EP4732025A1 - Cuvette cleaning workflow - Google Patents

Abstract

Systems and methods of flushing and washing a titer container. The container is drained, by a pump in fluid communication with an access port of the container via a multiport valve, of at least one of a biological or control sample comprising a first volume. The container is flushed, by the pump, with flushing agent comprising a second volume via the access port and the multiport valve. The container is filled, by the pump, with a cleaning agent comprising a third volume and rinsed, after a cleaning period, by the pump, with flushing agent comprising the second volume.

Description

CUVETTE CLEANING WORKFLOW

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application is being filed on June 14, 2024, as a PCT International application and claims the benefit of and priority to U.S. Application No. 63/509,243, filed on June 20. 2023, entitled CUVETTE CLEANING WORKFLOW, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] This disclosure generally relates to automated sample preparation and handling systems, and more particularly relates to improved cleaning of reusable sample analysis components. Some automated sample handling system incorporate permanently installed or otherwise reusable components, such as sample containers. Reusable components can reduce the lag time between sample reads by eliminating the need to continually load new sample containers, improving the efficiency of automated sample handing and analysis. Further, permanently installed or otherwise reusable components can reduce wear caused by continually removing and replacing consumable parts, but may require alternative maintenance procedures.

SUMMARY

[0003] Examples presented herein relate to a method of flushing a cuvette. The method includes draining, by a pump in fluid communication with an access port of the cuvette via a multiport valve, a first volume of at least one of a biological or control sample from the cuvette performing a flushing cycle. The flushing cycle includes filling, by the pump, the cuvette with a second volume of flushing agent via the access port and the multiport valve; and draining the second volume of flushing agent from the cuvette. The method further includes repeating the flushing cycle until a flushing criterion is satisfied.

[0004] In other aspects presented herein, the flushing criterion includes repeating the flushing cycle a predetermined number of times. In yet other aspects presented herein, the flushing criterion includes determining a level of contaminants in the cuvette is below a predetermined threshold.

[0005] In other aspects presented herein, the pump is a syringe pump. In further aspects presented herein, draining the sample from the cuvette includes pulling the sample into the syringe pump. In other further aspects presented herein, the method further includes emptying the syringe pump. In yet other further aspects presented herein, emptying the syringe pump includes fully draining the syringe pump to a waste receptable. In still other further aspects presented herein, the syringe pump is in fluid communication with the waste receptable via the multiport valve.

[0006] In other aspects presented herein, the second volume of flushing agent is deionized water. In yet other aspects presented herein, the second volume of flushing agent is greater than the first volume.

[0007] Other examples presented herein are directed to a method of washing a cuvette. The method includes draining, by a pump in fluid communication with an access port of the cuvette via a multiport valve, at least one of a biological or control sample comprising a first volume from the cuvette; flushing, by the pump, the cuvette with flushing agent comprising a second volume via the access port and the multiport valve; filling, by the pump, the cuvette with a cleaning agent comprising a third volume; and rinsing, after a cleaning period, by the pump, the cuvette with flushing agent comprising the second volume.

[0008] In other aspects presented herein, the cleaning period is at least 10 minutes. In yet other aspects presented herein, the first volume is less than the third volume and the third volume is less than the second volume.

[0009] In other aspects presented herein, the method further includes monitoring a level of background buildup associated with the cuvette. In still other aspects presented herein, the method further includes determining the level of background buildup exceeds a threshold level: and initiating, in response to the level of background buildup exceeding the threshold level, the method of washing the cuvette.

[0010] Still other examples presented herein are directed to a system for cleaning a cuvette. The system includes a cuvette including an access port; a multiport valve; a syringe pump in fluid communication with the access port of the cuvette via the multiport valve. The system further includes a controller having processing circuitry configured to operate the multiport valve and the syringe pump in concert to: drain a first volume of at least one of a biological or control sample from the cuvette; flush the cuvette with a second volume of flushing agent; and clean the cuvette with a third volume of a cleaning agent.

[0011] In other aspects presented herein, the system further includes a waste receptacle; a flushing agent source; and a cleaning agent source; wherein each of the waste receptacle, the flushing agent source, and the cleaning agent source is in fluid communication with the syringe pump via the multiport valve. In further aspects presented herein, each of the access port, the syringe pump, the waste receptacle, the flushing agent, and the cleaning agent source is in fluid communication with a dedicated port of the multiport valve.

[0012] In other aspects presented herein, the cuvette is integrated with an optical titer analysis system and a sample loaded into the cuvette is subject to an optical titer analysis. In still other aspects presented herein, the controller is further programmed to: determine when the optical titer analysis is complete; and initiate, in response to determining the optical titer analysis is complete, flushing the cuvette with the second volume of water.

[0013] A variety of additional inventive aspects will be set forth in the description that follows. The inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the forgoing general description and the following detailed description are exemplar}’ and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of the description, illustrate several aspects of the present disclosure. A brief description of the drawings is as follows:

[0015] FIG. 1 is an isometric view of an example of a sample analysis system that analyzes characteristics of samples.

[0016] FIG. 2 is another isometric view of the sample analysis system of FIG. 1 having a top cover removed from a housing of the sample analysis system.

[0017] FIG. 3 is a top view of a work platform supported inside the housing of the sample analysis system of FIG. 1.

[0018] FIG. 4 schematically illustrates an example of a titer module supported on the work platform of FIG. 3.

[0019] FIG. 5 is an isometric view of an example titer container assembly of the titer module of FIG. 4.

