Modular flow cytometry system and sample treatment method

Abstract

To provide a modular flow cytometry system and a method for treating a sample.SOLUTION: This system comprises a removable and replaceable automatic or half-automatic moule. A sample passage module may be removed, installed in a micro-fluid device cleaning moule for washing and then re-installed or stored for subsequent use. This system further comprises an optical module, an electronic module, and a mixing and collecting module. An assembly and a detection laser assembly included in the optical module to be damaged by light may be located on the same side as the plane or surface of a flow cytometry and a side opposite to the detection assembly. A beam west of a laser beam is wider in a direction perpendicular to a flowing direction. The mixing and collecting module can automatically mix collected samples in a sample pipe, and replace the sample pipe with another sample pipe when the other pipe is full.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 63 / 230,568, filed Aug. 6, 2021; U.S. Provisional Application No. 63 / 162,222, filed Mar. 17, 2021; U.S. Provisional Application No. 63 / 119,769, filed Dec. 1, 2020; and U.S. Provisional Application No. 63 / 117,104, filed Nov. 23, 2020, each of which is incorporated herein by reference in its entirety.

[0002] This application incorporates by reference in its entirety U.S. Application No. 17 / 525,125, filed Nov. 12, 2021, entitled MODULAR FLOW CYTOMETRY SYSTEMS AND METHODS OF PROCESSING SAMPLES (Klas et al.), docket number GS-38-2021-US2.

[0003] The present invention relates to automated and semi-automated systems and methods for processing samples. More particularly, the present invention relates to systems and methods in a flow cytometry-based system for analyzing, handling, and processing fluids and components or particles in a sample.

[0004] The present invention features a modular flow cytometry system and method for cleaning a sample passageway in a flow cytometry system.

[0005] The present invention also features an optical system for use in detecting and sorting particles in flow cytometry, including particle sorting using electromagnetic radiation and detection in a microfluidic device such as a microfluidic chip.

[0006] The present invention further features a mixing and collection system for mixing particles or components in a sample with a medium and maintaining a desired suspension, such as a homogeneous mixture, of the particles or components in the sample and the medium.

Background Art

[0007] In the field of flow cytometry, it is often necessary to identify and sort particles such as individual cells after either the particles are properly aligned within the flow cytometer and each droplet is dispensed as a series of droplets containing individual particles, or the particles are moved through the internal detection region of the flow cytometer. In some applications, it may further be necessary to remove specific particles from the flow stream, or to otherwise sort, inactivate, or deactivate particles in the flow stream.

[0008] In some systems, a microfluidic device such as a microfluidic chip is used to receive a particle sample and / or a sheath or directed fluid and to focus, arrange, or otherwise position individual particles from the particle sample within the fluid flow in one or more channels of the microfluidic device. The particles and fluid may flow through one or more interrogation and / or actuation sites in the channels of the microfluidic device where detection, sorting, and / or destruction processes are performed on the particles. The particles may then be released from the microfluidic device or otherwise removed, such as by fluid flow or pressure.

[0009] Existing systems and methods for processing particles or components in a sample by a flow cytometry-based system include a number of steps, components, and elements, many of which rely on human intervention and manual processes. Extensive use of operator intervention, calibration, and operation in flow cytometry increases the likelihood of inaccurate system parameters or calibration, sample mishandling, contamination, and overall error. Existing systems and methods that rely primarily on human operators in all aspects of the process also have extended downtime and longer adjustment and calibration steps based on the extensive specialized manual intervention required. Manual processes may include preparing the sample, configuring the flow rates of the sample and sheath fluid, configuring or calibrating the optical path of an electromagnetic radiation emitter (e.g., a laser module), configuring the laser module emission characteristics including power and duration, configuring the detector, configuring or programming the sample processing characteristics, capturing or diverting the sample, and mixing and storing the processed sample.

[0010] Existing systems, devices, and methods for processing a sample such as a biological fluid sample containing a plurality of cells in a flow cytometry device such as a microfluidic system include using a disposable microfluidic chip, cassette, or cartridge. Other systems and methods include a flow cytometry device that may flow a cleaning fluid or air through a sample processing passage. Still other systems and methods include a separate stand-alone device for cleaning a microfluidic chip, cassette, or cartridge.

[0011] However, existing systems do not completely isolate the sample passageways used by fluid samples from flow cytometry or microfluidic analysis devices. For example, in existing systems, a portion of the sample passageway, such as a microfluidic chip or cassette, may be removed, but other portions of the sample passageway that are contacted by the fluid sample are part of the flow cytometry device itself and cannot be removed from the system. These portions may include passageways between the sample storage or mounting area and the microfluidic chip itself. Between sample runs, extensive cleaning and flushing of the flow cytometry device must be performed. This results in extensive downtime between the processing of separate fluid samples, but it is necessary to eliminate the possibility of biological or other contamination and to comply with various rules and regulations related to the processing of biological samples. For example, after processing a first fluid sample, such as a semen sample from a first male animal, the system requires extensive cleaning to eliminate the possibility of cross-contamination with a second fluid sample, such as a semen sample from a second male animal. Cross-contamination elimination is necessary when samples contain different genetic material or traits and contamination renders the samples unsuitable for use.

[0012] An additional problem with existing systems and methods is that while parts of the system may be removed for cleaning, the cleaning process itself can be labor-intensive and may require extensive manual input and configuration. However, many systems treat components such as microfluidic chips, cartridges, and cassettes as disposable, single-use items, significantly increasing the amount of single-use plastic or glass waste generated by the laboratory.

[0013] What is needed is a removable and replaceable sample passage module that can be removed from the flow cytometry device and cleaned with a separate cleaning device. The sample passage module should be reusable and easily removable for the purpose of exchanging between sample passage modules on the flow cytometry device in order to operate the flow cytometry device for sample processing almost continuously.

[0014] Furthermore, existing systems and methods include means for determining one or more properties of particles based on the fluorescence of particles detected by a detector such as an avalanche photodiode. The particles may be caused to fluoresce by directed and focused radiation of electromagnetic radiation such as a laser. The detected fluorescence may be used as a criterion for sorting decisions or destruction decisions.

[0015] In a sorting decision, specific particles having specific fluorescence characteristics with specific properties, or satisfying specific thresholds detected by one or more detectors, are sorted based on those characteristics. This sorting may include directing particles having specific characteristics by means including a magnetic field, a microvalve, or pneumatic or hydraulic pulses or jets into different flow streams and / or into different collection containers.

[0016] Using either a sorting decision or a destruction decision to process particles in flow cytometry has both advantages and disadvantages. In a particular field of flow cytometry, the sorting of sperm cells, where the sperm cells are assumed to contain either X-chromosome or Y-chromosome DNA for the purpose of sex discrimination, it is important to preserve the viability or fertility of the sperm cells after being processed in a flow cytometer system. The sorting decision process or method may cause excessive stress to the selected or desired sperm cells and may reduce the viability or fertility of the collected sample of sperm cells. The destruction decision process or method may leave sperm cells that have not been completely inactivated in the collected sample and may reduce the viability or fertility potential of the entire sample.

[0017] In a destruction determination involving inactivation or damage of particles, certain particles having specific fluorescence characteristics, including specific properties, or meeting specific thresholds detected by one or more detectors may be damaged or inactivated. This destruction may include ablation or optical damage by directed and focused radiation of electromagnetic radiation by a laser. Living cells such as sperm may sometimes be referred to as a "kill event" or "cell kill event" because the determination of destruction or inactivation results in cell death.

[0018] Furthermore, with respect to the destruction determination process, aligning the laser or other electromagnetic radiation emitting device used in the destruction process is a particularly time-consuming process that requires extensive training and expertise to perform properly. This problem is particularly common when changing between samples of specific particles, such as when changing between untreated sperm samples from different male animals in semen processing. Existing systems and methods only adjust the interrogation or the height of the destruction or kill location within the flow cytometer's interrogation or action site, which is a function of laser divergence and is not width-controlled (e.g., the size of the kill beam or kill location in the longitudinal or flow direction). Also, existing laser-based flow cytometer systems may include multiple optical systems and paths for providing fluorescence and "kill" lasers, and associated detector systems, in a single or set of adjacent interrogations or action sites. These existing systems may require complex and time-consuming precise adjustments and configurations to function properly. If not adjusted correctly, these existing systems may not function as desired, if at all. Multiple optical paths may also cause problems of crosstalk and interference between separate optical systems.

[0019] What is needed is an improved system and method for the detection and destruction of particles at an interrogation or action location in a flow cytometer system that improves the rate, ratio, or proportion of destruction for particles having specific fluorescence characteristics, having specific properties, or meeting specific thresholds detected by one or more detectors. What is needed is a laser-based flow cytometer having an adjustable, tunable, or improved width for the laser kill location.

[0020] Furthermore, sample mixing in existing systems requires a manual mixing process where the operator periodically, for example, gently swirls the contents of the capture tube every 5 minutes. This process results in several forms of error in terms of what is defined as gentle swirling, differences between operators in achieving mixing every 5 minutes, and the interpretation of removing the tube from the path of the sample, e.g., cells, or splashing the sample out of the tube by overmixing. Off-the-shelf orbital shakers can be automated but have their own disadvantages. One is that they can occupy space if they are too tall to fit under the dispensing equipment. Another disadvantage is that the orbital movement can move the capture tube out of the path of the dispensed sample or remove it outside. More compact mixers, such as vortexers, induce rapid oscillatory motion and mix rapidly. However, these solutions can cause stress to the cells. What is needed is a system and method for improving the sample collection and mixing process.

[0021] Furthermore, at least partial or full automation of the processes required for sample handling in a flow cytometry system, including automation or semi-automation of at least the above-described manual processes, is needed to improve efficiency, reduce downtime, and reduce operator-induced errors.

[0022] Existing site meter systems are large, bulky, and non-modular. Their components are shipped separately or can be disassembled from each other, so adjustment and operation require experts or specialized training. Once assembled, the components are incorporated into the device itself and cannot be removed or repaired individually without extensively disassembling the entire device or the device's table. These systems are intricate and complex and do not assist relatively unskilled technicians in on-site repair or calibration. Further, these systems require long downtimes for maintenance, repair, or cleaning, resulting in interruptions of several hours or more in the absence of skilled technicians. Accordingly, it is desirable to have a site meter system with modular assemblies that can be easily adjusted, operated, and repaired by any operator without the need for specialized training.

[0023] The problems or issues described herein are illustrative and do not include all problems or issues that can be addressed. One of ordinary skill in the art will understand that a system, apparatus, or method that provides a solution to the problems defined above or addresses them may solve or address other similar, complementary, or undefined problems.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

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[0025] This specification provides automated systems, devices, and methods, as well as semi-automated systems, devices, and methods for processing samples in a flow cytometry-based system. In some aspects, the invention features a modular flow cytometry system for processing samples.

[0026] In some embodiments, the modular flow cytometry system can include a support structure, an electronics module, a sample pathway module, a fluidics module, an interrogation module, and a sample mixing and collection module. Each module can be configured to be separately removable and reinstallable from the support structure, or replaceable with another identical module, and disposed on the support structure. In some embodiments, the system can further include a microfluidics device cleaning module for cleaning the sample pathway module.

[0027] In some other embodiments, a modular flow cytometry system for processing samples includes a support structure, an electronics module having one or more computer processors and electronic ports, a sample passage module including a microfluidic module having at least one microfluidic channel, a fluidics module fluidly coupled to the sample passage module to circulate one or more fluids through the sample passage module, an interrogation module including a detector and operably coupled to the electronics module, a detection laser and a kill laser for processing a sample flowing through the microfluidic chip, and a sample mixing and collection module configured to collect and mix the processed sample exiting the microfluidic module. Each module may be configured to be removably and reinstallably separate from the support structure, or replaceable with another identical module, for substantially continuous operation of the modular flow cytometry system, and may be disposed on the support structure. The kill laser and the detection laser may be disposed on a common side of the interrogation module with respect to each other. The sample mixing and collection module may be rotatable about a pivot to move in an arcuate path, enabling mixing of the sample with controlled and uniform movement. In some embodiments, the system may further include a microfluidic device cleaning module for cleaning the sample passage module.

[0028] In some embodiments, the invention features a modular flow cytometry system for sample property discrimination. The system can include a chassis divided into multiple levels. At a first, lowest level of the multiple levels of the chassis, an electronics module is disposed that includes one or more computer processors and a plurality of electronics ports. At a second level of the chassis, a sample passage module is disposed that includes a microfluidic module for holding a microfluidic chip, and a fluidics module fluidly coupled to the sample passage module to circulate one or more fluids through the sample passage module. At a third level of the chassis, an interrogation module is disposed that is operably coupled to the electronics module, and the interrogation module includes a detection laser operably coupled to a detection assembly and a kill laser operably coupled to the detection assembly to process a sample flowing through the microfluidic chip, and the kill laser and the detection laser are disposed on a common side of the interrogation module relative to each other. Also, at the same second level of the chassis, below the microfluidic module, a sample mixing and collection module is disposed that is configured to collect and automatically mix the processed sample exiting the microfluidic chip. The sample mixing and collection module rotates about a pivot to move in an arcuate path to enable mixing of the sample with a controlled, uniform motion. Each module can be configured to be removably and reinstallably separate from a support structure, or replaceable with another identical module, for substantially continuous operation of the modular flow cytometry system, and can be disposed on the support structure.

[0029] In other embodiments, the sample passage module may further comprise a distribution block fluidly coupled to the microfluidic module. The distribution block can deliver a sample to the microfluidic module through a first fluidic line and sheath fluid to the microfluidic module through a second fluidic line. In other embodiments, the system does not require sheath fluid and the microfluidic module may receive inflow only from a single fluidic line that delivers the sample. In some embodiments, the distribution block comprises a fluidic manifold. In other embodiments, the distribution block further comprises a sample tube loader.

[0030] In some embodiments, the system may further comprise a microfluidic device cleaning module for cleaning the sample passage module. The microfluidic device cleaning system is adapted to clean the sample passage module by circulating fluid through the sample passage module. In some embodiments, the microfluidic device cleaning module may comprise a set of fluid reservoirs, a pump assembly, and a distribution block interface comprising a distribution block fixing element and a distribution block adjustment element. The distribution block interface further comprises a set of fluid interfaces at least one of which is in fluid communication with at least one of the set of fluid reservoirs, and a microfluidic device interface comprising a microfluidic device fixing element and a microfluidic device alignment element. The microfluidic device interface further comprises a fluid interface in fluid communication with at least one of the set of fluid reservoirs. The distribution block interface may be adapted to fix the distribution block thereon, and the microfluidic device interface may be adapted to fix the microfluidic module thereon.

[0031] According to some embodiments, the interrogation module may further include a laser heat sink for a laser that can draw in air by natural convection to cool the optical module. In one embodiment, the kill laser and the detection laser are disposed on a common side in relation to the main surface of the microfluidic chip. In another embodiment, the kill laser and the detection laser are each adapted to emit a laser beam including an elliptical beam profile at the focal plane. In a preferred embodiment, the interrogation components of the interrogation module can be removed and are replaceable or reconnectable.

[0032] In other embodiments, the sample mixing and collection module may include a rotation base, a set of collection tube holders disposed on the rotation base, a set of collection tubes configured to be disposed in the collection tube holders of the set of collection tubes respectively, and a fluid level sensor. One of the collection tubes can be disposed under the microfluidic chip such that the processed sample is collected in the collection tube. The sample mixing and collection module is configured to exchange with another collection tube to collect the processed sample when the fluid level sensor detects that the collection tube has reached a desired filling level. In some embodiments, the sample mixing and collection module may further include a mechanical diverter configured to prevent the processed sample exiting the microfluidic module from entering the sample mixing and collection module.

[0033] In other embodiments, the electronics module includes an electronics box with a removable lid for housing a computer processor and other electronic components. The electronic components are removable from the electronics box and can be replaced or reconnected. In some embodiments, the electronics box is disposed on rails for easy movement. In other embodiments, the electronics module includes one or more fans for cooling the electronic components.

[0034] In some embodiments, one or more identification elements may be disposed on or etched into the surface of the microfluidic chip. Examples of one or more identification elements include, but are not limited to, alphanumeric strings, barcodes, QR codes (registered trademark), fiducial markers, or combinations thereof. In one embodiment, one or more identification elements may be used to identify the chip currently mounted in the microfluidic module. In another embodiment, one or more identification elements may be used to automatically align and position the microfluidic chip with respect to the detection laser and the kill laser. For example, a microfluidic chip with identification elements may be attached to a multi-axis stage, which may be adjusted to reposition the microfluidic chip as needed based on reading of the identification elements by a detector, camera, scanner, or sensor.

[0035] In some embodiments, the system may further comprise an automatic sample level detector adapted to utilize the sample flow rate to determine the sample level. The sample flow rate is associated with the cell rate. If the cell rate is lower or higher than the target cell rate, or does not fall within the tolerance range, the automatic sample level detector can adjust the sample flow rate by adjusting the input sample pressure. When the input sample pressure exceeds a threshold pressure, the automatic sample level detector can provide a notification that the sample level is low or below the threshold level.

[0036] In one embodiment, the sample is a semen sample containing sperm cells from a non-human mammal. Non-limiting examples of non-human mammals include cows, pigs, horses, sheep, and goats.

[0037] According to other embodiments, the present invention provides a method for sex discrimination of sperm. In one embodiment, the method for sex discrimination of sperm includes classifying sperm cells as having a first characteristic, a second characteristic, or a third characteristic, and selectively damaging at least a part of the sperm cells based on those classifications. The first characteristic may be the presence of an X chromosome from one sperm cell, which is referred to as a singlet. The second characteristic may be the presence of a Y chromosome from one sperm cell. The third characteristic may be the presence of both an X chromosome and a Y chromosome, or a repetition of one of the chromosomes, also referred to as multiples, by multiple sperm cells. As a non-limiting example, the third characteristic may be an X chromosome and an X chromosome, an X chromosome and a Y chromosome, a Y chromosome and an X chromosome, or a Y chromosome and a Y chromosome by two sperm cells.

[0038] In another embodiment, the method for sex discrimination of sperm may include sending a response command signal to a stained sperm cell using a first radiation source to generate fluorescence radiation, detecting the fluorescence radiation, classifying the sperm cells based on the detected fluorescence radiation, and selectively damaging the sperm cells using a second radiation source based on these classifications.

[0039] In yet another embodiment, the method for sex discrimination of sperm includes delivering a fluid stream containing stained sperm cells to an interrogation position, sending a response command signal to the stained sperm cells using a first laser to generate fluorescence radiation, detecting the fluorescence radiation, classifying the sperm cells based on the detected fluorescence radiation, and selectively damaging one or more subpopulations of the classified sperm cells using a second laser based on these classifications. In other embodiments, the method may further include staining the sperm cells to generate stained sperm cells. The sperm cells may be stained using a fluorescent DNA-binding dye.

[0040] In some embodiments, when a response command signal is sent to the stained sperm cells, fluorescence emission corresponding to one sperm cell or fluorescence emission corresponding to multiple sperm cells is generated. In one embodiment, damaging the sperm cells can result in DNA damage and / or membrane damage to the cells. In one embodiment, damaging the sperm cells causes the sperm cells to become infertile sperm cells or dead sperm cells.

[0041] According to other embodiments, the present invention provides a method for generating a sex-discriminated semen product. In one embodiment, the method comprises the steps of providing a modular flow cytometry system comprising an electronic module, a sample passage module, a fluidics module, an interrogation module, and a sample mixing and collection module, wherein each module is configured to be removably reinstalled or replaced with another of the same module and is disposed within a support structure; flowing a semen sample having stained sperm cells through the fluidics module and the sample passage module; flowing sheath fluid through the fluidics module and the sample passage module; causing the stained sperm cells to fluoresce using a detection laser in the interrogation module; detecting the fluorescence of the stained sperm cells; generating a sex-discriminated semen product by damaging a subpopulation of the stained sperm cells using a kill laser in the interrogation module based on the detected fluorescence; collecting the sex-discriminated semen product flowing from the sample passage module into the sample mixing and collection module; and mixing the collected sex-discriminated semen products.

[0042] In other embodiments, the method may further include the step of staining a semen sample containing sperm cells to produce stained sperm cells. In still other embodiments, the method may further include the step of cleaning the sample passage module using a microfluidic system cleaning module. In some embodiments, the radiation from the detection laser and the kill laser may include an elliptical beam profile. In other embodiments, the sample mixing and collection module rotates about a pivot to move in an arcuate path, enabling mixing of the sex-discriminated semen product with a controlled uniform motion.

[0043] In another embodiment, a method for generating a sex-discriminated semen product includes the steps of providing the modular flow cytometry system described herein; staining a semen sample containing sperm cells to produce stained sperm cells; flowing the semen sample through a fluidics module and a sample passage module; flowing a sheath fluid through the fluidics module and the sample passage module, wherein the sheath fluid, one or more channels in the microfluidics chip, or a combination thereof, target the stained sperm cells as they flow through the microfluidics chip; fluorescing the stained sperm cells by radiation from a detection laser that includes an elliptical beam profile; detecting the fluorescence of the sperm cells by a detection assembly; and generating a sex-discriminated semen product by inactivating one or more subpopulations of the stained sperm cells by radiation from a kill laser based on the detected fluorescence, wherein the radiation from the kill laser includes an elliptical beam profile and the detection laser and the kill laser are disposed on the same side of the microfluidics chip; collecting the sex-discriminated semen product flowing from the microfluidics chip into a sample mixing and collection module; and automatically mixing the collected sex-discriminated semen product based on a set of defined sample mixing parameters associated with the sample to be processed. The sample mixing and collection module can rotate about a pivot to move in an arcuate path and enable mixing of the sex-discriminated semen product with controlled uniform movement.

[0044] In some embodiments, the method may further include diverting the flow of the sex-discriminated semen product exiting the microfluidic chip to individual collectors away from the sample mixing and collection module. In other embodiments, the method may further include automatically aligning and positioning the microfluidic chip with respect to the detection laser and the kill laser by using identification elements disposed on the microfluidic chip. In some other embodiments, the method may further include determining the sample level using an automatic sample level detector and notifying when the sample level is below a threshold level. In further embodiments, the method may include cleaning the sample passage module using a microfluidic device cleaning module.

[0045] In some embodiments, the method may further include diverting the flow of the sex-discriminated semen product away from the microfluidic chip when the collection tube of the sample mixing and collection module is filled to a desired level with the sex-discriminated semen product, rotating the rotating base of the sample mixing and collection module such that a second collection tube is positioned to collect the sex-discriminated semen product, and diverting the flow of the sex-discriminated semen product to the second collection tube.

[0046] According to other embodiments, the present invention features a system, method, and apparatus for providing a removable and replaceable sample passage module for use in a flow cytometry device, and a microfluidic device cleaning system for automatically or semi-automatically cleaning the removable and replaceable sample passage module. The sample passage module includes a distribution block that is in fluid connection or fluid communication with the microfluidic module. The distribution block of the removable and replaceable sample passage module, the microfluidic module, and the fluid connection therebetween comprise the entire fluid passage through which a fluid sample is processed by the flow cytometry device through the removable and replaceable sample passage module. The distribution block of the removable and replaceable sample passage module and the microfluidic module are connected to an interface using a corresponding interface of either the flow cytometry device or the microfluidic device cleaning system to receive one or more fluids for cleaning or processing. The removable and replaceable sample passage module is releasably fixed to the flow cytometry device or the microfluidic device cleaning module and can be used, removed, cleaned, and reused or replaced to process a fluid sample.

[0047] The microfluidic system device cleaning system comprises an automatic or semi-automatic system for cleaning sample passage modules such as the removable and replaceable sample passage module described above. The microfluidic system device cleaning system comprises a distribution block of the sample passage module and an interface for the microfluidic system module, and one or more fluids can be processed through the sample passage module to clean the sample passage module. A set of one or more internal or external reservoirs contains one or more cleaning fluids used in the cleaning process. An internal or external control system such as a computer, a tablet, or a dedicated microprocessor is used to control the cleaning process. The control system is used to control the operation of other internal devices including one or more internal valves and one or more internal pumps, and to process signals from one or more sensors such as proximity sensors, cameras or image sensors, and flow sensors for controlling the operation of valves or pumps.

[0048] In addition, without wishing to be bound by any theory or mechanism of the present invention, the methods and systems described herein are advantageous because the unusable time generally spent when exchanging a sample run or a microfluidic chip in a flow cytometry device used for processing particles in a fluid sample is eliminated or substantially reduced by the removable, replaceable, and cleanable modular sample passage module and the microfluidic system device cleaning system.

[0049] In a first embodiment, a removable and replaceable fluid passage module for use in a flow cytometer system is provided, the fluid passage comprising a distribution block having a set of fluid inlets and a set of fluid outlets, at least one of the set of fluid inlets being in fluid communication with at least one of the set of fluid outlets to form a first sample passage element; a microfluidic module having a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixing means, at least one of the set of fluid inlets being in fluid communication with at least one of the set of fluid outlets to form a second sample passage element; a fluid passage fluidly connecting the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, a portion of the fluid passage joining and fluidly connecting the first sample passage element and the second sample passage element to form a sample passage, and a sample passage through which a sample processed by the flow cytometer system through the removable and replaceable fluid passage module is confined within the sample passage during processing.

[0050] The microfluidic system device fixing apparatus is adapted to fix one of a microfluidic chip or a microfluidic cassette in a microfluidic system module. The microfluidic system device fixing apparatus may include one or more adjusting means adapted to adjust the position of the microfluidic chip or the microfluidic cassette. The distribution block may include a fluid system manifold. The distribution block may further include a sample tube loader. The fluid passage module may further include a distribution block, and another inlet of a set of fluid inlets is in fluid communication with another outlet of a set of fluid outlets to form a first buffer fluid path element, and another inlet of a set of fluid inlets of the microfluidic system module is in fluid communication with another outlet of a set of fluid outlets to form a second buffer fluid path element. The fluid passage further includes a second portion that joins and fluidly connects the first buffer fluid path element and the second buffer fluid path element to form a buffer fluid passage, and the buffer fluid processed through the removable and replaceable fluid passage module is confined in the buffer fluid passage during processing.

[0051] The distribution block and the microfluidic system module may each further include releasable fixing means adapted to fix the removable and replaceable fluid passage module to the flow cytometer system. The releasable fixing means may include at least one iron plate or at least one magnetic plate. The removable and replaceable fluid passage module may be removable from the flow cytometer system by releasing the fixing means. The releasable fixing means for the removable and replaceable fluid passage module may be further adapted to fix the removable and replaceable fluid passage module to the fluid passage cleaning system.

[0052] The sample may be a mammalian semen sample. In one embodiment, the sample may include sperm cells such as sperm cells of non-human animals including pigs, cows, horses, and sheep. The sperm cells may be stained by soaking them with a dye. The flow cytometer system may process the sample to identify subpopulations in the sample. This system may process the sample to perform a semen sex discrimination operation.

[0053] In some embodiments, the fluid passage module may comprise an identification element. The identification element may comprise an optical identification element or a wireless identification element. The optical identification element may comprise a set of features or criteria on a microfluidic chip in the microfluidic module.

[0054] In another embodiment, a microfluidic device cleaning system is provided, which comprises a set of fluid reservoirs, a pump assembly, and a distribution block interface comprising a distribution block fixing element and a distribution block adjusting element. The distribution block interface further comprises a set of fluid interfaces at least one of which is in fluid communication with a fluid reservoir, and a microfluidic device interface comprising a microfluidic device fixing element and a microfluidic device alignment element. The microfluidic device interface further comprises a fluid interface in fluid communication with at least one of the set of fluid reservoirs, and the microfluidic device cleaning system is adapted to circulate fluid through a removable and replaceable fluid passage module fixed to the distribution block interface and the microfluidic device interface.

[0055] The removable and replaceable fluid passage module is adapted for use in a flow cytometer system and comprises a distribution block having a set of fluid inlets and a set of fluid outlets, wherein at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a first sample passage element; a microfluidic module having a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixing means, wherein at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a second sample passage element; and a fluid passage fluidly connecting the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, wherein a portion of the fluid passage joins and fluidly connects the first sample passage element and the second sample passage element to form a sample passage, and the sample processed by the flow cytometer system through the removable and replaceable fluid passage module is confined to the sample passage during processing.

