Flow cytometry system that applies back pressure to waste liquid flow

Abstract

The flow cytometric evaluation system includes a sample outlet system including an effluent collection vessel having an outlet fluid inlet for receiving effluent of the fluid sample exiting the interrogation zone during flow cytometric evaluation, and an outlet fluid conduit from the interrogation zone to the outlet fluid inlet. A pressurized gas delivery system in fluid communication with the sample outlet system applies pressurized gas to the fluid sample outlet system to impede fluid flow through the outlet fluid conduit toward the outlet fluid inlet during flow cytometric evaluation.

Description

Background Art

[0001] Flow cytometry is an analytical technique for evaluating a fluid sample for the presence of target particles of interest. Flow cytometry involves exposing the flow of a fluid sample to a stimulus (typically light from a laser, etc.), detecting a response (typically response radiation), and analyzing that response to identify the presence of target particles. The response detection ability can include the detection of one or more radiation response characteristics, which may include the detection of one or more of the light scattering characteristics such as forward scattered light and / or side scattered light, and the detection of one or more fluorescence emission indicators of a fluorescent stain that can be added to the fluid sample to fluorescently label specific features of the target particles. Flow cytometry is a common technique used to evaluate the presence of cells and other similarly sized particles, which are often in the size range of 2 to 20 microns. Flow cytometers used for such applications generally include both light scattering detection using multiple light scattering detectors that enable the detection of different light scattering characteristics, and fluorescence emission detection ability using multiple fluorescence emission detectors that enable the detection of multiple different fluorescence emission indicators provided by different fluorescent stains. A flow cytometry evaluation system may also combine a flow cytometer with an autosampler, which can automatically process a sample tray containing many fluid samples for the continuous automatic delivery of fluid samples to the flow cytometer to perform continuous flow cytometry investigations of fluid samples. Such systems are widely used when analyzing cells and particles of similar size and provide a convenient and cost-effective technique for flow cytometry analysis of many fluid samples in a relatively short time.

[0002] More recently, flow cytometers have been developed that have the ability to analyze much smaller particles, such as viral particles (virions), virus-like particles, extracellular vesicles containing exosomes, and other particles of similar size. For convenience, these particles are generally referred to as virus-sized particles herein. Such virus-sized particles are often in the size range of 20 nanometers to 1 micron, and particle sizes of less than 200 microns or even less than 100 microns can be very common. When evaluating fluid samples for the presence of such virus-sized particles by flow cytometry, techniques and practices that work well for flow cytometry analysis of cells and particles of similar size often do not translate well for the analysis of virus-sized particles. An example of a flow cytometer designed for the analysis of such virus-sized particles is the Virus Counter 3100 flow cytometer (Sartorius Stedim Biotech), which processes much smaller fluid samples at much lower flow rates and uses fluorescence detection only without light scattering detection. Combining a flow cytometer with an autosampler that offers the flexibility to analyze virus-sized particles in a robust and accurate system that is easy to use, inspect, and maintain is challenging, and there remains a significant need for such a system. [Overview of the Initiative]

[0003] A first aspect of this disclosure relates to a flow cytometry evaluation system in which back pressure is applied to obstruct the flow of a fluid sample toward an effluent collection container (e.g., a waste container). In various implementations, such a flow cytometry evaluation system is A flow cytometry investigation system including an investigation zone, wherein the investigation zone is configured to receive the flow of a fluid sample during flow cytometry evaluation for flow cytometry investigation within the investigation zone regarding the presence of particles in the fluid sample flow, It is a sample leakage system, An effluent collection container having an effluent inlet for receiving the effluent of a fluid sample leaving the investigation zone during flow cytometry evaluation, A sample discharge system comprising an outflow fluid guideway from the investigation zone to the outflow fluid inlet, The system may comprise a sample outflow system and a pressurized gas delivery system in fluid communication, wherein the pressurized gas delivery system is configured to apply pressurized gas to pressurize at least a portion of the fluid sample outflow system with an applied gas pressure that provides positive back pressure within the outflow fluid conduit, thereby hindering the flow of fluid through the outflow fluid conduit toward the outflow fluid inlet during flow cytometry investigations.

[0004] The flow cytometry evaluation system of the first embodiment has been found to be conveniently adaptable in combination with an autosampler, providing flexibility to use the autosampler with a flow cytometer designed to analyze fluid samples for the presence of virus-sized particles, thereby providing an accurate and robust flow cytometry evaluation system, which is convenient to use, inspect, and maintain, and provides flexibility to various system configurations, including stacked configurations in which the autosampler is conveniently positioned higher in the stack structure than the flow cytometry evaluation system.

[0005] A second aspect of this disclosure relates to a method for flow cytometry evaluation, wherein an applied back pressure obstructs the flow of a fluid sample toward an effluent collection container (e.g., a waste container) during a flow cytometry investigation of the fluid sample. In various implementations, such a method is: This involves flowing a fluid sample through the investigation zone of a flow cytometry system, where the downstream end of the investigation zone is in fluid communication with a sample outflow system, and this sample outflow system is An effluent collection container having an effluent inlet for receiving the effluent of a fluid sample leaving the investigation zone during flow cytometry evaluation, A fluid outflow channel from the investigation zone to the fluid outflow inlet, and a fluid flow mechanism, Perform a flow cytometry study of the fluid sample flow within the survey zone, The fluid sample effluent leaving the survey zone is guided through the effluent conduit to an effluent collection container where the fluid sample effluent is collected. Fluid samples are flowed through the investigation zone. doing This may include applying a pressurized gas to pressurize at least a portion of the fluid sample discharge system by supplying a positive back pressure into the discharge fluid conduit, thereby obstructing the flow of fluid through the discharge fluid conduit toward the discharge fluid inlet of the discharge fluid recovery container.

[0006] The method of the second embodiment can be performed using the flow cytometry evaluation system of the first embodiment. Various other feature improvements and additional features are applicable to each of these and other aspects of the present disclosure, as disclosed in the following description (including numbered exemplary implementation combinations), drawings, and appended claims. These feature improvements and additional features may be used individually or in any combination within the subject matter of the aspects summarized above or other aspects disclosed herein. Any such feature improvement or additional feature may, but is not required, be used in conjunction with any other feature or combination of features disclosed herein. [Brief explanation of the drawing]

[0007] [Figure 1] This is a schematic diagram illustrating the general characteristics of an exemplary flow cytometry evaluation system according to a first aspect of the present disclosure. [Figure 2] Figure 1 is a perspective view of an exemplary instrument module, including an exemplary configuration of a flow cytometry evaluation system in a stacked structure where the autosampler is positioned higher than the flow cytometry survey zone. [Figure 3] Figure 2 is a partial perspective view of a portion of the equipment module with the side access panel removed to show the sliding shelf feature that supports the flow cytometry survey system. [Figure 4] Figure 2 is a partial perspective view of a portion of the equipment module, showing the connection configuration of several reagents and waste collection containers. [Figure 5] Figure 2 is a partial perspective view of the sliding shelf and flow cytometry survey system of the equipment module. [Figure 6] Figure 2 is a partial top view showing the features of the flow cytometry survey system in the equipment module. [Figure 7] Figure 2 is a fluid idix diagram of the flow cytometry evaluation system for the equipment module. [Figure 8] Figure 2 is a schematic diagram of the temperature control system of the flow cytometry evaluation system in the equipment module. [Figure 9] Figure 8 shows an example timeline for acquiring a temperature determination dataset in the temperature control system. [Figure 10] Figure 8 shows a side view of a common optical component mounting platform for mounting components of the optical processing system of the flow cytometry survey system of the equipment module shown in Figure 2, including the temperature sensor and resistance heating element of the temperature control system. [Modes for carrying out the invention]

[0008] Figure 1 shows an exemplary embodiment of a flow cytometry evaluation system 100, which has an applied gas pressure that provides positive back pressure in the outflow fluid conduit to partially or completely counteract gravity-driven fluid flow effects during flow cytometry evaluation, thereby hindering fluid flow from the flow cytometry investigation zone to the outflow fluid collection container. The flow cytometry evaluation system 100 shown in Figure 1 includes a flow cytometry investigation system 102 in which a fluid sample is investigated as part of a flow cytometry evaluation. The flow cytometry investigation system 102 includes an investigation zone 104 that provides a controlled flow conduit for the flow of a fluid sample for investigation, a radiation delivery system 106 that supplies input light 108 to the investigation zone 104 for investigation of the fluid sample, and a radiation detection system 110 that detects response radiation from the fluid sample exposed to the input light 108 as it passes through the investigation zone 104 as part of the flow cytometry evaluation. The radiation delivery system 106 may include one or more light sources for supplying one or more different light beams to the investigation zone 104. Such different light beams may have different properties (e.g., different wavelength bands) to investigate different properties of particles in a fluid sample flowing through the investigation zone 104. For example, the radiation delivery system 106 may include one or more other light sources, such as lasers and / or LEDs, that supply light having one or more specific wavelengths to stimulate one or more radiative responses that are to be detected by the radiation detection system 110. If the radiation delivery system 106 houses multiple different light sources, such light sources may be spaced apart along the investigation zone 104 and sufficiently shielded from each other to minimize interference between different light sources. The investigation zone 104 may be configured to receive the fluid sample flow itself, or to receive the hydrodynamically focused flow of a fluid sample surrounded by a sheath fluid. The investigation zone 104 may be provided as a passage through the flow cell of a flow cytometer.The investigation zone 104 may be a continuous transparent conduit or may consist of discontinuous transparent sections of a longer conduit system. The radiation detection system 110 may include one or more different radiation detectors to detect various different response radiation characteristics coming from the investigation zone 104. Such radiation detectors may be selected from the group consisting of, for example, photomultiplier tubes, silicon photomultiplier tubes, avalanche photodiodes, and selective photodiodes, with photomultiplier tubes often preferred when it is desirable to detect and process very weak signals. If multiple radiation detectors are included, the radiation detectors may detect signals in different wavelength ranges or may be positioned to receive signals from different directions. The response radiation detected by the radiation detection system 110 may include one or more fluorescent signals from fluorescent labels staining particles, and / or light scattering, such as forward scattered light and / or side scattered light. When detecting particles the size of a cell (e.g., about 2 to 20 microns), forward-scattered light detection and / or side-scattered light detection may be commonly used to aid in particle identification, and the detection of fluorescent signals from one or more fluorescent labels provides information for identifying specific characteristics of the particle. When detecting particles the size of a virus particle (e.g., about 20 nanometers to about 1 micron), detection by the radiation detection system 110 may consist only of detecting fluorescent signals from fluorescent labels stained with virus-sized particles to identify particle attributes. In the example shown in Figure 1, the radiation detection system 110 is shown to include four radiation detectors, including a first detector 112 for detecting a first fluorescent signal, a second detector 114 for detecting a second fluorescent signal, a third detector 116 for detecting forward-scattered light, and a fourth detector 118 for detecting side-scattered light. As understood, the radiation detection system 110 may include fewer or more radiation detectors than the four exemplary radiation detectors shown in Figure 1. Different radiation detectors may be appropriately oriented and spaced apart along the survey zone 104 for effective detection of the desired response radiation.