[0020] FIG. 6 is an exploded view of the titer container assembly of FIG. 5.

[0021] FIG. 7 is a schematic diagram of a liquid handling system associated with the titer container assembly of FIG. 6. [0022] FIG. 8 is a flowchart of an example workflow for flushing a titer container.

[0023] FIG. 9 is a flowchart of an example workflow for cleaning a titer container.

[0024] FIG. 10 schematically illustrates an example of the controller of the sample analysis system that can be used to implement aspects described herein.

DETAILED DESCRIPTION

[0025] Reference will now be made in detail to exemplary aspects of the present disclosure that are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0026] FIG. 1 is an isometric view of an example of an automated sample analysis system 100 that analyzes samples for various properties. For example, cell samples may be analyzed to determine the cell health of a plurality of cell samples. For a single cell sample, the sample analysis system 100 may measure cell count, cell viability⁷, antibodyconcentration (e.g., protein titer), and other cell characteristics with minimal interaction from a user of the system. Samples may be biological or control samples. Control samples may include, as some non-limiting examples, biological standards, reference beads that fluoresce, etc.

[0027] The sample analysis system 100 may include connectivity to automated bioreactors and other systems and devices. The sample analysis system 100 automates sample preparation, and minimizes sample volume requirements for measuring cell and other sample characteristics. The sample analysis system 100 provides remote access to data including the measured cell and other sample characteristics, supports multiple users at once, and is compatible with various information technology (IT) structures.

[0028] As shown in FIG. 1, the sample analysis system 100 includes a housing 102 that supports a work platform 300. The housing 102 includes a top cover 104 that can support a dispensing system 106 that will be described in more detail with reference to FIGS. 2 and 3.

[0029] FIG. 2 is another isometric view of the sample analysis system 100 having the top cover 104 removed from the housing 102, and thereby exposing the dispensing system 106. FIG. 3 is a top view of the work platform 300 supported inside the housing 102. Referring now to FIGS. 2 and 3, the work platform 300 supports one or more tube racks that hold a plurality of containers containing samples and various types of reagents. In some embodiments, other forms of sample and/or reagent containers are used, such as multi-well plates. In some instances, at least some of the containers are empty.

[0030] The dispensing system 106 includes a probe 108 that is movably mounted within the housing 102 in the space above the work platform 300. In some embodiments, probe 108 includes multiple probes which either move together as a unit for the purposes of aspirating and dispensing groups of adjacent samples from the containers, or may move independently for the purposes of aspirating and dispensing from a variety of non- adjacent locations. In some implementations, the probe tips are fixed, such that they are permanent until replacement during a service process, while in other implementations, probe tips are disposable and may be replaced after each use. Embodiments with multiple probes may include a combination of fixed and disposable tips.

[0031] The probe 108 is mounted for movement along three mutually perpendicular axes (e.g., X-axis, Y-axis, and Z-axis of a three-dimensional cartesian coordinate system). The three-dimensional movement allows a distal end of the probe 108, through which liquid aspiration and dispensing occurs, to access any container held on the work platform 300 inside the housing 102.

[0032] A proximal end of the probe 108 is fluidly connected to a bi-directional pump having a movable actuator that controls the mode of the pump. For example, a first mode can include aspirating and a second mode can include dispensing, and the movable actuator controls switching between the first and second modes, as well as a rate of liquid that is aspirated and dispensed under the first and second modes. As an example, the bidirectional pump can include a syringe pump. Movement of the probe 108 and the movable actuator is controlled by one or more step motors that operate under the control of a controller 900 that is programmable.

[0033] As shown in FIG. 2, the housing 102 is dimensioned to have a width W, a depth D, and a height H. As an illustrative example, the width W is about 24 inches to about 36 inches, the depth D is about 24 inches to about 36 inches, and the height H is about 24 inches to about 36 inches. In some examples, the housing 102 is dimensioned to have a cube shape.

[0034] As shown in FIG. 3, the work platform 300 supports a sample transfer module 302, one or more mixing plates 304, a cell health module 306, one or more tip racks 308, one or more titer plates 310, a tip waste bin 312, a diluent reservoir 314, sample inputs 316 such as tube trays and well plates, a metabolite module 318, and a titer module 400. The cell health module 306 measures cell health, including cell count and cell viability. The titer module 400 measures, for example, protein titer.

[0035] FIG. 4 schematically illustrates an example of the titer module 400 supported on the work platform 300. The titer module 400 includes optical components that are used to measure a concentration of an antibody present in a cell sample. For example, the titer module 400 measures a concentration of Immunoglobulin G (IgG) present in a sample of cells.

[0036] More specifically, the titer module 400 calculates a fluorescence polarization measurement for measuring the concentration of IgG protein in the sample of cells. Fluorescence polarization includes mixing the sample of cells with a fluorescence polarization assay, and subsequently measuring fluorescence polarization for determining the concentration of the IgG protein in the sample of cells. While the titer module 400 is described herein with reference to measuring IgG concentration, the titer module 400 and the measurement techniques described herein can be used to measure the concentrations of additional types of proteins and antibodies.

[0037] As shown in FIG. 4, the titer module 400 includes a light source 424 that emits light 436 toward a titer container 410 holding a solution of cells mixed with the fluorescence polarization assay. The fluorescence polarization assay binds with antibodies of interest (e.g., IgG) produced by the cells. As discussed above, the dispensing system 106 of the sample analysis system 100 is automated to mix the fluorescence polarization assay together with the sample of cells in the titer container 410, such that the user of the system does not need to manually mix the solution in the titer container 410. In some examples, the titer container 410 is a cuvette, tube, and the like. Examples discussed herein focus on titer containers configured as an open-topped cuvette, but other configurations are contemplated.