[0056] The set of fluid reservoirs comprises a cleaning fluid reservoir and a waste fluid reservoir. The set of fluid reservoirs may be disposed outside the housing of the system. The microfluidic device cleaning system may further comprise a plurality of releasable connections that provide fluid connection to the external set of fluid reservoirs. The pump assembly may comprise a liquid pump and an air pump. At least one of the pumps comprises a peristaltic pump. The flow cytometer system may further comprise a set of valves for controlling the internal fluid flow. The flow cytometer system may further comprise a set of fluid flow sensors adapted to supply a signal indicative of the flow rate in one or more elements of the system. The flow cytometer system may further comprise a set of proximity sensors adapted to supply a signal indicative of the presence or absence of a removable and replaceable fluid passage module. The flow cytometer system may further comprise an imaging device for capturing a set of images of a removable and replaceable fluid passage module, a distribution block interface, or a microfluidic device interface.

[0057] The microfluidic device cleaning system may further comprise a monitoring and interface system comprising a processor, a memory, and an input / output interface. The monitoring and interface system may be adapted to receive a set of inputs for selecting or configuring a cleaning protocol for the microfluidic device cleaning system. The set of inputs may include a set of user inputs. The set of inputs may be automatically determined based on the nature or characteristics of a removable and replaceable fluid passage module. The monitoring and input system may be adapted to receive the set of inputs from at least one sensor and change the cleaning protocol based thereon. The monitoring and input system may be adapted to transmit a set of control signals to one or more valves, actuators, or pumps based on the cleaning protocol. The microfluidic device cleaning system may further comprise a flow cytometry device. The flow cytometry device may comprise a fluorescence-based detection and laser ablation flow cytometry device.

[0058] In another embodiment, a method for replacement and cleaning of a sample passage module for a flow cytometry system is provided, the method comprising releasing a sample passage module comprising the entire fluid passage for processing a sample by the flow cytometry system from the flow cytometry device, removing the sample passage module from the flow cytometry device, connecting the sample passage module to a corresponding receptacle of the microfluidic device cleaning system, configuring a set of cleaning parameters for the microfluidic device cleaning system, and automatically cleaning the sample passage module by the microfluidic device cleaning system based on the set of cleaning parameters.

[0059] In some embodiments, the sample passage module is a distribution block adapted for use in a flow cytometry system and comprising a set of fluid inlets and a set of fluid outlets, wherein at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a first sample passage element; a microfluidic module comprising a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixing device, wherein at least one of the set of fluid inlets is in fluid communication with at least one of the set of fluid outlets to form a second sample passage element; and a fluid passage fluidly connecting the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, wherein a portion of the fluid passage joins and fluidly connects the first sample passage element and the second sample passage element to form the fluid passage, and a sample processed by a flow cytometer system through the sample passage module is confined to the fluid passage during processing. The method may further include removing the sample passage module from the microfluidic device cleaning system. The method may further include a set of cleaning parameters including the amount or volume of the cleaning fluid, the fluid pressure, and the duration of the cleaning procedure. The duration of the cleaning procedure may include the number of passages to be swept, which is the number of sample passage volumes to be cleaned. The duration of the cleaning procedure may include at least one drying cycle.

[0060] In some embodiments, the method may further include using an imaging sensor to detect an obstruction in the sample passage module and stopping the automatic cleaning. The step of stopping the automatic cleaning may further include supplying an error indication. The method may further include using at least one flow sensor to measure the volume of fluid used for the automatic cleaning. The measured volume may be used to determine a maintenance event for the microfluidic device cleaning system.

[0061] In other embodiments, the method may further include transmitting, from at least one proximity sensor, a signal indicating that the sample passage module is properly connected, to the microfluidic device cleaning system, and initiating automatic cleaning based on the transmitted signal. The sample passage module may be adapted for use in a flow cytometry system. The entire fluid passage for processing a sample may further include a sample passage having a fluid inlet and a fluid outlet in fluid communication, and a microfluidic chip having a set of fluid inlets and a set of fluid outlets, wherein the set of fluid inlets is in fluid communication with the set of fluid outlets to form at least one microfluidic channel, the microfluidic chip is disposed between the fluid inlet and the fluid outlet of the sample passage, at least one microfluidic channel includes elements of the sample passage, and a sample processed by a flow cytometry system through the sample passage module is confined within the sample passage during processing.

[0062] In another embodiment, a fluid passage module for processing a fluid sample containing a plurality of particles is provided, the fluid passage including a sample passage having a fluid inlet and a fluid outlet in fluid communication, and a microfluidic chip having a set of fluid inlets and a set of fluid outlets, wherein the set of fluid inlets is in fluid communication with the set of fluid outlets to form at least one microfluidic channel, the microfluidic chip is disposed between the fluid inlet and the fluid outlet of the sample passage, at least one microfluidic channel includes elements of the sample passage, and a fluid sample processed by a flow cytometry device through the fluid passage module is confined within the sample passage during processing.

[0063] The fluid passage module of the above embodiment may further include a sheath fluid passage having a fluid inlet and a fluid outlet in fluid communication. A microfluidic chip may be disposed between the fluid inlet and the fluid outlet of the sheath fluid passage, and at least one microfluidic channel includes elements of the sample passage. The fluid passage module is a distribution block including a set of fluid inlets and a set of fluid outlets, wherein at least one of the set of fluid inlets forms a first sample passage element in fluid communication with at least one of the set of fluid outlets; a microfluidic module including a microfluidic device fixing device adapted to fix the set of fluid inlets, the set of fluid outlets, and the microfluidic chip, wherein at least one of the set of fluid inlets forms a second sample passage element in fluid communication with at least one of the set of fluid outlets; and a fluid passage fluidly connecting the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, wherein a part of the fluid passage joins and fluidly connects the first sample passage element and the second sample passage element to form a sample passage. The fluid passage module may further include the fluid passage.

[0064] The dispensing block may comprise a fluid system manifold. The dispensing block may further comprise a sample tube loader. The fluid passage module may further comprise releasable fixing means adapted to fix the fluid passage module to a flow cytometer system. The releasable fixing means may comprise at least one iron plate or at least one magnetic plate. The fluid passage module may be removable from the flow cytometer system by releasing the fixing means. The releasable fixing means for the removable and replaceable fluid passage module may be further adapted to fix the removable and replaceable fluid passage module to a fluid passage cleaning system. The fluid sample may be a mammalian semen sample containing a plurality of sperm cells. The flow cytometer system may process the sample to identify a subpopulation in the fluid sample. The flow cytometer system may process the sample to perform a sex discrimination operation on the semen. The fluid passage module may comprise an identification element. The identification element may comprise an optical identification element or a wireless identification element. The optical identification element may comprise a set of features or references on a microfluidic chip in the microfluidic module.

[0065] According to another embodiment, the present invention provides a flow cytometer system comprising an improved optical path and combination of elements for delivering electromagnetic radiation to interrogation and / or action positions in a flow cytometer. The novel system of the present invention emits both a detection laser and a kill laser or an ablation laser from the same direction as the flow of the sample fluid.

[0066] The present invention addresses existing problems in known systems and methods for optically detecting and destroying particles in a flow cytometer system. In existing systems, when beams are directed from two or more lasers through separate beam paths, the potential for damage or degradation of components over time increases. Additionally, when electromagnetic radiation is emitted from separate sides of a flowing stream, complex beam paths and complex orientations and arrangements of elements for focusing and detecting signals derived from the electromagnetic radiation may be required. The present invention addresses these problems by emitting both a detection laser for causing fluorescent emission in particles such as those that have undergone a staining process, and a kill laser or ablation laser, from the same direction as the flow of the sample fluid.

[0067] In addition, the shape of the kill beam spot in the ablation region of a flow cytometer system used in the sperm sex discrimination process significantly affects the photon dose delivered to a specific point within the sample flow and affects the effect of the kill laser on individual cells. The width of the kill beam or kill position of the kill laser provided by the present invention is greater than the width of the flow channel or the droplet size in the flow cytometer system. The present invention provides for the adjustment of the width of laser beam emission at the beam waist or focal plane in a flow cytometer system, and this width is not a function of laser divergence such that it is determined by adjusting the height of the beam at the focal plane.

[0068] In addition, without wishing to be bound by any theory or mechanism of the present invention, the methods and systems herein are considered advantageous because they provide faster kill alignment, more reliable and uniform displacement of the particles to be sorted (i.e., more of the desired type of particles are collected at the end of the sorting process), and more uniform performance of the flow cytometer sorting system. The present invention may also provide faster or easier training and setup compared to existing systems and methods, based on a simplified optical path resulting from the positioning and emission of the detection laser and the kill laser from the same direction as the flow of the sample fluid. The simple systems of the present invention are applicable or usable in a wide range of microfluidic systems or flow cytometry systems, improving the flexibility of implementation compared to existing systems and methods.

[0069] In addition, without wishing to be bound by any theory or mechanism of the present invention, the combination of an adjustable beam expander, a cylindrical lens and an aspherical lens with a beam combiner such as a polarizing beam splitter results in the positioning of the kill laser assembly and the detection laser assembly on a common side with respect to the microfluidic system chip, while providing an adjustable elliptical beam waist that can be specifically configured for the type or size of the microfluidic system channel or chip at the focal plane, so the methods and systems herein are considered advantageous.

[0070] In a first embodiment, the present invention provides a laser-based flow cytometry system for particle detection, the system comprising a laser assembly comprising a kill laser assembly and a detection laser assembly, the kill laser assembly and the detection laser assembly being arranged on a common side with respect to the flow cytometry system. The flow cytometry system may comprise a microfluidic device such as a microfluidic chip or a microfluidic cassette. The kill laser assembly and the detection laser assembly are arranged on a common side with respect to the main surface of the microfluidic device. The main surface may be a top surface or a bottom surface. The laser assembly may further comprise a polarization beam splitter. Each of the kill laser assembly and the detection laser assembly may further comprise an optical path adapted to direct electromagnetic radiation to a focal plane. The optical path of the kill laser assembly and the optical path of the detection laser assembly each comprise a fast-axis beam expander and a slow-axis beam expander. The optical path of the detection laser may comprise a pair of cylindrical lenses. The fast-axis beam expander and the slow-axis beam expander of the optical path for the kill laser assembly may be adapted to adjust the fast-axis beam width and the slow-axis beam width of the kill laser assembly. The fast-axis beam expander and the slow-axis beam expander of the optical path for the detection laser assembly may be adapted to adjust the fast-axis beam width and the slow-axis beam width of the detection laser assembly. The cylindrical lenses of the optical path of the detection laser may be adapted to adjust the fast-axis beam width and the slow-axis beam width of the detection laser assembly and to shape the beam.

[0071] In some embodiments, the kill laser assembly and the detection laser assembly may be adapted to emit a laser beam including an elliptical beam profile at the focal plane. The elliptical beam profile of the kill laser assembly may have a fast-axis beam width of 0.5 to 5 μm when measured at the focal plane. The elliptical beam profile of the kill laser assembly may have a slow-axis beam width of 5 to 35 μm when measured at the focal plane. The elliptical beam profile of the kill laser assembly may have a fast-axis beam width of 3.45 to 5 μm and a slow-axis beam width of 5 to 35 μm when measured at the focal plane. The elliptical beam profile of the detection laser assembly may have a fast-axis beam width of 3.5 to 20 μm and a slow-axis beam width of 30 to 150 μm when measured at the focal plane. The kill laser assembly may operate with a pulse energy of 1 to 5 μJ per pulse. The kill laser assembly may comprise a pulsed laser module having a pulse duration of 1 to 500 nanoseconds. The kill laser assembly may comprise a pulsed laser module having a pulse duration of 5 to 30 nanoseconds. The detection laser assembly may comprise a continuous-wave laser module, a quasi-continuous-wave laser module, or a pulsed laser module. The system may further comprise a detection assembly. The detection assembly may be adapted to detect one or more fluorescences of particles excited by the radiation from the detection laser assembly, deactivation of particles by the radiation from the kill laser assembly, an image of a detection event, or an image of a kill event. The detection assembly may comprise at least one avalanche photodiode. At least one avalanche photodiode may be adapted to detect the fluorescence of particles excited by the radiation from the detection laser assembly. At least one avalanche photodiode may be adapted to detect the deactivation of particles by the radiation from the kill laser assembly. The detection assembly may comprise a CCD camera. The CCD camera may be adapted to capture an image of a detection event. The CCD camera may be adapted to capture an image of a kill event.

[0072] In another embodiment, the present invention provides a laser assembly comprising a laser module and an optical path, the optical path being adapted to shape the electromagnetic radiation beam emitted from the laser module into an elliptical profile at the focal plane.

[0073] In some embodiments, the laser assembly is one of a detection laser assembly and a kill laser assembly. The optical path may comprise a fast-axis beam expander and a slow-axis beam expander. The optical path may comprise a pair of cylindrical lenses. The fast-axis beam expander may be adapted to adjust the fast-axis beam width for beam emission. The slow-axis beam expander may be adapted to adjust the slow-axis beam width for beam emission. The pair of cylindrical lenses may be adapted to adjust the beam profile at the focal plane of the beam emission. The elliptical beam profile of the laser assembly may have a fast-axis beam width of 0.5 to 5 μm when measured at the focal plane. The elliptical beam profile of the laser assembly may have a slow-axis beam width of 5 to 35 μm when measured at the focal plane. The elliptical beam profile of the laser assembly may have a fast-axis beam width of 3.45 to 5 μm and a slow-axis beam width of 5 to 35 μm at the focal plane. The elliptical beam profile of the laser assembly may have a fast-axis beam width of 3.5 to 20 μm and a slow-axis beam width of 30 to 150 μm at the focal plane. The laser module may operate with a pulse energy of 1 to 5 μJ per pulse. The laser module may comprise a pulsed laser module having a pulse duration of 1 to 500 nanoseconds. The laser module may comprise a pulsed laser module having a pulse duration of 5 to 30 nanoseconds. The laser module may comprise a continuous-wave laser module, a quasi-continuous-wave laser module, or a pulsed laser module. The pair of cylindrical lenses may comprise an F25 cylindrical lens and an F150 cylindrical lens. The optical path may comprise an aspherical lens. The optical path may comprise a polarization beam splitter. The optical path may comprise a harmonic separator. The optical path may comprise a wave plate.

[0074] In another embodiment, the present invention provides an optical path for a laser assembly for use in flow cytometry, the optical path comprising a beam expander, a wave plate, an aspherical lens, and a polarization beam splitter, the beam expander being adapted to shape the electromagnetic radiation beam emitted from the laser module into an elliptical beam profile at the focal plane.

[0075] In some embodiments, the beam expander comprises a fast-axis beam expander and a slow-axis beam expander. The fast-axis beam expander may be adapted to adjust the fast-axis beam width for beam emission. The slow-axis beam expander may be adapted to adjust the slow-axis beam width for beam emission. The elliptical beam profile may have a fast-axis beam width of 0.5 to 5 μm and a slow-axis beam width of 5 to 35 μm when measured at the focal plane. The laser module may operate with a pulse energy of 1 to 5 μJ per pulse. The laser module may comprise a pulsed laser module with a pulse duration of 1 to 500 nanoseconds. The laser module may comprise a pulsed laser with a duration of 5 to 30 nanoseconds. The optical path may further comprise a harmonic separator.

[0076] In another embodiment, the present invention provides a sex-discriminated semen product of non-human mammals, which product comprises a collection of sex-discriminated sperm cells of non-human mammals derived from a sample of non-human semen that has not been sex-discriminated. The non-human semen that has not been sex-discriminated is subjected to the steps of staining a sample of non-human semen that has not been sex-discriminated, inserting the stained sample of non-human semen into a flow cytometry device, inserting sheath fluid into the flow cytometry device, wherein the sheath fluid and the channel of the flow cytometry device orient and position cells in the sample of non-human semen that has not been sex-discriminated, causing the dye in the stained sample of non-human semen to fluoresce by radiation from a first laser module, wherein the radiation from the first laser module comprises an elliptical beam profile, detecting the fluorescence for each sperm cell in the stained sample of non-human semen by a detector, and based on the detected fluorescence, inactivating a subset of the non-human semen by radiation from a second laser module to create sex-discriminated non-human semen, wherein the radiation from the second laser module comprises an elliptical beam profile and the first laser module and the second laser module are disposed on a common side of the flow cytometry device opposite the detector, and collecting the sex-discriminated non-human semen, and is sex-discriminated by a sex discrimination process.

[0077] In another embodiment, the present invention provides a method for sexing semen of non-human mammals, the method comprising the steps of: staining a sample of non-human semen that has not been sexed; inserting the stained sample of non-human semen that has not been sexed into a flow cytometry device; inserting a sheath fluid into the flow cytometry device, wherein the sheath fluid and the channel of the flow cytometry device orient and position cells in the sample of non-human semen that has not been sexed; causing the dye in the stained sample of non-human semen that has not been sexed to fluoresce by radiation from a first laser module, wherein the radiation from the first laser module includes an elliptical beam profile; detecting the fluorescence for each sperm cell in the stained sample of non-human semen that has not been sexed by a detector; inactivating a subset of the non-human semen by radiation from a second laser module based on the detected fluorescence to create sexed non-human semen, wherein the radiation from the second laser module includes an elliptical beam profile and the first laser module and the second laser module are disposed on a common side of the flow cytometry device opposite the detector; and collecting the sexed non-human semen.

[0078] According to yet another embodiment, the present invention provides an automated or semi-automated system for routing or re-routing and mixing a sample to be processed, for example, in a microfluidic chip-based flow cytometry system. By providing an automated or semi-automated system for handling the sample to be processed, manual or operator-intensive steps are eliminated from the sample processing, improving the efficiency of the process and the quality of the sample being processed. The likelihood of operator error is reduced by the automated routines or repeatable steps in the sample processing operation, through a more uniform operation enabled by automated sample routing and mixing.

[0079] Another object of the present invention is to provide a system and method that enable automated mixing, a more uniform mixing time, and a path to ensure that the inner diameter of the tube is always under the path of the cells. Embodiments of the present invention include a mixing system that gently mixes along a pivot, enabling automation of mixing for applying limited space. Embodiments of the present invention can reduce cell stress by controlling the mixing operation and can shorten the time that the cells are in an overly concentrated solution of the fluid.

[0080] In some aspects, the present invention features a sample mixing system. The sample mixing system is used with a flow cytometry system for processing a sample containing constituent molecules or particles. For example, the sample mixing system may be used with a microfluidic chip-based flow cytometry system, and a sample such as a biological sample or a gene sample containing a plurality of cells is processed by a set of radiation devices for electromagnetic beams such as a laser. Cells in the sample may be processed in a microfluidic chip such that a subset of the sample is inactivated, destroyed, or removed following processing in the microfluidic chip, and the sample is discharged from the microfluidic chip through one or more outlets at the terminal end of the chip. The processed sample is allowed to flow into a sample capture tube disposed on the sample mixing system or diverted to a waste collection container based on meeting a set of criteria or parameters.

[0081] In some embodiments, the sample mixing system may include a rotation base, a plurality of collection tube holders disposed on the rotation base, and a plurality of collection tubes. Each collection tube is configured to be disposed in a collection tube holder. The collection tube may contain a medium such as a buffer medium, with respect to which mixing maintains the buffer and controls the degree of buffering. The rotation base can rotate such that the collection tube holders and the collection tubes rotate or move in an arcuate path. This movement causes mixing of the contents of the collection tubes.

[0082] In some embodiments, the rotation base may be a disk disposed on a platform. In one embodiment, the rotation base is operably coupled to a motor that rotates the rotation base about a central axis. The rotation base is rotated based on one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotation speed. In some embodiments, the rotation base can rotate back and forth. The rotation base rotates about 10° to 180°.

[0083] In other embodiments, the rotating base may contain magnets that are spaced apart from each other. In this regard, the base of each collection tube holder contains a magnet. Each collection tube holder can be attached to the rotating base by the magnetic force between one of the magnets of the collection tube holder and one of the magnets of the rotating base.

[0084] In some embodiments, the sample mixing system may further include one or more Hall effect sensors. For example, the S pole of one of the magnets in the rotating base may be oriented towards one or more Hall effect sensors, and the N pole of another magnet may be oriented towards the same one or more Hall effect sensors.

[0085] In other embodiments, the sample mixing system may include about 2 to 6 collection tube holders. Each collection tube holder may include two or more arms that protrude upward from the base. The two or more arms are configured to hold a collection tube standing upright on the base. In some embodiments, the two or more arms are extendable to accommodate collection tubes of various sizes.

[0086] In some embodiments, the system may be disposed under the dispensing device such that the dispensing device dispenses a sample into one of the collection tubes. Preferably, when the collection tube moves in an arcuate path to mix the sample, the collection tube receiving the sample remains under the dispensing path of the dispensing device. The system automatically installs another collection tube under the dispensing device so as to dispense the sample into another collection tube after mixing the sample for a certain period (e.g., 5 to 10 minutes) in the collection tube.

[0087] In other embodiments, the sample mixing system may further include a sensor for detecting the liquid level in the collection tube. This sensor can be disposed on or near the outer surface of the collection tube. In still other embodiments, the sample mixing system may further include an automatic dump configured to prevent flow into the collection tube.

[0088] In another aspect, the sample mixing system described herein can be used in a method of mixing selected samples. This method can include collecting the selected samples into the collection tube of the sample mixing system, and rotating the rotation base by rotating the collection tube disposed in the collection tube holder in a controlled movement in an arcuate path to rotate or move uniformly to mix the collected samples, thereby automatically mixing the samples collected in the collection tube. In a further embodiment, this method can include redirecting the flow of the selected samples from the collection tube to an individual sampler when the collection tube is filled to a desired level, rotating the rotation base so that a second collection tube is arranged to collect the selected samples, and redirecting the flow of the selected samples to the second collection tube.

[0089] One of the unique and distinctive technical features of the present invention is the mixing automatically induced by the arcuate reciprocation of the sample tube. Without wishing to be limited to any theory or mechanism of the present invention, it is believed that the technical features of the present invention advantageously enable more uniform automatic mixing of the collection tube under dispensing devices such as microfluidic-based or droplet-based flow cytometers. The arcuate movement ensures a uniform concentration between the cell medium and the cells in the sample tube while gradually dripping the cells into the tube onto the medium. After the mixing of one tube is completed, due to the exchange ability, the motor can quickly move from the "in-use" tube to the "standby" tube. Also, the sample mixing system of the present invention is small enough to fit into the space allocated inside the sex discrimination device and requires less human intervention. There is no prior reference or publication currently known that has the unique and distinctive technical features of the present invention.

[0090] According to other embodiments, the present invention features a particle processing system comprising a flow cytometry assembly for processing particles or constituent molecules in a sample based on at least one characteristic, and an automatic sample mixing system configured to automatically mix a sample processed by the flow cytometry assembly based on a set of defined parameters. In one embodiment, the automatic sample mixing system can rotate about a pivot to move in an arcuate path, enabling mixing of the sample with a controlled and uniform movement. The set of defined parameters includes one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotational speed.

[0091] In one embodiment, the automatic sample mixing system further comprises a rotating base, a set of collection tube holders disposed on the rotating base, and a set of collection tubes configured to be disposed in the collection tube holders of the set of collection tubes, respectively. In another embodiment, the automatic sample mixing system further comprises a fluid level sensor. The automatic sample mixing system is configured to exchange a first tube and a second tube in the set of collection tubes when the fluid level sensor detects that the first tube has reached a desired filling level.

[0092] In some embodiments, the particle processing system further comprises a mechanical diverter configured to prevent an output stream containing the processed sample from the flow cytometer from entering the automatic sample mixing system.

[0093] According to another embodiment, the particle processing system includes a flow cytometry assembly for processing particles or constituent molecules in a sample based on at least one characteristic, and a mechanical diverter configured to prevent an output stream including the processed sample from the flow cytometer from entering a sample collection container based on a set of defined parameters. The mechanical diverter is configured to prevent an output stream including the processed sample from the flow cytometer from entering an automatic sample mixing system. The set of parameters defined for the mechanical diverter may include one or more of the purity of the sample, the number of particles in the sample, and the fill level of the collection container.

[0094] In other embodiments, the particle processing system may further include an automatic sample mixing system configured to automatically mix a sample processed by the flow cytometry assembly and collected in the sample collection container based on a set of defined parameters. The set of parameters defined for the automatic sample mixing system may include one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotation speed. In a non-limiting example, the automatic sample mixing system may include a rotation base, a set of collection tube holders disposed on the rotation base, and a set of collection tubes configured to be disposed in the collection tube holders of the set of collection tubes, respectively. The automatic sample mixing system is configured to exchange a first tube and a second tube in the set of collection tubes when a fluid level sensor detects that the first tube has reached a desired fill level. In some embodiments, the automatic sample mixing system further includes a fluid level sensor.

[0095] In a further embodiment, the present invention provides a method for generating a sex-discriminated, non-human semen product. The method includes processing a semen sample within a flow cytometry device to select a first sub-population of the sample that includes at least one desirable characteristic from a second sub-population of the sample that includes at least one undesirable characteristic, collecting a sample in a sample collection container that predominantly includes the first sub-population of the sample that includes at least one desirable characteristic, and automatically mixing the collected sample in the sample collection container using any of the sample mixing systems described herein. The mixing of the collected sample can be based on a set of defined sample mixing parameters associated with the sample being processed, such as one or more of a mixing arc defined in degrees, a mixing arc defined in steps, and a rotational speed. The sample mixing system can rotate about a pivot so as to move in an arcuate path, enabling the mixing of the sample with a controlled and uniform motion.

[0096] In some embodiments, the method further includes mechanically diverting a portion of the processed sample to a sample collection container after the processed sample exits the flow cytometry device. The step of mechanically diverting the processed sample can be based on one or more defined parameters, including the purity of the sample, the number of particles in the sample, and the fill level of the collection container. In other embodiments, the method includes exchanging a first tube and a second tube in a set of collection tubes when a fluid level sensor detects that the first tube has reached a desired fill level.

[0097] It will be technically apparent from the context, this specification, and the knowledge of one of ordinary skill in the art that any feature or combination of features described herein is included within the scope of the present invention as long as the features included in such a combination are not mutually inconsistent. Further advantages and aspects of the present invention will be apparent in the following detailed description and claims.

[0098] For the sake of facilitating a complete understanding of the present invention, reference is now made to the exemplary embodiments shown in the accompanying drawings, in which like elements are referred to by like numerals. These drawings are not to be construed as limiting the present invention, but are illustrative and are intended for reference purposes only.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0100] The present invention will now be described in more detail with reference to exemplary embodiments as shown in the accompanying drawings. It should be understood that the present invention is described herein with reference to the exemplary embodiments, but the present invention is not limited to such exemplary embodiments. Those having ordinary skill in the art and having the opportunity to utilize the teachings of this specification will recognize additional implementation forms, modifications, and embodiments, as well as other uses of the present invention, and these are fully contemplated herein as being within the scope of the present invention as disclosed and claimed herein, and in that regard, the present invention can be quite beneficial.

[0101] In one aspect, the present disclosure relates to a flow cytometry system and method capable of separating particle or cellular material, such as sperm and other particles or cells, into various components and parts. For example, various embodiments of the present invention isolate viable and motile sperm from non-viable or non-motile sperm, isolate sperm by means of a variant form that classifies genders and other genders, isolate stem cells from cells in a parent population, isolate one or more labeled cells from unlabeled cells to distinguish desirable / undesirable characteristics, isolate genes in nuclear DNA according to specified characteristics, isolate cells based on surface markers, isolate cells based on membrane integrity (viability), potential, or predicted reproductive state (fertility), ability to survive after freezing, etc., isolate cells from contaminants or debris, isolate healthy cells from damaged cells (i.e., cancer cells) (such as in bone marrow extraction), isolate red blood cells from white blood cells and platelets in a plasma mixture, and isolate any cell from any other cell component into a corresponding part, etc., to achieve the isolation of components in a mixture.

[0102] As used herein, it is understood that sperm samples should be provided from mammals other than humans. The sperm sample may be a newly collected sample from a source animal or a thawed sample of a previously cryopreserved sample from a source animal. Examples of source animals include, but are not limited to, cows, sheep, goats, horses, pigs, or rabbits, and other mammalian sources such as alpacas, dogs, cats, ferrets, rats, mice, or buffalos. For example, bovine sperm cells can include sperm cells from domestic cows, bison, African buffalos, water buffalos, or yaks. Further, the sample may be a single semen, multiple ejaculated semen from the same mammal, or multiple ejaculated semen from two or more animals.