[0009] The flow cytometry evaluation system 100 includes a sample outflow system for handling sample effluent (waste) leaving the investigation zone 104 of the flow cytometry investigation in the flow cytometry investigation system 102. The sample outflow system shown in Figure 1 includes an effluent collection container 120 that receives the fluid sample effluent from the investigation zone 104, and an effluent conduit 122 that conducts the fluid sample effluent from the investigation zone 104 to the effluent collection container 120. The effluent collection container 120 has an effluent inlet 124 into which the fluid sample effluent enters the effluent collection container 120. When referring to a sample outflow system, such a system is configured to guide and collect the fluid sample effluent from the investigation zone. However, the sample outflow system is not necessarily limited to guiding and collecting only fluid sample effluent, and other liquid effluents (e.g., waste liquids) may also be guided and collected, whether or not they leave the flow cytometry investigation zone, and whether or not they are mixed with the fluid sample effluent. As can be understood, if the flow cytometry survey in the flow cytometry evaluation system 100 in Figure 1 involves the use of a sheath fluid surrounding the fluid sample, the effluent from the survey zone 104 that is collected in the effluent collection container 120 will contain a mixture of the fluid sample effluent and the sheath fluid effluent. In addition, as the fluid sample is pushed into and passes through the survey zone 104 by the drive liquid, the effluent of the drive liquid leaving the survey zone 104 will also be collected as effluent in the effluent collection container 120.

[0010] As shown in Figure 1, the flow cytometry evaluation system 100 is in fluid communication with the effluent collection container 120 and includes a pressurized gas delivery system 126 that pressurizes the effluent collection container 120 by applying pressurized gas from a pressurized gas supply line 128. In the flow cytometry evaluation system 100 shown in Figure 1, the pressurized gas is delivered to the effluent collection container 120 through a gas inlet 130 located at the top of the effluent collection container 120, as well as the positioning of the effluent inlet 124. As shown in Figure 1, the effluent collection container 120 has a pressurized gas headspace 132 of the applied gas pressure supplied by the pressurized gas supply line 128 from the pressurized gas delivery system 126. As can be understood, as the effluent collection container 120 is filled with waste liquid 134, the size of the pressurized gas headspace 132 will decrease but will be adjusted by the pressurized gas delivery system 126 and maintained at the applied gas pressure provided by the pressurized gas supply line 128. The effluent recovery container 120 may be equipped with a pressure relief vent to allow pressurized gas to be released from the pressurized gas headspace 132 as the level of waste liquid 134 in the effluent recovery container 120 rises. Alternatively, the pressurized gas delivery system 126 may be configured to release pressure as needed to maintain a desired level of applied gas pressure in the effluent recovery container 120.

[0011] To continuously guide fluid samples into the investigation zone 104 and perform a continuous flow cytometry investigation of the fluid samples, the flow cytometry evaluation system 100 includes a sample delivery system in the form of an autosampler 140, as shown in Figure 1, to continuously draw fluid samples from multiple sample containers 146 and deliver the multiple fluid samples sequentially to the fluid sample pathway 142. In the example of Figure 1, the autosampler 140 has a sample receiving area 144 in the form of a platform, where multiple sample containers 146 can be received for continuous processing. The multiple sample containers 146 may be provided, for example, in a multi-container tray. Such a multi-container tray may be in the form of a multi-well plate, where the multiple fluid sample containers 146 are multiple wells of a plate. Such a multi-well plate may have any number of wells, for example, a plate with 24 wells, a plate with 48 wells, a plate with 96 wells, or more wells. Such a multi-container tray may alternatively take the form of a vial tray having multiple vials as sample containers 146 that are accepted within multiple receptacles of the tray. Such a vial tray may include any number of vial receptacles and any number of vials that are accepted into those vial receptacles. Such a vial tray may include, for example, 24, 48, 96, or more sample vials.

[0012] An exemplary autosampler 140 shown in Figure 1 includes a sample delivery probe 148, for example, in the form of a subcutaneous injection needle, which is inserted into multiple sample containers 146 at once and configured to sequentially withdraw fluid samples from the multiple sample containers 146 in order to deliver fluid samples to a fluid sample pathway 142 to lead fluid samples to the investigation zone 104 for continuous flow cytometry investigation of fluid samples. As is typical of autosamplers, the sample delivery probe 148 and the multiple fluid containers 146 are indexed and movable relative to each other to allow the sample delivery probe 148 to interact with each of the different containers among the multiple fluid containers 146. For example, the multiple fluid containers 146 may remain stationary, but the sample delivery probe 148 may move spatially over the area of ​​the multiple fluid containers 146 and move vertically up and down to allow insertion into each of the multiple sample containers 146 and sequentially withdraw fluid samples from the sample containers 146 one at a time. In another example, the sample delivery probe 148 may remain stationary, but the sample receiving area 144 moves relative to the sample delivery probe 148. The sample receiving area 144 may be configured to change height to allow the sample delivery probe 148 to be inserted into the sample container, or the sample delivery probe 148 may be on a mechanism that raises and lowers the sample delivery probe 148 to allow insertion into each sample container.

[0013] An exemplary autosampler 140 is further configured to have a sample holding zone 150 in which a fluid sample drawn from a sample container is first delivered from a sample delivery probe 148 through a multi-positional valve 152, the multi-positional valve 152 is positioned to fluidly connect the sample delivery probe 148 to the fluid sample holding zone 150 and to fluidly isolate the sample delivery probe 148 and the fluid sample holding zone 150 from the fluid passage 142 to the investigation zone 104. After the fluid sample is loaded into the sample holding zone 150, the multi-positional valve 152 may then be modified to fluidly isolate the fluid sample holding zone 150 from the sample delivery probe 148 and to fluidly connect the fluid sample holding zone 150 to the investigation zone 104, allowing the fluid sample to be pushed out from the sample holding zone 150 through the multi-positional valve 152 and the fluid sample passage 142 to the investigation zone 104 for flow cytometry investigation of the fluid sample.

[0014] By using the fluid sample holding zone 150 to first receive a fluid sample for processing, the operation of taking out a desired volume of fluid sample from the fluid container 146 and the separation and independent control of guiding and passing that desired volume of fluid sample through the investigation zone 104 become possible. As can be understood, when the multi-position valve 152 is positioned to direct the flow of the fluid sample from the fluid sample holding zone 150 to the investigation zone 104 for flow cytometry investigation, the flow path through the fluid sample holding zone 150 and the multi-way valve constitutes part of the fluid sample conduction path 142 to the investigation zone 104. Also, when the multi-position valve 152 is positioned to direct the fluid sample from the fluid sample holding zone 150 to the investigation zone 104, the fluid sample conduction path 142, the investigation zone 104, the effluent fluid conduction path 122, and the effluent collection container 120 all form a pressurized fluidics system during the flow cytometry investigation. The flow of fluid in the direction towards the effluent collection container 120 through this fluidics system is impeded by the positive backpressure from the applied gas pressure in the effluent collection container 120 provided by the pressurized gas supply line 128 from the pressurized gas delivery system 126.

[0015] The back pressure applied to the effluent recovery container 120 by the pressurized gas supply line 128 offers several advantages. Typical flow cytometry systems often have a survey zone positioned higher than the waste tank, and the fluid sample is recovered into the waste tank after leaving the survey zone. Flow from the survey zone to the waste tank is facilitated by gravity. However, such gravity-assisted drainage from the survey zone may, due to the nature of the siphon effect, introduce suction through the survey zone and the fluidicus upstream of the survey zone, potentially making it more difficult to control the flow rate through the survey zone. Small fluctuations in flow rate typically do not significantly affect the flow cytometry results, so this is usually not a major problem in flow cytometers designed primarily to detect and evaluate particles the size of cells. However, when operating at very low flow rates, such as around 400 nanoliters to 3000 nanoliters per minute, which are often used for flow cytometry evaluation of very small particles, such as virus-sized particles, such gravity-induced flow effects can become more problematic in terms of both flow control and the accuracy of flow cytometry results. By providing positive back pressure through the application of the pressurized gas supply line 128, such gravity effects can be largely reduced or eliminated in a system like the one shown in Figure 1. Preferably, the applied back pressure is at least the same magnitude as, and more preferably, greater than, the gravity-induced pressure in the system. As can be understood, such gravity-induced pressure in flow cytometry may be equal to the liquid head pressure exerted by the liquid mass between the rise and fall of the liquid mass in the fluid channel through the flow cytometry evaluation system 100 during flow cytometry evaluation. Such liquid head pressure can be exerted by the fluid sample, sheath fluid, and / or drive fluid in the fluid channel. Such fluids are typically aqueous liquids that have a density close to, if not equal to, that of water, and therefore exert a head pressure close to that of water.In one preferred implementation, the applied back pressure is at least as large as, and more preferably as large as, the head pressure of a water column at a vertical height equal to the height difference between the outflow fluid inlet 124 and the lowest height of the investigation zone 104. Even more preferably, the applied back pressure is at least as large as, and more preferably as large as, the head pressure of a water column at a vertical height equal to the height difference between the outflow fluid inlet 124 and the highest height in the fluid path, through which the fluid sample is guided to the investigation zone 104 in connection with the flow cytometry investigation and passed to the outflow fluid collection container 120. The siphon effect can be reduced by providing a pressure blockage immediately after the investigation zone, but greater control over the pressure effect is provided by applying positive back pressure to counteract the gravity-induced flow effect in a flow cytometry system, such as the flow cytometry system shown in Figure 1. Also, the gravity-induced suction effect in a fluidics system during a flow cytometry investigation can result in the generation of more and larger bubbles in the fluid sample, which can negatively affect uniform liquid flow and flow cytometry performance. To mitigate these problems, bubble removal devices have been used in flow cytometers; however, even with such devices, bubble formation remains a problem. By applying positive back pressure through this system, the gravity-induced suction effect can be counteracted, reducing the formation of more or larger bubbles. In addition, the added back pressure creates a higher pressure system within the investigation zone 104 and fluid sample passage 142 during flow cytometry evaluation, further reducing the likelihood of more or larger bubbles forming within the system. Furthermore, applying back pressure from the outflow system provides greater flexibility in flow cytometry system design. In typical designs for systems integrating an autosampler with a flow cytometer, the autosampler is positioned at a lower or nearly the same height as the flow cytometer.However, while positioning the autosampler at a higher height than the flow cytometer may offer operational and inspection advantages, doing so introduces fluidic issues in terms of gravity head pressure on the liquid column, which applies extra pressure to push the fluid sample, thereby complicating flow control. Addressing such gravity-induced flow effects through the application of positive back pressure, as shown in Figure 1, provides greater flexibility in flow cytometry system design in achieving other advantages.

[0016] One advantageous design facilitated by the use of positive back pressure, as shown in Figure 1, is a stack system design in which the autosampler 140 is positioned at a higher stack position compared to the flow cytometry survey system 102. The stack design offers the advantage of a smaller footprint compared to a side-by-side arrangement of the autosampler and flow cytometer. The natural positioning in a stack design is to position the autosampler at a lower height and the flow cytometer at a higher height, which is partly to partially avoid gravity-induced flow effects that negatively affect the flow of fluid samples from the autosampler to the survey zone. However, from the user's perspective, having the autosampler positioned at a higher position in a stack design allows for more convenient access to load the fluid sample tray for processing, remove the tray at the end of processing, observe and replace reagent containers in the autosampler as needed, and for a standing person to observe the performance of the autosampler as needed without bending or crouching. A flow cytometry survey system is typically accessed only for maintenance and inspection, which may include removing, replacing, and / or adjusting components of the flow cytometry survey system. Having its components at a lower level makes it easier to provide access for removing and maintaining such components. Figure 1 shows various heights within a flow cytometry survey system 100. In the example in Figure 1, E4 is the highest height within the fluid sample passage 142, which is located within the fluid sample holding zone 150, where the multi-position valve 152 is positioned to guide the fluid sample from the fluid sample holding zone 150 to the survey zone 104 for flow cytometry surveys. E3 is the height of the sample receiving area 144 of the autosampler 140, which is higher than the height E2 of the survey zone 104. Height E2 is higher than the height E1 of the outflow fluid inlet 124 of the outflow fluid recovery container 120. Generally, this design is well suited to stacked configurations where the autosampler 140 is positioned higher than the flow cytometry survey system 102.This promotes the diversity of the design of the orientation of the investigation zone 104. For example, the investigation zone 104 may be oriented to extend horizontally in the longitudinal direction of the flow path passing through the investigation zone 104, or the investigation zone 104 may be oriented inclined perpendicular to the longitudinal direction of the flow path, for example, when it is more convenient for the device design. Such a vertical inclination may be a slope that rises or falls vertically in the flow direction, or may be completely vertical (at an angle of 90° with respect to the horizontal) with an upward or downward flow. By applying back pressure as shown in FIG. 1, the gravity-induced flow effect due to the vertical inclination of the flow path passing through the investigation zone 104 can be canceled. As can be understood, when the investigation zone 104 is oriented with a horizontal flow path, there is little difference between the highest height and the lowest height within the investigation zone 104, but when the flow path of the investigation zone 104 includes a vertical inclination, the difference between the highest height and the lowest height within the investigation zone 104 can be much larger.