[0038] The light source 424 emits the light 436 without polarization such that the light 436 is unpolarized light. In some examples, the light source 424 is a light-emitting diode (LED). In some examples, the titer module 400 includes a focusing lens 426 and a spectral filter 428 that respectively focus and filter the light 436 emitted from the light source 424.

[0039] The light 436 passes through a first polarizing filter 444 that polarizes the light 436 in a first direction. In some examples, the first direction is in a linear direction. For example, for purposes of discussion of the example shown in FIG. 4. the first direction is vertically linear. In alternative examples, the first direction is horizontally linear. Additional polarization directions are possible.

[0040] The light 436, once polarized in the first direction, is absorbed by the solution of cells mixed with the fluorescence polarization assay in the titer container 410. This causes the solution in the titer container 410 to emit polarized fluorescence light 438.

[0041] The polarized fluorescence light 438 can pass through alens 430 for focusing the polarized fluorescence light 438 after emission from the solution in the titer container 410. Afterwards, the polarized fluorescence light 438 alternately passes through a second polarizing filter 446 and a third polarizing filter 448. The second and third polarizing filters 446. 448 are mounted to a structure 450 controlled by a controller to alternate placement of the second and third polarizing filters 446, 448 into the path of the polarized fluorescence light 438.

[0042] The second polarizing filter 446 is polarized in a direction parallel to the direction of the first polarizing filter 444 (i.e., in the first direction). The second polarizing filter 446 restricts passage of the polarized fluorescence light 438 to the first direction.

[0043] The third polarizing filter 448 is polarized in a second direction perpendicular to the first direction of the first polarizing filter 444. The third polarizing filter 448 restricts passage of the polarized fluorescence light 438 to the second direction. [0044] Thereafter, the polarized fluorescence light 438 passes through a spectral filter 432 before detection by a detector 434. In some examples, the detector 434 a photomultiplier tube (PMT). The detector 434 converts the polarized fluorescence light 438 into voltage values for input into a calculation to determine a measure of fluorescence polarization (FP).

[0045] The measured FP is correlated with the concentration of antibody in the sample of cells. For example, a higher detected FP correlates to a higher concentration of antibody, and a lower detected FP correlates to a lower concentration of antibody. This is because a higher concentration of antibody will have increased binding with the fluorescence polarization assay such that the voltage of parallel fluorescence is larger, and the voltage of perpendicular fluorescence is smaller, which results in a larger detected FP.

[0046] A photodiode 440 on the far side (opposite of light source 424) of the titer container 410 is used to monitor the direct output light 442 of the LED. This direct output light 442 is somewhat altered by whether the titer container 410 is empty, filled with water, filled with sample, etc. Output of the photodiode 440 may be used to monitor the intensity of the direct output light 442 available from the light source 424. The measured intensity of direct output light 442 can be used to diagnose loss of light, such as from actual decrease of the light source itself or from contamination by absorbing material in the path of the light. Sources of contamination of absorbing material include, for example, sample material that adheres to the surfaces of the titer container. For example, if the measured intensity of output light 442 drops below a predetermined threshold or minimum, or if a decreasing trend in intensity is observed, the system may provide an indication to an operator that the light source should be checked.

[0047] Photodiode 440 enables detection of the presence or absence of liquid in titer container 410. Using photodiode 440 in this way, the system determines whether the cuvette has successfully performed its intended drain sequence and/or wash sequence after a measurement and is fully empty and in a suitable condition to receive the next sample.

[0048] When the cuvette is drained of a sample, it may then be washed by refilling with water and draining from below multiple times. A final fill of water should be substantially devoid of sample material and/or residue of any cleaning agent, with the sample and/or any cleaning agent having been washed and drained. A measurement of the direct output light 442 straight through the water-filled titer container 410 by the photodiode 440 is performed. The titer container 410 may then be drained and a second measurement taken of the direct output light 442 straight through the empty titer container 410 by the photodiode 440.

[0049] When the titer container is empty, the intensity of the light straight through the entire titer container differs from when the titer container is full of water. In the case where the titer container is empty, there are two glass/air interfaces on the inside of the cuvette. In the case where the titer container is full of water, there are instead two glass/water interfaces.

[0050] Reflection, and associated loss of light, at two glass/air interfaces are higher than at two glass/water interfaces. The measurement of light immediately after draining, with the empty titer container, is expected to be lower than the measurement of light immediately before draining, when the titer container is full of water, according to Equation (1) for transmission from a surface between two different index materials: Where T is transmittance and N is the refractive index of the material. The refractive index of air is known to be 1. while the refractive index of the water and the titer container can be determined based on the material of the titer container and the wavelength of the direct output light 442.

[0051] In an example where the direct output light is known to be 488 nm (determined, for example, based on the configuration of light source 424), the refractive index of water can be determined to be 1.338. In this example, the titer container may be constructed with fused silica and with the output light is 488 nm, the refractive index of the titer container can be determined to be 1.46. Applying these values and Equation 1, a reflection from two inner surfaces in the titer container full of water can be determined to be 0.996:

A reflection from two inner surfaces in the titer container full of air can be determined to be 0.931 :

[0052] Comparing these two values demonstrates a difference of 6.5% between the expected readings from photodiode 440 based on whether titer container 410 contains water or air. This difference be used to set a predetermined threshold value. For example, the expected reading for the titer container being full of air may be set as a baseline, and a reading exceeding that baseline by 6.5% may exceed the threshold. Based on that reading exceeding the threshold, the system may determine that titer container has not yet drained or did not drain properly. In embodiments, the threshold may be adjusted up or down to account for effects, such as from multiple back reflections or stray light. Those of skill in the art will understand that the principals of this example may be applied to other systems, with different light sources and construction materials for the titer container, to determine an appropriate system-specific baseline and threshold for the determination.