[0103] As used herein, the term "passage" refers to a fluid line, channel, or pipe through which fluid can flow. For example, a sheath fluid passage refers to a line, channel, or pipe through which sheath fluid can flow, a sample passage refers to a line, channel, or pipe through which sample fluid can flow, and an air passage refers to a line, channel, or pipe through which air can flow, and so on. In the sample passage module described herein, the module comprises an overall sample passage through which a sample flows, thereby isolating the sample fluid from other modules during processing other than collection. This can prevent cross-contamination between different sample fluids and also allows the sample passage to be disconnected from other modules for cleaning. The overall sample passage refers to all passages through which sample fluid flows, including passages that are separate and physically separated from each other by other components. In a similar manner, the term "overall passage" can be applied to sheath fluid, air, or any other fluid, mainly in that the overall passage comprises all passages through which a particular fluid flows, including passages that are separate and physically separated from each other by other components.

[0104] As used herein, the term "path to be purged" refers to the number of sample passage volumes to be cleaned.

[0105] As used herein, the term "module" refers to a plurality of components that can be removed, reinstalled, or replaced as one unit. If an individual component can be removed, reinstalled, or replaced, the individual component is not a module.

[0106] According to some embodiments, the present invention features a modular flow cytometry system having a chassis and a plurality of modules disposed therein, as shown in FIGS. 1-2. These modules include, but are not limited to, electronic devices, and control modules, interrogation (or optical) modules, fluidic modules, sample channel modules, microfluidic device cleaning modules, and automated mixing and collection modules. By positioning ventilation holes or slots at multiple locations along the chassis, appropriate and sufficient airflow and cooling to the electronic devices can be enabled. While not wishing to limit the present invention to a particular theory or mechanism, the modular flow cytometry system can have automation mechanisms including, but not limited to, automatic adjustment of chip position via a chip stage, automatic adjustment of detection spots, automatic adjustment of kill spots, automatic sample and sheath throughput, automatic collection and agitation, automatic dump (e.g., from failure conditions in sample collection, such as too high a percentage of dead cells, too low resolution, contamination mistakes, alignment errors, etc.). The automation mechanisms can be controlled and operated by the computer system components of the cytometer.

[0107] As shown in FIG. 2, the sheath reservoir may be fluidically coupled to the modular flow cytometry system. The sheath reservoir may include a wheeled assembly for ease of transport and a scale for monitoring the amount of sheath fluid within the reservoir. Additional components that may be operably coupled to the modular flow cytometry include, but are not limited to, identification scanners such as a mouse, monitor, touch screen, keyboard, barcode, QR code (registered trademark), or RFID scanner.

[0108] Figure 3A shows the different components of the modular flow cytometry system in more detail. In some embodiments, the microfluidic module includes within it a chip cartridge and a microfluidic chip magnetically held with respect to the system. The sheath line is a piping path between the sheath inlet port and the fluid from the microfluidic chip sheath channel. The sample line is a piping path between the sample tube and the fluid from the microfluidic chip sample channel. The fluidics module guides the fluids from the sample inlet and the sheath inlet, as well as the fluid from the fluidics assembly, to their respective ports within the microfluidic chip. The fluidics access screw enables maintenance into the fluidics assembly. The liquid level sensor may be a capacitance sensor that detects the collection filling level within the sample tube. The collection module includes a rotatable system that includes a collection tube holder that holds a collection tube and switches between a "standby" position and a "collection" position. The visor is a swing cover that protects the chip module from environmental variations during collection. The sample tube is a reservoir that can hold a sample that includes a semen sample or a cleaning sample and discharge it into the microfluidic sample channel. The storage tray can hold additional tubes and caps during operation for convenience.

[0109] Referring to Figure 3B, the backside components of the modular flow cytometry system include a control box module that includes a control panel and a cover, a fluidics port that includes a quick disconnect to the sheath inlet, a waste output, an air input, an air output to the sheath tank, an electronics box module that includes a power supply and a main computer, an electronics power port for plugging into a cable to supply power to the electronics box, and a status indicator that indicates the overall system state.

[0110] The optical system assembly is involved in the alignment and shaping of the beam path from the kill laser and the detection laser to the microfluidic system chip. The chip can be viewed through the focusing lens into the camera. The detection laser emits light through each individual cell when the cells are flowing through the microfluidic system chip and is involved in collecting the emission through the APD detection sensor to evaluate the gender of the cells. The kill laser then transmits a pulse of light in each cell that is not gate-controlled in the software, slicing and inactivating each of the non-gate-controlled cells. From the software, the operator can optimize the cell gender detection resolution and the kill slice accuracy by controlling the movement of the chip stage and the detection beam.

[0111] In a non-limiting example of the gender discrimination method implemented in the present invention, the sperm sample is taken into the microchannel, surrounded by the sheath fluid, and concentrated in all four directions. When the modular flow cytometry system is operating, the stained sperm cells in the sample are illuminated by a laser (either pulsed wave or continuous wave) to cause fluorescence emission. When the light or photons from the fluorescence emission of the cells hit an avalanche photodiode ("APD"), the determination process starts in the sensor within the system. The signal from the APD is processed and sent to a field programmable gate array ("FPGA"). In the FPGA, if the appropriate time dead time has elapsed since the last detection event, then a new event state starts; otherwise, no event can occur at all.

[0112] At the start of event state determination, if the signal value exceeds the threshold value and the signal value exceeds the falling hysteresis value, at this time, the event counter is incremented. Next, it is determined whether the event is a multi-peak event or a single-peak event. Regarding the signal or peak, the direction of the refractive change of the signal is determined, and it is determined whether the signal is the first peak in a multi-peak event (doublet) or the second, i.e., subsequent peak in a multi-peak event. If the event is a multi-peak event, the peak count, peak voltage, and peak time are used to process the multi-peak event. Since singlets or doublets have approximately the same peak height, they appear at the same location on the 1-D histogram of peak height ("intensity"). However, because the regions are significantly different, they appear at different locations on the 2-D histogram having peak height and region.

[0113] If the event is a multi-peak event, the following data, namely peak voltage, peak time, integration region, and span value for the event, are updated for each data point within the event as the signal is processed until the end of the event is determined. If the span value exceeds the minimum value for the event, the event is a valid event, and the FPGA enters "gate mode", determines the gating result, and decides whether to start using this result. Peaks corresponding to already dead cells are determined not to start. The FPGA results, including peak voltage, region, and span, are sent to a digital signal processor ("DSP"). The DSP can make additional gating decisions, or it can decide whether to start using the FPGA results. In DSP gating, the event data from the FPGA is processed to determine whether the event data points are within a pre-defined gating polygon.

[0114] After the FPGA transmits the signal to the DSP, it waits for the DSP gating result. Based on the DSP gating result, one or more peaks are added to the kill schedule based on the time stamps of the peaks within the event. In the case of a single peak, one event is added to the laser trigger schedule. In the case of a multi-peak event, one or all of the peaks within the multi-peak event are added to the laser trigger schedule based on the current settings. For the laser trigger, the EPGA continuously checks to see if a new trigger request has been added to the schedule. If a trigger request is added to the schedule, a delay is set based on the request. After the delay, the output from the FPGA causes the laser to start in the spermatid corresponding to the specific trigger request.

[0115] In some embodiments, the fluidics module is a liquid handling device that precisely controls the flow rate between the sheath line and the sample line from the fluidics manifold to the microfluidics chip, enabling an optimal cell flow rate through the microfluidics chip and allowing for the maximum acceptable spacing between cells within the chip. The fluidics module is removable from the system.

[0116] In some embodiments, the fluid system module includes a fluid regulator for controlling the flow rate of sheath fluid and sample fluid through the system, and a fluid valve. The fluid system module may also include a dual air regulator assembly near the back of the system that can drop the enclosure air pressure in the instrument to about 55 psi and the air pressure to the sheath tank to less than 25 psi. The fluid system module houses a fluid control PCB and controllable devices to accurately control the fluid flow rate through the instrument. The fluid system module can be accessed by activating a release mechanism, such as by releasing a latch or turning a knob until it loosens, and then sliding the module out. Referring to FIG. 9A, once the fluid system module is pulled out of the instrument, maintenance staff can quickly access the module by first unplugging the plug from the electrical port to the fluid panel and then pulling up on the unit while holding the handle located at the top of the module. The fluid system module can be disconnected from and removed from the system, for example, for maintenance or diagnosis, and replaced with a backup fluid system module, and the system can still remain operational. For example, the fluid system module may be removed to replace the sheath line filter once a month for preventive maintenance.

[0117] For some fluid to flow within the system, both the microfluidic module and the fluidic module need to be properly installed at their respective spots. There is a sensor to detect that the manifold is in a predetermined location to reduce the chance of accidental leakage. Both manifolds have handles attached to facilitate gripping. In some embodiments, both the chip and the fluidic manifold are intended to be handled simultaneously. To mount the fluidic module and the chip module, the vise is lifted and then the modules are moved to their intended positions on the system. In the case of the fluidic module, two dowel pins from the module are aligned with holes in the fluidic module base and then pushed into place. In the case of the chip module, the positioning balls on the back of the module are aligned with the positioning holes in the chip module base and then pushed forward to slide into place at a predetermined location on the system. Both modules should click into place when they are magnetically held. The sheath line and the sample line are inserted into their respective slots on the clip directly below the vise to hold the lines in place. Finally, the vise is closed. Removing the fluidic module and the chip module from the system is the reverse of the mounting procedure.

[0118] In some embodiments, a 5 mL tube sample with the desired fluid is loaded onto the fluidic module by placing the tube on the sample tube loader and positioning the loaded sample tube loader vertically below the fluidic module against the instrument face. Once the sample tube is aligned with the center of the sample tube, the sample tube loader is pushed up until the top face contacts the fluidic module. The sample tube loader locks into place within a predetermined location by the magnetic force between the loader and the fluidic module. To remove the sample tube, the sample tube loader is twisted to break the magnetic force with the fluidic module and pulled down to remove it from the fluidic manifold.

[0119] In other embodiments, the collection module can automate some parts of the system's collection process. It has sensors for detecting when the collection tube is full. The liquid level sensor is calibrated to activate when the sample tube is full to a pre-defined level. The collection module can switch a full collection tube to a waiting empty collection tube and automatically mix the contents inside the tube every five minutes. In some embodiments, the automated collection module includes a turntable base therein, which holds two sample tubes. The collection base also includes magnets therein to hold the sample tube holder in a specific orientation at a predetermined location during collection. While collection is taking place, the collection module can mix the contents frequently. When the sample collection in the tube is finished, the module switches the tube collecting the sample to a waiting tube. Finally, there is an automated system for redirecting the sample flow to the waste line without the need to stop the flow.

[0120] During the monitoring of the sex discrimination system, the operator mainly interacts with the collection module. When the software starts up, the collection module returns to a predetermined location. Except when the collection module moves, there is always one sample tube holder in line with the sample collection zone called the "collection" position. Directly across from the sample tube holder under the sample collection zone is the second sample tube holder. The second sample tube holder is called the "waiting" position.

[0121] During sample collection, the collection tube is automatically mixed. The mixing protocol reciprocates the collection tube back and forth for a few seconds and then returns it to the collection position. When a new collection tube is switched to the collection position, the finished tube is switched to return to the waiting position, the full collection tube is removed and placed in the collection tray. A new collection tube is placed in the collection tube holder and mounted to return to the waiting position.

[0122] Without wishing to be bound by any particular theory or mechanism, a novel and progressive mechanism of a modular flow cytometry system is the configuration and positioning of the modules relative to each other to enable efficient use of space. The system can be divided into three separate levels or floors. On the first floor (e.g., the bottom floor), the electronic modules can slide in and out of the chassis. It includes a partition plate or plate for protection from the fluid at the upper level. Thus, the electronic modules are physically isolated from other modules. On the second floor (e.g., the middle floor), the fluidic modules are provided at the front part to facilitate access by the operator. The third floor (e.g., the top floor) houses the optical module that contains a laser, detectors, and other optical components. The optical module is isolated from the moving air so as not to interfere with the optical components. Since the laser generates the most heat, the optical module is at the top level for optimal ventilation. Instead of using fans that could disrupt the air around the optical components, a heat sink for the laser can draw in air via natural convection to cool the components. The optical module must be separated from the electronic modules because these modules generate heat together. To cool the electronic components, fans may be placed on the first floor and / or near the electronic modules. The layout of the modules further enables access to areas for frequent maintenance.

[0123] Another unique and progressive mechanism of the present invention advantageously enables easy assembly of various modules and removal for maintenance and / or replacement, resulting in reduced downtime and near continuous operation, which is the modular nature of the entire system. For example, the computer may be placed on rails for easy movement and can be replaced with a similar electronic module. In the optical module, the laser can be removed, replaced, or reconnected. Other optical components, detectors, cameras, lenses, etc. can also be removed, replaced, or reconnected. The sample passage module can be removed and cleaned using a cleaning module. The fluid system module can also be removed and cleaned. During maintenance, the fluid system module and the sample passage module can be replaced with other cleaned modules. Replacement of the fluid system module or its components can enable continuous operation of the flow cytometer. The system can be easily operated and maintained without requiring a professional technician or special training. Furthermore, the modular nature also enables easy shipping of the system with little or no assembly required after delivery.

[0124] Another unique and further progressive feature of the present invention is the automated or semi-automated nature of the modular flow cytometry system, which results in reduced downtime of the system, reduced product contamination, and improved product uniformity and purity. For example, the physical location of the microfluidic chip can be adjusted relative to where it requires the use of an automated chip stage adjustment procedure. Mixing of the sample tube containing the processed sample can also be automated by rotating the tube while the sample is being collected, thereby reducing sample loss and damage to sperm cells. Further examples of the automated mechanisms of the system include an automatic dump module that enables redirecting the processed sample flow towards waste collection and an automated sample level detection that notifies the operator when the sample level is low.

[0125] The following sections describe the various modules of the modular flow cytometry system in more detail. Alternatives and equivalents are within the scope of the present invention.

[0126] Sample passage module and microfluidic device cleaning module

[0127] In the field of commercial semen processing, for example, in semen sex determination operations, there is a requirement to clean microfluidic chips, cassettes, or cartridges to prevent contamination between sample runs.

[0128] In semen sex determination, the process is a fluid sample change sub-process, for example, a bull change sub-process, where the processing of one fluid sample (e.g., a semen sample) is completed and the system must then prepare for the processing of subsequent samples. During bull change, the microfluidic chip used in the sex determination process for the semen sample from the first bull must be cleaned and all of the semen from the previous bull must be removed to perform the next sex determination process using the semen of the new bull. Alternatively, a new or separate microfluidic chip may be used. Contamination between bulls, or more generally between animals, or between genetic fluid samples, is a possible result of a microfluidic chip that has not been perfectly cleaned. Cross-contamination is a major problem because if the contaminated product reaches the farmer, it can result in incorrect breeds or genders among the offspring.

[0129] In existing systems and methods, reducing contamination among bulls is achieved by fixing and maintaining a microfluidic system chip within a flow cytometry device or instrument. Chip cleaning procedures are performed on the flow cytometry device, where a cleaning solution or wash fluid, such as PARVOSOL, and a sheath fluid or buffer are processed through the microfluidic system chip within the flow cytometry device during an approximately 10-minute cleaning process during periods of bull changeover. The procedure is a semi-autonomous process, where the operator sets the pressure for cleaning, configures the flow of the wash fluid, and sets a 10-minute cleaning timer.

[0130] However, in commercial semen processing settings, the flow cytometry device loses valuable time for semen processing if that valuable time is instead used to clean the chip. In the semi-autonomous cleaning procedure, there is also a possibility that the microfluidic system chip may not be fully cleaned. The semi-autonomous cleaning procedure performed on the flow cytometry device leaves it to the operator's discretion to accurately set the cleaning flow, accurately set the timer, ensure that the cleaning cycle does not end prematurely, and monitor and guarantee that sufficient cleaning solution, such as PARVOSOL, is present in the sample tube throughout the cleaning cycle.

[0131] Accordingly, an object of the present invention is to provide a flow cytometry system including a replaceable or removable sample passage module including a microfluidic system module having a microfluidic system chip, cassette, or cartridge and further including a distribution block or fluidic manifold. In some embodiments, the flow cytometry system may include a microfluidic device cleaning system that performs automatic cleaning of the sample passage module using a pre-programmed or configured cleaning protocol.

[0132] Without wishing to limit the present invention to any theory or mechanism, the methods and systems described herein are believed to be advantageous because removable and replaceable fluid or sample passage modules include a microfluidic chip and a distribution block or a microfluidic module holding a fluidic manifold, are portable, and can be carried to a chip-cleaning station, thereby enabling a fully autonomous cleaning cycle and enabling substantially continuous operation of a flow cytometry device.

[0133] In an exemplary embodiment, a removable and replaceable fluid or sample passage module that includes a microfluidic chip and a distribution block or a microfluidic module holding a fluidic manifold is releasably held (e.g., magnetically) in a flow cytometry system and can be releasably removed by an operator, such as by an outward force, by removing the sample passage module from the flow cytometry system. The sample passage module can then be carried to a chip-cleaning station, which is a microfluidic device cleaning system, where magnetic forces may releasably secure the sample passage module to the microfluidic device cleaning system so that the operator can easily attach the sample passage module to the microfluidic device cleaning system.

[0134] Upon being on the microfluidic device cleaning system, the operator removes the sample tube loader used to press the sample tube against the distribution block and replaces it with a sample reservoir on the microfluidic device cleaning system. The operator initiates the cleaning procedure by selecting a cleaning protocol or cleaning parameters through the user interface of an integrated or attached control system or computer. This initiates an automated cleaning procedure that draws a cleaning solution (e.g., PARVOSOL) from a container or reservoir connected to the back of the microfluidic device cleaning system and fills the sample reservoir to a pre-determined level (e.g., 4.5 mL) according to the cleaning protocol. Thereafter, the microfluidic device cleaning system primes with deionized water or microbiological grade water and flows it through the flow path of the sheath line or buffer line or sample passage module. Thereafter, pressure is applied to the sample reservoir to push the cleaning solution (e.g., PARVOSOL) through the sample line or flow path and thus through the sample passage module. The procedure then flows both the cleaning solution and deionized water through the sample passage module for an amount of time as determined by the cleaning protocol, e.g., for 10 minutes. Finally, air is blown through both the sample line and the sheath line through the chip.

[0135] In some embodiments, the microfluidic device cleaning system comprises a set of manifolds and valves within the microfluidic device cleaning system that enable switching of various liquids. This also enables the microfluidic device cleaning system to perform the same procedure as on a flow cytometry system for starting and stopping chip cleaning.

[0136] A removable and replaceable fluid or sample passage module comprising a microfluidic chip and a microfluidic module holding a distribution block or manifold is portable here as opposed to being fixed. This enables an operator, after flowing a biological fluid sample such as a bovine sample, to remove the sample passage module from the flow cytometry system, carry the sample passage module to a microfluidic device cleaning system (i.e., away from the flow cytometry device) without downtime for unnecessary or excessive system processing to clean the instrument, obtain a clean sample passage module from a storage, and carry and install it onto the flow cytometry device. The chip cleaning station is automated, where parameters of the cleaning protocol, such as flow rate and timer, are preconfigured or programmed as part of the cleaning protocol. The microfluidic device cleaning system and the cleaning protocol comprise built-in logic and algorithms to ensure that the flow rate matches and that the manifold is in a predetermined position to achieve a perfect cleaning of the microfluidic chip within the sample passage module.

[0137] Using an automated system and a removable and replaceable fluid or sample passage module comprising a microfluidic chip and a microfluidic module holding a distribution block or manifold results in a cleaning process that is at least as good as or better than existing systems and methods that require the cleaning process to be done in or on the flow cytometry system, while reducing the downtime of the flow cytometry system. In some embodiments, it may take an operator approximately 13 to 16 minutes to perform a bovine change cleaning process on the microfluidic device cleaning system. The duration of the cleaning process may be further optimized through future optimization and training.

[0138] Generally, a removable and replaceable fluid or sample passage module comprising a microfluidic system device cleaning system, as well as a microfluidic chip and a microfluidic module holding a distribution block or manifold, eliminates downtime or substantially reduces the downtime by an order of magnitude for fluid sample exchange for a flow cytometry system, which downtime has heretofore occurred due to the requirements for cleaning the microfluidic chip on the flow cytometry device.

[0139] The self-cleaning cycle achieves complete cleaning and requires little monitoring by the operator. Cleaning solutions, such as PARVOSOL, are shared in one or more larger reservoirs instead of requiring the operator to fill individual 5 mL tubes for each cleaning routine. Additionally, a removable and replaceable fluid or sample passage module comprising a microfluidic chip and a microfluidic module holding a distribution block or manifold is easy to replace or substitute in case of clogging, reducing the downtime for replacing the microfluidic chip in existing systems.

[0140] With reference to FIG. 5, a front perspective view is provided illustrating one embodiment of a removable and replaceable sample passage module 100 and a flow cytometry system 10 for processing and analyzing a sample. The flow cytometry system 10 may be any type of particle processing system capable of distinguishing between one or more particles based on the observed or determined properties of the particles. For example, the flow cytometry system 10 may be a laser-based detection and inactivation system that uses one or more lasers and detectors to determine the properties of particles, such as cells, in a sample flow and to inactivate a subset of the particles. Other methods of particle sorting, particle separation, or particle inactivation, such as fluidic sorting, jet sorting, laser manipulation, holographic optical trapping, or electrophoresis, may also be used in the flow cytometry system 10.

[0141] The removable and replaceable sample passage module 100 comprises the entire fluid passage for the sample being processed by the flow cytometry system 10. The sample passage module 100 comprises a distribution block 110 and a microfluidic module 150. The distribution block 110 comprises a body 112, a device face 111, a handle 114, a set of alignment pins or rods 116 and a sample straw 118. Using a set of releasable pipe joints including a sample passage pipe joint 103 and a sheath fluid pipe joint or buffer fluid pipe joint 105, the sample fluid passage and the sheath fluid passage or buffer fluid passage within the distribution block 110 are joined to corresponding passages within the microfluidic module 150 via respective sample fluid piping 102 (e.g., the first fluidic line) and sheath piping or buffer fluid piping 104 (e.g., the second fluidic line). In an alternative embodiment, this does not require sheath fluid and the sample fluid passage within the distribution block 110 may be coupled to the corresponding passage within the microfluidic module 150 via the sample fluid piping 102 while eliminating the need for buffer fluid piping 104. The sample fluid piping 102 and the sheath fluid piping or buffer fluid piping 104 may be fixed to the microfluidic module 150 by a set of releasable pipe joints similar to those used on the distribution block 110. The set of releasable pipe joints used on the distribution block 110 and on the microfluidic module 150 may be fixtures such as barbed fittings, lock collar fittings, screw-in fittings, or other suitable fittings capable of fixing the piping 102 and 104 under operable and pressurized conditions of the sample being processed by the flow cytometry system 10.

[0142] The microfluidic system module 150 includes a body 152, a handle 154, and a microfluidic device holder 156. The microfluidic device holder 156 is used to fix and position a microfluidic chip or cassette within the microfluidic device holder 156. As shown in FIG. 14, a knob, handle, threaded rod, or other positioning device may be used to fix and adjust the position of the microfluidic chip or cassette within the microfluidic device holder 156 to align the chip or cassette with the detection and / or inactivation optical system within the flow cytometry system 10.

[0143] The microfluidic device holder and distribution block 110 may each include one or more first iron, ferromagnetic, or other magnetic plates or blocks used to releasably secure the sample passage module 100 to the flow cytometry system 10. Additionally, or in an alternative embodiment using a magnet-based fixation system, other latches, locks, or fasteners may be used to releasably secure the sample passage module 100 to the flow cytometry system 10. However, a fixation system that enables quick and easy fixation of the sample passage module 100, as well as quick and easy release of the sample passage module 100 from the flow cytometry system 10, such as a magnet-based fixation system, may provide advantages related to ease of use and simplified operation.

[0144] When fixed to the flow cytometry system 10, the sample passage module 100 comprises an overall fluid passage for the sample being processed by the flow cytometry system 10. For example, a sample housed in the sample tube 12 is pumped, pushed up, or supplied through the sample passage module 110 via the sample straw 118 by the action of a fluid pressure, such as pneumatic pressure or gas pressure in the sample tube 12, when the sample tube 12 is mounted on or fixed to the distribution block, for example, to push the sample into the distribution block 110 through the sample straw 118. The fluid pressure to the sample tube 12 is provided from the flow cytometry system 10 via one or more fluid inlets, such as a set of fluid inlets on the device surface 111 of the distribution block 110. A pump, such as a peristaltic pump, or an external pneumatic pressure source within the flow cytometry system 10 provides pneumatic pressure to the sample tube 12.

[0145] The fluid sample may contain particles such as sperm cells and passes through the distribution block 110 and via the sample fluid pipe 102 to the microfluidic module 150. In the microfluidic module 150, a microfluidic chip or cassette is used to process the fluid sample. After processing, the fluid sample may be collected within the automatic fluid collection system 14 or its path may be automatically changed to enter a waste collection tube, reservoir, or container. During processing, the fluid sample from the sample tube 12 never comes into contact with any part of the flow cytometry system 10 other than the waste collection reservoir after processing. In this way, the sample passage module 100 may be removed from the flow cytometry system 10 after the fluid sample has been processed without any risk of contamination of other samples to be processed subsequently.

[0146] For example, when a first fluid sample, such as a semen sample having sperm cells, is processed by a flow cytometry system 10 via a sample passage module 100, the sample passage module 100 is released from, or removed from, the flow cytometry system 10 after processing and replaced with another similar or identical sample passage module to process a second fluid sample without any risk of cross - contamination between the first and second fluid samples. This is particularly important when processing biological samples where cross - contamination is not only undesirable but may also violate laws, regulations, or guidelines for processing and distributing biological samples. Cross - contamination can further pose problems when genotyping or genetic sequencing is performed on the sample. The sample passage module 100 provides a removable sample passage module that is isolated, completely enclosed, and used in or with a flow cytometry device that effectively eliminates or at least greatly reduces the potential for cross - contamination between individual samples.

[0147] Next, referring to FIG. 6, a front perspective view is provided that illustrates an example of a removable and replaceable sample passage module 100 and a microfluidic device cleaning system 20 (or cleaning module) for cleaning the removable and replaceable sample passage module 100. As shown in FIG. 6, the sample passage module 100 may be removed from the flow cytometry system 10 as shown in FIG. 5, aligned with, placed on, secured to, or fastened to the microfluidic device cleaning system 20.

[0148] The microfluidic system device cleaning system 20 of FIG. 6 (the block diagram embodiments of which are shown in FIGS. 12 and 13) includes a housing 22, a front portion 24, a back portion 26, a microfluidic module or chip interface 28, and a distribution block or fluidic manifold interface 30. A waste collection tube 32 guides waste from a microfluidic chip or cassette within the microfluidic module 150 through a grommet or opening 34 in the front portion 24 of the housing 22 into a waste collection reservoir. Fluid, such as cleaning fluid or pressurized air, is provided to a cleaning fluid reservoir 38 from one or more internal or external fluid reservoirs through a connection passing through an opening or grommet 36.

[0149] In one embodiment, and with reference to the flow cytometric system 10 of FIG. 5 and the microfluidic system device cleaning system 20 of FIG. 6, a set of valve fixtures may be used at the distribution block 110 of the sample passage module 100 or the interface of the microfluidic module 150. The valve fixture includes a fixture that opens the valve when the fixture is connected and closes the valve when the fixture is disconnected so that fluid within the flow cytometric system 10, the microfluidic system device cleaning system 20, or the sample passage module 100 does not leak from or exit the system when the connection is formed or disconnected. This prevents biological contamination and further prevents the device or operator from being exposed to dangerous waste or substances such as cleaning solvents. The valve or port assumes a closed position when removed or disconnected and opens to permit fluid flow only when the connection is made. In one embodiment, the valve fixture further includes a fixture that is actuated by an actuator or motor or by manual operation of the valve.

[0150] The operation of a flow cytometry system 10, such as a flow cytometry system using a removable and replaceable sample passage module 100, and a microfluidic device cleaning system 20, such as a microfluidic device cleaning system, will be described next with reference to the process 300 shown in the flowchart provided in FIG. 10. The process 300 describes an example method of operating a flow cytometer using a removable and replaceable sample passage module and removing the sample passage module from the flow cytometer for cleaning in a microfluidic device cleaning system.