[0017] Here, refer to FIGS. 2 to 7 in combination with FIG. 1. FIGS. 2 to 7 show the features of an exemplary single-unit device module 200 that includes one exemplary configuration of the general flow cytometry evaluation system 100 of FIG. 1, where the autosampler 140 and the flow cytometry investigation system 102 are in a stack configuration. A single unit means that the device module is one integrated structure that can be moved as a single component and is not composed of separate units that are physically connected together, and preferably, all the autosampler and flow cytometer components are supported on a common support frame and within a common housing. Specifically, the autosampler 140 is positioned at a higher position in the stack than the flow cytometry investigation system 102 and includes a pressurized gas delivery system 126 for providing back pressure to the flow of fluid through the fluidics system during the execution of the flow cytometry investigation of the fluid sample. The reference numbers for the same features shown in FIGS. 2 to 7 are the same as the reference numbers used for the features in FIG. 1.

[0018] First, referring to Figures 2-4 in combination with Figure 1, the flow cytometry instrument module 200 includes a housing 202, within which an upper compartment 204 houses components of the autosampler 140, and a lower compartment 206 (which can be seen in Figure 3) houses components of the flow cytometry investigation system 102. The autosampler 140 located in the upper compartment 204 includes a receiving area 144 for receiving multiple sample containers 146, and a sample delivery probe 148 configured to interface with the sample containers 146 and extract fluid samples for continuous flow cytometry investigations. The autosampler 140 located in the upper compartment 204 may also include one or more containers containing liquid reagents, such as washing or rinsing solutions, used in conjunction with the operation of the autosampler 140. The housing 202 includes a hinged door 208, which provides the user access to load a tray of fluid samples into the autosampler 140 for processing, remove the processed tray after flow cytometry evaluation of the fluid samples, and replace or refill the containers with reagents used by the autosampler 140. The door 208 has a window that allows the user to observe the operation of the autosampler 140 while the flow cytometry evaluation is being performed. The flow cytometry investigation system 102 in the lower compartment 206 is not normally accessed by the user during a flow cytometry investigation. The housing 202 includes a removable component in the form of a removable side access panel 210, which can be moved to provide access into the lower compartment 206, for example, to perform inspection or maintenance of the flow cytometry investigation system 102. Figure 3 shows the instrument module 200 with the access panel 210 removed to provide access into the lower compartment 206. In an alternative configuration, the access panel 210 may be hinged rather than fully removable. Removal of the side access panel 210 also provides access to the upper compartment 204 for inspection and maintenance of components within the upper compartment 204.Furthermore, an access door or panel may be provided on the side of the housing 202 opposite the access panel 210 to provide additional access to the upper compartment 204 for convenient access to the autosampler 140 for inspection and maintenance of the autosampler 140. To provide convenient inspection and maintenance access to the autosampler 140 within the upper compartment 204, a movable cover member, such as a separate access panel, may be provided on the side of the housing 202 opposite the access panel 210. It should be understood that the references to the upper and lower compartments refer only to separate spaces within the housing, and the space of the upper compartment is located higher within the housing than the space of the lower compartment. Referring to these spaces as compartments does not necessarily mean that the spaces of each compartment are isolated from each other within the housing 202 by physical barriers between them.

[0019] The instrument module 200 also includes a front compartment 212 located in front of the lower compartment 206, and within the front compartment 212 are fluid containers for holding reagents for use during flow cytometry evaluation and for receiving waste liquid from the operation of the flow cytometry evaluation system 100. A first container 214 may be a reagent container for holding sheath fluid for hydrodynamically focusing the fluid sample for flow cytometry investigation in the investigation zone 104. A second container 216 may be a reagent container for holding drive fluid for pushing and passing the fluid sample into the investigation zone 104 during flow cytometry investigation. A third container may be a waste container in the form of an effluent recovery container 120 for collecting effluent of the fluid sample leaving the investigation zone 104 during flow cytometry investigation. A fourth container 220 may be a waste container for collecting waste liquid used in the operation of the autosampler 140 to wash away and clean the components of the autosampler 140 between fluid samples. The spillage recovery container 120 is pressurized by the applied gas pressure from a pressurized gas supplied from a pressurized gas delivery system 126 through a pressurized gas line 128, which in the exemplary equipment module 200 includes a pressurized tank 222 pressurized by a gas compressor 224. The pressurized gas is typically air, but may preferably be another pressurized gas such as nitrogen. Alternatively, the pressurized gas delivery system 126 may include a connection to an external pressurized gas source instead of having an onboard compressor, or it may operate with only a pressurized gas container. As shown in Figure 4, the pressurized gas is delivered to the first container 214 through the gas supply line 226, and the sheath fluid is pushed out of the first container 214 through the outlet line 228. The second container 216 and the fourth container 220 are not pressurized. The second container 216 is connected to an air inlet line 230 to allow filtered air to enter the second container 216 for pressure equalization as the drive fluid is discharged from the second container 216 through the outlet line 232 during processing.Pressurized gas is delivered to the effluent collection container 120 through the pressurized gas supply line 128, and waste liquid, including fluid samples and sheath fluid effluent, is delivered to the effluent collection container 120 from the investigation zone 104 through the effluent fluid conduit 122. The fourth container 220 is not pressurized and receives waste liquid from the autosampler 140 through two waste inlet lines 238 and 240.

[0020] The front compartment 212 provides receiving locations for receiving each of the vessels 214, 216, 120, and 220 at different receiving positions for fluid connections within the flow cytometry evaluation system 100. As best seen in Figure 2, the equipment module 200 includes a lighting system to illuminate the internal space inside each of the vessels 214, 216, 120, and 220. As seen in Figure 2, each of the vessels 214, 216, 120, and 220 is rear-illuminated by separate lighting elements 215, 217, 121, and 221 of the lighting system. Such lighting elements may include, for example, light-emitting diodes (LEDs) (preferably), incandescent lamps, fluorescent lamps, or other light sources. As shown in Figure 2, each of the illumination elements 215, 217, 121, and 221 is positioned inside the front compartment 212 behind each container, illuminating the internal space of each of the containers 214, 216, 120, and 220. This conveniently allows the user to observe the instrument module 200 from the front and easily identify the liquid level inside the containers. Specifically, the user can quickly and easily identify the extent to which the containers are filled with liquid or empty, and predict the need for maintenance, either by filling the containers with reagents (sheath fluid or drive fluid) or emptying the waste liquid (fluid sample effluent or autosampler waste). As can be understood, the light illumination system may be configured differently from that shown in Figure 2, provided that the light illumination system adequately illuminates the internal space of the containers 214, 216, 120, and 220, allowing a person to easily observe the liquid level inside these containers. For example, the illumination system may include an illuminated light strip extending behind all the containers. In other examples, the illuminating elements may be oriented to illuminate upward from below into the container, downward from above into the container, or at an angle to illuminate upward from a forward illuminating element near the bottom of the container. As should be understood, containers 214, 216, 120, and 220 must be made from a sufficiently transparent material to allow for easy observation of the liquid level.In addition, the front compartment 212 is covered at least in its forward portion by an optically transparent (e.g., light-transmitting plastic material) housing so that an observer positioned in front of the equipment module 200 can easily observe the containers 214, 216, 120, 220 and the liquid levels within them. In the example shown in Figure 2, the front compartment 212 is conveniently positioned in front of the lower compartment 206 and below the height of the upper compartment 204, providing convenient visual observation of the containers 214, 216, 120, 220 without impeding access to the upper compartment 204.

[0021] Furthermore, as is best seen in Figure 4, each of the gas supply line 128, air inlet line 230, and gas supply line 226 has in-line filters 121, 231, and 227, respectively. Filters 227 and 231 filter the pressurized gas stream (typically air) delivered to the first container 214 and the second container 216 to prevent the sheath fluid and the drive fluid from being contaminated by dust particles that may be carried by the gas, respectively.

[0022] The filter 129 filters the pressurized gas flow (typically air) entering and leaving the effluent collection container 120. During normal operation, as the effluent collection container 120 is filled with effluent from the flow cytometry survey, the gas flow is generally directed out of the effluent collection container 120, and the filter 129 filters out any viral particles that may be present in the exit gas and could otherwise pose a safety hazard. The gas flow entering the effluent collection container 120 may occur, for example, during the initial pressurization of the effluent collection container 120 to apply a desired level of back pressure in preparation for performing the flow cytometry assessment. Furthermore, the filter 129 can provide an additional safety feature by being made of a hydrophobic material that acts as an obstruction to the flow of liquid through the filter 129 when the effluent collection container is filled with aqueous effluent up to the level of the filter 129. A flow sensor that monitors the fluid flow through the fluidic system of the flow cytometry survey system 102 will detect the obstruction to the flow and guide the control system to stop all additional fluid flow toward the effluent collection container 120 through the flow cytometry evaluation system 100 until the obstruction is removed, for example, by emptying or replacing the effluent collection container 120.

[0023] As shown in Figure 3, the components of the flow cytometry survey system 102 are supported on a slidably mounted member in the form of a sliding shelf 244, which is slidably supported on a sliding system, for example, on a sliding rail such as a sliding rail commonly used for cabinet drawers or sliding cabinet shelves. The sliding shelf 244 is slidably movable between a first position in which the sliding shelf 244 is fully housed within the internal space of the lower compartment 206, and a second position in which the sliding shelf 244 extends at least partially outside the lower compartment 206, and at least a portion of the flow cytometry survey system 102 is located outside the internal space of the lower compartment 206. As can be understood, the first position is the normal position in which the flow cytometry survey system 102 is fully housed in the internal space within the lower compartment 206 for normal use of the flow cytometry evaluation system 100, and the side access panel 210 is in a fixed closed position as shown in Figure 2 to protect the flow cytometry survey system 102 during use. The sliding shelf 244 may be locked in place in the first position, for example, by a latch or thumbscrew. The sliding shelf 244 may also be locked in place in the second position, for example, by another latch or thumbscrew. The second position provides improved access to the components of the flow cytometry survey system 102 for inspection and maintenance.

[0024] The position, mounting, and structure of the flow cytometry survey system 102 on the sliding shelf 244 in the lower compartment 206 offer several operational advantages. As described above, mounting the components of the flow cytometry survey system 102 on the sliding shelf 244 provides convenient access for inspection and maintenance. From a usability standpoint, positioning the autosampler 140 in the upper compartment 204 is advantageous for users who need to access and observe the autosampler 140 during normal operation. Combining the positioning of the autosampler 140 in the upper compartment 204 with mounting the flow cytometry survey system 102 on the sliding shelf 244 in the lower compartment 206 provides a favorable combination of improved user usability of the flow cytometry evaluation system 100 and improved access for inspection and maintenance of the flow cytometry survey system 102.