[0053] Referring now collectively to FIGS. 5 and 6, the titer container assembly 402 of the titer module 400 will be discussed in more detail. FIG. 5 is an isometric view⁷ of an example titer container assembly 402 of the sample analysis system 100. FIG. 6 is an exploded view of the titer container assembly 402 of FIG. 5. Titer container assembly 402 includes a body 404, a base 406, and a top retainer 408.

[0054] Body 404 supports a titer container 410 and includes an upper seal 412, a lower seal 414, and an insert 416. Titer container 410 is held mechanically in place by the supporting structure of body 404 and insert 416. Titer container 410 is sealed as the top and bottom, by upper seal 412 and lower seal 414, each sealing against upper or lower surfaces of titer container 410. Upper seal 412 and lower seal 414 may be formed by any appropriate sealing means and, in some embodiments, may each be an O-ring. In some embodiments, upper seal 412 and lower seal 414 may be formed from materials with different properties and/or have dimensions different from each other.

[0055] In addition to support, body 404 may provide thermal control for samples loaded into titer container 410. Thermal control of samples aid in providing constant temperature for measurements taken by titer module 400. Titer measurements are sensitive to temperature and consistency in sample temperature improves the accuracy of readings determined according to standard curves, which may be temperature specific. In some embodiments, body 404 is made of aluminum.

[0056] In embodiments, titer container 410 is a hollow, open-topped and open- bottomed container for liquid. In some embodiments, titer container 410 has a fourwalled construction, and may have, for example, a square or rectangular cross-section. Titer container 410 may be formed of fused silica or other optically compatible material, such as quartz. Titer container 410 may have internal dimensions configured based on desired sample volumes to use and the resulting fill level in titer container 410 when the sample is loaded, to ensure adequate sample presence for effective analysis. In some embodiments, the internal dimensions of the titer container may be 3 mm x 3mm. In some embodiments, the internal dimensions may be larger or smaller, or the two dimensions may differ from one another, e.g., a rectangular container.

[0057] Titer container 410 is oriented vertically in operation with an upper opening, lying inside of upper seal 412, available to receive sample liquid dispensed by the pipette of a liquid handler, such as probe 108 of FIG. 2.

[0058] Upper seal 412 mates against a fill port to prevent any liquid from spilling over the top edge of titer container 410 and contaminating the exterior optical surfaces of titer container 410. Liquid may be either dispensed into the titer container, such as a sample, or pushed into the titer container during flushing and/or cleaning. [0059] Lower seal 414 mates against a drain outlet port that has a access port. The access port may extend through insert 416. The dimensions of the access port may be kept relatively small, e.g., on the scale of about 0.7 mm, such as specifying dimensions of about 0.5-0.9 mm or 0.3-1.1 mm, etc. In some embodiments, samples may be loaded through the top of titer container 410 with other fluid exchanges associated with titer container 410 generally performed using the access port in the lower portion of titer container 410. Using a single access port for both draining the titer container and filling of flushing and cleaning agents provide manufacturing advantages by limiting the number of holes drilled or otherwise formed during the construction of the titer container. Further, use of a combined access port may simplify the assembly and arrangement of the components of the titer module 400 and the liquid handling system 500.

[0060] In some embodiments, the titer container may be configured with a drain port and a separate fill port. For example, a fill port may be provided towards the top of one or more sidewall of titer container 410. In another example, a fill port is provided from the interior of top retainer 408.

[0061] Base 406 secures and retains titer container 410 within the support of body 404. Base 406 includes optical input aperture 418 and output aperture 420 to permit the beam of the optical interrogation system to pass through a sample loaded into titer container 410.

[0062] Top retainer 408 secures upper seal 412. Top retainer may include tip guide 422, which may assist in guiding probe 108 to access titer container 410, such as for sample loading. In embodiments, top retainer 408 may be removable to provide access to titer container 410, such as for maintenance.

[0063] FIG. 7 is a schematic diagram of a liquid handling system 500 associated with titer container 410. Liquid handling system 500 includes a bi-directional pump 502 and a multiport valve 504. Liquid handling system 500 may be fluidly in communication with the interior of titer container 410 via an access port integrated with titer container 410 and insert 416.

[0064] The access port of titer container 410 is attached via tubing to bi-directional pump 502, which can pull fluid out of the cuvette through the access port and subsequently dispense the discarded fluid to a waste container. The bi-directional pump can further provide a variety of flushing and/or cleaning liquids from bottled sources into the titer container through the same access port. [0065] Bi-directional pump 502 may include a movable actuator 506 that controls the mode of the pump. For example, a first mode can include aspirating and a second mode can include dispensing, with the movable actuator 506 controlling switching between the first and second modes. as the movable actuator 506 may control the rate of aspiration and or dispensation of liquid that is manipulated under the first and second modes. As an example, the bi-directional pump can be a syringe pump including a moveable actuator 506 and a reservoir 508. In the first mode, when aspirating, fluid is drawn into the reservoir 508 by the action of actuator 506. In the second mode, fluid is dispensed from the reservoir 508 by the action of actuator 506.