[0151] The process 300 begins at step 302 where the sample passage module is ready to execute processing on the flow cytometry device. Preparing the sample passage module for processing may include installing or fixing the sample passage module to the flow cytometry device. Fixing the sample passage module to the flow cytometry device may include aligning the sample passage module with one or both of a distribution block interface and a microfluidic device or chip interface on the flow cytometry device. In one embodiment, aligning the microfluidic device may include using alignment pins or rods to accurately position it relative to the flow cytometry device for sample passage module installation. Other alignment devices may be used, and may include a set of fiducial marks on the sample passage module and the flow cytometry device, complementary geometrically corresponding surfaces or faces of the sample passage module and the flow cytometry device, or alignment channels, guides, or rails for aligning and positioning the sample passage module relative to the flow cytometry device for installation.

[0152] In some embodiments, the sample passage module may be fixed to the flow cytometry device by a first iron, iron or ferromagnetic, or other magnetic material plate on the surface of the sample passage module or the flow cytometry device, and a corresponding magnetic plate on the opposing surface. For example, in one embodiment, a set of four plates may be disposed on the surface of the distribution block, and a corresponding set of four magnets may be disposed on the interface surface of the flow cytometry device to align and releasably fix the distribution module of the sample passage module to the flow cytometry device. One or more magnets and corresponding plates may be used to fix either or both of the microfluidic system module and the distribution block of the sample passage module. In another embodiment, a set of fasteners such as a set of latches, a set of lock tabs, a set of bolts, a spring load maintaining pin, or a maintaining or locking lug may be used to fix the sample passage module to the flow cytometry device.

[0153] Additionally, the sample tube may be fixed manually or automatically to the sample tube loader on the distribution block. For example, in one embodiment, the sample tube is removed or taken out from the storage area, the lid or cover is removed, the sample tube is positioned and pushed into the sample tube loader, and is fixed by a set of fixing lugs or tabs that are displaced by the action of the sample tube, for example, and return to their original position by the action of a biasing spring that fixes the sample tube in a predetermined location on the distribution block. In another embodiment, the sample tube is screwed into or onto the distribution block through a corresponding threaded neck of the sample tube and an opening in the distribution block. In another embodiment, an actuator-driven loader moves the sample tube from the storage area and fixes the sample tube to the distribution block, for example, by a spring-loaded sample tube loader.

[0154] Once fixed to the flow cytometry system, the sample passage module may be primed to process the fluid sample. The priming operation may include flushing or priming the sample passage module with a fluid such as air, buffer, or both, prior to processing the fluid sample. In step 304, the sample is processed through the sample passage module by the flow cytometry device. The sample tube mounted on the dispensing block is pressurized via a pressurized gas source within the flow cytometry device such as a compressor, air pump, or by an external pressurized air source. The pressurized gas is passed through the dispensing block to the sample tube, and the pressurized gas within the sample tube causes the fluid sample to pass through the sample straw of the dispensing block. Simultaneously with the pressurization of the sample tube, buffer or sheath fluid is drawn from an internal or external reservoir of the flow cytometry device by a pump or pressurized gas. The sheath fluid or buffer and the fluid sample from the sample tube move through the dispensing block to the microfluidics module via a fluid connection, such as a flexible hose or tube held in place by a releasable clamp. In the microfluidics module, the fluid sample and the sheath fluid or buffer pass through or flow through a microfluidics chip, cartridge, or cassette.

[0155] Particles in the fluid sample are analyzed by a flow cytometry device to determine one or more qualities or characteristics of the particles for the purpose of identifying one or more subgroups or subsets of the particles, such as by the methods described above. After processing, the sheath fluid or buffer and the fluid sample may be collected in a sample collection tube and collected within a sample collection medium in the sample collection tube. If undesirable characteristics or situations are identified, such as contamination, inappropriate identification, poor calibration of the equipment, or other system or sample problems that create a non-ideal sorting situation, then at this time, instead of being collected in the sample collection tube, a portion or all of the fluid sample or sheath fluid or buffer may be diverted to a waste collection reservoir. After all of the fluid samples in the sample tubes mounted on the distribution block have been processed, the processing of the samples by the flow cytometry device is complete.

[0156] Next, in step 306, the sample tube is removed from the sample passage module, and the sample passage module is removed from the flow cytometry system. If the sample passage module is held in place by magnetic attraction, it may simply be pulled from the flow cytometry system, or a set of latches or fasteners may be removed or released to enable the sample passage module to be disconnected from the flow cytometry system. After the sample passage module has been removed from the flow cytometry system, the sample tube loader, which may be a spring-loaded mechanism, may optionally be removed from the distribution block. This step may not be necessary if the cleaning fluid reservoir used by the microfluidic device cleaning system in the cleaning step is compatible with the sample tube loader.

[0157] In step 307, a replacement sample passage module having the same or a different configuration as the one just removed is installed or connected to the flow cytometry system in the same manner as described above. The fluid sample processed by the first sample passage module does not contact any part of the flow cytometry system itself, thereby ensuring that there is no cross-contamination between sample processing runs or between sample passage modules, and thus no additional cleaning or sanitization of the flow cytometry system is required prior to processing of the sample by the replaceable sample passage module. After being installed on the flow cytometry system, the replacement sample passage module may be used immediately to process a new fluid sample.

[0158] In step 308, the first sample passage module removed from the flow cytometry system is installed or connected to a cleaning station, which in one embodiment is a microfluidic device cleaning module. The connection or installation on the microfluidic device cleaning module of the sample passage module is similar to the installation on the flow cytometry system of the sample passage module. However, instead of connecting the sample tube to the distribution block, a cleaning fluid reservoir is connected to the distribution block. If compatible, a sample tube loader may be used to secure the cleaning fluid reservoir, or otherwise the cleaning fluid reservoir may be secured by a set of fasteners or by magnetic attraction.

[0159] In step 310, a cleaning protocol for cleaning the sample passage module is selected. In semi-automatic operation, the cleaning protocol is selected by the operator based on the configuration of the sample passage module, including the microfluidic chip, cassette or cartridge type within the microfluidic module of the sample passage module, and further based on the fluid sample type processed by the sample passage module. The cleaning protocol is selected using a user interface that provides a selection of one of a plurality of cleaning protocols and provides a configuration of one or more parameters of the cleaning protocol. For example, in one embodiment, a display and a set of user interface elements (such as switches, buttons, joysticks, knobs, etc.) are used to select the cleaning protocol. In another embodiment, a touch screen display and one or more graphical user interface elements are used to select the cleaning protocol. In another embodiment, the display and interface elements may be presented on a graphical user interface, presented as physical control devices, on a microfluidic device cleaning system, or integrated with the microfluidic device cleaning system. In another embodiment, a separate control computer or system with a display and interface elements is provided. In one embodiment, a common control system is provided for the flow cytometry device and the microfluidic device cleaning system.

[0160] In fully automatic operation, the cleaning protocol for the sample passage module is automatically determined by the microfluidic device cleaning system. The identifier on the sample passage module is used to identify the type of the sample passage module and may further be used to identify the type of the fluid sample last processed by the sample passage module. In one embodiment, an optically readable identifier is used on the sample passage module. The optically readable identifier may be a barcode, a quick response ( "QR") code, or any other optically readable code or identifier capable of identifying an individual sample passage module. In another embodiment, a radio wave identifier is used on the sample passage module. The radio wave identifier may be a radio frequency identification ( "RFID") tag or chip, a near field communication ( "NFC") tag or chip, a Bluetooth® wireless-based device, or any other radio wave identifier capable of identifying an individual sample passage module.

[0161] For any type of identifier used with the sample passage module, the type of the sample passage module may be determined locally on the microfluidic device cleaning system, or the identifier read from the sample passage module may be transmitted or sent to a central system or server for processing or retrieval. For example, the identifier read from the sample passage module may be compared with a database or table of known sample passage modules to identify a particular sample passage module. A set of characteristics may be read from at least one database entry, field, or record associated with the sample passage module, where one of the database entry, field, or record includes one cleaning protocol, or a set of cleaning protocol configuration information.

[0162] "Cleaning Protocol" includes a set of parameters that define the cleaning operations of a microfluidic device cleaning system for a sample passage module. "Cleaning Protocol" includes the type of cleaning fluid used (e.g., PARVOSOL, quaternary ammonium cleaning solution, biocide (i.e., antiviral, antibacterial, antifungal) cleaning solution, bleach (e.g., sodium hypochlorite solution), alcohol (e.g., isopropyl alcohol)), the volume of cleaning fluid used, the type of rinse fluid used (e.g., deionized water, molecular biology grade water, biologically neutral solvent), the duration of cleaning (e.g., 0 - 5 minutes, at least 5 minutes, 5 minutes, 5 - 10 minutes, at least 10 minutes, 10 minutes, 10 - 15 minutes, at least 15 minutes, 15 minutes, 15 - 20 minutes, at least 20 minutes, 20 minutes, 20 - 25 minutes, at least 25 minutes, 25 minutes, 25 - 30 minutes, at least 30 minutes, 30 minutes), the volume of the sample path within the sample passage module, the number of sample passage volumes to be cleaned ("paths to be swept"), the pressure used (e.g., 5 PSI, 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, at least 20 PSI, at least 30 PSI, at least 40 PSI, at least 50 PSI), the number of cleaning cycles (e.g., at least once, once, more than once, continuously), the temperature used (e.g., at least 20°C, at least 30°C, at least 40°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C, at least 100°C), clog detection procedures, warning procedures, drying procedures (e.g., drying the entire fluid passage with pressurized gas for a specified amount of time, drying the entire fluid passage until the detected moisture content drops below a specified level), and a set of maintenance procedures or logging procedures (e.g., logging all cleaning protocol parameters and sensor data for each cleaning cycle or operation).

[0163] Additionally, the "cleaning protocol" may include one or more of disinfection procedures or operations, sanitization procedures or operations, or cleaning procedures or operations. Cleaning procedures may include the physical removal of contaminants or debris, with or without a reduction in the amount of "microorganisms", bacteria, fungi, or viruses. Sanitization procedures may include a reduction in the amount of "microorganisms", bacteria, fungi, or viruses, with or without the physical removal of contaminants or debris. Disinfection procedures or sanitization procedures completely kill all "microorganisms", bacteria, fungi, or viruses, with or without the physical removal of contaminants or debris. The cleaning protocol may include specific parameters or configurations for achieving one or more of the cleaning procedures or operations, sanitization procedures or operations, or disinfection procedures or operations. The set of parameters described above with respect to the cleaning protocol may further include irradiation (e.g., by ultraviolet light) of one or more portions of the sample passage module, or an increase in the temperature of one or more portions of the sample passage module, in addition to fluid-based disinfection or sanitization and in addition to disinfection or sanitization achieved through cleaning.

[0164] For example, in one embodiment, the cleaning protocol may include specifying a cleaning fluid (e.g., PARVOSOL), a cleaning duration (e.g., 10 minutes), the number of passages to be cleaned (e.g., 20), a temperature (e.g., 24 °C), a pressure (e.g., 138 kPa), a warning procedure (e.g., stopping the cleaning and an audible alarm), a clog detection procedure (e.g., an increase in fluid pressure up to a specified limit), a rinse or flushing fluid (e.g., deionized water or microbiology grade water), and a drying procedure (e.g., flowing pressurized air through the sample passage module for a specified period). Those skilled in the art will understand that these are exemplary cleaning protocol parameters and that other configurable cleaning protocol parameters are contemplated within the scope of the present disclosure.

[0165] Determining the cleaning protocol to be used may be based on identifying the sample passage module being cleaned. In one embodiment, the sample passage module is identified based on one or more identification elements on the microfluidic system chip. For example, the identification element may be a feature or criterion on the microfluidic system chip that is visually identified by processing a signal or image from an image sensor. In one embodiment, the sample passage module is identified based on one or more identification elements on the sample passage module. For example, to identify the sample passage module, an optical or wireless identifier on the outer surface of the sample passage module is scanned by a scanning device. In any embodiment, based on the identification of the sample passage module, one cleaning protocol or a set of cleaning protocol parameters may be automatically specified by the system. Alternatively, or in addition to the automatic identification, a set of information associated with the sample passage module may be presented to the user within a graphical user interface. In one embodiment, the user may select from a set of cleaning protocols based in part on the information presented within the graphical user interface.

[0166] In one embodiment, to identify any flow resistance downstream of an arbitrary device, an algorithm for clog detection monitors signal data from a flow sensor within a fluid passage. For example, a flow sensor downstream of a component transmitting a signal indicating a flow rate lower than the predicted flow rate may be interpreted as indicating a clog, blockage, or other obstruction in the component upstream of the flow sensor. The system responds based on a particular flow sensor indicating a flow rate lower than the predicted flow rate. In one embodiment, a flow sensor downstream of a microfluidic module comprising a microfluidic chip transmits a signal indicating a low flow rate. The system identifies, based on the signal transmitted from the downstream flow sensor, that the source of the low flow rate appears to be the microfluidic chip. In one embodiment, based on a determination that a clog exists within the microfluidic chip, the flow rate or pressure is increased in an attempt to clear the clog. In another embodiment, visual, audible, or audiovisual alarms, warnings, or notifications are provided to the user so that the user can take appropriate action to clear the clog. In another embodiment, operation is halted to enable removal or manual cleaning of the clogged component. In another embodiment, an image sensor may be used to visually identify a clog, such as within a transparent component such as a microfluidic chip, in addition to or as an alternative to the use of a flow sensor. In another embodiment, a secondary operation mode is initiated when a clog or blockage is detected. In another embodiment, a manual override mode is initiated when a clog or blockage is detected.

[0167] In step 312, after a cleaning protocol is determined and selected, the cleaning protocol is used by the microfluidic device cleaning system to perform a cleaning operation on the sample passage module according to the parameters of the cleaning protocol.

[0168] In some embodiments, alternative cleaning protocols may be used for samples used in specific applications or jurisdictions. For example, for samples to be manufactured, used, or sold in a particular jurisdiction, or for applications requiring higher cleaning or safety standards, system elements such as flow cytometry devices, microfluidic device cleaning systems, or sample passage modules specific to that application or jurisdiction may be used. System components specific to a jurisdiction or application are not used in the processing or cleaning of any other components used in any other jurisdiction or application, or for the cleaning thereof. In one embodiment, a system specific to a jurisdiction or application may comprise one or more flow cytometry devices, microfluidic device cleaning systems, and sample passage modules used to process fluid samples for a particular jurisdiction or application. In one embodiment, a jurisdiction may be a geographically, economically, or politically organized region. In one embodiment, an application may be for processing a specific fluid sample from a specific source. In one embodiment, an application may be for processing a semen sample from a mammalian animal associated with a particular jurisdiction.

[0169] After the cleaning protocol is completed, in step 314, the sample passage module is removed from the microfluidic device cleaning system and either placed in a sanitized secure storage location in step 316, or installed on the same or a different flow cytometry system in step 307.

[0170] Referring now to FIGS. 11A - 11B, a block diagram 400 illustrating an example of a system and method for using a removable and replaceable sample passageway 100 in a flow cytometry system 10 and for cleaning the removable and replaceable sample passageway 100 using a microfluidic device cleaning system 20, and a block diagram 500 illustrating an example of a system and method for using a removable and replaceable sample passage module 502 in a flow cytometry system or device 10 and for cleaning the removable and replaceable sample passage module 502 using a microfluidic device cleaning system 20 are provided, respectively.

[0171] In FIG. 11A, a sample passage module 100 is shown positioned or installed on a flow cytometry system 10. The sample passage module 100 may be installed and operated as described in the flowchart 300 and as controlled by a control system 402. In one embodiment, the control systems 402 and 404 are a common system or are remote or "thin" clients connected to a central server or data center that performs information storage, management, and processing. A sample tube 12 provides a fluid sample to the sample passage module 100 for processing by the flow cytometry system 10. After processing of the fluid sample in the sample tube 12 by the flow cytometry system 10, the sample passage module 100 may be removed from the flow cytometry system 10. The sample passage module 100 may then be positioned, connected, or installed on the microfluidic device cleaning system 20. A cleaning fluid reservoir 38 is connected to the distribution block of the sample passage module 100. The cleaning fluid reservoir 38 holds a small amount of cleaning fluid required for a single cleaning operation of the sample passage module 100 and is filled from one or more larger internal or external fluid reservoirs.

[0172] In FIG. 11B, the sample passage module 502 is shown positioned or installed on the flow cytometry system 10. The sample passage module 502 may be installed and operated as described in the flowchart 300 and as controlled by the control system 402. The sample passage module 502 is different from the sample passage module 100 shown in FIG. 11A in that the sample passage module 502 combines the microfluidic system module and the distribution block or manifold into a single element. The sample passage module 100 includes a separate microfluidic system module 150 joined by one or more fluid connections and a distribution block 110, but these components are combined into a single element within the sample passage module 502. This configuration of the sample passage module 502 enables a simpler design and operation of the sample passage module 502, in which case the configuration of the flow cytometry system 10 allows the combined microfluidic system module and distribution block to be used. The sample tube 12 provides a fluid sample to the sample passage module 502 for processing by the flow cytometry system 10. After processing the fluid sample in the sample tube 12 by the flow cytometry system 10, the sample passage module 502 may be removed from the flow cytometry system 10. The sample passage module 502 may then be positioned, connected, or installed on the microfluidic device cleaning system 20. A cleaning fluid reservoir 38 is connected to the distribution block of the sample passage module 502. The cleaning fluid reservoir 38 holds a small amount of cleaning fluid required for a single cleaning operation of the sample passage module 502 and is filled from one or more larger internal or external fluid reservoirs.

[0173] Referring now to FIG. 12, a block diagram is provided that illustrates one embodiment of a microfluidic device cleaning system 600 with an external fluid reservoir. The microfluidic device cleaning system 600 includes a primary housing or body 602 to which a set of fluid reservoirs (fluid reservoir 1 610a, fluid reservoir 2 610b, and fluid reservoir n 610n) and a waste reservoir 650 are releasably and fluidly connected. The fluid reservoirs 610a - 610n hold one or more cleaning fluids or other fluids, which may include PARVOSOL, 7X, deionized water, microbiology grade water, isopropyl alcohol, ethanol, or other suitable cleaning, wetting, or rinsing solutions or solvents.

[0174] Computing or control system 690 communicates electronically with one or more elements of the microfluidic device cleaning system 600 and includes a processor 692, which can be a commercially available microprocessor, FPGA, or other suitable processor (e.g., RISC, ARM, x86, x64, etc.), a memory 693, a networking interface or transceiver 694, and an input / output ("I / O") interface 695. A display 691 may be integral with or in electrical communication with the computing system 690 and displays output from the computing system 690, including any user interface, graphical user interface, warnings, system information, or other output required by or desired by the system or the user. The computing or control system 690 stores in the memory 693 a set of cleaning protocols or procedures that control or manage the operation of the microfluidic device cleaning system 600. The protocols may include the parameters or mechanisms described above, but may be modified or adjusted based on user input or on sensor signals from flow sensors, proximity sensors, or image sensors of the microfluidic device cleaning system 600. Additionally, the cleaning protocols may include algorithms or processes for controlling fault detection and response, such as when a clog or low fluid flow is detected. The fault response may include a temporary increase in fluid flow or pressure, cessation of operation, or the provision of audible, visual, or audiovisual alarms.

[0175] The fluid reservoirs 610a - 610n are releasably connected to ports or openings in the body 602 of the microfluidic device cleaning system 600. A set of valves 612a, 612b - 612n control the fluid flow from their respective reservoirs and are actuated based on control signals received from the computing system 690. A set of flow sensors 614a, 614b - 614n provide signals to the computing system 690 related to the volume or flow rate of the flow from their respective fluid reservoirs into the microfluidic device cleaning system 600. The pump assembly 620 may be a single pump or multiple pumps including a liquid pump and an air pump, one or more of which may be peristaltic pumps, and pumps fluid from the reservoirs 610a - 610n to the dispensing interface 630. The fluid pumped by the pump assembly 620 may include pressurized gas including pressurized air. The pump assembly 620 pumps fluid through one or more fluid passages and may have separate control valves 621 and 622 and flow sensors 623, 624, and 625. The valves 621 and 622 effect fluid flow control and may be used, such as in valve 621, to divert the path of fluid flow between one or more fluid flow passages.

[0176] In one embodiment, the dispensing interface 630 includes a set of magnets 632 for retaining the dispensing block or manifold of the sample channel module. Other securing devices such as latches, locking lugs, or fasteners may be used. The proximity sensor 634 may be an optical, ultrasonic, or magnetic proximity sensor configured to provide a signal to the computing system 690 indicating that the dispensing block is properly aligned, positioned, or secured relative to the dispensing interface 630. A set of fluid inlets from the pump side of the dispensing interface 630 lead to a set of fluid outlets 631. The fluid outlets 631 allow fluids such as compressed gas or cleaning liquid to pass from the microfluidic device cleaning system 600 into the dispensing block. The fluid passes through the dispensing block of the sample channel module and through or is processed through the microfluidic chip, cassette, or cartridge within the microfluidic module of the sample channel module during the cleaning process to be controlled by the cleaning procedure.

[0177] In one embodiment, the chip interface 640 includes a set of magnets 642 for retaining the microfluidic module of the sample passage module. Other securing devices such as latches, locking lugs, or fasteners may be used. The proximity sensor 644 may be an optical, ultrasonic, or magnetic proximity sensor configured to provide a signal to the computing system 690 indicating that the microfluidic module is properly aligned, positioned, or secured relative to the chip interface 640. The image sensor 646 may be a charge-coupled device (CCD) sensor or an active pixel sensor, such as a complementary metal-oxide semiconductor (CMOS) sensor, and captures image information that can be processed by the computing system 690 to determine whether a clog exists within the microfluidic chip, cassette, or cartridge within the microfluidic module. Simpler sensors, such as photoresistors or photodiodes, may also be used to determine whether a clog exists. For any sensor type, the image sensor 646 provides image data indicating a clog within the fluid passage to the computing system 690. Such image data may include a reduced light level, an increased black level, an on signal, or an off signal, which may indicate that some object or debris is blocking the fluid flow path. The computing system 690 may process this input data to initiate a troubleshooting or clog removal process. The image sensor 646 may also be used to capture an image of identification markings on the microfluidic chip, such as features, reference markers, reference images, or reference markings, which may be processed by the computing system 690 to identify a particular microfluidic chip and provide calibration or adjustment information for the optical or sensor components. Optically identifying the microfluidic chip may be used to automatically determine a cleaning protocol or a set of cleaning parameters for the microfluidic chip or sample passage module.

[0178] Valve 652 and flow sensor 654 may be used to control and monitor, respectively, the flow of fluids, including gas, cleaning solution, and rinse solution or other solutions, as they exit the microfluidic system chip, cassette, or cartridge and flow to waste reservoir 650. In one embodiment, an ultraviolet (UV) light-emitting sterilization device 648, which may be a UV light-emitting diode ("LED"), is used in combination with fluid-based cleaning and sterilization to further sterilize and clean the microfluidic system chip.

[0179] A set of valves, an image sensor, and a proximity sensor provide signal outputs that are processed by computing system 690 to determine one or more states or conditions of microfluidic device cleaning system 600 and to compare the determined or detected states to an ideal or desired system state. For example, a flow sensor may be used to determine the amount of fluid flowing from one or more reservoirs, which may then be used to determine the amount of fluid remaining in the reservoir and signal to an operator that the reservoir fluid is low and replacement is needed. The flow sensor may also be used to determine the amount of fluid flowing through a particular component, tube, or piping run that should be used to determine a maintenance event for that component, such as replacing a pump or tube at the end of its maintenance life or cycle. For example, a peristaltic pump may have a predetermined life and need to be replaced at the end of its operating life. By measuring the fluid flow before or after the pump, an accurate determination can be made as to when the pump should be replaced.

[0180] To provide an indication when a component or device needs to be replaced, similar measurements and feedback may be provided for any system component for which a measured value can be compared to a stored value such as a maintenance period or useful life. In another example, an image sensor may be used alone or in combination with one or more flow sensors to detect or determine when a component of the microfluidic device cleaning system 600 is clogged. Based on the type of clogging determined by the measured system conditions, a signal may be sent to the operator or a fault condition operation may be triggered, such as increasing the flow rate or pressure to break the clog or abort the operation. Sensors, including flow sensors, may also be used to track the volume of the sample passage module that is cleaned (e.g., the number of paths that are cleaned) to accurately determine when the sample passage module is clean enough to end the cleaning operation.

[0181] Valves within the microfluidic device cleaning system may be used to control the flow of fluid within the microfluidic device cleaning system 600, for example, to divert or stop the flow of fluid to a particular component of the microfluidic device cleaning system 600, or to divert or stop the flow of fluid from a particular component. The valve may be a solenoid valve, an actuator control valve, or a pneumatic or hydraulic control valve that is actuated by a control signal from the computing system 690.

[0182] FIG. 13 provides a block diagram illustrating an example of a microfluidic device cleaning system 700 with an internal fluid reservoir and a control system. The microfluidic device cleaning system 700 is substantially similar to the microfluidic device cleaning system 600, except that all system elements including the computing system 690, the display 691, the waste reservoir 650, and the fluid reservoirs 610a, 610b to 610n are arranged within the main body or housing 702. In both the microfluidic device cleaning systems 600 and 700, the number and location of the reservoirs, as well as the configuration of the computing system 690, may be modified (e.g., included, omitted, or internal or external) based on specific implementation forms or processing requirements. For example, in one embodiment, the microfluidic device cleaning system may include a single external fluid reservoir 660 used for both the cleaning fluid and the waste fluid, in which the fluid is filtered or reused. In another embodiment, the microfluidic device cleaning system may include an internal waste reservoir and one external fluid reservoir. In yet another embodiment, the microfluidic device cleaning system may include two internal fluid reservoirs and one external waste reservoir. In any of the embodiments, the microfluidic device cleaning system may include an internal gas compressor or pump, or a connection to an external pressurized gas source.

[0183] Referring now to FIG. 14, a block diagram is provided that illustrates an example of a removable and replaceable sample passage module 800 that includes a microfluidic module 850 and a distribution block 810. The sample passage module 800 includes a sample fluid passage 830 and a sheath fluid passage 832 that are continuous from the distribution block 810 to the microfluidic module 850. The sample passage module 800 may be used with a flow cytometric device or may be used with a microfluidic device cleaning system. As represented in the example of FIG. 14, the sample passage module 800 is to be understood as having a configuration that is common to either a flow cytometric device or a microfluidic device cleaning system.

[0184] The dispensing block 810 includes a body or manifold 811 having a sample passage element 814, a gas passage element 815, a sheath fluid or buffer fluid passage element 817, and a connection passage element 816. The spring-loaded sample tube loader 813 abuts against a surface such as the bottom surface of the dispensing block body 811 to hold and seal the fluid reservoir 822. Alternatively, in the case of a microfluidic device cleaning system, etc., the sample tube loader 813 may be removed, and the fluid reservoir 822 may be held in place relative to the dispensing block 810 by other suitable means. In one embodiment, the fluid reservoir 822 is a cleaning fluid reservoir that abuts against and is held to the dispensing block 810 by corresponding screws or lugs and a set of magnets. In one embodiment, the fluid reservoir 822 is a sample fluid reservoir that is sufficiently held and retained within the sample tube loader 813, and the sample tube loader 813 is releasably fixed to the dispensing block 110 by a set of magnets. In another embodiment, the sample tube loader 813 further includes a spring or spring biasing means for applying pressure to the fluid reservoir 822 such as a sample tube, so that the seal abutting against the sample straw 812 extends into the fluid reservoir 822 and to the fluid 824 contained therein. In a flow cytometry system, when processing a biological fluid sample, the fluid 824 may be a semen sample, and the fluid reservoir 822 may be a sample tube. In a microfluidic device cleaning system, the fluid 824 within the fluid reservoir 822 may be a cleaning fluid, solution or solvent, or water such as deionized water or microbiology grade water.

[0185] A pressurized fluid such as a pressurized gas enters the sample tube through port or opening 820. The pressure from the pressurized gas propels or flows fluid 824 through sample straw 812 and into sample passage element 814. A sheath fluid or buffer, or a flushing or rinsing fluid or solvent, is flowed or pumped through sheath fluid or buffer path element 817. The pressurized gas may also be routed from gas path 815 through connection path element 816 to sheath fluid or buffer path element 816, for example, by an internal or external valve, to provide pressurized gas within sheath fluid passage 832 for purposes such as flushing or drying. Similarly, the pressurized gas may be advanced through sample passage element 814 to clean or dry sample fluid path 830. Hoses, tubes or pipes 831 and 833 are releasably held in place by fixture sets 819 and 852, and 818 and 853, to join distribution block 810 and microfluidic module 850 within a fluid connection, forming respective portions of sample fluid path 830 and sheath fluid or buffer path 832.