[0025] Referring primarily to Figures 3, 5, and 6, the flow cytometry survey system 102 includes an optical processing system 250 mounted on a common optical component mounting member in the form of a common optical component mounting platform 252. In Figure 3, many of the components of the common optical component mounting platform 252 and the optical processing system 250 are not visible because they are obscured from view by a protective cover 253. However, Figure 5 shows the optical processing system 250 and the common optical component mounting platform 252 with the protective cover 253 removed. The sliding shelf 244 includes a front edge 256 positioned closer to the side access panel 210 when the sliding shelf 244 is in a first position, and a rear edge 258 located opposite the front edge 256 and positioned further away from the side access panel 210 when the sliding shelf 244 is in a second position. The common optical component mounting platform 252 is positioned near the leading edge 256 and is supported by two support members 262, 264 at a higher position above the sliding shelf 244. The sliding shelf 244 also has a circuit board 288 mounted on it, which contains electronics for operating the various components of the flow cytometry survey system 102. To clearly illustrate the various features of the flow cytometry survey system 102, the electrical connections between the components of the flow cytometry survey system 102 and the circuit board 288 are not shown in the figure.

[0026] As shown in Figures 5 and 6, the optical processing system 250, supported on a common support platform 252, includes a flow cell 268, which includes an investigation zone 104 through which a fluid sample surrounded by a sheath fluid flows for flow cytometry investigation. Input light 108 from a radiation delivery system 106 is delivered to the investigation zone 104 of the flow cell 268 as focused light from a focusing element 270, illuminating the flow of the fluid sample within the flow cell 268 with focused light. The input light 108 is transmitted to the focusing element 270 through an optical conduction path including an optical conduit 284 between the radiation delivery system 106 and the focusing element 270. In the exemplary configurations shown in Figures 2 to 6, the radiation source 106 is in the form of a laser, and the optical conduit 284 includes an optical fiber. The light-gathering element 270 may be, for example, an optical component or a combination of optical components (e.g., a focusing lens, a focusing mirror, a tapered light guide) that focuses the input light 108 to the desired extent for delivery to the flow cell 268, or may include such a combination. Within the optical processing system 250, there is a focusing lens that focuses the emitted light to a photodetector. As an example, the optical processing system 250 includes two photodetectors 272, 274, indicated for example as photomultiplier tubes, and a response radiation conduction path from the flow cell 268 to the photodetectors 272, 274. The response radiation conduction path is located within a housing 276 and includes a spatial filter (not shown) and a dichroic mirror (not shown) that subsequently separates the response radiation by wavelength and delivers it to either the first photodetector 272 or the second photodetector 274. An optical filter may be placed before the photodetectors 272, 274 to allow wavelengths of light that are to be detected by each of the photodetectors 272, 274 to pass through. For example, before photodetectors 272 and 274, there may be different optical filters that allow light of different wavelengths to pass through to each of the photodetectors 272 and 274, corresponding to different fluorescence emission indices from various different fluorescent stains that are to be detected.

[0027] The laser of the radiation delivery system 106 is the primary heat-generating element of the components of the flow cytometry survey system 102 located in the lower compartment 206, and the laser is thermally coupled to a heat sink 280 having cooling fins for dissipating the heat generated by the light source. Heat removal is facilitated by an exhaust fan (not shown) mounted on the rear panel of the housing 202 of the equipment module 200, which can expel warm air from the second compartment 206 and draw in cooler ambient air. Preferably, such an exhaust fan is located on the rear panel adjacent to the heat sink 280. The performance of the optical processing system 250 mounted on the common optical component mounting platform 252 is susceptible to changes in component spacing and alignment due to temperature changes caused by the expansion and contraction of the material, particularly the expansion and contraction of the common optical component mounting platform 252 on which the components of the optical processing system 250 are mounted. The common optical component mounting platform 252 may be made from any desired rigid material, but metallic materials (e.g., steel, aluminum) are preferred. Changes resulting from thermal expansion and contraction of the common optical component mounting platform 252 are often not a significant issue for flow cytometry systems designed primarily to evaluate fluid samples for the presence of particles larger than cell size. However, when evaluating very small particles, such as virus-sized particles, even relatively small changes can have a greater impact, for example, because the optical signals produced by smaller particles are generally weaker. To counteract the potential adverse effects of temperature changes on performance, the flow cytometry investigation system 102 includes advantageous design features in addition to the exhaust fan described above.

[0028] One advantageous design feature is the provision of some thermal isolation between the light source 278 and the optical components of the optical processing system 250. In this regard, the light source 278 is not mounted on the common optical component mounting platform 252. In the example shown in Figures 2 to 6, the laser of the radiation delivery system 106 is mounted on a heat sink 280, which is mounted on a sliding shelf 244, reducing direct conduction heating of the common support platform 252 and providing a large exposed area to the airflow around the laser and heat sink 280, so that the heat generated by the laser is removed and efficiently discharged from the second compartment 206 by the exhaust fan described above.

[0029] Another advantageous design feature is the use of an optical fiber sealed within a protective optical tube 284 to supply input light 108 from the light source 278 to the flow cell 104 through an input optical conduction path between the light source 278 and the focusing element 270. The optical fiber in the optical tube 284 has a first end optically coupled adjacent to the light source 278 to receive input light 108 from the light source 278, and a second end optically coupled adjacent to the focusing element 270. By using an optical fiber to conduct input light 108 from the light source 278 to the focusing element 270, the input optical conduction path can be made inherently insensitive to temperature changes, and in addition, it simplifies the optical system alignment problem that would normally occur with optical input conduction paths that utilize one or more mirrors to direct light to the flow cell, as is common in some flow cytometers.

[0030] A further advantageous design feature is the provision of temperature control for the common optical component mounting platform 252 through the use of a temperature control system with reduced self-heating, which enables effective temperature control of the common optical component mounting platform. This temperature control system is described separately below with reference to Figures 8 to 10.

[0031] Here, we refer to Figure 7, which contains a fluidic diagram of an exemplary fluid system configuration of equipment module 200. The fluidic diagram in Figure 7 is sometimes called a system piping diagram or fluid flow diagram. Figure 7 shows the fluid flow and fluid handling characteristics in an example of a flow cytometry system 100 contained within equipment module 200, including within the autosampler 140, the flow cytometry survey system 102, and the pressurized gas delivery system 126. Only the fluid characteristics are shown in Figure 7; optical characteristics are not shown.

[0032] Referring to Figure 7, the processing of a fluid sample for a flow cytometry study begins with drawing the fluid sample from one of several fluid containers 146 using a sample delivery probe 148. As shown in Figure 7, the autosampler 140 includes three multi-component valves 152, 300, and 302, also designated as V1, V2, and V3 in Figure 7, respectively. To draw the fluid sample from the fluid container 146, valve 152 is set so that position 4 is connected to position 5, valve 300 is set so that position 4 is connected to position 5, and valve 302 is set so that position 2 is connected to syringe 304. Once valves 152, 300, and 302 are set as described above, the plunger 306 of syringe 304 is retracted, applying fluid suction to the sample delivery probe 148 through valves 302, 300, and 152, and drawing the fluid sample from the sample container 146. The plunger 306 is retracted just enough to draw a desired volume of fluid sample into the sample holding zone 150, which is in the form of a coil of tube as shown in Figure 7, for flow cytometry investigation. After the desired volume of fluid sample has been drawn into the sample holding zone 150, the retraction of the plunger 306 then stops. Prior to the initiation of the retraction of the plunger 306 to draw the fluid sample into the sample delivery probe 148 for delivery to the sample holding zone 150, the fluid path from the syringe 304, through valve 302, fluid line 310, valve 300, fluid line 312 (including the sample holding zone 150), fluid line 314, and the sample delivery probe 148, is filled with a drive fluid pre-supplied from a second container 216. After the plunger 306 stops retracting, the fluid line 312 will be filled with the fluid sample through valve 152 and a portion of the tube coil in the sample holding zone 150, while the remaining portion of the fluid line 312 up to valve 300 will be filled with the drive fluid.A fluid sample of the desired volume is drawn into the sample holding zone 150, and after the plunger 306 stops retracting, the positions of valves 300 and 152 are then changed so that only positions 5 and 6 of each valve are open, and pressurized gas from the pressurized gas delivery system 126 is supplied at a controlled pressure from the gas pressure regulator 316, passing through the fluid line 318 and through the open valve positions 6 and 5 of valve 300, pushing the drive fluid and the fluid sample in front of the drive fluid. These fluids then pass through the open positions 5 and 6 of valve 152 and through the fluid sample guideway 142 to reach and pass through the investigation zone 104 in the flow cell 268 for flow cytometry investigation, and then through the outflow fluid guideway 122 to reach the outflow fluid inlet 124 for recovery as part of the waste liquid 134 in the outflow fluid recovery container 120.

[0033] In order to push the drive fluid and expel the fluid sample from the sample holding zone 150 through the investigation zone 104 to the effluent collection container 120, it is necessary to apply sufficient gas pressure behind the drive fluid to overcome the effect of positive back pressure from the applied gas pressure in the fluidix system through the pressurized gas supply line 128 to the effluent collection container 120.

[0034] Following a flow cytometry study of the fluid sample, the fluid component of the autosampler 140 is subjected to a rinse cycle using a rinse reagent from the reagent container 320, and subsequently, the fluid path from syringe 304 through valve 302, valve 300, sample holding zone 150, valve 152, fluid line 314, and sample delivery probe 148 is filled with a drive fluid from a second container 216, in preparation for drawing the next fluid sample from another of the multiple fluid containers 146 via the sample delivery probe 148 for the next flow cytometry study. Details of the rinse cycle are not described here. Following the rinse cycle, with valve 302 in position 1 open, the syringe plunger 306 is retracted from its forward position to draw the drive fluid from the second container 216 into the syringe 304 via the fluid line 322, thereafter valve 302 is closed in position 1, valve 302 is opened in position 2, and with valves 300 and 152 in positions 4 and 5 respectively open, the plunger 306 is then advanced to push the drive fluid through the fluid path to the tip of the sample delivery probe 148, which is positioned to remove excess drive fluid and enter the rinse station 358, where the excess drive fluid is collected in the fourth container 220 via the waste inlet line 238. As shown in Figure 7, the drip pan 322 is positioned to capture any liquid leakage from the autosampler 140 and guide it to the fourth container 220 via the waste inlet line 240.

[0035] As shown in Figure 7, an exemplary configuration of the flow cytometry evaluation system 100 of the instrument module 200 includes a sheath fluid surrounding a fluid sample for flow cytometry investigation in an investigation zone 104 within a flow cell 268. As shown in Figure 7, during the flow cytometry investigation 104 of the fluid sample, the sheath fluid is delivered from a first container 214 to a focusing zone 324 of the flow cell 268, where the sheath fluid is introduced around the flowing fluid sample, hydrodynamically focusing the flowing fluid sample for flow cytometry investigation in the investigation zone 104. The sheath fluid effluent exits the investigation zone 104 together with the fluid sample effluent and is collected as waste in an effluent collection container 120 together with the fluid sample effluent.

[0036] Several other components are also shown in Figure 7. An air inlet 330 provides an air intake to the compressor 224. A gas pressure regulator 316 includes pressure control valves 334, 336, and 338 for regulating the pressure at which pressurized gas is delivered to different parts of the system. Filters 340, 342, 344, 346, and 348 are positioned in various gas lines to filter various gas flows. Shut-off valves 350 and 352 allow for selective isolation of the effluent recovery container 120 and the first container 214 from the rest of the system. A sample flow sensor 354 measures the flow of a fluid sample, and a sheath flow sensor 356 measures the flow of sheath fluid to the flow cell 268 for flow cytometry investigations. A sample delivery probe 148 is movable between the sample container 146, the reagent bottle 320, and its position in the rinse station 358 to perform various different operations through the sample delivery probe 148.