[0066] Multiport valve 504 selectively connects the bi-directional pump 502 to one or more liquid sources or container locations, depending on the current operation. Bidirectional pump 502 moves liquids between sources and other containers based on the orientation of multiport valve 504. Movement of multiport valve 504 and the movable actuator 506 of bi-directional pump 502 is controlled by one or more step motors that operate under the control of a controller 900 that is programmable.

[0067] Multiport valve 504 may have any number of ports according to the associated workflows and number of required agents. In the example of FIG. 7, multiport valve 504 has eight total ports and four “active” ports, with the active ports associated with agents and elements for executed workflows. A first port 510 aligns with the access port of the titer container 410. A second port 512 aligns with a waste receptacle. A third port 514 aligns with a source of a flushing agent. A fourth port 516 aligns with a source of a cleaning agent. Accordingly, multiport valve 504 may selectively align bi-directional pump 502 to be in fluid communication with any of ports 510, 512, 514, and 516 according to the orientation of multiport valve 504, enabling bi-directional pump 502 to move fluid in and out of the associated sources and containers.

[0068] As discussed above, titer container 410 is integrated with titer module 400, which may include an optical titer analysis system. Sample loaded into titer container 410 may be subjected to an optical titer analysis by the components of titer module 400. [0069] An example sample dispensed into the titer container contains in part cells, cell culture media, IgG protein, and a fluorescent probe. After a fluorescence polarization measurement is made, draining the titer container removes the bulk of the material, but a small amount of undrained residual fluid inevitably remains, as well as adhering residue on the inner surfaces of the titer container. A cleaning process to remove undrained residual fluid and any accumulated residue on the inner surfaces of the cuvette is required to ensure satisfactory continued operation of the sample analysis system and that subsequent sample are not contaminated.

[0070] FIG. 8 is a flowchart of an example workflow 700 for flushing the titer container. In some embodiments, workflow 700 may be executed following every sample read. Workflow 700 may be initiated automatically, such as following a completed analysis of a sample, or may be manually initiated by a user. For example, a controller such as controller 900. may be programmed to determine when an optical titer analysis is complete and initiate, in response to determining the optical titer analysis is complete, flushing the titer container. In some instances, workflow 700 may initiated in response to detection of a threshold level of protein or other debris in the titer container.

[0071] At operation 702, the titer container is drained. In some embodiments, a bidirectional pump, such as bi-directional pump 502 of FIG. 7 which may be a syringe pump, drains the titer container by pulling the sample into the syringe. The bi-directional pump is in fluid communication with an access port, such as an access port, of the titer container via a multiport valve. The sample may be, as some non-limiting examples, a biological sample, such as a cell sample, or a control sample, such as a biological standard.

[0072] In some instances, workflow 700 may be performed with an empty' titer container. If the titer container is empty, this operation may instead pull air.

[0073] In some cases, a second flush may be performed if an operator is dissatisfied with a first flush or if a threshold level of remaining proteins or debris are detected. A threshold level of proteins or debris may be evaluated based on monitored background buildup, such as tracking an increase in buildup over a time elapsed between periodic flushes or cleanings. Accumulated debris may be evaluated using a "test" sample of water. Background may be detected using the optical components of titer module 400. In some embodiments, optical detection may be accomplished by observing decreased throughput intensity for straight-through detection of light diminished by the absorption and obscuration by contamination. In other implementations, optical detection may be accomplished by observation of increased off-axis intensity by increased scatter of light by contamination. In some embodiments, draining of the titer container may be confirmed, such as by comparing a reading of direct output light from the titer container to a predetermined threshold. The reading of the direct output light and/or the determination of whether the titer container is empty may be performed automatically or manually initiated. [0074] At operation 704, the multiport valve is reoriented and the bi-directional pump is dispensed to waste. In embodiments where the bi-direction pump is a syringe pump, the syringe empties fully by draining the contents to waste. The bi-directional pump is in fluid communication with the waste receptable via the multiport valve so that by reorienting, the multiport valve aligns the pump with a waste receptacle.

[0075] At operation 706, the multiport valve is reoriented and the bi-directional pump draws a predetermined amount of a flushing agent. In embodiments, the flushing agent may be water, such as deionized water.

[0076] At operation 708, the multiport valve is reoriented and the bi-directional pump fills the titer container with the predetermined amount of the flushing agent. The predetermined amount of the flushing is determined according to a desired fill level of the titer container. The amount of the flushing agent is determined as an amount sufficient to fill the titer container to a level higher than any sample level or any cleaner level. In an example where the dimensions of the titer container may be 3 mm x 3 mm, and a sample size dispensed into the titer container may generally be 53 pL. The sample volume may be determined such that the sample level is just high enough to cover input aperture 418 and output aperture 420.

[0077] In some embodiments, the sample level desired is enough to cover the input aperture with sufficient margin that light rays from the illumination coming into the aperture do not intersect with and/or reflect off the sample-air interface. In the case of dispensed liquid, such as a sample, into a cuvette this sample-air interface is generally an upward concave meniscus. The light rays from the illumination are not necessarily parallel, and therefore a liquid level that is at precisely the same height as the top of the input aperture may reflect unwanted light. In some implementations, the sample volume includes a margin to account for tolerances in the inner dimensions of the cuvette, tolerances in the dimensions of the drain, tolerances in the placement of the input aperture, tolerances in the length and diameter of the fluidic supply line, etc.