[0186] The microfluidic system module 850 includes a body 851 in which a microfluidic chip (which may be a microfluidic cassette or cartridge) 880 is positioned and held by a microfluidic device holder 856. The device holder 856 is used to fix and position the microfluidic chip 880 within the body 851. The device holder 856 is used to adjust the relative position and angle of the microfluidic chip 880 by, for example, a knob screw, a threaded rod, or other suitable positioning means to align the microfluidic chip 880 with a detection optical system or a laser kill optical system within a flow cytometric device or with an image sensor within a microfluidic device cleaning system. In another embodiment, the position of the microfluidic chip 880 is fixed within the microfluidic module 850 by pins or by corresponding geometries of the microfluidic module 850, and the positions of the kill laser, the detection laser, or other optical devices are adjusted in the X, Y, or Z directions relative to the microfluidic chip 880 within a flow cytometer or within a microfluidic device cleaning system.

[0187] In an exemplary embodiment, the microfluidic chip 880 includes a body 881 and a set of internal fluidic channels. The channels 855 in the microfluidic module body 851 direct sheath fluid, cleaning fluid, or pressurized gas into the sheath input 882, through the sheath channel 883, into the common channel 886, through the detection region 887, and out through one or more outlets 888 and 889 to be collected as waste fluid 892 in the waste reservoir 890. The channels 854 in the microfluidic module body 851 direct sample fluid, cleaning fluid, or pressurized gas into the sample input 884, through the sample channel 885, into the common channel 886, through the detection region 887, and out through one or more outlets 888 and 889 to be collected as waste fluid 892 in the waste reservoir 890.

[0188] The sample passage module 800 is a fully modular device that can be removed from a flow cytometry device and placed on a microfluidic device cleaning system for a fully automated cleaning process. Components of the sample passage module 800, such as the distribution block 810 and the microfluidic module 850, etc., may be separately removed, separated, and replaced for cleaning or maintenance purposes. The sample fluid passage 830 of the sample passage module 800 comprises the entire sample fluid passage for the sample fluid when the sample passage module 800 is used with a flow cytometry device. The sample fluid processed by the sample passage module 800 within the flow cytometry device does not contact any part of the flow cytometry device except when the sample is collected in the sample collection tube or diverted to the waste collection container. This enables the easy and rapid replacement of the sample passage module 800 between sample runs, for example, for changing the source of a semen sample. The replaceable, removable, reusable, and modular nature of the sample passage module 800 reduces or eliminates the downtime of commercial flow cytometry equipment and further reduces the waste that may be generated by the use of disposable or single-use microfluidic devices.

[0189] Referring now to FIG. 16A, a block diagram is provided that illustrates a side view of one embodiment of a microfluidic module 950 of a removable and replaceable sample passage module. The microfluidic chip 880 may be a microfluidic chip having internal sample fluid and buffer or sheath fluid flow channels as depicted, or may have any configuration of sample and buffer or sheath fluid flow channels that would be apparent to one of ordinary skill in the art, and is disposed within a chip fixing device 955 of a body 951 of the microfluidic module 950. The sample line is connected to a connection 952 and is supplied to the microfluidic chip 880 through an internal passage 954, and the sheath fluid line or buffer line is connected to a connection 953 and is supplied to the microfluidic chip 880 through an internal passage 955. The connections 952 and 953 may be releasable connections as described above, and the characteristics and functions of the microfluidic chip 880 may be as described above and may be subject to modifications known to one of ordinary skill in the art.

[0190] Additional details and examples of microfluidic chips that may be used in accordance with the systems and methods of the present invention can be found in the following co-owned patents and patent applications, namely US8961904, US9588100, US10488320, US10928298, US10532357, US16 / 279430, US16 / 419756, US16 / 597235, US16 / 704175, and US16 / 741608, the contents and specifications of which are incorporated herein by reference.

[0191] The microfluidic system chip 880 is retained within the chip fixture 955 by a plate and clamp 959 on the surface of the chip fixture 955, by a set of retaining pins 960, by a set of corresponding geometry mechanisms 961, or by any combination of these elements. The front, front face, or front plate element 956 of the microfluidic module 950 is generally orthogonal to the chip fixture 955. The front portion 956 includes one or more magnets 956 used to releasably secure the microfluidic module 950 to a flow cytometer or a microfluidic device cleaning system.

[0192] Referring now to FIG. 17, a block diagram is provided that illustrates an example of a modular flow cytometry system 1000 including a flow cytometry device 1010, a microfluidic device cleaning system 1020, a sample passage module 1100, a local control system 1400, and a remote server 1500. In system 1000, a removable and replaceable sample passage module 1100 is used with the flow cytometry device 1010 and the microfluidic device cleaning system 1020. In one embodiment, the flow cytometry device 1010 includes a distribution block interface 1018, a microfluidic module interface 1016, a set of untreated sample tubes 1012 retained within a sample tube holder, and a mixing station 1014 for the automatically processed samples. In one embodiment, the microfluidic device cleaning system 1020 includes a distribution block interface 1630, a microfluidic device interface 1640, and a cleaning fluid reservoir 1038. The cleaning fluid may be supplied from one or more internal or external fluid tanks to the reservoir 1038, and the waste cleaning fluid may be returned to the microfluidic device cleaning system 1020 for internal or external storage.

[0193] The microfluidic system module interface 1016 and the microfluidic device interface 1640 each include an adjustable element for positioning an optical imaging device (e.g., a camera, an imaging device for a microscope), a detector (e.g., a photomultiplier tube, an avalanche photodiode), or an irradiation device (e.g., a kill laser or a detection laser) with respect to a microfluidic chip within the microfluidic system module of the sample passage module 1100. The position and angle of the adjustable element may be adjusted manually (e.g., by a set of screws, etc.) or by an element (e.g., an actuator, a stepper motor) of the flow cytometry system 1010 or the microfluidic device cleaning system 1020. This enables any optical or detection element of the flow cytometry device 1010 or the microfluidic device cleaning system 1020 to be accurately positioned with respect to a part (e.g., an active region, a detection region, or a calibration marking) of the microfluidic chip within the sample passage module 1100. The adjustable element may be moved in the X, Y, or Z directions, or in any combination of those directions, or may be tilted with respect to the microfluidic chip to properly align the adjustable element.

[0194] The sample passage module 1100 is held and fastened to the holding or storage station 1700 by a set of magnets or the like, and this station is physically adjacent to one or both of the flow cytometry device 1010 and the microfluidic device cleaning system 1020. Using a scan device 1720, which is a device capable of reading or scanning an optical or wireless identification tag (such as a barcode scanner or RFID reader), to read or scan the identification element 1956 on the sample passage module 1100 before it is connected to or installed for use in processing a fluid sample by the flow cytometry device 1010. This information is transmitted to the local control system 1400. The local control system 1400 is one or more computing devices such as a desktop computer, laptop computer, tablet computer, thin client computer, or other suitable computer, which may be outside the flow cytometry device 1010 and the microfluidic device cleaning system 1020, may be integrated with one or both of the flow cytometry device 1010 and the microfluidic device cleaning system 1020, may be shared by the flow cytometry system 1010 and the microfluidic device cleaning system 1020, or may comprise separate stations or terminals for each of the flow cytometry system 1010 and the microfluidic device cleaning system 1020.

[0195] The local control system 1400 receives and processes the identification information scanned by the scan device 1720 from the sample passage module 1100. The graphical user interface 1410 displays information associated with the sample passage module 1100, such as configuration, type of microfluidic chip, appropriate flow cytometry or cleaning protocol or parameters, maintenance information or warning information, to inform the user of how the sample passage module 1100 is used and configured. Additionally, this information may be used by the flow cytometry system 1010 and the microfluidic device cleaning system 1020, respectively, to automatically configure the flow cytometry protocol or the cleaning protocol based on the configuration of the sample passage module 1100. Additionally, or alternatively, scanning is used to track the location or status of the sample passage module 1100, and similar identification elements are scanned on the flow cytometry device 1010 and the microfluidic device cleaning system 1020 to specifically track when and how the sample passage module 1100 was used and to identify where the sample passage module 1100 is at any given time.

[0196] The information used by the local control system 1400 may be stored locally or may be stored in a remote server 1500 having one or more databases. The databases stored within the remote server 1500 include information regarding the particular sample passage module 1100, which includes usage logs, maintenance information, configuration information, flow cytometry protocols, and cleaning protocols. This information may alternatively be stored in the local control system 1400. In one embodiment, the information stored in the local control system 1400 or the remote server 1500 may be shared among multiple systems, i.e., between two or more systems. For example, in one embodiment, the configuration information and usage information from one flow cytometry device 1010 may be shared with other flow cytometry systems, the configuration information and usage information from one microfluidic device cleaning system 1020 may be shared with other microfluidic device cleaning systems, and the information regarding the sample passage module 1100 may be transmitted and shared among any connected devices or systems.

[0197] The user interface 1410 provides the user with a GUI and a set of user-operable or controllable elements, such as graphical user interface elements, to enable viewing and control of parameters or information associated with any aspect of the system 1100. For example, in one embodiment, a set of user interface elements within the user interface 1410 are provided to the user for configuring a flow cytometry protocol for a particular sample passage module 1100 and for a particular sample being processed, such as a semen sample. In another embodiment, a set of user interface elements within the user interface 1410 are provided to the user for configuring a cleaning protocol for a particular sample passage module 1100. The user interface 1410 is further configured to provide warnings or information regarding the current operating status of the flow cytometry device 1010 or the microfluidic device.

[0198] In one embodiment, any flow cytometry system (e.g., flow cytometry system 10), microfluidic device cleaning system (e.g., microfluidic device cleaning system 20), and sample passage module (e.g., sample passage module 100), as well as any fluid passage, i.e., any component that physically contacts the fluid, must be chemically inert. For example, silicate glass, polyvinylidene fluoride ("PVDF"), polytetrafluoroethylene ("PTFE"), perfluoroalkoxy alkane ("PFA"), polyether ether ketone ("PEEK"), or other suitable materials may be used to prevent damage, corrosion, or other undesirable reactions within components that may be exposed to corrosive substances (e.g., acids, oxidants, bases) or other hazardous substances used in the cleaning process or procedure. Specifically, the selection of materials for components within any fluid passage should prioritize materials that can be safely disposed of even when in contact with any chemical substances used in the cleaning operation or sample processing operation or process, and materials that are substantially chemically inert should be used.

[0199] Referring now to FIG. 18, there is provided a block diagram 2000 illustrating an example of fluid flow within a microfluidic device cleaning system, such as microfluidic device cleaning systems 20, 600, 700 or 1020. FIG. 2000 illustrates an example of the movement and control of fluid entering, passing through, and exiting a microfluidic device cleaning system. A set of fluid sources, including a sheath line source 2010, a sample line source 2012, and a gas source 2014, each of which may be an internal or external source, may be provided from a fluid reservoir (e.g., bag, container, tank), a compressed gas source or tank, and may include a cleaning solution, a solvent such as water, and compressed air, and comprises a set of inputs for the fluid system. Overall, three fluid passages are provided, a sheath fluid line or path 2002, a gas line or path 2006, and a sample line or path 2004. In the sheath fluid line 2002, the flow of fluid from one of the sources 2010 is controlled by a corresponding valve 2020 to allow or restrict flow to the sheath line manifold 2030. In the sample fluid line 2004, the flow of fluid from one of the sources 2012 is controlled by a corresponding valve 2022 to allow or restrict flow to the sample line manifold 2032. One or more fluid inputs, such as a specific cleaning liquid or solution, a solvent such as water, or other fluids, may be shared or provided to both the sheath fluid line 2002 and the sample line 2004. Control of the fluid path for the shared fluid is achieved by the operation of appropriate corresponding valves within the set of valves 2020 or 2022. With respect to the gas line 2006, the gas pressure from the gas or air input 2014, which may be a pressurized external or internal gas source, is regulated by an air regulator 2040 to permit or adjust the pressurized gas within the fluid system.

[0200] In sheath line 2002, sheath line pump 2050 may be a peristaltic pump or other suitable pump that draws or pumps one or more fluids from source 2010 into main flow system manifold 2080. The fluid flow drawn by sheath line pump 2050 is measured by flow sensor 2060 downstream of the pump and controlled by valve 2070. In sample line 2004, sample line pump 2052 may be a peristaltic pump or other suitable pump that draws or pumps one or more fluids from source 2010 or 2012 into main flow system manifold 2080. The fluid flow drawn by sample line pump 2052 is measured by flow sensor 2060 and may be controlled by the operation of a valve downstream of the flow sensor. Flow sensor 2060 is used to measure the amount of fluid that has moved through the flow line or the velocity of the fluid flow through the flow line and may be used to detect or identify that it is lower than the desired fluid flow rate, which may indicate a blockage or obstruction in the fluid flow line. Compressed gas from air input 2014 is regulated by air regulator 2040, and the flow of compressed gas is controlled by valves 2072 and 2074 to allow the gas to flow into flow system manifold 2080 or into sheath fluid line 2002. The gas is used to clean sample line 2004 or sheath fluid line 2002 and is used to dry the fluid line after the cleaning operation.

[0201] The fluid, including gas, flows into the fluid system manifold 2080 and flows through the sample passage module before returning in the chip manifold 2090. The waste fluid, including the cleaning solution and the rinsing agent, flows to the waste output 2100, where it is received, and this output directs the waste fluid to an internal or external waste reservoir or drain line. The waste valve 2102 is used to divert any fluid from the sample line 2004, the sheath fluid line 2002, or the gas line 2006 directly into the waste output 2100 without passing through the sample passage module. The diversion of the fluid may be performed manually or automatically as a result of an error or fault condition, including those based on detected blockages or obstructions.

[0202] On-chip identification

[0203] Referring now to FIGS. 16A and 16B, the microfluidic chip may include identification elements 957 for the identification and reference of on-system objects to automatically focus the microfluidic chip. For example, one or more identification elements 957 may be disposed at the front portion 956 of the chip. The identification elements 957 may be visual or optical identification elements such as QR codes (registered trademarks) or barcodes, or may be wireless identification elements such as RFID or NFC tags, or may be a combination of optical or visual elements and wireless identification elements.

[0204] Each microfluidic chip is intended to be geometrically identical, but may vary slightly due to manufacturing variations. The identification elements can enable comparison of the chips with respect to device performance, which is useful for eliminating chips with inferior performance and identifying high-performance chips. The identification elements can also automatically track how long the chip has been used to inform the user when the chip needs to be removed for cleaning.

[0205] In some embodiments, the identification element 957 is used to identify a unique microfluidic system module 950 and a microfluidic chip 880 installed or disposed within the module 950. This is for the purpose of avoiding cross - contamination, for cleaning, storage and retrieval, and for facilitating the identification of elements for the configuration of flow - cytometry or cleaning parameters or protocols based on the type and configuration of the microfluidic chip 880 and the microfluidic system module 950 as identified by the identification element 957.

[0206] In other embodiments, the identification element can also be used to automatically align and position a microfluidic chip within a flow - cytometry device. This can save the operator's time by eliminating the need to manually focus and align the microfluidic chip. As shown in FIG. 16C, in one embodiment, a microfluidic chip with an identification element is placed on a three - axis stage, which is a stage that can move along three axes. Light sources such as a camera and a bright - field LED light can be used to image the microfluidic chip and provide feedback to the three - axis stage to properly align the chip. The camera and the light source are fixed in place and alternative levels of illumination may be required. The dimensions of the identification element should be small enough to be fully visible within the field of view of the camera. The three - axis stage allows the chip, and thus the etching pattern, to be re - positioned to move the chip into focus by moving the chip instead of refocusing and re - positioning the camera.

[0207] In some embodiments, the chip may have one or more identification elements 957. For example, a number of identification elements 957 may be required to achieve accurate focusing and positioning. In some embodiments, the identification elements 957 can be laser-etched within each chip. The identification element can be any pattern that can be decoded through image analysis. The pattern can include, but is not limited to, alphanumeric sequences, barcodes, QR codes (registered trademarks), etc., which are applicable and equivalent.

[0208] In another application, the identification element 957 may be used to indicate which chip is currently mounted on the microfluidic system module. Since the etching pattern can store information, the flow cytometry device may be able to read the etching pattern on the chip to indicate which chip is on the device.

[0209] Optical system module and electronic system module

[0210] Without wishing to limit the present invention to a particular use or example, the systems, devices, and methods described herein, such as the removable and replaceable sample passage module 100, may be used with a particle processing system such as a semen sorting system (e.g., a flow cytometry system 10). In one embodiment, the semen sorting system may comprise a flow cytometry device or a microfluidic chip or device comprising a flow chamber configured to direct a fluid flow containing sample particles through a particle interrogation location. The sample particles may be cells such as sperm that have been stained or otherwise processed prior to entering the flow cytometry device or microfluidic device. The flow cytometry device or microfluidic device may further comprise a laser configured to emit electromagnetic radiation along a beam path to the particle interrogation location and a detector configured to detect radiation from the particles. After detection, the particles may be sorted or inactivated, for example, by ablation with electromagnetic radiation, before or after exiting the flow cytometry device or microfluidic device. An additional detector may be used to determine the effect of inactivating the particles, for example, to determine or detect the amount or dose of electromagnetic radiation to which individual particles have been exposed and to determine whether individual particles have been sufficiently inactivated or damaged. The particles may be X-chromosome or Y-chromosome sorted or sex-discriminated semen, for example, X-chromosome sex-discriminated semen, in which case they are collected in one or more collection containers.

[0211] The removable and replaceable sample passage module 100 may also be used in a method for analyzing particles, such as sperm cells, contained in a fluid flow as the particles flow through an interrogation location within a flow cytometer device or within a microfluidic chip or microfluidic device, or in such a method. Such a method includes illuminating the fluid flow and the particles contained therein using electromagnetic radiation emitted from a laser. A detector detects the electromagnetic radiation emitted from the interrogation location by the particles, and a processor determines the characteristics of the particles in the fluid flow based in part on the signal from the detector. After detection, the particles may be sorted or inactivated by a physical sorting and removal process, such as before or after exiting the flow cytometer device or microfluidic device, or by ablation with electromagnetic radiation or inactivation by photodamage. The particles may be semen that has been sorted for the X or Y chromosome or sex discriminated, for example semen that has been sex discriminated to have the X or Y chromosome, and may then be collected in one or more collection containers.

[0212] The removable and replaceable sample passage module 100 may also be used in a method for evaluating the total amount of DNA in the nucleus of sperm cells using a flow cytometry device or a microfluidic chip or device, or in such a method. The method may include staining the DNA in the nucleus of sperm cells, and then irradiating the stained DNA in the nucleus of sperm cells with an electromagnetic radiation device such as a laser when the sperm cells pass through the laser in the corresponding region of the flow cytometry device or the microfluidic device. A detector such as an avalanche photodiode detects the fluorescent light emitted from the irradiated and stained DNA in the nucleus of sperm cells at a detection location or interrogation location within the flow cytometry device or the microfluidic device. The method may further include first determining the sex of the sperm cells using the detected total amount of DNA in the nucleus of the sperm cells, and then distinguishing a plurality of sperm cells based on the determination of the sex, thereby distinguishing the X chromosome-bearing sperm cells from the Y chromosome-bearing sperm cells. The characteristics of the sperm cells may include the total amount of the corresponding DNA in the nucleus of the sperm cells. The method may further include inactivating a given sperm cell based on the determined total amount of DNA in the nucleus of the given sperm cell. The inactivating may include photodamaging or excising a given sperm cell by irradiation with electromagnetic radiation. The method may further include forming a plurality of droplets each having one of the captured sperm cells, separately charging each of the droplets based on the sex differentiation characteristics of the sperm cells captured in the droplets, deflecting one of the droplets, and separately collecting each of the droplets differently based on the sex differentiation characteristics of the sperm cells captured in the droplets. The method may further include collecting the sperm cells after determination, sorting or inactivation when the sperm cells exit the microfluidic device. The particles may be semen sorted for the X or Y chromosome or sex-discriminated, for example, sex-discriminated semen having the X or Y chromosome, and are collected in one or more collection containers.

[0213] In some embodiments, the invention is a larger emitted electromagnetic radiation kill beam having an adjustable elliptical beam waist profile at the focal plane, the height and width of which are independently adjustable and not factors of each other or derived from each other, and provides optical and optomechanical modules for both positioning and emitting a detection laser and a kill laser from the same direction with respect to the flow of the sample fluid or from a common side with respect to the flow cytometry device.

[0214] FIG. 19 shows an embodiment of an interrogation or optical system module 7020 that includes a detection optical system and a detection laser and a kill laser therein. The optical system module floats separately from the rest of the module system and may be isolated by rubber legs, i.e., removable from the chassis. FIG. 20 shows the internal arrangement of the optical system module. Both detection and kill come from one side in combination with the use of a beam cube. The optical system module combines "S" and "P" polarized beams of the same wavelength. A filter in front of the object protects it from damage. An aspherical lens focuses the kill laser rather than the object. Both lasers have shaped lenses to control the spot size at the location of the chip.

[0215] FIGS. 20A-20B and 21A-21B show an electronic system module 7010 that includes an electronic box that houses electrical components and a control panel. The box includes a removable lid that protects the interior of the box. The electronic system module can likewise be removed from the chassis. FIG. 22 is a schematic diagram of the electronic components of a modular flow cytometry system.

[0216] Referring now to FIG. 23, there is provided a diagram illustrating the beam width of the electromagnetic radiation emission of a kill laser having an elliptical profile at the focal plane according to an embodiment of the present invention. The width of the beam is configured, calibrated, or adjusted such that the width in the X direction is from 5 μm to 35 μm, where the Y direction is longitudinal along the flow path or channel length in a microfluidic device in which a sample containing a set of particles and / or sheath fluid flows. The width of the kill beam as shown is 20 μm, which is wider than the core width of the illustrated example. The core width of 5 - 20 μm is the width of a fluid such as the sample fluid, bounded by other focusing fluids and not necessarily by a physical channel wall. The physical channel in a flow cytometer may exceed 100 μm in width but is a restricted fluid, and the core in which the particles or sample are placed has a width of 5 - 20 μm. For example, a single sheath design microfluidic chip ("SSMC") channel may have a core width of 5 - 20 μm, and a double sheath design microfluidic chip ("DSMC") may have a core width of 5 - 15 μm. The width of the kill beam is configured by adjustment of one or more optical or optomechanical components, as provided, for example, in FIGS. 25A, 25B, and 26B, to provide a beam width equal to or greater than the core width in the working or interrogation region of the microfluidic channel.

[0217] The kill beam has an elliptical profile in the focal plane, has a Gaussian intensity profile, in which case the highest concentration of electromagnetic radiation emission, e.g., photons, is at the center of the kill beam. The factor of the effect of the kill beam when inactivating or destroying particles passing through the focal plane in the action or interrogation area of the microfluidic system or in droplets in a droplet-based flow cytometer is the amount of electromagnetic radiation emission, e.g., photons, per unit space that hits particles passing through that space, e.g., sperm cells. The kill beam needs to have sufficient intensity over the surface area of the particles to ensure that the particles are sufficiently inactivated, e.g., by light damage or ablation. The laser module used to generate the kill beam is a pulsed laser module, e.g., a pulsed laser module having a pulse duration of 1 to 500 nanoseconds and an available pulse energy of 1 to 5 μJ per pulse. In another embodiment, the pulsed laser module has a pulse duration of 5 to 30 nanoseconds. Increasing the pulse energy increases the size of the kill beam. In existing systems and methods, this can cause the beam to have an increased beam waist in a circularly shaped beam, e.g., by increasing the radius and outer perimeter of the beam in the focal plane. However, using a kill beam with an elliptical profile is more effective, as it can output the same amount of electromagnetic radiation emission per unit area with a lower pulse energy and over a wider area in the focal plane than existing circular profile beams. Providing an elliptical profile by expanding the width rather than the height of the kill beam provides an increased kill width or effective area at the same pulse energy level of the laser module compared to a circular kill beam of the same area.

[0218] Referring next to FIG. 24A, the figure illustrates, in μm, for a particular beam shape according to an embodiment of the present invention, for a beam increasing in beam width, how much the pulse energy increases in μJ. The kill width, i.e., the effective area, at the focal plane where there is sufficient electromagnetic radiation to inactivate or destroy particles in the extended or elliptical beam shape of the present invention, increases as the laser pulse energy increases, but is higher at any given pulse energy level compared to a standard circular beam. The extended, elliptical beam has a kill width that is larger in μm than a standard circular beam where there is sufficient electromagnetic radiation at the focal plane to completely inactivate the particles. In the exemplary embodiment provided in the figure in FIG. 24A, this difference between the extended beam and the standard beam is approximately 2.5 μm at a lower pulse energy, e.g., 1.8 μJ, but can be up to about 7.5 μm at a higher pulse energy, e.g., 3 μJ. The extended beam has a kill beam width that is larger in μm than a standard beam where there is sufficient electromagnetic radiation at the focal plane to partially inactivate or slice the particles. This difference between the extended beam and the standard beam is approximately 2.5 μm at a lower pulse energy, e.g., 1.8 μJ, but can be up to about 5 μm at a higher pulse energy, e.g., 3 μJ. Similar results are achieved at lower pulse energies, e.g., 1 μJ, and higher pulse energies, e.g., 5 μJ.

[0219] When the beam width of an elliptical kill beam, such as the beam shown in FIG. 23, is increased up to 30 μm, the difference between the elliptical kill beam and the standard circular kill beam can be 1.33 + / - 0.06% in the sliced particle width and 1.29 + / - 0.15% in the killed or completely inactivated particle width. This difference exists at laser radiation pulse energies from 1 μJ to 5 μJ and may become even larger as the laser radiation pulse energy increases.

[0220] Without wishing to limit the present invention to a particular theory or mechanism, the optical and optomechanical systems of the present invention increase the kill width of the beam emitted by the kill laser and provide similar kill performance or effect with respect to the kill volume in the X dimension or the Z dimension (where the Y dimension is the dimension of the flow direction in the flow cytometer system).

[0221] For example, referring to the chart shown in FIG. 24B, in one non-limiting exemplary embodiment, a 30 μm wide kill beam having an elliptical profile at the focal plane was used at a radiant energy of 2.4 μJ. However, the emitted pulse energy may be 1 - 5 μJ per pulse, the pulse duration may be 1 - 500 nanoseconds, or in one embodiment may be 5 - 30 nanoseconds. In this exemplary embodiment, the 30 μm wide kill beam showed improved kill counts as a percentage of particles excised or photodamaged by the electromagnetic radiation of the kill laser. The 30 μm wide kill beam having an elliptical profile at the focal plane provided different improvements at relative positions of + / - 10 μm from the ideal or determined kill or action location. A typical kill beam or kill location less than 18 μm wide having a circular profile at the focal plane was unable to effectively kill, inactivate, significantly photodamage, or excise particles beyond + / - 10 μm from the ideal or determined kill location, whereas the improved 30 μm wide kill beam having an elliptical profile at the focal plane achieved inactivation, killing, destruction, or significant photodamage or excision of at least 90% of the particles subjected to the kill laser. Comparing particles such as sperm cells killed, destroyed, or sliced by a narrower circular beam, the improvement with the wider elliptical beam is readily apparent with respect to the killing effect or slicing.

[0222] Without wishing to limit the invention to a particular embodiment, the broader kill beam described above may be used with existing flow cytometry systems and in existing flow cytometry optical configurations. However, the broader kill beam described herein provides additional advantages when used with a flow cytometry system having a detection laser and a kill laser or inactivation laser positioned on a common side relative to a major or principal surface of a flow cytometry device such as a microfluidic chip. These advantages include additional adjustment and configuration capabilities, and the ease of doing so with respect to beam size, shape, and positioning, such as provided by having optical components or optical paths positioned on a common side of the system.