[0037] Referring here to Figure 8, Figure 8 shows an exemplary temperature control system 400 located in a lower compartment 206 of an equipment module 200 within a housing 202, preferably a temperature control system 400 in which all components are supported on a sliding shelf 244. As shown in Figure 8, the exemplary temperature control system 400 includes an electric heating unit 402 that can be selectively operated to heat the environment within the lower compartment 206, preferably the electric heating unit 402 is positioned to directly heat the common optical component mounting platform 252 by conduction, thereby maintaining the common optical component mounting platform 252 at a temperature near a target setpoint temperature. In this regard, the electric heating unit 402 may be mounted on the common optical component mounting platform 252, or embedded within the common optical component mounting platform 252, or form part of the common optical component mounting platform 252. As shown in Figure 8, the electric heating unit 402 includes a resistance heating element. The temperature control system 400 includes a temperature sensor 404, preferably including a thermistor, to supply temperature sensor readings corresponding to the temperature state in the lower compartment 206 of the housing 202. Preferably, the temperature sensor 404 is positioned to directly sense the temperature of the common optical component mounting platform 252. In this regard, the temperature sensor 404 may be mounted on the common optical component mounting platform 252, embedded within the common optical component mounting platform 252, or form part of the common optical component mounting platform 252. The temperature control system 400 includes a reference resistor 406 to supply a reference reading for use in combination with the corresponding temperature sensor reading for making a temperature determination. The analog-to-digital converter 408 can be selectively connected to the temperature sensor 404 to receive the temperature sensor reading, or to the reference resistor 406 to receive the reference reading, in either case supplying a digital output corresponding to the respective reading.The current source 410 is configured to selectively supply AC current to the temperature sensor 404 to obtain a sensor reading, or to the reference resistor 406 to obtain a reference reading. A switch unit 412, for example in the form of a multiplexer, can be operated to selectively switch the direction of current from the current source 410 to either the temperature sensor 404 or the reference resistor 406, and to selectively switch the input to the analog-to-digital converter 408 to receive the temperature sensor reading or the reference reading. The switch unit 412 may include any desired number of switches. In the example in Figure 8, the switch unit 412 has three switches 432, 434, and 436, each switchable between a first position (position 1) and a second position (position 2). The controller unit 414 is configured to control the operation of the temperature control system 400, which includes periodically collecting temperature determination datasets, each dataset including a first digital output from the analog-to-digital converter 408 corresponding to a sensor reading and a second digital output from the analog-to-digital converter 408 corresponding to a reference reading; periodically making temperature determinations based at least in part on such temperature determination datasets; and instructing the operation of an electric heating unit as needed to provide heat to heat a controlled environment (e.g., a common optical component mounting platform 252) toward a setpoint temperature and to maintain the temperature of the controlled environment near the setpoint temperature. As shown in Figure 8, the current source 410 is provided by a digital-to-analog converter that supplies AC current from a DC power supply according to instructions from the controller unit 414.

[0038] The temperature control system 400 also includes a first timer 420 and a second timer 422, which trigger a first interrupt service routine and a second interrupt service routine, respectively, that provide chopper-stabilized operation. The temperature control system 400 also includes a pulse width modulation (PWM) unit 424 that receives temperature control commands from a controller unit 414 based on a temperature determination, and the PWM unit 424 outputs heater drive commands to a driver unit 424 to operate an electric heating unit at a level and duration for heating a controlled environment toward maintaining a setpoint temperature, which is preferably a fixed setpoint temperature. The driver unit 426 may be a power supply that can switch between on and off modes to supply or not supply power to the electric heating unit 402 in accordance with commands from the PWM unit 424 to heat or not heat the controlled environment as instructed by the controller unit 414, or may include such a power supply. The driver unit 426 may have a variable power output that supplies different levels of power to operate the electric heating unit 402 to heat a controlled environment at various different speeds, or the driver 426 may have a fixed power output that operates the electric heating unit 402 at a fixed speed for the duration that power is supplied from the driver unit 426 to the electric heating unit 402.

[0039] A significant problem associated with precise tolerance temperature control of an environment within a closed space is the potential for errors arising from the effects of local temperature changes and from the self-heating of the temperature sensor due to the heat generated by its operation. Existing techniques for temperature measurement include absolute measurement techniques and ratiometric measurement techniques. Absolute temperature measurement is simple but has the problem of measurement errors introduced by changes in the supply voltage and / or reference voltage of the temperature sensor, as well as the problem of self-heating of the temperature sensor due to the current applied for the operation of the temperature sensor, which is typically continuously supplied to the temperature sensor. Ratiometric temperature measurement eliminates voltage fluctuations as measurement errors, but also has problems with offset, noise, and drift with respect to temperature, as well as with the self-heating of the temperature sensor. An important aspect of the temperature control system 400 in Figure 8 is a chopper stabilization operation that includes interrupt service routines triggered by a first timer 420 and a second timer 422 associated with the periodic collection of a temperature determination dataset used by a controller unit 414 to periodically perform temperature determinations and provide temperature control commands as needed. The chopper stabilization operation significantly reduces the self-heating of the temperature sensor while obtaining temperature sensor readings at a desired sampling interval and operating the semiconductor electronic equipment at high frequencies to reduce so-called 1 / f semiconductor noise. In this regard, the power-related noise of semiconductor characteristics is often proportional to the reciprocal of the power frequency (1 / f) in the lower frequency range (e.g., frequencies below approximately 10 to 100 Hz) and transitions to being almost frequency-independent in the higher frequency range (e.g., frequencies above approximately 100 Hz). In preferred operation, the power from the current source 410 and the operation of the analog-to-digital converter 408 are within such higher frequency ranges.

[0040] Conveniently, as shown in Figure 8, the functions of the analog-to-digital converter 408, current source 410, switch unit 412, controller unit 414, first timer 420, second timer 422, and PWM unit 424 may all be contained within a single microchip 430, facilitating a simplified circuit board design for the equipment module 200 and significantly reducing the footprint and cost of the electronic components. The driver unit 426 and reference resistor 406 can be conveniently mounted as electronic components on circuit board 288, which is a common circuit board with the microchip. Figure 5 shows an exemplary location of the temperature sensor 404 and the microchip 430 on circuit board 288. The reference resistor 406 and driver unit 426 may also be components mounted on circuit board 288.

[0041] Referring to Figures 8 and 9, an exemplary procedure for collecting a temperature determination dataset using the temperature control system 400 at the direction of the controller unit 414 is illustrated in the timeline shown in Figure 9. Figure 9 shows the timeline for collecting a temperature determination dataset, which includes a first digital output from the analog-to-digital converter 408 corresponding to the temperature sensor reading and a second digital output from the analog-to-digital converter 408 corresponding to the reference reading. Time zero (t0) is triggered by the first timer 420 to initiate a first interrupt service routine (I 1,1 ) corresponds to. The first interrupt service routine, which starts at t0, includes restarting the first timer 420 and executing over the first duration (Δt1) of the first timer 420, setting switches 332, 334, and 336 of the switch unit 412 to position 1 according to the value of the loop counter for the switch unit 412, directing the current from the current source 410 to the temperature sensor 404, and starting the second timer 422 and executing over the second duration (Δt2) of the second timer 422. After the expiration of the second duration (Δt2) at t1, the interrupt (I 2,1An interrupt occurs, initiating a second interrupt service routine triggered by the second timer 422. The second interrupt service routine, which begins at t1, includes turning off the second timer 422 and starting the analog-to-digital converter 408. During the subsequent third duration (Δt3), when the digital output result becomes available from the analog-to-digital converter, an interrupt is triggered, the first digital output corresponding to the temperature sensor reading is acquired and stored by the controller unit 414, the analog-to-digital converter 408 is turned off, and the loop counter (specifying the measurement point as either the temperature sensor 404 or the reference resistor 406) is incremented. As shown in Figure 8, the temperature sensor reading includes the voltage drop across the temperature sensor 404, and the temperature sensor reading is input to the analog-to-digital converter 408. At the end of the first duration (Δt1) at t2, an interrupt (I) of the first timer 420 occurs. 1,2 An interrupt (I) occurs, triggering the next first interrupt service routine, which is initiated by the first timer 420. The first interrupt service routine, which starts at t2, includes restarting the first timer 420 and executing over the first duration (Δt1) of the first timer 420, setting switches 432, 434, and 436 of the switch unit 412 to position 2 according to the value of the loop counter for the switch unit 412, directing the current from the current source 410 to the reference resistor 406, and starting the second timer 422 and executing over the second duration (Δt2) of the second timer 422. After the second duration (Δt2) expires at t3, the interrupt (I) of the second timer is initiated. 2,2An interrupt occurs, triggered by the second timer 422, which starts a second interrupt service routine. The second interrupt service routine, which starts at t3, includes turning off the second timer 422 and starting the analog-to-digital converter 408. During the subsequent third duration (Δt3), when the digital output result becomes available from the analog-to-digital converter, an interrupt is then triggered, the second digital output corresponding to the reference reading is acquired and stored by the controller unit 414, the analog-to-digital converter 408 is turned off, and the corresponding loop counter is incremented. As shown in Figure 8, the reference reading is the voltage drop across the reference resistor 406, and the reference reading is input to the analog-to-digital converter 408. When the first duration (Δt1) expires at t4, an interrupt (I) of the first timer 420 is triggered. 1,3 This occurs, initiating the next first interrupt service routine to similarly acquire the next temperature determination dataset. As can be understood, the temperature determination dataset may alternatively include a pair of digital outputs in which a second digital output corresponding to a reference reading is acquired prior to a first digital output corresponding to a temperature sensor reading.

[0042] After collecting a temperature determination dataset, the controller unit 414 may perform a temperature determination, which may be performed each time a temperature determination dataset is collected, or only after a certain number of temperature determination datasets have been collected since the previous temperature determination. The temperature determination dataset includes first data supplied by a first digital output corresponding to the temperature sensor reading with respect to the voltage drop at the temperature sensor (Vsensor), and second data supplied by a second digital output corresponding to the reference reading with respect to the voltage drop at the reference resistor (Vref). The temperature determination may be performed using the following relationship between Vsensor and Vref.

[0043] Vsensor = Isensor × Rsensor Vref=Iref×Rref Here, Isensor is the current flowing through the temperature sensor 404 corresponding to the temperature sensor reading, Rsensor is the resistance of the temperature sensor 404 corresponding to the temperature sensor reading, Iref is the current flowing through the reference resistor 406 corresponding to the reference reading, and Rref is the resistance of the reference resistor 406 corresponding to the reference reading. Since the current supplied from the current source 410 is essentially constant, Isensor is essentially equal to Iref, and the value of Rref is known for the reference resistor 406, so the unknown variable Rsensor can be calculated as follows.

[0044] Rsensor = (Vsensor / Vref) × Rref The calculated Rsensor value is then compared by the controller unit 414 to a temperature-versus-Rsensor correlation table for the temperature sensor 404 to determine the temperature corresponding to the temperature sensor reading. This temperature is used by the controller unit 414 to compare with the setpoint temperature to determine whether and how much the electric heating unit 202 needs to be operated to maintain the controlled environment temperature (e.g., the temperature of the common optical component mounting platform 252) near the setpoint temperature. The correlation table may be stored in the controller unit 414's non-temporary memory, accessible, for example, by the controller unit 414's computer processor. The controller unit 414 may also analyze trends in multiple temperature determinations when determining whether and how much the electric heating unit 402 needs to be operated. As understood, it is important to select a setpoint temperature higher than the expected ambient air temperature in the lower compartment 206 during the operation of the flow cytometry survey system 102 of the instrument module 200.