[0078] In the present example, the predetermined amount of the flushing agent may be determined to fill the titer container to a level equivalent to 100-120 pL. The flushing agent volume may be determined such that the flushing agent level in the titer container is approximately twice as high as the sample level to ensure all exposed surfaces are adequately rinsed.

[0079] The amount of liquid that the pump dispenses is dependent on the tube length from the pump to the titer container. To prevent overfilling or underfilling with the intended amount of flushing agent, the length and inner diameter of the tubing line from the pump to the outlet port of the cuvette must be controlled in manufacture. Like the inner dimensions of the titer container, the length and inner diameter of the tubing line are know n at the time the predetermined amount of the flushing agent is determined.

[0080] At operation 710, the bi-directional pump draws the flushing agent back out of the titer container, draining the titer container. In some embodiments, draining of the titer container may be confirmed, such as by comparing a reading of direct output light from the titer container to a predetermined threshold. The reading of the direct output light and/or the determination of whether the titer container is empty may be performed automatically or manually initiated.

[0081] Operations 704-710, together forming a flushing cycle, are immediately repeated multiple times to successively lower the amount of residual contaminants from the titer container, and from any residual contaminants material that may cling to the multiport valve or the bi-directional pump components after operation 702. The flushing cycle may be repeated successively until a flushing criterion is satisfied. The flushing criterion may be any number of criteria to evaluate the cleanliness of the titer container. For example, flushing criterion may refer to a number of repetitions of the flushing cycle and/or a threshold level of detected contaminants.

[0082] In embodiments, flushing operations 704-710 are repeated twice, three times, four times, etc. Flushing operations 704-710 may be repeated until detected contaminants fall below a threshold level. At operation 712, the determination is made whether to repeat the process. The determination is made based on, for example, a number of times the flushing operations 704-710 have already been performed and/or the level of detected contaminants.

[0083] For example, the determination at operation 712 may be based on whether the flushing operations 704-710 have yet been performed three consecutive times. If the flushing operations 704-710 have not yet been performed three consecutive times, the determination is made, at operation 712, to repeat the flushing operations and return to operation 704. If the flushing operations 704-710 have already been performed three consecutive times, the determination is made, at operation 712, not to repeat the flushing operations and to end the flushing method 700, at operation 714.

[0084] The number of repetitions, or the threshold level of contaminants, is a tradeoff between the acceptable carryover and the duration of the flushing cycle which affects total system sample throughput. The number of flushes can be increased at the expense of time and the amount of flushing agent used. A user may manually initiate any number of flushes.

[0085] FIG. 9 is a flowchart of an example workflow 800 for cleaning the titer container. In some embodiments, workflow 800 may be executed following flushing the titer container, such as by method 700 of FIG. 8.

[0086] Workflow 800 may be initiated automatically, such as at a particular time of day or following a certain number of sample reads and/or flushes of the titer container. Cleaning workflow 800 may be manually initiated by a user. In some instances, workflow 800 may initiated in response to detection of a threshold level of protein or other debris in the titer container. A threshold level of proteins or debris may be evaluated based on monitored background buildup, such as tracking an increase in buildup over a time elapsed between periodic flushes or cleanings. Accumulated debris may be evaluated using a “test” sample of water. Background may be detected using the optical components of titer module 400. In embodiments, cleaning workflow 800 may be initiated in response to a higher threshold value than a threshold set to trigger initiation of flushing workflow 700.

[0087] At operation 802, the titer container is drained. At the initial draining, the titer container may contain a sample, flushing agent, or may be empty. In some embodiments, draining of the titer container may be confirmed, such as by comparing a reading of direct output light from the titer container to a predetermined threshold. The reading of the direct output light and/or the determination of whether the titer container is empty may be performed automatically or manually initiated.

[0088] The titer container is drained by a bi-directional pump in fluid communication with an access port of the titer container via a multiport valve. When draining a sample, the sample may be a biological or control sample. The sample may be a first volume, e.g., ~ 53 pL, rising to a first level in the titer container. Whatever fluid is drained from the titer container may be dispensed to a waste receptable. The bidirectional pump may alternatively be aligned with the access port of the titer container or the waste receptable according to the orientation of the multiport valve. The multiport valve may be repositioned automatically, according to the workflow, in response to communication from a control unit, such as controller 900.

[0089] At operation 804, the titer container is flushed. The titer container may be flushed by backfilling the titer container with a flushing agent by the bi-directional pump. The bi-directional pump may alternatively be in fluid communication with a source of the flushing agent and the access port of the titer container according to the orientation of the multiport valve. The flushing agent may generally be water, such as deionized water. In some embodiments, operation 804 may include execution of all or some of method 700 of FIG. 8. In some instances,

[0090] The flushing agent may be provided at a second volume, e.g., ~ 100-120 pL, that is greater than the first volume of samples. Further, the second volume must also be sufficient to rise to a second level in the titer container, sufficiently above the first level to fully cover any interior surface which may come into contact with sample material and thereby become a potential surface to which contaminants may adhere. The second volume also accounts for filling the length and inner diameter of tubing or another conduit running from the bi-directional pump to the access port of the titer container, which sufficient volume entering the titer container to reach the second level.

[0091] At operation 806, the multiport valve is reoriented and the bi-directional pump draws a predetermined amount of a cleaning agent. In embodiments, the cleaning agent may be an enzymatic cleaner, though other more caustic cleaning agents may also be suitable. The predetermined amount of the cleaning agent may include a third volume, where the third volume is greater than the first volume of the sample but less than the second volume of the flushing agent.