[0223] Referring now to FIG. 25A, there is provided a block diagram of an optical-mechanical system 3400 for positioning a set of laser assemblies 3410 comprising a detection laser assembly 3430 and a kill laser assembly 3420 on the same side of a main (e.g., top or bottom) surface of a microfluidic chip, and a detection assembly 3450 on the opposite side. The optical-mechanical system 3400 is used to detect certain characteristics of particles and to inactivate, kill, photodamage, or excise particles such as sperm cells passing through an action or interrogation region 3404 in a flow path 3402 of a flow cytometer system such as within a channel of a microfluidic chip. The optical-mechanical system 3400 may further be used to detect the result (e.g., success, failure, partial success) of a kill action or photodamage excision event. The kill laser assembly 3420 comprises a kill laser 3422 which may be a pulsed laser module having a pulse duration of 1 to 500 nanoseconds, a set of one or more beam expander optics 3424, and a cylindrical lens pair 3426. The profile or shape and configuration of the lenses of the beam expander 3424 and the cylindrical lens pair 3426 achieve compensation for misalignment up to + / -5°. This enables a simpler alignment and calibration process and further provides some margin against operator error in alignment and calibration. The detection laser assembly 3430 comprises a detection laser 3432 which may be a coherent continuous wave (“CW”) laser, a quasi-CW laser, or a pulsed laser, a set of one or more beam expander optics 3434, and a cylindrical lens pair 3436. A mirror 3428 directs the electromagnetic radiation emission (“beam”) of the kill laser 3422 towards a beam combiner 3412 to combine the beam emission of the kill laser 3422 with the beam emission of the detection laser 3432 and further direct both beam emissions towards a focal plane 3452 within the action or interrogation region 3404 of the flow path 3402. Here, “combining” does not refer only to the physical combination or overlap of beam emissions, but may also refer to the reorientation of one or more of the beams to be positioned in close physical proximity such that they are directed in the same direction and the beam emissions do not coincide.

[0224] On the detector side of the optomechanical system 3400, the detection assembly 3450 includes one or more filters such as a short or long pass filter 3454, a mirror 3456, a near ultraviolet ("NUV") objective lens 3458, a mirror 3460, a mirror 3462, a kill sensor 3464, a camera 3468, and a detection sensor 3470. The kill sensor 3464 and the detection sensor 3470 may be avalanche photodiode type sensors, and the camera 3468 may be a charge coupled device ("CCD") camera. In another embodiment, the kill sensor 3464 and the detection sensor 3470 may each include a photomultiplier tube ("PMT"), a complementary metal oxide semiconductor ("CMOS") sensor, or an electron multiplying charge coupled device ("EMCCD") type sensor. In another embodiment, the camera 3468 may include a complementary metal oxide semiconductor ("CMOS") or an electron multiplying charge coupled device ("EMCCD") type sensor.

[0225] Without wishing to limit the present invention to a particular use or example, the systems, apparatuses, and methods of the present invention, e.g., the optomechanical system 3400, may be used with a particle processing system such as a semen sorting system. In one example, the semen sorting system may include a flow cytometer device or a microfluidic chip or device having a flow chamber configured to direct a fluid stream containing sample particles through a particle interrogation location (e.g., the actuation or interrogation location 3404). The sample particles may be cells such as sperm that have been stained or otherwise processed prior to entering the flow cytometer device or microfluidic device. The flow cytometer device or microfluidic device may further include a laser (e.g., the detection laser assembly 3430) configured to emit electromagnetic radiation along a beam path to the particle interrogation location, and a detector (e.g., the detection assembly 3450) configured to detect radiation from the particles. After detection, the particles may be sorted or inactivated, e.g., by ablation with electromagnetic radiation (e.g., by the kill laser assembly 3420), before or after exiting the flow cytometer device or microfluidic device. An additional detector may be used to determine, e.g., to determine or detect the total or fractional amount of electromagnetic radiation to which individual particles have been exposed, and to determine whether individual particles have been sufficiently inactivated or damaged. The particles may be X chromosome or Y chromosome sorted or sex-discriminated semen, e.g., X chromosome sex-discriminated semen, and may then be collected in one or more collection containers.

[0226] The optomechanical system 3400 may also be used with, or in, a method for analyzing particles such as sperm cells included in a fluid flow as the particles flow through an interrogation location (e.g., the actuation or interrogation location 3404) within a flow cytometer device or a microfluidic chip or device. Such a method includes illuminating the fluid flow and the particles included therein using electromagnetic radiation emitted from a laser (e.g., the detection laser assembly 3430). A detector (e.g., the detection assembly 3450) detects the electromagnetic radiation emitted from the interrogation location by the particles, and a processor determines the characteristics of the particles in the fluid flow based in part on the signal from the detector. After detection, the particles may be sorted and removed, or inactivated, by a physical sorting and removal process, or by ablation or photodamage by electromagnetic radiation (e.g., by the kill laser assembly 3420), before or after exiting the flow cytometer device or microfluidic device. The particles may be X-chromosome or Y-chromosome sorted, or sex-discriminated, semen, e.g., sex-discriminated semen having an X chromosome or a Y chromosome, and are then collected in one or more collection containers.

[0227] The optomechanical system 3400 may also be used with, or in, a method for assessing the amount of DNA in the nucleus of sperm cells using a flow cytometry device or a microfluidic chip or device. The method may include staining the DNA in the nucleus of the sperm cells and then irradiating the stained DNA in the nucleus of the sperm cells with an electromagnetic radiation device such as a laser (e.g., the detection laser assembly 3430) when the sperm cells pass through the laser within a corresponding region (e.g., the action or interrogation location 3404) of the flow cytometry device or the microfluidic device. A detector (e.g., the detection assembly 3450), such as an avalanche photodiode, detects the fluorescent light emitted from the irradiated and stained DNA in the nucleus of the sperm cells at a detection or interrogation location (e.g., the action or interrogation 3404) within the flow cytometry device or the microfluidic device. The method may further include first determining the sex of the sperm cells using the detected amount of DNA in the nucleus of the sperm cells and then differentiating a plurality of sperm cells based on the sex determination to distinguish sperm cells having an X chromosome from sperm cells having a Y chromosome. The characteristics of the sperm cells may include the corresponding amount of DNA in the nucleus of the sperm cells. The method may further include inactivating a given sperm cell based on the determined amount of DNA in the nucleus of the given sperm cell. The inactivating may include damaging or excising the given sperm cell by irradiation with electromagnetic radiation (e.g., by the kill laser assembly 3420). The method may further include forming a plurality of droplets each having one of the captured sperm cells, separately charging each of the droplets based on the sex differentiation characteristics of the sperm cells captured in the droplets, deflecting one of the droplets, and separately collecting each of the droplets based on the sex differentiation characteristics of the sperm cells captured in the droplets. The method may further include collecting the sperm cells after determination, sorting, or inactivation when the sperm cells exit the microfluidic device.The particles may be semen that has been sorted for the X chromosome or the Y chromosome, or sex-discriminated, for example, semen that is sex-discriminated and has the X chromosome or the Y chromosome, and is collected in one or more collection containers.

[0228] Without wishing to limit the present invention to a particular use or example, the optomechanical system 3400 interrogates, detects, and provides improvements to existing systems and methods for inactivating particles by positioning a kill laser assembly 3420 and a detection laser assembly 3430 of a set of laser assemblies 3410 on a common side of a flow cytometry device such as a microfluidic system chip. For example, the kill laser assembly 3420 and the detection laser assembly 3430 may be positioned or arranged on the same, or a common, side with respect to the top or front surface of the microfluidic system chip, or with respect to the bottom or back surface of the microfluidic system chip, and the detection assembly 3450 may be positioned or arranged on the opposite side with respect to the laser assembly 3410. This configuration provides advantages over configurations where the kill laser assembly and the detection laser assembly are positioned on opposite sides of the flow surface of the flow cytometry device.

[0229] For example, in a configuration that includes positioning the kill laser assembly and the detection laser assembly on opposite sides of the flow surface of the flow cytometry device, the radiation beam of the kill laser assembly may need to pass through the objective lens of the detection assembly. This can cause damage or degradation of one or more optical components of the detection assembly. By positioning the kill laser assembly 3420 on the same side as the detection laser assembly 3430, the radiation beam of the kill laser assembly 3420 does not need to first pass through any optical components of the detection assembly 3450 that could cause unnecessary or undesirable degradation, damage, or wear.

[0230] Additionally, positioning the kill laser assembly 3420 and the detection laser assembly 3430 on a common side of the flow cytometer simplifies the setup, alignment, and assembly of the optical and mechanical elements of the system 3400. This provides excellent stability of the system 3400 and further enables improvement of detection resolution and reduction of the signal-to-noise ratio. Positioning the kill laser assembly 3420 and the detection laser assembly 3430 in common provides an even better ability to eliminate unwanted wavelengths on the detection assembly 3450 side of the system 3400. Further, when the kill laser assembly 3420 and the detection laser assembly 3430 are positioned in common, the set of laser assemblies 3410 may share optical components that reduce the complexity of the system and provide inherent compensation for physical vibration or movement of the system when the assemblies share a subset of components within the optical path. If the kill laser assembly 3420 and the detection laser assembly 3430 do not share optical components and are not positioned in common, vibrations of the system 3400 may cause the radiation beams from both assemblies 3420 and 3430 to move independently or vibrate, causing problems with the accuracy and effectiveness of detection and inactivation. However, when positioned in common, vibrations cause the radiation beams of the kill laser assembly 3420 and the detection laser assembly 3430 to vibrate in common, reducing losses in the effectiveness and accuracy of detection and inactivation.

[0231] Continuing to refer to the optical-mechanical system 3400 in FIG. 25A, not only the configuration of the kill laser assembly 3420 provides the advantages described above, but it may also be used with a broader kill beam as shown and described in FIGS. 1-3. For example, the electromagnetic radiation emission or laser beam is emitted from a laser head that can be a kill laser module 3422 (e.g., a pulsed laser module having a pulse duration of 1 to 500 nanoseconds and a pulse energy of 1 to 5 μJ). The beam passes through a series of optical components, namely a beam expander 3424, to shape the beam into a desired shape and size, which is typically circular. The beam then passes through a pair of cylindrical lenses 3426 that continue to shape the beam from a circular profile to an elliptical profile. The beam is then directed by a mirror 3428 towards the beam combiner 3412, where the beams from the kill laser module 3422 and the detection laser module 3432 are combined and projected towards the focal plane.

[0232] Additionally, the positions of the kill laser assembly 3420 and the detection laser assembly 3430 may be exchanged or reversed, such that the beam or radiation from the kill laser assembly 3420 passes directly through the polarization beam splitter or combiner 3412, and the beam or radiation from the detection laser assembly 3430 is redirected 90 degrees towards the focal plane. The radiation from the kill laser assembly 3420 and the detection laser assembly 3430 may or may not be fully combined, i.e., the radiation may not fully coincide at the focal plane 3452, depending on the flow cytometer and particle processing operations in which the assemblies are used.

[0233] Referring now to FIG. 25B, there is provided a block diagram of an optical-mechanical system 3500 for positioning a set of laser assemblies 3510 including a detection laser assembly 3530 and a kill laser assembly 3520 on the same side of a main (e.g., top or bottom) surface of a microfluidic chip and on the opposite side of the detection assembly 3550, according to an embodiment of the present invention. The optical-mechanical system 3500 is used to detect specific properties of particles and to inactivate, kill, photodamage, or excise particles such as sperm cells passing through an action or interrogation region 3504 in a flow path 3502 of a flow cytometer system, such as within a channel of a microfluidic chip. The optical-mechanical system 3500 may further be used to detect the result (e.g., success, failure, partial success) of a kill action or photodamage excision event. The kill laser assembly 3520 includes a kill laser 3522, which may be a pulsed laser module having a pulse duration of 1 to 500 nanoseconds, a set of one or more beam expander optics 3524, a wave plate 3526, and an aspherical lens 3528. The detection laser assembly 3530 includes a detection laser 3532, which may be a coherent continuous wave ("CW") laser, a quasi-CW laser, or a pulsed laser, and a cylindrical lens 3534. A mirror 3512 directs the electromagnetic radiation emission ("beam") of the detection laser 3532 toward a beam combiner 3514, which is a polarization beam splitter, to combine the beam emission of the kill laser 3522 with the beam emission of the detection laser 3532 and further direct both beam emissions toward a focal plane 3552 within the action or interrogation region 3504 of the flow path 3502. Here, "combining" does not refer only to the physical combination or overlap of beam emissions, but may also refer to the reorientation of one or more of the beams to be directed in the same direction and positioned in close physical proximity.

[0234] On the detector side of the optomechanical system 3500, the detection assembly 3550 includes filters such as a high-pass filter 3554, a NUV objective lens 3556, a dichroic mirror 3558, a kill sensor 3564, a camera 3560, a beam splitter 3562, and a detection sensor 3566. The kill sensor 3564 and the detection sensor 3566 may be avalanche photodiode type sensors, and the camera 3560 may be a CCD camera. In another embodiment, the kill sensor 3564 and the detection sensor 3566 may each include a PMT, a CMOS sensor, or an EMCCD type sensor. In another embodiment, the camera 3560 may include a CMOS or an EMCCD type sensor.

[0235] Without wishing to limit the present invention to a particular application or example, the systems, devices, and methods of the present invention, such as the optomechanical system 3500, may be used with a particle processing system, such as the semen sorting system described above with respect to the system 3400 shown in FIG. 25A.

[0236] The optomechanical system 3500 may also be used with, or in, a method for analyzing particles, such as sperm cells, contained in a fluid flow as the particles flow through an interrogation location (e.g., an actuation or interrogation location 3504) within a flow cytometer device or within a microfluidic chip or device, such as the method described above with respect to the system 3400 shown in FIG. 25A.

[0237] The optomechanical system 3500 may also be used with, or in, a method for evaluating the amount of DNA in the nucleus of a sperm cell using a flow cytometer device or a microfluidic chip or device, such as the method described above with respect to the system 3400 shown in FIG. 25A.

[0238] Without wishing to limit the invention to a particular use or example, the optomechanical system 3500 provides improvements to existing systems and methods for interrogating, detecting, and inactivating particles by positioning a set 3510 of laser assemblies, a kill laser assembly 3520 and a detection laser assembly 3530, on a common side of a flow cytometry device, such as a microfluidic system chip. For example, the kill laser assembly 3520 and the detection laser assembly 3530 may be positioned or arranged on the same, or a common side, with respect to the top or front face of the microfluidic system chip, or with respect to the bottom or back face of the microfluidic system chip, and the detection assembly 3550 may be positioned or arranged on the opposite side with respect to the laser assembly 3510. This configuration provides advantages over configurations where the kill laser assembly and the detection laser assembly are positioned on opposite sides of the flow surface of the flow cytometry device.

[0239] For example, in a configuration that includes positioning the kill laser assembly and the detection laser assembly on opposite sides of the flow surface of the flow cytometry device, the emission beam of the kill laser assembly may need to pass through an objective lens. This can cause damage or degradation to one or more optical components of the detection assembly. By positioning the kill laser assembly 3520 on the same side as the detection laser assembly 3530, the emission beam of the kill laser assembly 3520 does not need to first pass through any optical components of the detection assembly 3550 that could cause unnecessary or undesirable degradation, damage, or wear.

[0240] Additionally, positioning the kill laser assembly 3520 and the detection laser assembly 3530 on a common side of the flow cytometer simplifies the setup, alignment, and assembly of the optical and mechanical elements of the system 3500. This provides excellent stability of the system 3500 and further provides improved detection resolution and reduced signal-to-noise ratio. Positioning the kill laser assembly 3520 and the detection laser assembly 3530 in common provides an even better ability to eliminate unwanted wavelengths on the detection assembly 3550 side of the system 3500. Further, when the kill laser assembly 3520 and the detection laser assembly 3530 are positioned in common, the set of laser assemblies 3510 may share optical components that reduce the complexity of the system and provide inherent compensation for physical vibration or movement of the system. If the kill laser assembly 3520 and the detection laser assembly 3530 do not share optical components and are not positioned in common, vibrations of the system 3500 may cause the radiation beams from both assemblies 3520 and 3530 to move independently or vibrate, causing problems with the accuracy and effectiveness of detection and inactivation. However, when positioned in common, vibrations cause the radiation beams of the kill laser assembly 3520 and the detection laser assembly 3530 to vibrate in common, reducing losses in the effectiveness and accuracy of detection and inactivation.

[0241] Continuing to refer to the optical-mechanical system 3500 in FIG. 25B, not only the configuration of the kill laser assembly 3520 provides the advantages described above, but it may also be used with a broader kill beam as shown and described in FIGS. 1-3. For example, the electromagnetic radiation emission or laser beam is emitted from a laser head that can be a kill laser module 3522 (e.g., a pulsed laser module having a pulse duration of 1 to 500 nanoseconds). The beam passes through a series of optical components, namely a beam expander 3524, to shape the beam into a desired shape and size, which is typically circular. The beam then passes through a wave plate 3526 that changes the polarization state of the beam as the beam passes through the plate, and then through an aspherical lens 3528 that changes the profile from circular to elliptical. These components may be adjusted to independently adjust the height and width of the elliptical profile of the beam at the focal plane 3552. The beam is then directed to pass through a beam combiner 3514, where the beams from the kill laser module 3522 and the detection laser module 3532 are combined and projected towards the focal plane. The emissions from the kill laser assembly 3520 and the detection laser assembly 3530 may or may not be fully combined, i.e., the emissions may not be perfectly coincident at the focal plane 3552 depending on the flow cytometer and particle processing operations in which the assemblies are used.

[0242] Optical paths for the detection laser assemblies (e.g., detection laser assemblies 3430 and 3530) and the kill laser assemblies (e.g., kill laser assemblies 3420 and 3520) may be used with systems 3500 and 3400 and are provided in the exemplary embodiments shown in FIGS. 26A and 26B, respectively. The laser-based particle detection and inactivation system in the exemplary embodiment includes a detection laser assembly having an optical path 3600 shown in FIG. 26A and a kill laser assembly having an optical path 3700 shown in FIG. 26B. The optical path 3600 for the detection laser includes a detection laser module 3602, a first harmonic separator 3604, a second harmonic separator 3606, an F150 cylindrical lens 3608, an F25 cylindrical lens 3610, a polarization beam splitter 3612, and ends at a focal plane or a microfluidic system chip.

[0243] The optical path 3700 for the kill laser assembly includes a kill laser module 3702, a first harmonic separator 3704, an F50 cylindrical lens 3706, an F25 cylindrical lens 3708, a wave plate 3710, a second harmonic separator 3712, an F-25 cylindrical lens 3714, an F150 cylindrical lens 3716, a third harmonic separator 3718, an aspherical lens 3720, a polarization beam splitter 3722, and ends at a focal plane or a microfluidic system chip.

[0244] Without wishing to limit the present invention to a particular use or example, the improved systems and optical paths of the present invention, the optomechanical system 3400 shown in FIG. 25A, the optomechanical system 3500 shown in FIG. 25B, and the optical paths 3600 and 3700 shown in FIGS. 26A and 26B respectively, improve existing systems by incorporating a spherical lens into at least the kill laser beam path and through the use of a polarization beam splitter for combining the emissions from the kill laser module and the detection laser module. Specifically, using an aspherical lens in a configuration such as that shown in the exemplary embodiment, beam emission from a higher pulse energy laser is shaped without risk of damage to the optical elements including the aspherical lens. The aspherical lens further enables precise adjustment and / or calibration of the beam emission to obtain an optimal beam shape and size at the focus. The use of such optical components further enables independent adjustment of the height and width of the beam waist for beam emission at the focal plane. Additionally, in the exemplary embodiment, a polarization beam splitter may be used to combine the beam emissions from the kill laser module and the detection laser module, in which case both beam emissions are of a common wavelength or close to a common wavelength and the emissions of both modules are polarized. Other methods for combining beam emissions, such as dichroic mirrors, may not be suitable or optimal in a flow cytometer system having a kill laser module and a detection laser module positioned on a common side of the flow cytometer and on opposite sides of the detection module or assembly. This type of exemplary configuration achieves superior results compared to other beam combining systems or methods through the unconventional use of a polarization beam splitter.

[0245] Referring now to FIG. 28, a diagram of the radiation from the detection spot 4001 and the kill spot 4003 is provided. The detected radiation 4014 is generated at the detection spot 4001 at the focal plane 4002 or interrogation location, and the kill radiation 4012 is generated at the kill spot 4003 of the focal plane 4002. Optical elements of the optical path, such as those shown in FIG. 27, shape and direct both the detected radiation 4014 and the kill radiation 4012 so that both can be separately analyzed, detected, and / or captured by one or more sensors or devices, such as the system 4400 provided in FIG. 27.

[0246] Referring next to FIG. 27, a diagram of the optical, opto-mechanical, and electrical elements of a detection assembly 4400 according to an embodiment of the present invention is provided. The detection assembly 4400 may include a filter 4404, which may be a high-pass filter, for filtering, splitting, and / or redirecting any electromagnetic radiation emitted from either particle detection laser excitation (detection radiation 4414) or particle kill-laser excitation (kill radiation 4412); an objective lens 4406, which may be a NUV 20x objective lens; a tube lens 4408, which may be a 100 mm or 200 mm tube lens; and one or more filters or splitters, such as dichroic mirror 4420 and splitter 4422. The detection radiation 4414 is generated at a detection spot at the focal plane 4402 or interrogation location, and the kill radiation 4412 is generated at a kill spot at the focal plane 4402. At least the filter 4404, objective lens 4406, and tube lens 4408 shape and direct both radiations so that both the detection radiation 4414 and the kill radiation 4412 can be separately analyzed, detected, and / or captured by one or more sensors or devices, such as a CCD camera 4430, a kill avalanche photodiode (“APD”) or kill detector 4432, and detector 4434. The CCD camera 4430 is used to capture images of the radiations 4412 and 4414 and may be used in determining the type or quality of the radiation, such as determining the amount of fluorescence emission of particles caused by the electromagnetic radiation of the kill laser or detection laser. The dichroic mirror 4420 is used to reflect a portion or a very small amount of the radiations 4412 and 4414 towards the CCD camera 4430 and to allow the remainder of the radiation to pass towards the kill detector 4432 and detector 4434. The kill detector 4432 is used to detect whether a kill event by the kill laser for inactivating the particles has been successful. The detector 4434 is used to determine the type and quality of the particle radiation, such as within the particle radiation 4414, and to determine the quality or characteristics of the particles, such as the DNA content of sperm cells, based on the determined level of fluorescence emission of the cells.

[0247] "Example 1"

[0248] Although it is not desirable to limit the present invention to a specific use or example, the following exemplary examples will be described.

[0249] The flow cytometry particle sorting system comprises a detection laser assembly, a microfluidic chip, a detection sensor, a set of signal processing electronics, a graphical user interface, a CCD camera, a kill laser module, and a kill sensor. The detection laser assembly is configured to excite a dye such as a hext dye within particles that may be individual sperm cells. The beam from the detection laser is focused to a specified size such as an ellipse having a fast axis of 3.5 to 20 μm and a slow axis of 30 to 150 μm. The microfluidic chip receives a particle sample such as an ejaculate sample containing sperm and uses a sheath fluid within one or more microchannels in the chip to focus or orient the particles. The detection sensor collects the radiation at the detection laser location. The signal processing electronics may include digital electronic components including a processor and a memory, or analog electronic components, or a combination of digital and analog processing components. The signal processing electronics receives one or more signals from one or more sensors or cameras, converts or interprets the signals, and makes a determination based on the input signals. This determination may be a determination of the sex or gender of sperm cells based on fluorescence detected based on the amount of DNA in the cells. The graphical user interface ("GUI") displays information regarding the status of the system including processing rate, laser pulse energy, kill rate, detection rate, fluid level, sample collection level, and further provides one or more user interface elements for configuring system elements such as laser pulse energy, flow rate, temperature, pressure, and distortion of the desired sample. The CCD camera captures an image of one or both of the kill location and the detection location and provides feedback via the GUI for user alignment of the optical and optomechanical elements of the system. The kill laser kills or inactivates unwanted particles. The kill laser radiation is focused to a small location or spot in order to effectively slice, excise, photodamage, or otherwise kill particles such as sperm cells. The focused laser radiation may have an elliptical profile shape having a fast axis of 3.5 to 5 μm and a slow axis of 5 to 35 μm.

[0250] The detection laser assembly includes a CW laser module, a laser shutter, a set of one or more harmonic separators, a horizontally oriented F150 cylindrical lens, a vertically oriented F25 cylindrical lens, and a polarization beam splitter. The CW laser module emits a CW laser beam that is used to cause fluorescence emission in the particles. The laser shutter prevents the laser from reaching the user's access point when the safety switch is activated. The harmonic separator reflects 355 nm light while transmitting light of other wavelengths and is used to remove the 352 nm and 1064 nm carrier wavelengths from the laser beam. The F150 cylindrical lens shapes the horizontal dimension of the detection laser at the chip or focal plane and is disposed on an adjustable base or stage that can adjust the beam position horizontally. The F25 cylindrical lens shapes the vertical dimension of the detection laser at the chip or focal plane and is disposed on a base or stage that can adjust the beam position vertically. The polarization beam splitter reflects S polarization while transmitting P polarized laser light, enabling the detection laser beam to pass through the beam splitter onto the chip or focal plane.

[0251] The kill laser assembly comprises a kill laser module, a set of one or more UV mirrors, a vertically oriented F-25 cylindrical lens, a vertically oriented F+150 cylindrical lens, an F25 aspherical lens, and a polarization beam splitter. The kill laser module is a pulsed laser module that emits a pulsed laser beam at 1 to 5 μJ. The set of one or more UV lenses are each mounted on a tilting / tipping mount for reflecting the laser beam by 90 degrees and steering or directing the kill laser beam. The F-25 cylindrical lens, in combination with the F+150 cylindrical lens, expands the kill laser beam vertically. By adjusting the separation of the F-25 cylindrical lens and the F+150 cylindrical lens, the height of the kill laser at the chip or focal plane can be controlled. The F-25 and F+150 cylindrical lenses can expand the kill laser beam from 1 mm to 6 mm. The F25 aspherical lens may be used to focus the kill laser beam on all axes into a small elliptical spot at the focal plane in the microfluidic system chip. The aspherical lens reduces spherical aberration compared to standard or common spherical lenses.

[0252] The cell detection assembly includes an objective lens, a 390 long-pass filter, a first F100 spherical tube lens, a 90:10 beam splitter, a detection sensor, a kill sensor, a second F100 spherical tube lens, and a CCD camera. The objective lens collects dye emissions from both the detection location and the kill location at the focal plane of a fluid path (e.g., a microchannel of a microfluidic chip) within a flow cytometry device, and further collects a portion of the laser wavelength from the kill laser and the detection laser to measure the size of the laser spot. The 390 long-pass filter transmits wavelengths longer than 390 nm, reflects wavelengths less than 390 nm, reflects a portion of the light towards the CCD camera, and the remaining light can pass through to the kill sensor and the detection sensor. The 90:10 beam splitter transmits 90% of all light and reflects 10%, transmitting 90% of the light passing through the 390 long-pass filter to the detection sensor and reflecting 10% of the light to the kill sensor. The detection sensor is an avalanche photodiode that collects the light emitted at the detection location. The kill sensor is an avalanche photodiode that collects the light emitted at the kill location. The first F100 spherical tube lens focuses the light collected from the objective lens onto the detection sensor and the kill sensor, and the second F100 spherical tube lens focuses a portion of the light collected from the objective lens onto the CCD camera as reflected by the 390 long-pass filter. The CCD camera images the light collected by the objective lens and is a source of user feedback for system alignment and measurement of the size of the laser spot.

[0253] "Example 2"

[0254] Although it is not desirable to limit the present invention to a specific application or example, the following exemplary examples are described.

[0255] Typically, pulsed lasers are used with fluorescent dyes in particles in a flow cytometry system. For example, existing systems for sex discrimination of semen in sperm use a pulsed laser to fluoresce a Hoechst dye such as Hoechst 33324 in sperm cells to determine the amount, and thus the type, of DNA in the sperm cells in the sex discrimination process. However, the use of a CW laser offers advantages in terms of efficiency, calibration, and adjustment compared to conventional pulsed lasers. The use of the CW laser in the present invention provides the above improvements over existing pulsed detection lasers and detection laser assemblies while maintaining the same fertility or viability of sperm cells sorted in this manner within a statistically significant error range. The fertilization rate as a measure of the fertility of cells irradiated by the CW laser was at least as good, if not better in some situations, than that of cells irradiated by the pulsed laser. Specifically, the difference in fertility was about 1%, within the range of measurement error. Therefore, the use of the CW laser in particle detection has no measurable effect on the viability or fertility of sperm cells treated using the CW laser compared to the control.