[0045] The second duration (Δt2) should be selected to be long enough to allow signal settling following the switching of current to the temperature sensor 404 or the reference sensor 406. The first duration (Δt1) should be extended for at least a time equal to the second duration (Δt2), plus a time appropriate for obtaining the digital output from the analog-to-digital converter 408 based on the cycle time of the analog-to-digital converter 408.

[0046] A key advantage of the chopper stabilization operation of the temperature control system 400 for collecting temperature determination datasets is that current is not continuously supplied to the temperature sensor 404, which greatly reduces the possibility of adverse self-heating of the temperature sensor, which could lead to serious temperature sensor reading errors. At the same time, the current source 410 and the analog-to-digital converter 408 can operate at high frequencies outside the 1 / f noise region.

[0047] One exemplary set of operating variables for the temperature control unit 400 is provided, which includes a second duration of 1 millisecond for the second timer 422 and a conversion frequency of 600 Hz (cycle time of 1.66 milliseconds) for the analog-to-digital converter. The first duration of the first timer 420 is at least 2.66 milliseconds (1 millisecond + 1.66 milliseconds), and preferably somewhat longer to ensure the acquisition of the digital conversion from the analog-to-digital converter 408.

[0048] Referring now to Figure 10, one preferred configuration for positioning the temperature sensor 404 and the resistance heating elements of the electric heating unit 402 relative to a common optical component mounting platform 252 on which the components of the optical processing system 250 are mounted. Figure 10 shows a common optical component mounting platform 252 having a mounting side 450 and an opposite side 452 on which the components of the optical processing system are mounted. The temperature sensor 404 is mounted on the mounting side 450, and two resistance heating elements 454, 456 (e.g., printed or adhesive films made of resistance heating material) are positioned adjacent to each other on the opposite side 452. The temperature sensor 404 is positioned near the longitudinal center of the common optical component mounting platform 252, while the resistance heating elements 454, 456 are positioned closer to the longitudinal end of the common optical component mounting platform 252 to provide a certain separation distance of thermocouples between the temperature sensor 404 and the resistance heating layers 454, 456 via the common optical component mounting platform 252. The resistance heating elements 454 and 456 are shown to protrude from adjacent portions on opposite sides 452 of the common optical component mounting platform 252; however, in an alternative configuration, the common optical component mounting platform 252 may have recesses on the opposite side 452 in which a resistance heating layer can be positioned.

[0049] Example implementation combinations With or without additional features as disclosed above or elsewhere in this specification, some other intended embodiments of combinations of implementations for various aspects of this disclosure can be summarized as follows:

[0050] (Section 1) A flow cytometry evaluation system, A flow cytometry investigation system including an investigation zone, wherein the investigation zone is configured to receive the flow of a fluid sample during flow cytometry evaluation for flow cytometry investigation within the investigation zone regarding the presence of particles in the flow of the fluid sample. It is a sample leakage system, An effluent collection container having an effluent inlet for receiving effluent of a fluid sample leaving the investigation zone during flow cytometry evaluation, A sample discharge system comprising an outflow fluid guideway from the investigation zone to the outflow fluid inlet, A flow cytometry evaluation system comprising a sample outflow system and a pressurized gas delivery system in fluid communication, wherein the pressurized gas delivery system is configured to apply pressurized gas to pressurize at least a portion of the fluid sample outflow system with an applied gas pressure that supplies positive back pressure into the outflow fluid conduit, thereby obstructing the flow of fluid through the outflow fluid conduit toward the outflow fluid inlet during a flow cytometry investigation.

[0051] (Section 2) The flow cytometry evaluation system according to item 1, wherein a pressurized gas is applied to the sample discharge system at a height within the sample discharge system that is lower than the lowest height within the investigation zone.

[0052] (Section 3) The flow cytometry evaluation system according to item 2, wherein the applied gas pressure is a gauge pressure that is at least the same magnitude, preferably greater, more preferably at least 0.1 psi (0.69 kPa), and even more preferably at least 0.2 psi (1.38 kPa), compared to the head pressure of a water column at a vertical height equal to the height difference between the height at which the pressurized gas is applied to the sample outflow system and the lowest height of the investigation zone.

[0053] (Section 4) The flow cytometry evaluation system according to claim 2 or 3, wherein the height difference between the lowest height of the investigation zone and the height in the sample outflow system to which pressurized gas is applied is at least 15 centimeters, preferably at least 30 centimeters, optionally 120 centimeters or less, or preferably 80 centimeters or less.

[0054] (Section 5) A flow cytometry evaluation system according to any one of items 1 to 4, wherein a pressurized gas is applied to the effluent recovery container.

[0055] (Section 6) The flow cytometry evaluation system according to item 5, wherein the outflow fluid inlet is located at a height lower than the lowest height of the investigation zone, and the applied gas pressure in the outflow fluid recovery container is a gauge pressure that is at least the same magnitude as, preferably larger than, more preferably at least 0.1 psi (0.69 kPa), and even more preferably at least 0.2 psi (1.38 kPa), compared to the head pressure of a water column at a vertical height equal to the height difference between the outflow fluid inlet height and the lowest height of the investigation zone. Optionally, the gauge pressure of the applied gas pressure is not greater than 1.0 psi (6.89 kPa) compared to the head pressure of a water column at a vertical height equal to the height difference between the outflow fluid inlet height and the lowest height of the investigation zone.

[0056] (Section 7) The flow cytometry evaluation system according to item 6, wherein the height difference between the lowest height of the survey zone and the height of the outflow liquid inlet is at least 15 centimeters, preferably at least 30 centimeters, optionally 120 centimeters or less, or preferably 80 centimeters or less.

[0057] (Section 8) A flow cytometry evaluation system according to any one of items 1 to 7, comprising a fluid sample guideway to an investigation zone for supplying a fluid sample to the investigation zone for flow cytometry investigation.

[0058] (Section 9) The flow cytometry evaluation system according to item 8, wherein the fluid sample conduit, investigation zone, outflow fluid conduit, and outflow fluid collection container are configured to form a pressurized fluid system during the flow cytometry investigation, and the fluid flow toward the outflow fluid collection container through the fluid system is obstructed by back pressure from the applied gas pressure.

[0059] (Section 10) The flow cytometry evaluation system described in Section 9, wherein the highest height in the fluidic system is located within the fluid sample guideway.

[0060] (Section 11) A flow cytometry evaluation system as described in any one of items 8-10, wherein the highest height in the fluid sample conduit is higher than the highest height in the survey zone and higher than the height of the outflow fluid inlet.

[0061] (Section 12) A flow cytometry evaluation system according to item 11, wherein the applied gas pressure is applied to the effluent recovery container at a gauge pressure that is at least the same magnitude, preferably greater, more preferably at least 0.1 psi (0.69 kPa), and even more preferably at least 0.2 psi (1.38 kPa), compared to the head pressure of a water column at a vertical height equal to the height difference between the highest point in the fluid sample passage and the height of the effluent inlet. Optionally, the gauge pressure of the applied gas pressure is not greater than 1.0 psi (6.89 kPa) compared to the head pressure of a water column at a vertical height equal to the height difference between the highest point in the fluid sample passage and the height of the effluent inlet.

[0062] (Section 13) A flow cytometry evaluation system according to either item 11 or 12, wherein the height difference between the highest point in the investigation zone and the highest point in the fluid sample conduit is at least 15 centimeters, preferably at least 30 centimeters, optionally 120 centimeters or less, or preferably 80 centimeters or less.

[0063] (Section 14) The system includes an autosampler configured to receive several fluid samples contained in multiple sample containers and sequentially deliver several fluid samples to a fluid sample pathway in order to continuously guide them to an investigation zone for continuous flow cytometry evaluation of the fluid samples. A flow cytometry evaluation system according to any one of items 8 to 13, wherein the flow cytometry survey system and the autosampler are in a stack relationship, and the autosampler is located in a first stack location above a second stack location where the flow cytometry survey system is located.

[0064] (Section 15) The flow cytometry evaluation system according to item 14, comprising a housing in which a flow cytometry survey system and an autosampler are housed, wherein a first stacking location is located in a first compartment within the housing, and a second stacking location is located in a second compartment within the housing, below the first compartment.

[0065] (Section 16) The autosampler includes a sample receiving area configured to accept multiple sample containers, each containing several fluid samples for continuous flow cytometry evaluation. The autosampler includes a sample delivery probe configured to draw fluid samples from a sample container to sequentially deliver several fluid samples to the investigation zone for continuous flow cytometry evaluation. The sample receiving area is located at a height higher than the highest point in the survey zone, preferably at least 15 centimeters higher, more preferably at least 30 centimeters higher, and optionally at a height of 120 centimeters or less, preferably 80 centimeters or less, higher than the highest point in the survey zone. A flow cytometry evaluation system as described in either item 14 or item 15.

[0066] (Section 17) A flow cytometry evaluation system according to any one of items 14 to 16, comprising a waste container in fluid communication with an autosampler to receive waste liquid from the autosampler, wherein the waste container is not pressurized.

[0067] (Section 18) A flow cytometry evaluation system according to any one of claims 1 to 17, wherein the pressurized gas delivery system comprises a gas pressure regulator in fluid communication with a sample discharge system, the gas pressure regulator being configured to receive a pressurized gas input and supply a regulated gas output to provide an applied gas pressure to the sample discharge system, preferably to a discharge fluid recovery container.

[0068] (Section 19) The flow cytometry evaluation system according to item 18, wherein the pressurized gas delivery system comprises a gas compressor in fluid communication with a gas pressure regulator.

[0069] (Section 20) A flow cytometry evaluation system as described in any one of sections 1 to 19, wherein the flow cytometry survey system is part of a single-unit instrument module.

[0070] (Section 21) A flow cytometry evaluation system according to item 20, comprising a gas compressor as specified in item 19, wherein the gas compressor is part of an equipment module.

[0071] (Section 22) A flow cytometry evaluation system according to either item 20 or 21, comprising an autosampler as described in any one of items 14 to 17, wherein the autosampler is part of an instrument module.

[0072] (Section 23) A flow cytometry evaluation system according to item 20, comprising a gas compressor as described in item 19 and an autosampler as described in any one of items 14 to 17, wherein the autosampler and the gas compressor are part of an equipment module and are located within a common housing of the equipment module.

[0073] (Section 24) A flow cytometry evaluation system according to any one of items 1 to 23, wherein the flow cytometry survey system comprises an optical processing system supported on a common optical component mounting member (optionally, a platform), the optical processing system comprising a flow cell having a survey zone, a focusing element for focusing input light prior to the survey zone, and a photodetection system for detecting response radiation from the survey zone.

[0074] (Section 25) The flow cytometry evaluation system according to item 24, wherein the flow cytometry survey system comprises a light source that supplies input light, and the light source is optically connected to a focusing element by an inlet optical conduction path comprising an optical fiber.

[0075] (Section 26) The flow cytometry evaluation system according to item 24, wherein the light source is a laser optically coupled to an optical fiber, and the laser is supplied to the optical fiber to conduct input light through an input optical conduction path to a focusing element.

[0076] (Section 27) A flow cytometry evaluation system according to either item 25 or 26, wherein the light source is not part of an optical processing system supported on a common optical component mounting member.

[0077] (Section 28) The flow cytometry evaluation system according to item 27, wherein the optical fiber has a first end adjacent to the light source for receiving input light from the light source, and a second end supported on a common optical component mounting member for supplying input light to a focusing element.

[0078] (Section 29) A housing containing an internal containment space, wherein a flow cytometry survey system is placed within the internal containment space during flow cytometry evaluation. A flow cytometry evaluation system according to any one of claims 24 to 28, comprising: a posable member on which a flow cytometry survey system is supported, which is posable to provide improved maintenance access to the flow cytometry survey system between a first position in which the flow cytometry survey system is located within a housing internal space and a second position in which at least a portion, preferably all, of the flow cytometry survey system is located outside the housing internal space.

