37 to 38, further comprising a source of the first fluid, the system being configured to place the first fluid source in fluid communication with the sample line.

[0126] Aspect 41. The system according to any one of Aspects 33 to 40, wherein the system is configured to contact the first fluid with the sample.

[0127] Aspect 42. The system according to aspect 41, wherein the sample comprises cells, cellular components, or both.

Claims

1. A method for estimating the volume enclosed within a sample line, the method comprising: A calibration volume of the first fluid is placed into the sample line such that the calibration volume completely fills the sample line, and the filled sample line defines a volume SL of the first fluid therein. The calibration volume of the first fluid and the displacement volume D of the second fluid are transferred to the sample area, wherein the calibration volume of the first fluid and the displacement volume D of the second fluid define a known total fluid volume; The known total fluid volume from the sample area is delivered at a known flow rate to a sensor configured to identify the front boundary of the second fluid and the boundary between the first fluid and the second fluid; as well as The volume SL of the sample pipeline is estimated based on the known total fluid volume and the time taken to identify the boundary.

2. The method of claim 1, wherein the sensor detects changes in electrical signals, changes in acoustic signals, or any combination thereof.

3. The method according to any one of claims 1 to 2, further comprising delivering a volume of the first fluid exceeding the volume that completely fills the sample line into the sample line and removing the first fluid exceeding the volume that fills the sample line, so that the sample line is completely filled.

4. The method according to any one of claims 1 to 3, further comprising measuring the volume of the second fluid based on changes in electrical signals, changes in acoustic signals, or any combination thereof.

5. The method according to any one of claims 1 to 4, wherein the first fluid and the second fluid are immiscible.

6. The method according to any one of claims 1 to 5, wherein the method is performed in an automated manner.

7. The method according to any one of claims 1 to 6, wherein the second fluid comprises air.

8. The method according to any one of claims 1 to 7, further comprising operating the sample region to analyze one or more fluid samples, the sample volume of each of the one or more fluid samples being the volume SL.

9. A system comprising: A sample line that surrounds the volume; A sample area, configured to receive a first fluid from the sample line; A fluid delivery column, the fluid delivery column being configured to: (a) deliver a volume of the first fluid into the sample line; (b) A calibration volume CV of the first fluid that completely fills the sample line is delivered from the sample line into the sample region; and (c) A displacement volume D of the second fluid is delivered into the sample region such that the calibration volume CV of the first fluid and the displacement volume D of the second fluid define a known total fluid volume TV; A sensor region configured to receive a first fluid and a second fluid from the sample region at a known flow rate and to detect the leading boundary of the second fluid and the boundary between the first fluid and the second fluid. A shunt array, the shunt array being configured to: (a) place the sample line in fluid communication with the sample region; (b) place the sample region in fluid communication with the sensor; or both (a) and (b); as well as Optionally, the processor is configured to determine the volume surrounded by the sample line based at least on the difference between the volume D of the displacement volume of the second fluid and the known total fluid volume TV.

10. The system of claim 9, further comprising a container in fluid communication with an end of the sample line, the container being configured to receive fluid delivered through the sample line.

11. The system of claim 10, wherein the fluid delivery column is further configured to remove fluid from the container while fluid is retained within the sample line such that the sample line is completely filled.

12. The system according to any one of claims 9 to 11, wherein the shunt column consists of a single valve.

13. The system of claim 12, wherein the single valve is characterized as a rotary valve.

14. The system according to any one of claims 9 to 13, wherein the distributor array comprises a plurality of valves.

15. The system according to any one of claims 9 to 14, wherein the sensor region includes a bubble sensor.

16. The system according to any one of claims 9 to 15, further comprising an instrument configured to analyze a sample disposed in a first fluid received by the instrument from the sample region.

17. The system of claim 16, wherein the system is configured to operate the instrument based at least in part on the volume surrounded by the sample tubing.

18. The system of claim 17, further comprising an autosampler.

19. The system of claim 18, wherein the automatic sampler is positioned at a distance from the instrument.

20. The system of claim 19, wherein the sample line positions the autosampler in fluid communication with the instrument.

21. The system according to any one of claims 9 to 20, wherein the system includes a region configured to contact the sample and the first fluid.

22. The system of claim 21, wherein the sample comprises cells, cellular components, or both.

23. The system according to any one of claims 9 to 22, wherein the volume surrounded by the sample line is in the range of 30 μL to 2.5 mL.

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