[0256] "Example 3"

[0257] Although the present invention is not to be limited to a particular use or example, the following exemplary examples are described.

[0258] A laser-based particle detection and inactivation system comprises a kill laser assembly and a detection laser assembly. The kill laser assembly may operate at a power of 350 mW to 2 W. The power of the detection laser assembly may be set from 10 mW to 50 mW. The power output by the detection laser assembly may incur a power loss of about 10% and a measured power drift of less than + / - 1% over 24 hours. Accurate and stable detection laser power may be achieved by increasing the laser power in the first minute of operation and then decreasing the laser power to provide a stable detection power at a temperature rise of about 4 degrees Celsius.

[0259] Referring now to FIGS. 29A and 29B, there are shown respective diagrams of a representative graphical user interface output for measuring the size and intensity of the detection laser beam (FIG. 29A) and the size and intensity of the kill laser beam (FIG. 29B) at the focal plane. The outputs shown in FIGS. 29A and 29B may be achieved using the laser module power settings described in Example 3 above in this specification. The major axis of the ellipse of the detection laser is between 95 and 110 μm, and the minor axis of the ellipse is between 12 and 15 μm. Specifically, as shown in FIG. 29A, the major axis or slow axis is 104.2 μm, and the minor axis or fast axis is 11.2 μm. The major axis of the ellipse of the kill laser is between 20 and 25 μm, and the minor axis of the ellipse is between 2 and 4 μm. Specifically, as shown in FIG. 29B, the major axis or slow axis is 20.9 μm, and the minor axis or fast axis is 3.45 μm. However, the placement and positioning of the optical elements may be adjusted or calibrated, and the pulse energy for the laser module may be adjusted to shape the beam emission as desired.

[0260] "Example 4"

[0261] Although it is not desired to limit the present invention to a specific application or example, the following exemplary examples will be described.

[0262] The laser-based particle detection and inactivation system includes a kill laser assembly and a detection laser assembly. The detection laser assembly includes an annular light-emitting diode ("LED") and a power sensor. The annular LED has an inner diameter of 6 mm and is disposed between a set of cylindrical lenses in the optical path of the detection laser. The power sensor has a TO-5 packaging and is disposed on a printed circuit board. The power sensor is disposed within or adjacent to the optical path of the detection laser by means of a polarizing beam splitter or the like to detect the power of the detection laser beam. This is used to continuously monitor the output power and provide feedback to the operator. The detected power may also be used in a feedback loop to automatically or semi-automatically adjust the output power.

[0263] "Example 5"

[0264] The following is a non - limiting example for determining the velocity of particles in the flow of samples within a microfluidic system chip and can be used for kill - trigger delay timing, flow rate adjustment, etc.

[0265] In cell velocity calibration, the cell velocity is calculated as follows. Velocity = (Detection Spot Y - Kill Spot Y) / Trigger Delay

[0266] When the current velocity deviates significantly from the target velocity, the following equation is used to calculate the new flow rate. (Flow 1 / Velocity 1)=(Flow 2 / Velocity 2) In this equation, Flow 1 and Velocity 1 are the current values, Velocity 2 is the target velocity, and Flow 2 is the new flow rate.

[0267] The cell rate (cells / second) equal to events / second is a fluid parameter that is desirably maintained constant. The flow cytometer tracks the number of cells or events reported by the detector in the last 1 second. Since this fluid parameter is controlled via a pressure regulator in the fluidic system module, if the cells / second is too low, the pressure rises, and if the cells / second is too high, the pressure drops. Since the pressure is applied to the sample, the higher the pressure, the more of the sample is pushed through the flow cytometer. The sample is mixed with sheath fluid, and the sheath fluid is kept at a constant flow rate by a PID controller operating in the fluidic system module. In some embodiments, the sheath flow is controlled by opening and closing valves such as a pressure regulator.

[0268] Alignment Procedure

[0269] Existing systems require an operator to align a kill laser with the flow of cell cores in order to effectively kill target cells. The operator uses a two-camera system, where one camera monitors the cell location and another camera monitors the kill laser, to align the flow of the core in three dimensions with the laser. Each spot is given the location of the center of gravity in the image. The positional relationship between spots is then maintained during acquisition. Alignment is achieved using images, a two-axis stage, and a time delay. The two-axis stage enables alignment at the center of the laser, and the time delay enables the laser to target cells passing through the microfluidic system channel. By focusing the flow of the core, most of the laser energy is concentrated on the cells, and a high-purity product is obtained. After alignment, the operator gates an appropriate population, and cells outside that population are targeted by the kill laser. Problems that occur during operation include laser drift and cell velocity drift, which cause misalignment of the system where the target cells are not effectively killed or are missed. The operator needs to identify and correct the misalignment.

[0270] However, the operator is unreliable when identifying and correcting misalignment at the required frequency to maintain product purity. Furthermore, the operator cannot correct for drift between cameras, i.e., both measurement devices themselves drift, and the measurement accuracy is not accurate enough to maintain alignment within an acceptable range. Automating the alignment process reduces labor costs, improves product consistency, and improves product quality by not relying on the operator.

[0271] Accordingly, in some embodiments, the present invention features automatic alignment correction by measuring misalignment and gradually correcting the stage position or delay until the misalignment is within an acceptable range.

[0272] Referring to FIG. 30, a procedure for aligning the microfluidic chip stage with the detection laser and the kill laser to achieve the desired resolution is shown. First, the microfluidic chip stage is aligned with the detection laser to achieve the desired detection resolution. Next, the position of the microfluidic chip stage is adjusted so that the kill laser is aligned with the center of the sample flow, thereby achieving the desired resolution. If the kill count is below the kill count threshold, the microfluidic chip stage is repositioned relative to the kill laser until the kill count threshold is reached or exceeded.

[0273] FIG. 31 shows a procedure for aligning the instrument by adjusting the position of the core flow relative to the kill laser and measuring the characteristics of the fluorescence generated by the kill laser and the cell core flow. During the initial alignment, the operator optimizes the position of the core flow relative to the core laser. A crosshair is placed on this spot in the 2D image space. The orange crosshair is fixed and used as the position to maintain during acquisition. The red crosshair is the measured activity value of the spot on the image. If the spot moves, the red crosshair follows, but the orange crosshair does not.

[0274] In some embodiments, left / right adjustment may be required if the core flow is shifted left or right from its original position. The distance between the crosshairs is measured, and the chip / core flow is moved left or right until the distance between the crosshairs is zero. In other embodiments, the focus may be optimized by sweeping in one direction from one extreme until the zero-slope condition is met as the focus optimization measurement criterion.

[0275] In some embodiments, delay adjustment may be required when the cell velocity or the distance between the detection laser and the kill laser changes. After lateral and focus optimization, the location of the spot on the image may be marked as the target location. The cell is fired, and the current location of the cell in the image is measured relative to the target location. After measuring the distance between the crosshairs, the delay is adjusted until the current location relative to the target location becomes zero. For example, the delay is decreased or increased accordingly until the distance between the crosshairs becomes zero.

[0276] Although it is not desirable to limit the present invention to a particular theory or mechanism, the alignment procedure has the advantage of not requiring additional hardware to measure the laser position. Further, the system is optically stable, and the laser only moves 0 - 1 μm during acquisition. Thus, it is possible to measure the location of the moving kill laser and maintain the initial alignment even after verification without the need to frequently realign to maintain product quality.

[0277] Detection of Depleted Samples

[0278] In semen processing, in order to maintain the viability of the semen, it is necessary to create several samples over the course of the operation. Thus, a volume of 3 - 5 mL is processed at a time and replaced with newly created samples when the previous sample runs out. The operator visually checks the level of the sample tube to identify when the sample is low and determine when it should be replaced. This can lead to the sample being completely depleted and a long downtime.

[0279] Therefore, the present invention solves this problem by providing an indicator when the sample is depleted before it is completely depleted, helping to reduce the burden on the operator and reduce the downtime of the equipment. The uniqueness of this solution is to monitor the output of the controller as a means of detecting known change patterns.

[0280] Referring to FIG. 32, the sample is flowed from the 5 mL container through the small-diameter pipe by applying pressure above the fluid. Since the sample in the pipe is assumed to have a uniform concentration, the rate at which cells leave the container (or cell rate) is proportional to the applied pressure. As the pressure increases, the cell rate increases. Ideally, the cell rate is kept constant to maintain a constant instrument performance. Due to local variations in concentration and downstream pressure, it is necessary to slightly adjust the pressure to maintain the cell rate at the target.

[0281] In reality, the concentration does not remain uniform throughout the duration of the sample. Sedimentation occurs, and the cells fall towards the bottom, leaving the lower part of the sample more concentrated than the upper part. As the fluid level decreases, the local concentration near the pipe outlet continuously decreases. To maintain a constant throughput, the required pressure increases. This pressure increases exponentially as the sample begins to deplete. The method of the present invention requires monitoring this change in pressure during collection. The operator is then notified when the change in pressure exceeds a threshold.

[0282] As shown in FIG. 32, the sample container contains sperm cells in a solution. The pressure regulator receives a constant pressure from the source. The pressure setpoint is received from the PC / GUI. The pressure is output to the sample pipe containing sperm cells and applied above the fluid level. The small-diameter pipe has its end provided near the bottom of the container and is placed inside the sample container. The other end of the pipe is inserted into a microfluidic system chip used to focus the cells within a flow cytometer for cell rate measurement. The cells are driven outside the container through the small-diameter pipe when pressure is applied. The flow cytometer measures the cell rate and gender. The cell rate is input into a feedback loop to maintain a constant cell rate.

[0283] The feedback controller may be a PID-based controller that uses the cell rate as input and the pressure as output. The purpose of the PID controller is to maintain a constant cell rate, which may be user-specified. When a constant cell rate deviates from the baseline, for example, when the cell rate decreases, the feedback controller increases the pressure output from the pressure regulator into the sample container to maintain a constant cell rate. When the pressure exceeds the pressure setpoint, this indicates that the amount of sample in the container is low, and the operator is notified of the low level.

[0284] The above solution has the advantage of not requiring additional hardware to determine the sample fluid level. However, in an alternative embodiment, the present invention may use one or more additional components, such as a liquid level sensor, to directly measure the level. Examples of liquid level sensors are described herein.

[0285] Particle processing

[0286] Referring to FIG. 33, a sample, which may be a semen sample containing sperm cells, enters the microfluidic system chip 850 through a sample passage, and the sheath fluid enters the chip through one or more sheath fluid passages. The sample and the sheath fluid may be pumped into the chip at a rate and pressure suitable for the individual sample by a fluid pump, a syringe pump, or other suitable pumping means.

[0287] In some embodiments, one or more electromagnetic radiation emitters are directed at or into the chip using an optical pathway. The laser detection module may be based on a continuous wave laser and may be configured to cause fluorescence emission in a dye injected into particles or components in the sample. The laser kill module may be based on a pulsed or micropulsed laser and may be configured to inactivate one or more particles or components in the sample, such as by photoablation, based on the characteristics of the sample detected by the laser detection module. The detection module collects or detects radiation from particles or components in the sample to identify one or more characteristics of the particles or components, such as the amount or type of DNA, and provides feedback to the system and / or operator. The detection module may comprise one or more of a camera, a photomultiplier tube, or an avalanche photodiode-based detector. Based on the detected characteristics, the kill laser in the emission module emits a kill laser beam to inactivate or destroy a subset of the particles or components in the sample.

[0288] The processed sample exits the chip 850 through one or more openings at the end of the chip. The automatic dump assembly 5160 includes an actuator and a mechanical diverter. The actuator may be a two-position linear actuator or other suitable mechanical or electromechanical actuation means. The actuator moves the diverter inside and outside the path of the processed sample exiting the chip to either allow the sample to pass into the collection tube 5130 or divert the sample into a waste collection container or area. The determination is based on one or more qualities of the sample, such as purity, or on operator input. The determination may also be based on whether a sample tube is present in the sample collection path.

[0289] In some embodiments, the sample mixing assembly or module 5100 comprises one or more sample collection containers or tubes 5130 held in one or more collection tube holders 5120 integrated or fixed to a rotating base 5110. The rotating base 5110 is magnetically fixed to the platform 5360 or held by bolts or threaded caps. The rotating base 5110 is rotated, vibrated, or moved by an actuator or motor 5340, which may be a stepper motor, a linear actuator, a brushless motor, or other suitable driving means capable of rotating the rotating base 5110. The rotating base 5110 rotates in an arcuate path to mix the samples collected in the sample collection tubes 5130 or to rotate the sample collection tubes on the base into the sample collection path.

[0290] Although optical means for detecting samples have been described, it is understood that other detection means may be used in the modular flow cytometry system of the present invention. Instead of or in addition to the embodiments related to the detection of samples, the samples may be detected using electrodes, cameras, magnets, electroporation, impedance, or conductivity.

[0291] Although a kill laser for damaging samples has been described, it is understood that other means for damaging cells may be used in the modular flow cytometry system of the present invention. Instead of or in addition to the embodiments related to damaging cells, the cells may be inactivated or killed using physical deformation or electrical means such as the use of electrodes or electroporation.

[0292] In other alternative embodiments, the sex-discriminated sperm cells may be separated into two or more partial subpopulations using separation means including, but not limited to, physical barriers, droplet separation, acoustic means, laser manipulation, electrodes, or combinations thereof.

[0293] Automated Mixing and Collection Module

[0294] Referring to FIG. 34 here, the present invention features a sample mixing system 5100 comprising a rotating base 5110, a plurality of collection tube holders 5120 disposed on the upper part of the rotating base, and a plurality of collection tubes 5130. Each tube is configured to fit within and be disposed in the collection tube holders of the plurality of collection tubes. While not wishing to limit the present invention to a particular theory or mechanism, the rotating base 5110 can rotate such that the collection tube holders 5120 and collection tubes 5130 disposed therein rotate or move along an arcuate path, and the contents in the collection tubes 5130 are mixed by the above movement.

[0295] In some embodiments, the rotating base 5110 can reciprocally rotate on the platform 5360 on which it is disposed. The rotating base 5110 may be operably coupled to a motor 5340 that rotates the rotating base 5110 about its central axis. The rotating base 5110 can rotate about its central axis in a first direction, a second direction, or both directions. For example, the rotating base 5110 can rotate in a first direction, a second direction, or both directions to change from an "in-use" collection tube to the next or adjacent "standby" collection tube. For switching purposes, in some embodiments, the rotating base 5110 can rotate from about 45° to 360°.

[0296] In some embodiments, the motor is a stepper motor used to automatically induce an arcuate reciprocating mixing of the sample tubes. From other mixing solutions, this is the movement of a pivot base. The motor moves along an arc, whereby the collection tubes 5130 can remain within the dropping path under the microfluidic system module. For mixing purposes, in some embodiments, the rotating base 5110 can rotate about 10° - 40°.

[0297] In other embodiments, the rotary base 5110 can move along an orbital path to induce mixing of the sample tube in a swirling motion, similar to the motion of an orbital shaker, i.e., orbital movement. Instead of, or in addition to, axial rotation, the axis of the rotary base can move along a circular path having a minor axis such that the collection tube 5130 also remains under the droplet path of the microfluidic system module while moving in a circular path.

[0298] In some preferred embodiments, the mixing technique allows for gentle stirring or mixing of the collection tube. In some embodiments, the mixing technique is performed periodically. For example, the collection tube is mixed periodically for a duration. For example, the collection tube is mixed every about 3 - 8 minutes for a duration in the range of about 10 - 120 seconds. In some embodiments, the system 5100 allows for automatic switching from an "in-use" collection tube to a "standby" collection tube while focusing on the main task of mixing the internal contents. In some embodiments, the design is optimized such that the mixing device fits within the space provided within a modular flow cytometry system.

[0299] In one embodiment, the hybrid profile for the stepper motor 5340 may include left, center return, and right movement of + / - 50 steps out of the possible 1600 steps. In a non-limiting example, for instance, a hybrid profile of + / - 50 steps for each of the left, center return, and right movements may include an arc of + / - 11.25 degrees for a total arcuate movement of 22.5 degrees. However, this range may increase or decrease based on the size of the sample output stream, the size of the sample collection container or tube, and the degree of mixing required. For example, a stronger mixing profile can be obtained using smaller steps that cause shorter and faster movements. This causes an impulse-type movement to be imparted to the agitated or mixed sample instead of a rocking movement. In another example, a hybrid profile having a movement of more than 50 steps in each direction and an arcuate movement of more than 22.5 degrees may be desired or required for any of a larger sample container, a more viscous sample, a sample requiring more thorough or uniform mixing, or a larger sample size. Additionally, other means for rotating the rotary base 5110 may be used to rotate or move the base, such as a linear actuator that rotates the base or moves the base in a reciprocating motion, a brushed or brushless electric motor that rotates or moves the base, a fluid actuator such as a pneumatic actuator that rotates or moves the base, or a piezoelectric actuator that stirs or moves the base.

[0300] The configuration of the specified mixed profile may be manually configured by the user and unique to each individual sample being processed, or the configuration of the specified mixed profile may be automatically determined based on the sample type. For example, a code read from an information storage means such as a barcode, QR code (registered trademark), or RFID tag may be read by a machine from a sample container and used to configure, search for, or otherwise identify a set of configuration information that defines the mixed profile for the sample. The information storage means may include the mixed profile information or may include a location, pointer, or address where such information can be found. The mixed profile entered into or read by the system of the present invention is used by the mixed profile to automatically mix, agitate, or stir the samples processed by the sample mixer, regardless of the presence or absence of minimal additional input or action from the user or operator.

[0301] In some embodiments, the rotation base 5110 is a disk. Referring to FIG. 35A, the rotation base 5110 may include magnets 5112 that are disposed separately from each other. The base 5122 of each collection tube holder also includes a magnet 5124. The collection tube holder 5120 can be attached to the rotation base 5110 by the magnetic attraction between one of the magnets 5124 of the collection tube holder and the magnet 5112 of the rotation base. In another embodiment, instead of or in addition to the use of the magnet 5124, threaded nuts or knobs may be used to secure the rotation base 5110 to corresponding threaded rods or bolts.

[0302] In some embodiments, the system may include about 2 to 6 collection tube holders 5120. The number and location of the rotation base magnets 5112 may be determined by the number of collection tube holders. For example, if the system includes two collection tube holders 5120, the tube holders are diametrically opposed or 180° apart. For four collection tube holders 5120, the arrangement may be 90° apart, and for six collection tube holders 5120, the arrangement may be 60° apart.

[0303] In other embodiments, the collection tube holder may include two or more arms 5126 projecting upward from the base 5122. The two or more arms 5126 are configured to hold the collection tube 5130 upright. In some embodiments, the tube holder base may be circular or square. In a non-limiting example, FIG. 34 shows a collection tube holder having two arms 5126 disposed on both sides of the tube holder base 5122 and projecting upward therefrom. Another embodiment may include four arms 5126 that are equidistant from each other, for example, 90° apart, or at the corners of a square base. In one embodiment, the arms 5126 may be connected to the edge of the tube holder base 5122, or alternatively, the arms 5126 may be attached to the upper surface of the tube holder base 5122. In another embodiment, the arms 5126 may be curved when viewed from above and straight when viewed from the side. Alternatively, the arms 5126 can be straight when viewed from above and when viewed from the side. In yet other embodiments, the collection tube holder 5120 may include a circular or square tube having a completely enclosed side surface. In another embodiment, the collection tube holder may include at least one arm or protrusion having fixing means for fixing the collection tube or container to the collection tube holder. The fixing means may be a friction fit opening, a set of magnets, an element of a magnet and a magnetic receiving material, a metal or plastic clip or fastener, a surface fastener, or a retaining ring or collar.

[0304] In a preferred embodiment, two or more arms 5126 are expandable to accommodate collection tubes 5130 of various sizes. In one embodiment, the volume capacity of the collection tube ranges from about 50 ml to about 300 ml. For example, the collection tube may have a volume of 50 ml or 250 ml. In another embodiment, the diameter of the collection tube may range from about 100 mm to about 200 mm. In another embodiment, the diameter of the collection tube may range from about 50 mm to about 150 mm. The height of the collection tube may range from about 150 mm to about 250 mm. In other embodiments, the height of the tube holder may be shorter than that of the collection tube. The height of the tube holder may range from 50 mm to 100 mm. However, any suitable collection tube or container fixed to a substrate rotatable or movable by any suitable means as described herein may be used.

[0305] In some embodiments, the sample mixing system 5100 may further include one or more Hall effect sensors. In a non-limiting example, as shown in FIG. 35B, one of the magnets 5112 in the rotating base 5110 may have its south pole oriented towards one or more Hall effect sensors, and the other magnets 5112 may be oriented such that their north poles are directed towards one or more Hall effect sensors. The north ends or poles of the magnets 5112 at the top and bottom of the base 5110 are configured to hold or retain the sample tube carrier in a fixed position on the base 5110. The sample tube carrier holds and positions the sample collection tube or container at the position of the magnet. In another example, one or more sample tube holders are integrated into the base 5110. In another example, the sample tube holder is retained by press-fitting into a corresponding opening in the base, one or more plastic clips, spring-loaded clamps, or one or more fasteners such as screws, bolts, or rivets. In one example, a magnet 5112 having a south end or pole disposed at the top of the base 5110 is used to track or return to a return position or position 0. A Hall effect sensor is used to detect or identify this magnet to determine position 0, and then the base 5110 may be rotated or turned to a collection position. An optical sensor and a corresponding mark or physical feature of the base 5110, an electrical switch, a corresponding conductive area on the base 5110 and a conductive area in a fixed position, a mechanical switch, a hard stop such as a physical lip, edge or engagement tab on the base 5110, or other sensors such as an absolute encoder may be used in place of the Hall effect sensor and corresponding magnet to track or return to a return position or position 0. Any of these sensor types may be used to identify a return position or position 0 and move the base 5110 to a designated or set position.

[0306] According to some embodiments, the sample mixing system 5100 may be implemented in a method of mixing samples. For example, the present invention can be used for the treatment of semen samples, such as the mixing of sperm cells. When used in such a sample processing system, the sample mixing system 5100 may be a component of a flow cytometry system, such as one that employs a microfluidic chip and one or more optical components in the processing of components or particles in the sample. This may be, for example, the determination of the characteristics of particles such as the amount or type of DNA in sperm cells. A sample processed by such a flow cytometry system exits the processing region and is collected in a sample collection container or tube, and may be collected with an additional medium or fluid that is required to remain in a suspension such as a suspension in which the sample is uniformly mixed.

[0307] As shown in FIGS. 36A and 36B, in one embodiment, the system 5100 can be placed under the dispensing device. The dispensing device can dispense a sample into one of the sample collection tubes 5130 as the rotation base moves along an arcuate path. In a preferred embodiment, the sample collection tube 5130 that receives the sample remains under the dispensing path of the dispensing device as the collection tube 5130 moves along the arcuate path, thereby mixing the sample in the collection tube 5130. In a non-limiting embodiment, the sample may contain sperm cells, and the collection tube 5130 can contain a medium. While not wishing to limit the present invention to a particular theory or mechanism, this method can reduce the time that cells are in an over-concentrated solution of the medium. For example, the medium may be a buffer medium that is already in the tube before the sample is added or is added together with the sample. The medium is mixed or agitated to maintain buffering and control the degree of buffering.

[0308] In some embodiments, the sample may be dropped into the collection tube. In other embodiments, the sample may be continuously dispensed into the collection tube. To force the sample through a flow cytometer device such as a microfluidic chip, a fluid pump such as a syringe pump or other suitable hydraulic or pneumatic pump may be used. The sample is inserted into the flow cytometer, processed by one or more systems or elements of the flow cytometer, and discharged from one or more outlets at the end of the flow cytometer. For example, in one embodiment, the sample and at least one sheath fluid are inserted into a microfluidic chip, and the sample is processed in an active or interrogation region of the microfluidic chip, such as a set of electromagnetic emission devices (e.g., laser modules and associated optical systems), and the sample is discharged from one or more outlets at the end of the microfluidic chip. The processed sample is then dripped, dropped, or drained into the sample collection tube through one or more channels or openings by the action of fluid pressure and / or gravity.

[0309] After mixing the sample in one collection tube 5130, e.g., the "in-use" tube, for a certain period of time, the system 5100 may automatically place another collection tube 5130, e.g., the "standby" tube, under the dispensing device so that the sample is dispensed into the above-mentioned collection tube 5130. In some embodiments, the time for mixing is about 3 to 8 minutes.

[0310] Liquid level sensor

[0311] In some embodiments, the sample mixing system includes a sensor for detecting the liquid level inside the collection tube. The sensor may be disposed on or near the surface of the collection tube. For example, the sensor is disposed 1 to 10 mm from the surface. In one embodiment, FIGS. 38A and 38B show an example of a square capacitance sensor for liquid level detection. In another embodiment, FIGS. 39A and 39B show an example of a tubular capacitance sensor for liquid level detection. In other embodiments, the liquid level sensor is an infrared sensor.

[0312] While not wishing to be bound by a particular theory or mechanism, the liquid level sensor can detect when the desired level of fluid has been reached in the collection tube. When the liquid level is full, the system 5100 automatically places another collection tube 5130 under the dispensing device and the liquid level sensor then detects the liquid level inside this collection tube. This may be done, for example, by rotating the base 110 to position another collection tube under the sample collection location or the sample dispensing path. When it is determined that the first tube is filled to the desired level or the sensor detects a particular level of fluid, for example, if the fluid is splashed or raised to a particular point by mixing the sample fluid in the first collection tube, the first collection tube may be rotated out of the sample dispensing path and the second tube rotated into the path. When there is no collection tube under the sample dispensing path or at the sample dispensing location, the sample being processed may be discarded into or diverted to the waste collection location as described below.

[0313] Automatic dump module

[0314] Referring to FIGS. 40A and 40B, in some embodiments, an automatic dump subsystem may be coupled to a sample mixing system. An "automatic dump" system includes a mechanical diverter configured to direct the path of a sample processed by a flow cytometer, such as a microfluidic chip-based system. The mechanical diverter of the automatic dump is used to automatically divert, re-direct, or change the path of the processed sample based on a determined set of parameters. For example, the mechanical diverter of the automatic dump may be configured to automatically divert or re-direct the path of the processed sample based on a determination that the sample contains components or particles (e.g., cells such as sperm cells) that were not properly processed by the flow cytometry system. In one embodiment, this may be one or more sperm cells in a semen sample that were not completely or properly inactivated by a laser-based "laser kill" system of the flow cytometer. If cells that were to be inactivated are present in the processed sample, the sample may be diverted into a waste collection location.

[0315] Without wishing to limit the invention, the mechanical diverter or automatic dump is configured to prevent flow from entering the collection tube through the autonomous system at the discretion of the software. In some embodiments, the automatic dump may include a cap that separates and disperses sheath fluid from the collection chute. The automatic dump cap may be coated with a fluoropolymer such as CYTOP (trademark). In other embodiments, fluid droplets cannot roll under the cap. In one embodiment, the cap may include an undercut to further ensure that fluid does not enter under the cap.

[0316] In one embodiment, a flow cytometry system, such as a microfluidic chip-based system, processes samples continuously. The entire processed sample is collected in a sample collection tube, and the sample does not change its path to another collection location, such as a waste collection location, within the microfluidic chip. However, in certain situations, it may be desirable for the processed sample to have its path changed after being released from an outlet in the microfluidic chip. For example, if a component being processed, such as a laser optical component, is not calibrated or is misaligned, the processed sample may contain undesirable elements that should be removed or inactivated by the processing system. This may also be a subset of the sample, such as cells in the sample, that should be removed or inactivated. If this occurs, the improperly processed sample is mechanically diverted so that it is not collected in the sample collection tube. This prevents the processed sample containing undesirable characteristics from mixing with or contaminating the sample containing desirable characteristics.

[0317] This may be referred to as "filtering" or "not filtering". For example, the default position may be a mechanical diverter that is normally closed to "filter" all samples from the microfluidic chip unless a set of desired conditions is met. This may be the case if the sample contains only or primarily cells or particles having certain characteristics, such as a DNA moiety, or if a suitable sample collection tube is placed to collect the processed sample. When the desired conditions are met, the mechanical diverter is opened to not filter the processed sample. When the diverter is opened, the processed sample may be collected by a sample collection tube or the like. The filtered sample may be discarded or diverted into a waste collection location, such as a large waste collection bin or a sample collection tube designated for waste collection.