[0092] At operation 808, the titer container is filled with the predetermined amount of the cleaning agent. The cleaning agent may be held in the titer container for a cleaning period. At operation 810, a determination is made whether a cleaning period has elapsed. The cleaning period is a predetermined amount of time during which the cleaning agent is held in titer container to allow the cleaning agent to act on any contaminants which may be clinging to the interior surfaces of the titer container. The cleaning period may account for factors such as the cleaning agent used, the degree of debris or buildup, time elapsed since the last cleaning, time elapsed since the last flushing, number of sample loaded, etc. The cleaning period may be, for example, less than minute, 1 minute, 2 minutes, 3 minutes. 5 minutes, 10 minutes, 15 minutes, 30 minutes, etc. In some embodiments, the cleaning period may be 10 minutes or about 10 minutes. If the determination is made that the cleaning period has not elapsed, no action is taken and the determination is made again whether the cleaning period has elapsed.

[0093] If the cleaning period has elapsed, the cleaning agent is drained from the titer container, at operation 812. In some embodiments, draining of the titer container may be confirmed, such as by comparing a reading of direct output light from the titer container to a predetermined threshold. The reading of the direct output light and/or the determination of whether the titer container is empty may be performed automatically or manually initiated.

[0094] At operation 814, the titer container is rinsed. Rinsing the titer container may include filling the titer container with the flushing agent at a predetermined volume such as the second volume. In embodiment, rinsing the titer container may include a flushing cycle or a flushing workflow, such as workflow 700 of FIG. 8.

[0095] FIG. 10 schematically illustrates an example of the controller 900 of the sample analysis system 100 that can be used to implement aspects described herein, including features of the titer module 400 and liquid handling system 500. As shown in FIG. 10, the controller 900 includes one or more processing devices 902. a memory storage device 904, and a system bus 906 that couples the memory storage device 904 to the one or more processing devices 902. The one or more processing devices 902 can include central processing units (CPU). In some instances, the one or more processing devices 902 are part of a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to perform the various aspects, features, and functionalities described herein.

[0096] As shown in FIG. 10, the memory' storage device 904 can include a randomaccess memory (“RAM"’) 908 and a read-only memory (“ROM"’) 910. Basic input and output logic having basic routines that help to transfer information between elements within the controller 900, such as during startup, can be stored in the ROM 910.

[0097] The controller 900 can also include a mass storage device 912 that can include an operating system 914 and store software instructions and data 916. The mass storage device 912 is connected to the processing device 902 through the system bus 906. The mass storage device 912 and associated computer-readable data storage media provide non-volatile, non-transitory storage for the controller 900.

[0098] Although the description of computer-readable data storage media contained herein refers to the mass storage device 912, it should be appreciated by those skilled in the art that computer-readable data storage media can be any available non-transitory. physical device or article of manufacture from which the controller 900 can read data and/or instructions. The computer-readable storage media can be comprised of entirely non-transitory media. The mass storage device 912 is an example of a computer-readable storage device. [0099] Computer-readable data storage media include volatile and non-volatile, removable, and non-removable media implemented in any method or technology for storage of information such as computer-readable software instructions, data structures, program modules or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory' or other solid-state memory technology, or any other medium which can be used to store information, and which can be accessed by the device.

[0100] The controller 900 can operate in a networked environment using logical connections to the other devices through the network 920. The controller 900 connects to the network 920 through a network interface unit 918 connected to the system bus 906. The network interface unit 918 can also connect to additional types of communications networks and devices, including through Bluetooth, Wi-Fi, and cellular telecommunications networks including 4G and 5G networks. The network interface unit 918 can connect the controller 900 to additional networks, systems, and devices. The controller 900 also includes an input/output unit 922 for receiving and processing inputs and outputs from peripheral devices.

[0101] The mass storage device 912 and the RAM 908 can store software instructions and data. The software instructions can include an operating system 914 suitable for controlling the operation of the sample analysis system 100. The mass storage device 912 and/or the RAM 908 can also store the software instructions and data 916, which when executed by the processing device 902, provide the functionality of the sample analysis system 100 discussed herein.

[0102] Embodiments of the disclosure are described with reference to the following numbered clauses:

[0103] Clause 1. A method of flushing a cuvette, the method comprising: draining, by a pump in fluid communication with an access port of the cuvette via a multiport valve, a first volume of at least one of a biological or control sample from the cuvette; performing a flushing cycle comprising: filling, by the pump, the cuvette with a second volume of flushing agent via the access port and the multiport valve; and draining the second volume of flushing agent from the cuvette; and repeating the flushing cycle until a flushing criterion is satisfied.

[0104] Clause 2. The method of clause 1, wherein the flushing criterion comprises repeating the flushing cycle a predetermined number of times. [0105] Clause 3. The method of clause 1, wherein the flushing criterion comprises determining a level of contaminants in the cuvette is below a predetermined threshold.

[0106] Clause 4. The method according to any one of clauses 1 -3, wherein the pump is a syringe pump.

[0107] Clause 5. The method of clause 4, wherein draining the sample from the cuvette comprises pulling the sample into the syringe pump.

[0108] Clause 6. The method of clause 5, further comprising emptying the syringe pump.

[0109] Clause 7. The method of clause 6, wherein emptying the syringe pump comprises fully draining the syringe pump to a waste receptable.

[0110] Clause 8. The method of clause 7, wherein the syringe pump is in fluid communication with the waste receptable via the multiport valve.

[OHl] Clause 9. The method according to any one of clauses 1-8, wherein the second volume of flushing agent is deionized water.