[0079] (Section 30) The flow cytometry evaluation system according to paragraph 29, wherein the housing comprises an access member, the access member being movable to open the housing, and providing access to a movable member to move the movable member from a first position to a second position.

[0080] (Section 31) The flow cytometry evaluation system according to paragraph 30, wherein when the access member is in the closed position, the translatable member is fully housed within the internal space of the housing.

[0081] (Section 32) A flow cytometry evaluation system according to either item 30 or 31, wherein the access member comprises a removable access panel.

[0082] (Section 33) A flow cytometry evaluation system according to any one of claims 29 to 32, wherein a common optical component mounting member is spaced apart from a sliding member by at least one support member on which the common optical component mounting member is supported, and a light source is attached to the support member.

[0083] (Section 34) The optical processing system is equipped with a temperature control system that controls the temperature inside the housing where it is located, and this temperature control system, The system includes a controller unit configured to periodically collect a temperature determination dataset, which includes a first digital output and a second digital output corresponding to the temperature sensor reading and the reference reading, respectively, and the collection of the temperature determination dataset is performed as follows: The current is directed in a first direction, and the first digital output corresponding to the sensor reading is obtained after a first signal settling period following the commencement of the first direction; The process includes, after obtaining the first digital output described above, directing the current to a second direction, and after a second signal settling period following the commencement of the second direction, obtaining the second digital output corresponding to the reference reading described above. Optionally, the temperature control system is An electric heating unit located within the housing and capable of selectively operating to heat the environment within the housing, A temperature sensor located inside the housing and capable of supplying temperature sensor readings corresponding to temperature conditions, A reference resistor located inside the housing and capable of operating to supply a reference reading, An analog-to-digital converter that can selectively receive either the temperature sensor reading from the temperature sensor or the reference reading from the reference resistor, and supply the corresponding digital output, A current source that selectively supplies current to the temperature sensor to generate the temperature sensor reading, or to the reference resistor to generate the reference reading, A switch unit selectively switches the direction of the current to a temperature sensor or a reference resistor, and selectively switches the input to an analog-to-digital converter to receive the temperature sensor reading or the reference reading, A controller unit configured to control the operation of a temperature control system, wherein this operation is Periodically collect a temperature determination dataset including the first digital output and the second digital output from the analog-to-digital converter corresponding to the sensor reading and the reference reading. The above temperature determination dataset will be used to periodically perform temperature determination, This includes instructing the operation of the electric heating unit based at least partially on the above temperature determination, Collecting a temperature determination dataset is The first method involves using a switch unit to direct the current to the temperature sensor, and the resulting sensor reading to the analog-to-digital converter. Following a first signal settling period that begins with the commencement of the first orientation, the controller unit acquires the first digital output corresponding to the sensor reading, After obtaining the first digital output described above, a switch unit is used to direct the current to a reference resistor, and the resulting reference reading to an analog-to-digital converter, thus providing a second direction. A flow cytometry evaluation system according to any one of paragraphs 24 to 33, comprising: obtaining the second digital output corresponding to the reference reading by the controller unit after a second signal settling period following the commencement of the second orientation.

[0084] (Section 35) The temperature control system A first timer that measures a first duration between the start of the first orientation and the start of the second orientation in order to collect the above temperature determination dataset, The flow cytometry evaluation system according to item 34, comprising: a second timer for timing a second duration of a first signal settling period.

[0085] (Section 36) The flow cytometry evaluation system according to item 35, wherein the first signal settling period and the second signal settling period are each equal to a second duration and are each timed by a second timer.

[0086] (Section 37) A flow cytometry evaluation system according to either item 35 or 36, wherein the duration between the start of a second orientation for collecting a temperature determination dataset and the start of the next first orientation for collecting the next temperature determination dataset is equal to the first duration and is timed by a first timer.

[0087] (Section 38) A flow cytometry evaluation system according to any one of items 35 to 37, wherein a current source, an analog-to-digital converter, a controller unit, a first timer, and a second timer are located on a single microchip.

[0088] (Section 39) The flow cytometry evaluation system according to Section 38, comprising a pulse width modulation unit on a microchip that communicates with a controller unit, which receives temperature control commands from the controller unit and instructs drive commands to drive the operation of a heating unit to heat the environment.

[0089] (Section 40) A flow cytometry evaluation system according to any one of sections 34 to 39, configured to collect the above temperature determination dataset at a frequency ranging from 10 to 150 times per second.

[0090] (Section 41) A flow cytometry evaluation system according to any one of items 34 to 40, wherein a temperature sensor is arranged to detect the temperature of a common optical component mounting member.

[0091] (Section 42) A flow cytometry evaluation system according to any one of items 34 to 41, wherein a temperature sensor is arranged to detect the temperature on the mounting side of a common optical component mounting member to which the optical processing system is attached.

[0092] (Section 43) The flow cytometry evaluation system according to item 42, wherein the electric heating unit comprises an electric heating element adjacent to the surface of the common optical component mounting member on the side opposite to the mounting side of the common optical component mounting member.

[0093] (Section 44) A flow cytometry evaluation system according to any one of items 34 to 43, wherein the temperature sensor comprises a thermistor.

[0094] (Section 45) A flow cytometry evaluation system according to any one of claims 34 to 44, wherein the controller unit is configured to maintain a setpoint temperature in the range of 25°C to 45°C, more preferably in the range of 28°C to 33°C.

[0095] (Section 46) A receiving location for receiving at least one fluid container at a receiving position to accommodate the fluid related to the operation of the flow cytometry evaluation system, A lighting system configured to illuminate the internal space within the fluid container when it is in the receiving position, A flow cytometry evaluation system according to any one of items 1 to 45, comprising:

[0096] (Section 47) The flow cytometry evaluation system according to item 46, comprising an illumination element, optionally comprising LEDs, wherein the fluid container in the receiving position is observable from the front of the fluid container, and the light illumination system is positioned to illuminate the fluid container through the rear side opposite to the front.

[0097] (Section 48) A flow cytometry evaluation system according to either item 46 or 47, wherein the receiving area is configured to receive a plurality of fluid containers, each at a separate receiving position, with the internal space being illuminated by a light illumination system.

[0098] (Section 49) The flow cytometry evaluation system according to item 48, wherein the light illumination system comprises separate illumination elements for illuminating each of the multiple fluid vessels.

[0099] (Section 50) A flow cytometry evaluation system according to any one of claims 46 to 49, wherein the receiving area is located in a container compartment having an optically transparent housing portion, and when illuminated by a light illumination system, each of the fluid containers at the receiving area is observable through the optically transparent housing portion.

[0100] (Section 51) A flow cytometry evaluation system according to any one of claims 46 to 50, wherein the light illumination system comprises at least one illuminating element, which is optionally located in the container compartment of claim 50 and oriented to illuminate the interior space of the fluid container when the fluid container is placed in the receiving location at the receiving position.

[0101] (Section 52) A flow cytometry evaluation system according to any one of claims 46 to 51, comprising at least one of the above-mentioned fluid containers, optionally a plurality of the above-mentioned fluid containers, each of which is placed in a receiving location at the above-mentioned receiving position.

[0102] (Section 53) The flow cytometry evaluation system according to item 52, comprising an effluent recovery container placed at the receiving location at the above receiving position.

[0103] (Section 54) A flow cytometry evaluation system according to either item 52 or 53, comprising a sheath fluid container positioned at the receiving location at the above receiving position, wherein the sheath fluid container contains sheath fluid for use in the flow cytometry evaluation and is fluidly connected to the investigation zone.

[0104] (Section 55) The flow cytometry evaluation system according to any one of paragraphs 52 to 54, further comprising a drive fluid container positioned at the receiving location in the above receiving position, wherein the drive fluid container contains a drive fluid for pushing and passing a fluid sample into the investigation zone during the flow cytometry evaluation, and is fluidly connected to a fluid sample guideway to the investigation zone.

[0105] (Section 56) An autosampler, which is optionally an autosampler described in any one of items 14 to 17, A flow cytometry evaluation system according to any one of claims 52 to 55, further comprising a waste container placed at the receiving location in the above receiving position for collecting waste liquid from the operation of an autosampler.

[0106] (Section 57) Further comprising an autosampler in any one of the combinations described in items 14 to 17, A flow cytometry evaluation system according to any one of items 52 to 56, wherein the above-mentioned receiving position is located in front of the second compartment at a lower height than the first compartment.

[0107] (Section 58) A method for evaluating flow cytometry, This involves flowing a fluid sample through the investigation zone of a flow cytometry system, where the downstream end of the investigation zone is in fluid communication with a sample outflow system, and this sample outflow system is An effluent collection container having an effluent inlet for receiving effluent of a fluid sample leaving the investigation zone during flow cytometry evaluation, A fluid outflow channel from the investigation zone to the fluid outflow inlet, and a fluid flow mechanism, Perform a flow cytometry study of the fluid sample flow within the survey zone, The fluid sample effluent leaving the survey zone is guided through the effluent conduit to an effluent collection container where the fluid sample effluent is collected. Fluid samples are flowed through the investigation zone. doing A method comprising, in between, applying a pressurized gas to pressurize at least a portion of the fluid sample discharge system by providing a positive back pressure within the discharge fluid conduit and thereby obstructing the flow of fluid through the discharge fluid conduit toward the discharge fluid inlet of the discharge fluid collection container.

[0108] (Section 59) The method according to item 58, comprising supplying a fluid sample to an investigation zone through a fluid sample conduit, wherein during a flow cytometry investigation, the fluid sample conduit, the investigation zone, the outflow fluid conduit, and the outflow fluid collection container constitute a pressurized fluid system, and the flow of fluid in the direction toward the outflow fluid collection container through the fluid system is obstructed by back pressure from the applied gas pressure.

[0109] (Section 60) The process includes several sequential flow cytometry evaluations of multiple fluid samples, wherein the sequential flow cytometry evaluations include sequentially drawing several fluid samples from multiple sample containers by an autosampler and sequentially delivering several fluid samples to a fluid sample pathway for sequential delivery to an investigation zone for performing a flow cytometry investigation for each of the fluid samples. The method described in Section 59, wherein during the withdrawal, some of the multiple sample containers are at a height higher than the highest point in the investigation zone.

[0110] (Section 61) The method according to any one of claims 58 to 60, wherein the flow cytometry survey system comprises a light source that supplies input light and an input light conduction path that conducts the input light from the light source to the survey zone for flow cytometry surveying.

[0111] (Section 62) The method of paragraph 61, wherein the input optical conduction path comprises an optical fiber. (Section 63) The method according to any one of claims 58 to 62, wherein the flow cytometry survey system includes an optical processing system supported on a common optical component mounting member, the optical processing system comprising a flow cell having a survey zone, an inlet optical focusing element of an input optical conduction path for focusing input light prior to the survey zone, and an optical detection system for detecting response radiation from the survey zone.