[0318] The mechanical diverter may be operated by a fluid-actuated rotary system, such as a pneumatically actuated rotary system that includes a 2-position linear actuator, a linear actuator such as a solenoid, or a rotary cap having at least one opening through which a processed sample can be collected into a sample collection tube.

[0319] In some embodiments, the automatic dump subsystem can tolerate all the chemicals used in the process. The subsystem can also determine whether the collection chute is open or closed. In other embodiments, the subsystem can be disassembled for maintenance inside the instrument and reassembled. For example, two screws, which are the areas circled in FIG. 40A, can be removed to remove the actuator of the automatic dump subsystem 5160.

[0320] Although it is not desired to limit the present invention to a particular application or embodiment, the systems, devices, and methods of the present invention may be used in combination with a particle processing system, such as a semen discrimination system demonstrated in FIG. 41. In one embodiment, the semen discrimination system may include a flow cytometer device or a microfluidic chip or device comprising a flow chamber configured to direct a fluid stream containing sample particles to a particle interrogation location. The sample particles may be cells, such as sperm, that have been stained or otherwise processed prior to entering the flow cytometer device or microfluidic device. The flow cytometer device may further include a laser configured to emit electromagnetic radiation along a beam path to the particle interrogation location and a detector configured to detect radiation from the particles. Following detection, the particles may be sorted or destroyed, such as by ablation with electromagnetic radiation, before or after exiting the flow cytometer device or microfluidic device. The sample mixing system of the present invention may be used to mix the sample of particles and fluid collected following the interrogation, determination, and sorting or destruction process.

[0321] The sample mixing system of the present invention may be used in combination with, or in this method, for analyzing particles such as sperm cells contained in a fluid flow when the particles flow through an interrogation site in a flow cytometer device or a microfluidic chip or device. Such a method includes illuminating the fluid flow and the particles contained therein using electromagnetic radiation emitted from a laser. A detector, such as an avalanche photodiode, detects the electromagnetic radiation emitted from the interrogation site by the particles, and a processor determines the characteristics of the particles in the fluid flow based at least in part on the signals from the detector. Following detection, the particles may be sorted or destroyed, such as by ablation or photodamage by electromagnetic radiation, before or after exiting the flow cytometer device or microfluidic device. The sample mixing system of the present invention may be used to mix the particles and fluid samples collected following the interrogation, determination, and sorting or destruction processes.

[0322] The sample mixing system of the present invention may be used in combination with, or used in, a method for evaluating the amount of DNA in the nucleus of sperm cells using a flow cytometry apparatus or a microfluidic chip or device. This method may include staining the DNA in the nucleus of sperm cells and then irradiating the stained DNA in the nucleus of sperm cells with an electromagnetic radiation device such as a laser when the sperm cells pass through a corresponding region of a flow cytometry apparatus or a microfluidic device. A detector such as an avalanche photodiode detects fluorescence emitted from the irradiated and stained DNA in the nucleus of sperm cells at a detection or interrogation location within the flow cytometry apparatus or microfluidic device. This method may further include first determining the sex of sperm cells using the detected amount of DNA in the nucleus of sperm cells and then differentiating between a plurality of sperm cells based on the sex determination to distinguish X-chromosome-bearing sperm cells and Y-chromosome-bearing sperm cells. The characteristics of sperm cells may include a corresponding amount of DNA in the nucleus of sperm cells. This method may further include inactivating a given sperm cell based on a determined amount of DNA in the nucleus of the given sperm cell. The inactivation may include photodamaging or excising a given sperm cell by radiation of electromagnetic radiation.

[0323] In other embodiments, this method may further include forming a plurality of droplets each having one of the sperm cells carried therein, differentially charging each of the droplets based on the sex-distinguishing characteristics of the sperm cells carried in the droplets, deflecting each of the droplets, and differentially collecting each of the droplets based on the sex-distinguishing characteristics of the sperm cells carried in the droplets. This method may further include collecting sperm cells after determination, sorting, or inactivation when the sperm cells exit the microfluidic device. The sample mixing system of the present invention may be used to mix samples of particles and fluids collected following an interrogation, determination, and sorting or destruction process.

[0324] Additional automation features

[0325] In some embodiments, in addition to automating the determination of the sample path using a mechanical diverter or an automatic dump, and in addition to automating the sample mixing process using a sample mixing system, additional elements of the flow cytometry-based particle processing system may be automated. For example, parameters related to the fluid pressure used with the sample, the amount, volume or velocity of the sheath fluid used, the power or duration of the laser module, or the desired characteristics identified in particles such as cells may be automatically determined based on the sample type detected by the flow cytometry system or input by the operator. In one embodiment, this may include the operator scanning a tag or identifier on the sample. The tag or identifier may be a barcode, QR code®, RFID tag, or similar information encoding tag on the sample, but may also include a set of configuration information for the flow cytometry system, or may include an address, pointer, or link to where such information is stored. Based on the information scanned from the tag or identifier on the sample, the flow cytometry system may be automatically configured with parameters optimal for the individual sample or sample type.

[0326] For example, semen samples from individual Holstein bulls may vary in consistency, sperm cell density, pH (e.g., acidity), fertility, or susceptibility, and may require specific parameters to successfully process the semen sample, such as identifying and inactivating sperm cells from within the sample including the amount or type of DNA such as X chromosome-containing sperm cells or Y chromosome-containing sperm cells. The specific processing requirements may be stored on a tag or identifier on the sample, or in a database record or location identified by the tag or identifier, and those processing requirements may include the configuration requirements of the flow cytometry-based system.

[0327] The flow rate and / or pressure of the sample and sheath fluid, the chromosome type detected, the laser force and / or duration, the pH of the medium, the amount or type of additive automatically introduced by the system, and other parameters may be set and automatically executed by the system for the specified sample. In this way, the sample may be processed under ideal conditions for the specified sample without the need for a human operator to use this information piecemeal to search, configure, or calibrate the flow cytometry system.

[0328] Examples

[0329] The following examples are illustrative only and are intended not to be limiting.

[0330] Example Set A:

[0331] "Example A1" A modular flow cytometry system for processing a sample, - a support structure, - an electronic module, - a sample passage module, - a fluidics module, - an interrogation module, - a sample mixing and collection module In a system comprising, - each module is configured to be removably reinstalled separately or replaced with another of the same module and is disposed on the support structure, System.

[0332] "Example A2" The system according to Example A1, further comprising a microfluidics device cleaning module for cleaning the sample passage module.

[0333] "Example A3" A modular flow cytometry system for processing samples, - a support structure, and - an electronic module comprising one or more computer processors and electronic ports, - a sample passage module comprising a microfluidic module having at least one microfluidic channel, - a fluidic module fluidly coupled to the sample passage module to circulate one or more fluids through the sample passage module, - an interrogation module operably coupled to the electronic module, the interrogation module including a detector, and a detection laser and a kill laser for processing a sample flowing through the microfluidic chip, - a sample mixing and collection module configured to collect and mix the processed samples exiting the microfluidic module In a system comprising: - each of the modules is configured to be separately removed and reinstalled or replaced with another of the same module for substantially continuous operation of the modular flow cytometry system, and is disposed on the support structure, System.

[0334] "Example A4" The system according to Example A3, wherein the kill laser and the detection laser are disposed on a common side of the interrogation module with respect to each other.

[0335] "Example A5" The system according to Example A3 or Example A4, wherein the sample mixing and collection module rotates about a pivot so as to move in an arcuate path, enabling mixing of the samples with controlled and uniform movement.

[0336] "Example A6" A modular flow cytometry system for sex discrimination of samples, - A chassis divided into a plurality of levels, and - An electronic module disposed at the lowest first level of the plurality of levels of the chassis, the electronic module including one or more computer processors and a plurality of electronic ports, - A sample passage module disposed at the second level of the chassis, the sample passage module including a microfluidic module for holding a microfluidic chip, - A fluidic module disposed at the second level of the chassis, the fluidic module being fluidically coupled to the sample passage module so as to circulate one or more fluids through the sample passage module, - An interrogation module including a detection laser and a kill laser operably coupled to a detection assembly for processing a sample flowing through the microfluidic chip, the interrogation module being disposed at the third level of the chassis and operably coupled to the electronic module, wherein the kill laser and the detection laser are disposed on a common side of the interrogation module with respect to each other, - A sample mixing and collection module configured to collect and automatically mix the processed sample exiting the microfluidic chip and disposed under the microfluidic module at the same second level of the chassis, the sample mixing and collection module rotating about a pivot so as to move in an arcuate path and enabling mixing of the sample with a controlled and uniform movement In a system comprising: - Each module is configured to be separately removed and reinstalled or replaced with another of the same module for substantially continuous operation of the modular flow cytometry system. System.

[0337] "Example A7" The sample passage module further comprises a distribution block fluidly coupled to the microfluidic module, the distribution block delivering a sample to the microfluidic module through a first fluidic line and delivering sheath fluid to the microfluidic module through a second fluidic line, the system according to any one of Examples A3 - A6.

[0338] "Example A8" The system according to Example A7, wherein the distribution block comprises a fluidic manifold.

[0339] "Example A9" The system according to Example A7 or Example A8, wherein the distribution block further comprises a sample tube loader.

[0340] "Example A10" The system according to any one of Examples A3 - A9, further comprising a microfluidic device cleaning module.

[0341] "Example A11" The microfluidic device cleaning module comprises - a set of fluid reservoirs, - a pump assembly, - a distribution block interface comprising a distribution block fixing element and a distribution block adjusting element, further comprising a set of fluid interfaces, at least one of the set of fluid interfaces being in fluid communication with at least one of the set of fluid reservoirs, the distribution block interface, - a microfluidic device interface comprising a microfluidic device fixing element and a microfluidic device alignment element, further comprising a fluid interface in fluid communication with at least one of the set of fluid reservoirs, the microfluidic device interface and comprising a microfluidic device cleaning module, - The dispensing block interface is adapted to secure a dispensing block thereon, the microfluidic system device interface is adapted to secure a microfluidic system module thereon, and the microfluidic system device cleaning system is adapted to clean a sample passage module that circulates fluid through the sample passage module. The system according to Example A10.

[0342] "Example A12" The system according to any one of Examples A6 to A11, wherein the kill laser and the detection laser are disposed on a common side in relation to the main surface of the microfluidic chip.

[0343] "Example A13" The system according to any one of Examples A3 to A12, wherein the kill laser and the detection laser are each adapted to emit a laser beam including an elliptical beam profile at the focal plane.

[0344] "Example A14" The sample mixing and collection module - A rotating base, - A set of collection tube holders disposed on the rotating base, - A set of collection tubes configured to be respectively disposed in the collection tube holders of the set of collection tubes, - A fluid level sensor and comprising a system, - One of the collection tubes is disposed under the microfluidic chip such that the processed sample is collected in the collection tube, - The sample mixing and collection module is configured to exchange with another collection tube to collect the processed sample when the fluid level sensor detects that the collection tube has reached a desired filling level. The system according to any one of Examples A1 to A13.

[0345] "Example A15" The system according to any one of Examples A1 to A14, further comprising a mechanical diverter configured to prevent the processed sample coming out of the microfluidic system module from entering the sample mixing and collection module.

[0346] "Example A16" The system according to any one of Examples A1 to A15, wherein the interrogation module includes a laser heat sink for the laser, and the laser heat sink can draw in air by natural convection to cool the interrogation module.

[0347] "Example A17" The system according to any one of Examples A1 to A16, wherein the sample is a semen sample containing sperm cells.

[0348] "Example A18" The system according to any one of Examples A1 to A17, wherein the electronic module includes an electronic box with a removable lid for housing a computer processor and other electronic components, and the electronic components are removable and can be replaced or reconnected.

[0349] "Example A19" The system according to Example A18, wherein the electronic box is disposed on rails so as to be easily movable.

[0350] "Example A20" The system according to any one of Examples A1 to A19, wherein the electronic module includes one or more fans for cooling the electronic components.

[0351] "Example A21" The system according to any one of Examples A1 to A20, wherein the interrogation components of the interrogation module are removable and can be replaced or reconnected.

[0352] "Example A22" The system according to any one of Examples A6 to A21, wherein one or more identification elements are disposed on the surface of the microfluidic system chip or etched on the surface.

[0353] "Example A23" The system according to Example A22, wherein one or more identification elements include an alphanumeric string, a barcode, a QR code (registered trademark), a reference marker, or a combination thereof.

[0354] "Example A24" The system according to Example A22 or Example A23, wherein one or more identification elements are used to identify the chips currently mounted on the microfluidic system module.

[0355] "Example A25" The system according to any one of Examples A22 to A24, wherein the identification element is used to automatically align and place the microfluidic system chip with respect to the detection laser and the kill laser.

[0356] "Example A26" The system according to any one of Examples A22 to A25, wherein the microfluidic system chip with identification elements is attached to a multi-axis stage for readjusting the position of the microfluidic system chip.

[0357] "Example A27" The system according to any one of Examples A1 to A26, further comprising an automatic sample level detector adapted to determine the sample level using the sample flow rate and notify when the sample level is below a threshold level.

[0358] "Example A28" A method for sex discrimination of sperm, comprising: - classifying sperm cells as having a first characteristic, a second characteristic, or a third characteristic; - selectively damaging at least a part of the sperm cells based on these classifications; A method including

[0359] "Example A29" The method according to Example A28, wherein the first characteristic is the presence of an X chromosome from one sperm cell, the second characteristic is the presence of a Y chromosome from one sperm cell, and the third characteristic is the presence of both an X chromosome and a Y chromosome, or a repetition of one of the chromosomes by a plurality of sperm cells.

[0360] "Example A30" A method for sex discrimination of sperm, comprising: - sending a response command signal to the stained sperm cells using a first radiation source to generate fluorescence radiation; - detecting the fluorescence radiation; - classifying the sperm cells based on the detected fluorescence radiation; - selectively damaging the sperm cells using a second radiation source based on these classifications A method including

[0361] "Example A31" A method for sex discrimination of sperm, comprising: - delivering a fluid flow containing stained sperm cells to an interrogation position; - sending a response command signal to the stained sperm cells using a first laser to generate fluorescence radiation; - detecting the fluorescence radiation; - classifying the sperm cells based on the detected fluorescence radiation; - selectively damaging one or more sub-populations of the classified sperm cells using a second laser based on these classifications A method including

[0362] "Example A32" The method according to any one of Examples A28 to A31, further comprising staining sperm cells to generate stained sperm cells.

[0363] "Example A33" The method according to any one of Examples A28 to A32, wherein the sperm cells are stained with a fluorescent DNA-binding dye.

[0364] "Example A34" The method according to any one of Examples A30 to A33, wherein a fluorescence emission corresponding to one sperm cell or a fluorescence emission corresponding to a plurality of sperm cells is generated by sending a response command signal to the stained sperm cells.

[0365] "Example A35" The method according to any one of Examples A28 to A34, wherein the sperm cells are damaged to cause DNA damage and / or membrane damage to the cells.

[0366] "Example A36" The method according to any one of Examples A28 to A35, wherein the sperm cells are damaged to make the sperm cells infertile sperm cells or dead sperm cells.

[0367] "Example A37" A method for generating a sex-discriminated semen product, comprising: - preparing a modular flow cytometry system comprising an electronic module, a sample passage module, a fluidics module, an interrogation module, and a sample mixing and collection module, wherein each module is configured to be removably reinstalled separately or replaced with another of the same module and disposed in a support structure; - flowing a semen sample having stained sperm cells through the fluidics module and the sample passage module; - flowing a sheath fluid through the fluidics module and the sample passage module; - causing the stained sperm cells to fluoresce using a detection laser in the interrogation module; - detecting the fluorescence of the stained sperm cells; - Based on the detected fluorescence, generating a sex-discriminated semen product by damaging a sub-population of the stained sperm cells using a kill laser in an interrogation module; - Collecting the sex-discriminated semen product flowing from the sample passage module into a sample mixing and collection module; - Mixing the collected sex-discriminated semen product; and a method comprising the steps.

[0368] "Example A38" The method according to Example A37, wherein the radiation from the detection laser and the kill laser includes an elliptical beam profile.

[0369] "Example A39" The method according to Example A37 or Example A38, further comprising the step of staining a semen sample containing sperm cells to generate stained sperm cells.

[0370] "Example A40" The method according to any one of Examples A37 to A39, wherein the sample mixing and collection module rotates around a pivot so as to move in an arcuate path, enabling mixing of the sex-discriminated semen product with a controlled and uniform movement.

[0371] "Example A41" The method according to any one of Examples A37 to A40, further comprising the step of cleaning the sample passage module using a microfluidic device cleaning module.

[0372] "Example A42" A method for generating a sex-discriminated semen product, comprising: - Providing a modular flow cytometry system according to any one of Examples A6 to A27; - Staining a semen sample containing sperm cells to generate stained sperm cells; - flowing a semen sample through a fluidic system module and a sample passage module; - flowing a sheath fluid through the fluidic system module and the sample passage module, wherein the sheath fluid, one or more channels in the microfluidic system chip, or a combination thereof, target the stained sperm cells when flowing through the microfluidic system chip; - causing the stained sperm cells to fluoresce by radiation from a detection laser including an elliptical beam profile; - detecting the fluorescence of the sperm cells by a detection assembly; - generating a sex-discriminated semen product by inactivating one or more sub-populations of the stained sperm cells by radiation from a kill laser based on the detected fluorescence, wherein the radiation from the kill laser includes an elliptical beam profile and the detection laser and the kill laser are dispose...

Claims

1. 1. A modular flow cytometry system for processing samples, comprising: a. a support structure; b. an electronics module; c. a sample passage module; d. a fluidics module; e. a query module; f. A sample mixing and collection module; 1. A modular flow cytometry system comprising: A modular flow cytometry system, wherein each module is configured to be separately removed and reinstalled or replaced with another of the same module, and disposed on the support structure.

2. 2. The system of claim 1, wherein the interrogation module comprises a detector, a detection laser, and a kill laser, the kill laser and the detection laser being disposed on a common side of the interrogation module.

3. The system of claim 2 , wherein each of the kill laser and the detection laser are adapted to emit a laser beam that includes an elliptical beam profile at a focal plane.

4. The system of claim 1 , wherein the sample mixing and collection module rotates about a pivot to move in an arcuate path to enable mixing of the sample in a controlled, uniform motion.

5. The system further comprises a chassis divided into a plurality of levels, a. the electronics module is disposed in a first, lowest level of the chassis and includes one or more computer processors and a plurality of electronics ports; b. the sample passage module is disposed on a second level of the chassis and includes a microfluidic module for holding a microfluidic chip; c. the fluidics module is disposed in the second level of the chassis and is fluidly coupled to the sample path module to circulate one or more fluids through the sample path module; d. the interrogation module is disposed on a third level of the chassis and operably coupled to the electronics module, the interrogation module including a detection laser operably coupled to a detection assembly and a kill laser operably coupled to the detection assembly for processing a sample flowing through the microfluidic chip, the kill laser and the detection laser being disposed on a common side of the interrogation module; e. the sample mixing and collection module is configured to collect and automatically mix the processed samples exiting the microfluidic chip, disposed below the microfluidic module on the same second level of the chassis, and rotates about a pivot to move in an arcuate path to enable mixing of the samples in a controlled, uniform motion; In the system, each module being configured to be independently removed and reinstalled or replaced with another of the same module for substantially continuous operation of the modular flow cytometry system; The system of claim 1 .

6. 6. The system of claim 5, wherein the sample passage module further comprises a distribution block fluidly coupled to the microfluidic module, the distribution block delivering the sample to the microfluidic module through a first fluidic line and delivering sheath fluid to the microfluidic module through a second fluidic line.

7. 11. The system further comprising a microfluidic device cleaning module for cleaning the sample passage module, the microfluidic device cleaning module comprising: a. a set of fluid reservoirs; b. a pump assembly; c. a distribution block interface comprising a distribution block securing element and a distribution block adjusting element, the distribution block interface further comprising a set of fluid interfaces, at least one of the set of fluid interfaces in fluid communication with at least one of the set of fluid reservoirs; d. a microfluidic device interface comprising a microfluidic device securing element and a microfluidic device alignment element, the microfluidic device interface further comprising a fluid interface in fluid communication with at least one of the set of fluid reservoirs; Equipped with the distribution block interface is adapted to secure the distribution block thereon, the microfluidic device interface is adapted to secure the microfluidic module thereon, and a microfluidic device cleaning system is adapted to clean the sample path module which circulates fluid through the sample path module. The system of claim 6.

8. The system of claim 5, wherein one or more identification elements are disposed on or etched into a surface of the microfluidic chip and are used to identify a chip currently mounted in the microfluidic module and to automatically align and position the microfluidic chip relative to the detection laser and the kill laser.

9. The system of claim 8 , wherein the microfluidic chip with the identification element is mounted on a multi-axis stage that uses the identification element to reposition the microfluidic chip.

10. 2. The system of claim 1, wherein the sample passage module is configured to process particles or constituent molecules in a sample based on at least one characteristic, the sample mixing and collection module is configured to automatically mix the sample processed by the sample passage module based on a defined set of parameters, and the automated sample mixing system rotates about a pivot to move in an arcuate path, thereby enabling mixing of the sample in a controlled, uniform motion.

11. the sample mixing and collection module comprising: a. a rotating base; b. a set of collection tube holders disposed on the rotating base; c. a set of collection tubes, each configured to be disposed in a collection tube holder of the set of collection tubes; The system of claim 1 further comprising:

12. 12. The system of claim 11, wherein the sample mixing and collection module further comprises a fluid level sensor and is configured to replace the first tube with a second tube in the set of collection tubes when the fluid level sensor detects that a first tube has reached a desired fill level.

13. 1. A laser-based flow cytometry system for particle detection adapted to distinguish particles in a sample stream flowing in a channel of the flow cytometry system, comprising: a. the flow cytometry system comprising a laser assembly, the laser assembly comprising: i. a kill laser assembly; ii. A detection laser assembly; wherein the kill laser assembly and the detection laser assembly are disposed on a common side of the flow cytometry system.

14. The system of claim 13 comprising a microfluidic device.

15. The system of claim 14 , wherein the kill laser assembly and the detection laser assembly are disposed on a common side relative to a major surface of the microfluidic device.

16. The system of claim 13 , wherein the laser assembly further comprises a polarizing beam splitter.

17. The system of claim 13 , wherein the kill laser assembly and the detection laser assembly each comprise an optical path adapted to direct electromagnetic radiation emissions to a focal plane.

18. 20. The system of claim 17, wherein the optical path of the kill laser assembly comprises a fast axis beam expander and a slow axis beam expander.

19. 20. The system of claim 18, wherein the fast axis beam expander and the slow axis beam expander of the optical path for the kill laser assembly are adapted to adjust a fast axis beam width and a slow axis beam width of the kill laser assembly.

20. 20. The system of claim 17, wherein the optical path for the detection laser assembly comprises a pair of cylindrical lenses adapted to adjust a fast axis beam width and a slow axis beam width for the detection laser assembly.

21. 14. The system of claim 13, wherein each of the kill laser assembly and the detection laser assembly is adapted to emit a laser beam that includes an elliptical beam profile at a focal plane.

22. 14. The system of claim 13, further comprising a detection assembly adapted to detect one or more of fluorescence of a particle excited by radiation from the detection laser assembly, deactivation of a particle by radiation from the kill laser assembly, an image of a detection event, or an image of a kill event.

23. a. a flow cytometry assembly for processing particles or constituent molecules in a sample based on at least one characteristic; b. an automatic sample mixing system configured to automatically mix the samples processed by the flow cytometry assembly based on a defined set of parameters, the automatic sample mixing system rotating about a pivot to move in an arcuate path, thereby enabling mixing of the samples in a controlled, uniform motion; The system of claim 13 further comprising:

24. The automated sample mixing system comprises: a. a rotating base; b. a set of collection tube holders disposed on the rotating base; c. a set of collection tubes, each configured to be disposed in a collection tube holder of the set of collection tubes; 24. The system of claim 23, further comprising:

25. 25. The system of claim 24, wherein the automated sample mixing system further comprises a fluid level sensor and is configured to replace the first tube with a second tube in the set of collection tubes when the fluid level sensor detects that a first tube has reached a desired fill level.

26. 1. A flow cytometry system comprising a removable and replaceable fluid path module, the fluid path comprising: a. a distribution block comprising a set of fluid inlets and a set of fluid outlets, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a first sample path element; b. a microfluidic module comprising a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixture, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a second sample path element; c. a fluid passageway providing fluid communication between the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, a portion of the fluid passageway joining and fluidly communicating the first sample path element with the second sample path element to form a sample path; A flow cytometry system comprising: a sample being processed by the flow cytometer system through the removable and replaceable fluid path module is confined to the sample path during processing; Flow cytometry systems.

27. 27. The system of claim 26, wherein the microfluidic device fixing apparatus is adapted to fix one of a microfluidic chip or a microfluidic cassette in the microfluidic module.

28. 28. The system of claim 27, wherein the microfluidic device fixing apparatus comprises one or more adjustment means adapted to adjust the position of the microfluidic chip or the microfluidic cassette.

29. another fluid inlet of the set of fluid inlets of the distribution block in fluid communication with another fluid outlet of the set of fluid outlets of the distribution block to form a first buffer fluid path element; another fluid inlet of the set of fluid inlets of the microfluidic module in fluid communication with another fluid outlet of the set of fluid outlets of the microfluidic module to form a second buffer fluid path element; the fluid path further comprises a second portion joining and in fluid communication with the first buffer fluid path element and the second buffer fluid path element to form a buffer fluid path; a buffer fluid processed through the removable and replaceable fluid passage module is confined to the buffer fluid passage during processing; 27. The system of claim 26.

30. 27. The system of claim 26, wherein each of the distribution block and the microfluidic module further comprises releasable fastening means adapted to fasten the removable and replaceable fluid passage module to the flow cytometer system, and wherein the removable and replaceable fluid passage module is removable from the flow cytometer system by releasing the fastening means.

31. The system of claim 26 , wherein the fluid path module comprises an identification element.

32. a. a set of fluid reservoirs; b. a pump assembly; c. a distribution block interface comprising a distribution block securing element and a distribution block adjusting element, the distribution block interface further comprising a set of fluid interfaces, at least one of the set of fluid interfaces in fluid communication with at least one of the set of fluid reservoirs; d. a microfluidic device interface comprising a microfluidic device securing element and a microfluidic device alignment element, the microfluidic device interface further comprising a fluid interface in fluid communication with at least one of the set of fluid reservoirs; The system further comprises: the microfluidic device cleaning system is adapted to circulate fluid through the distribution block interface and a removable and replaceable fluid passage module secured to the microfluidic device interface.

27. The system of claim 26.

33. the removable and replaceable fluid path module is adapted for use in the flow cytometer system; a. a distribution block comprising a set of fluid inlets and a set of fluid outlets, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a first sample path element; b. a microfluidic module comprising a set of fluid inlets, a set of fluid outlets, and a microfluidic device fixture, at least one of the set of fluid inlets in fluid communication with at least one of the set of fluid outlets to form a second sample path element; c) a fluid passageway providing fluid communication between the set of fluid outlets of the distribution block and the set of fluid inlets of the microfluidic module, a portion of the fluid passageway joining and fluidly communicating the first sample path element with the second sample path element to form a sample path; 1. A system comprising:

33. The system of claim 32, wherein samples processed by the flow cytometer system through the removable and replaceable fluid path module are confined to the sample path during processing.

34. a. a flow cytometry assembly for processing particles or constituent molecules in a sample based on at least one characteristic; b. an automatic sample mixing system configured to automatically mix the samples processed by the flow cytometry assembly based on a defined set of parameters, the automatic sample mixing system rotating about a pivot to move in an arcuate path, thereby enabling mixing of the samples in a controlled, uniform motion; 27. The system of claim 26, further comprising:

35. The automated sample mixing system comprises: a. a rotating base; b. a set of collection tube holders disposed on the rotating base; c. a set of collection tubes, each configured to be disposed in a collection tube holder of the set of collection tubes; 35. The system of claim 34, further comprising:

36. 36. The system of claim 35, wherein the automated sample mixing system further comprises a fluid level sensor and is configured to replace the first tube with a second tube in the set of collection tubes when the fluid level sensor detects that a first tube has reached a desired fill level.

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