[0112] Clause 10. The method according to any one of clauses 1-9. wherein the second volume of flushing agent is greater than the first volume.

[0113] Clause 11. The method according to any one of clauses 1-10, further comprising: reading an intensity of direct output light from the titer container; and determining, based on the intensity of direct output light and a predetermined threshold, that the titer container is empty.

[0114] Clause 12. A method of washing a cuvette, the method comprising: draining, by a pump in fluid communication with an access port of the cuvette via a multiport valve, at least one of a biological or control sample comprising a first volume from the cuvette; flushing, by the pump, the cuvette with flushing agent comprising a second volume via the access port and the multiport valve; filling, by the pump, the cuvette with a cleaning agent comprising a third volume; and rinsing, after a cleaning period, by the pump, the cuvette with flushing agent comprising the second volume.

[0115] Clause 13. The method of clause 12, wherein the cleaning period is at least 10 minutes.

[0116] Clause 14. The method according to any one of clauses 12-13, wherein the first volume is less than the third volume and the third volume is less than the second volume.

[0117] Clause 15. The method according to any one of clauses 12-14, further comprising monitoring a level of background buildup associated with the cuvette. [0118] Clause 16. The method of clause 15, further comprising determining the level of background buildup exceeds a threshold level; and initiating, in response to the level of background buildup exceeding the threshold level, the method of washing the cuvette. [0119] Clause 17. The method according to any one of clauses 12-16, further comprising: reading an intensity of direct output light from the titer container; and determining, based on the intensity of direct output light and a predetermined threshold, that the titer container is empty.

[0120] Clause 18. A system for cleaning a cuvette, the system comprising: a cuvette comprising an access port; a multiport valve; a syringe pump in fluid communication with the access port of the cuvette via the multiport valve; a controller having processing circuitry configured to operate the multiport valve and the syringe pump in concert to: drain a first volume of at least one of a biological or control sample from the cuvette; flush the cuvette with a second volume of flushing agent; and clean the cuvette with a third volume of a cleaning agent.

[0121] Clause 19. The system of clause 18, further comprising: a waste receptacle; a flushing agent source; and a cleaning agent source; wherein each of the waste receptacle, the flushing agent source, and the cleaning agent source is in fluid communication with the syringe pump via the multiport valve.

[0122] Clause 20. The system of clause 19, wherein each of the access port, the syringe pump, the waste receptacle, the flushing agent, and the cleaning agent source is in fluid communication with a dedicated port of the multiport valve.

[0123] Clause 21. The system according to any one of clauses 18-20, wherein the cuvette is integrated with an optical titer analysis system and a sample loaded into the cuvette is subject to an optical titer analysis.

[0124] Clause 22. The system of clause 21, wherein the controller is further programmed to: determine when the optical titer analysis is complete; and initiate, in response to determining the optical titer analysis is complete, flushing the cuvette with the second volume of the flushing agent.

[0125] The various embodiments described above are provided by way of illustration only and should not be construed to be limiting in any way. Various modifications can be made to the embodiments described above without departing from the true spirit and scope of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of flushing a cuvete, the method comprising: draining, by a pump in fluid communication with an access port of the cuvete via a multiport valve, a first volume of at least one of a biological or control sample from the cuvete; performing a flushing cycle comprising: filling, by the pump, the cuvette with a second volume of flushing agent via the access port and the multiport valve; and draining the second volume of flushing agent from the cuvete; and repeating the flushing cycle until a flushing criterion is satisfied.

2. The method of claim 1, wherein the flushing criterion comprises repeating the flushing cycle a predetermined number of times.

3. The method of claim 1, wherein the flushing criterion comprises determining a level of contaminants in the cuvete is below a predetermined threshold.

4. The method according to any one of claims 1-3, wherein the pump is a syringe pump.

5. The method according to any one of claims 1-4. wherein draining the sample from the cuvete comprises pulling the sample into the pump.

6. The method according to any one of claims 1-5, wherein emptying the pump comprises fully draining the pump to a waste receptable.

7. The method according to any one of claims 1-6, wherein the pump is in fluid communication with the waste receptable via the multiport valve.

8. The method according to any one of claims 1-7. wherein the second volume of flushing agent is deionized water.

9. The method according to any one of claims 1-8, wherein the second volume of flushing agent is greater than the first volume.

10. The method according to any one of claims 1-9, further comprising: reading an intensity’ of direct output light from the titer container; and determining, based on the intensity⁷ of direct output light and a predetermined threshold, that the titer container is empty.

11. A method of washing a cuvette, the method comprising: draining, by a pump in fluid communication with an access port of the cuvette via a multiport valve, at least one of a biological or control sample comprising a first volume from the cuvette; flushing, by the pump, the cuvette with flushing agent comprising a second volume via the access port and the multiport valve; filling, by the pump, the cuvette with a cleaning agent comprising a third volume; and rinsing, after a cleaning period, by the pump, the cuvette yvith flushing agent comprising the second volume.

12. The method according to claim 11, wherein the first volume is less than the third volume and the third volume is less than the second volume.

13. The method according to any one of claims 11-12, further comprising monitoring a level of background buildup associated with the cuvette.

14. The method of claim 13, further comprising determining the level of background buildup exceeds a threshold level; and initiating, in response to the level of background buildup exceeding the threshold level, the method of washing the cuvette.

15. The method according to any one of claims 11-14, further comprising: reading an intensity⁷ of direct output light from the titer container; and determining, based on the intensity of direct output light and a predetermined threshold, that the titer container is empty.