[0112] (Section 64) The control includes controlling the temperature of a common optical component mounting member using a temperature control system as instructed by a controller unit, and the control includes the controller unit periodically collecting a temperature determination dataset including the first digital output and the second digital output corresponding to the temperature sensor reading and the reference reading, respectively, and the collection of the temperature determination dataset is The current is directed in a first direction, and the first digital output corresponding to the sensor reading is obtained after a first signal settling period following the commencement of the first direction; The process includes, after obtaining the first digital output described above, directing the current to a second direction, and obtaining the second digital output corresponding to the reference reading after a second settling period following the second commencement of the second direction, Optionally, the temperature control system is An electric heating unit is also located within a housing that also houses an optical processing system, and the electric heating unit is capable of selectively operating to heat the environment within the housing. A temperature sensor located inside the housing and capable of supplying temperature sensor readings corresponding to temperature conditions, A reference resistor located inside the housing and capable of operating to supply a reference reading, An analog-to-digital converter that can selectively receive either a temperature sensor reading from a temperature sensor or a reference reading from a reference resistor, and supply a corresponding digital output. A current source that selectively supplies current to either a temperature sensor to obtain a temperature sensor reading, or to a reference resistor to obtain a reference reading, A switch unit that selectively switches the direction of current to a temperature sensor or a reference resistor, and selectively switches the input to an analog-to-digital converter to receive the temperature sensor reading or the reference resistor reading, A controller unit configured to control the operation of a temperature control system, wherein this operation is Periodically collect a temperature determination dataset including the first digital output and the second digital output from the analog-to-digital converter corresponding to the sensor reading and the reference reading, The above data set is used to periodically perform the above temperature determination, This includes instructing the operation of the electric heating unit based at least partially on the above temperature determination, Collecting a temperature determination dataset is The first method involves using a switch unit to direct the current to the temperature sensor, and the resulting sensor reading to the analog-to-digital converter. Following a first signal settling period that begins with the commencement of the first orientation, the controller unit acquires the first digital output corresponding to the sensor reading, After obtaining the first digital output described above, a switch unit is used to direct the current to a reference resistor, and the resulting reference reading to an analog-to-digital converter, thus providing a second direction. The method of paragraph 63, comprising obtaining the second digital output corresponding to the reference reading by the controller unit after a second settling period following a second commencement of the second orientation.

[0113] (Section 65) The method according to item 64, further comprising heating a common optical component mounting member by heat supplied by the operation of an electric heating unit, as instructed by the controller unit.

[0114] (Section 66) The method described in any one of items 58 to 65, performed in a flow cytometry evaluation system described in any one of items 1 to 45.

[0115] (Section 67) The method described in any one of sections 58 to 66, including the operation of a flow cytometry evaluation system as described in any one of sections 1 to 57.

[0116] (Section 68) Use of a flow cytometry evaluation system described in any one of sections 1 to 57 to perform flow cytometry evaluation of each of multiple fluid samples, of any choice.

[0117] The foregoing description of the present invention and its various embodiments is provided for illustrative and explanatory purposes only. Furthermore, the description is not intended to limit the present invention to the forms disclosed herein. Accordingly, variations and modifications corresponding to the above teachings, as well as the art and knowledge of the related art, are within the scope of the present invention. The embodiments described above illustrate known modes of carrying out the present invention and are further intended to enable those skilled in the art to utilize the present invention with various modifications required by the particular use or application of the present invention in such or other embodiments. The appended claims are intended to be construed as including alternative embodiments to the extent permitted by the prior art.

[0118] A description of one or more features in a particular combination does not preclude the inclusion of one or more additional features in variations of that particular combination. Processing steps and orderings are for illustrative purposes only, and such examples do not preclude the inclusion of other steps or other orderings of steps to the extent that they are not necessarily incompatible. Additional steps may be included between any illustrated processing steps, or before or after any illustrated processing steps, to the extent that they are not necessarily incompatible.

[0119] The terms “comprising,” “containing,” “including,” and “having,” as well as their grammatical variations, are intended to be inclusive and non-restrictive in that their use indicates the existence of a stated condition or feature, but does not exclude the existence of any other condition or feature. The use of the terms “comprising,” “containing,” “including,” and “having,” as well as their grammatical variations, is intended to include and disclose more specific embodiments in which, when referring to the existence of one or more components, subcomponents, or materials, the terms “comprising,” “containing,” “including,” or “having” (or variations of such terms) are, in some cases, replaced by one of the narrower terms “consisting essentially of,” “consisting of,” or “consisting of only” (or any appropriate grammatical variation of such narrower terms). For example, the statement “contains” a stated element(singular or plural) is also intended to include and disclose more specific and narrower embodiments of what “essentially consists of” the stated element(singular or plural) and what “consists of” the stated element(singular or plural). Examples of various features are provided for illustrative purposes, and terms such as “example” and “for example” indicate exemplary examples that are not limiting and should not be interpreted as limiting one or more features to any particular example. The term “at least” (e.g., “at least one”) means that number or a number greater than that. The term “at least a portion” means all or less than all. The term “at least a part” means all or less than all.

Claims

1. A flow cytometry evaluation system, A flow cytometry investigation system including an investigation zone, wherein the investigation zone is configured to receive the flow of a fluid sample during flow cytometry evaluation for flow cytometry investigation of the presence of particles in the flow of the fluid sample within the investigation zone, It is a sample leakage system, An effluent recovery container having an effluent inlet for receiving the effluent of the fluid sample leaving the investigation zone during the flow cytometry evaluation, The sample discharge system includes an outflow fluid guideway from the investigation zone to the outflow fluid inlet, The system comprises a pressurized gas delivery system that is in fluid communication with the aforementioned sample outflow system, A flow cytometry evaluation system wherein the pressurized gas delivery system is configured to supply positive back pressure into the outflow fluid conduit, thereby pressurizing at least a portion of the sample outflow system with an applied gas pressure that obstructs the flow of fluid through the outflow fluid conduit toward the outflow fluid inlet during the flow cytometry investigation.

2. The flow cytometry evaluation system according to claim 1, wherein pressurized gas is delivered to the sample discharge system at a height in the sample discharge system that is lower than the lowest height in the survey zone.

3. To supply the fluid sample to the investigation zone for the flow cytometry investigation, the system further comprises a fluid sample guideway to the investigation zone, The flow cytometry evaluation system according to claim 1 or 2, wherein the fluid sample passage, the investigation zone, the outflow fluid passage, and the outflow fluid recovery container are configured to form a pressurized fluid system during the flow cytometry investigation.

4. The flow cytometry evaluation system according to claim 3, wherein the highest height in the pressurized fluidic system is higher than the outflow fluid inlet in the fluid sample conduit.

5. The flow cytometry evaluation system according to claim 4, wherein the applied gas pressure is applied to the effluent collection container at a gauge pressure that is at least the same magnitude as the head pressure of a water column with a vertical height equal to the height difference between the highest point in the fluid sample passage and the height of the effluent inlet.

6. The aforementioned flow cytometry evaluation system is An autosampler configured to receive some of the multiple fluid samples contained in multiple sample containers and to deliver some of the fluid samples to the fluid sample pathway for flow cytometry evaluation, The system further comprises a housing in which the flow cytometry survey system and the autosampler are arranged in a stacked relationship. A flow cytometry evaluation system according to any one of claims 3 to 5, wherein the first stacking location is in a first compartment within the housing in which the autosampler is located, and the second stacking location is in a second compartment within the housing in which the flow cytometry survey system is located, and the second compartment is located below the first compartment.

7. The autosampler includes a sample receiving area configured to receive a plurality of sample containers containing some of the plurality of fluid samples for flow cytometry evaluation, The autosampler includes a sample delivery probe configured to draw the plurality of fluid samples from the plurality of sample containers to deliver them to the investigation zone for flow cytometry evaluation, The flow cytometry evaluation system according to claim 6, wherein the sample receiving area is located at a height higher than the highest point within the survey zone.

8. The flow cytometry survey system includes an optical processing system supported on an optical component mounting member, the optical processing system comprising a flow cell having the survey zone, a focusing element that focuses input light prior to the survey zone, and a photodetection system that detects response radiation from the survey zone. The flow cytometry survey system includes a light source that supplies the input light, The flow cytometry evaluation system according to any one of claims 1 to 7, wherein the light source is optically connected to the light-collecting element by an entrance optical conduction path comprising an optical fiber.

9. The flow cytometry survey system includes an optical processing system supported on an optical component mounting member, the optical processing system comprising a flow cell having the survey zone, a focusing element that focuses input light prior to the survey zone, and a photodetection system that detects response radiation from the survey zone. The aforementioned flow cytometry survey system, During flow cytometry evaluation, the internal space in which the flow cytometry survey system is located, The flow cytometry evaluation system according to claim 8, further comprising: a movably mounted member on which the flow cytometry survey system is supported, the movably mounted member being movably mounted between a first position in which the flow cytometry survey system is positioned within the internal space and a second position in which at least a portion of the flow cytometry survey system is positioned outside the internal space.

10. The optical processing system further comprises a temperature control system that controls the temperature inside a housing located within it. The temperature control system, The system includes a controller unit configured to periodically collect a temperature determination dataset, which includes a first digital output and a second digital output corresponding to the temperature sensor reading and the reference reading, respectively. The collection of the aforementioned temperature determination dataset is Directing the current to a first direction, and obtaining the first digital output corresponding to the temperature sensor reading after a first signal settling period following the commencement of the first direction; A flow cytometry evaluation system according to claim 8 or 9, comprising: after acquiring the first digital output, directing the current to a second direction, and after a second signal settling period following the commencement of the second direction, acquiring the second digital output corresponding to the reference reading.

11. The temperature control system, A first timer for timing a first duration between the start of the first orientation and the start of the second orientation in order to collect the temperature determination dataset, A second timer that measures the second duration of the first signal settling period, A current source, an analog-to-digital converter, the controller unit, the first timer and the second timer, all located on a single microchip. The flow cytometry evaluation system according to claim 10, comprising: a pulse width modulation unit on the microchip that communicates with the controller unit, the pulse width modulation unit receiving a temperature control command from the controller unit and instructing a drive command to drive the operation of a heating unit to heat the environment inside the housing.

12. A method for evaluating flow cytometry, The fluid sample is flowed through the investigation zone of a flow cytometry investigation system, wherein the downstream end of the investigation zone is in fluid communication with a sample outflow system, and the sample outflow system is An effluent collection container having an effluent inlet for receiving the effluent of the fluid sample leaving the investigation zone during flow cytometry evaluation, The system includes an outflow fluid guide passage from the investigation zone to the outflow fluid inlet, and allows fluid to flow. Perform a flow cytometry survey of the fluid sample flow within the survey zone, The outflow of the fluid sample leaving the survey zone is guided through the outflow fluid guide passage to the outflow fluid collection container where the outflow of the fluid sample is collected. A method comprising supplying positive back pressure into the outflow fluid conduit while the fluid sample is flowing through the investigation zone, thereby pressurizing at least a portion of the sample outflow system with an applied gas pressure that obstructs the flow of fluid through the outflow fluid conduit toward the outflow fluid inlet of the outflow fluid collection container during the flow cytometry investigation of the fluid sample flow.

13. The system further comprises supplying the fluid sample to the investigation zone through a fluid sample guideway, The method according to claim 12, wherein the fluid sample guide passage, the investigation zone, the outflow fluid guide passage, and the outflow fluid recovery container constitute a pressurized fluid system, and the flow of fluid in the direction toward the outflow fluid recovery container through the pressurized fluid system is obstructed by back pressure from the applied gas pressure.

14. The flow cytometry survey system includes an optical processing system supported on an optical component mounting member, the optical processing system includes a flow cell having the survey zone, an inlet optical focusing element of an input optical conduction path that focuses input light prior to the survey zone, and an optical detection system that detects response radiation from the survey zone. The aforementioned method, The controller unit further provides a system that controls the temperature of the optical component mounting member using a temperature control system, The above control is The controller unit periodically collects a temperature determination dataset, which includes a first digital output and a second digital output corresponding to the temperature sensor reading and the reference reading, respectively. Collecting the aforementioned temperature determination dataset is Directing the current to a first direction, and obtaining the first digital output corresponding to the temperature sensor reading after a first signal settling period following the commencement of the first direction; The method according to claim 12 or 13, further comprising: after obtaining the first digital output, directing the current to a second degree, and obtaining the second digital output corresponding to the reference reading after a second settling period following a second commencement of the second degree of direction.

15. The method according to any one of claims 12 to 14, which is performed in a flow cytometry evaluation system according to any one of claims 1 to 11.

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