Flow cytometry systems and methods for use

Abstract

The present disclosure provides a flow cytometry system. The flow cytometry system includes a flow cell, a fluid path in fluid communication with the flow cell, and a probe in fluid communication with the fluid path. The probe is configured to inject aliquots of a plurality of samples and a separation gas between successive ones of the plurality of samples into the fluid path. The flow cytometry system also includes two or more lasers positioned such that an illumination spot of each of the two or more lasers is directly over the flow cell, and two or more side scatter detection modules in communication with the two or more lasers. The flow cytometry system also includes a processor in communication with the two or more side scatter detection modules and a non-transitory computer-readable medium having stored thereon instructions executable to cause the processor to perform several functions when the flow cytometry system is used. These functions include determining a time delta between a first laser of the two or more lasers and a second laser of the two or more lasers based on a difference between a plurality of first timestamps detected by a first side scatter detection module and a plurality of second timestamps detected by a second side scatter detection module.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 18 / 146,560, filed December 27, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Flow cytometry is a technique used, for example, in cell counting, cell sorting, biomarker detection, and protein engineering, performed by suspending cells in a fluid stream and passing them through an electronic detection device. Flow cytometry enables simultaneous multiparameter analysis of the physical and / or chemical properties of up to tens of thousands of particles per second. Traditionally, flow cytometers are standalone instruments designed to measure biological samples in aqueous suspension contained in assay plates or vials, and may be capable of actively separating and isolating particles with properties of interest. As such, these flow cytometers are typically independent laboratory instruments. An operator (or robotic device) presents samples to the cytometer, which performs the measurements and reports the results to the operator. Traditionally, flow cytometry has been used for low-throughput sample analysis by placing samples one by one under the cytometer's sampling port.

[0003] More recently, high-throughput flow cytometry systems have been developed that rapidly deliver samples in microliter volumes to a flow cytometry engine. High-throughput flow cytometry systems use a pump system to fill sample tubing lines with individual sample particle suspension streams aspirated from the wells of a microplate and separated from each other by air gaps. The entire sample stream is continuously delivered to the flow cytometer so that data from all samples in the microplate are acquired and stored in a single data file. High-resolution time parameters are also recorded during data acquisition. The passage of the air gap creates time gaps in particle detection in the data stream, which, when plotted in conjunction with the time parameter, allow individual particle suspensions to be distinguished and evaluated separately. Based on this time distribution, data peaks are identified and assigned to individual wells of the microplate. The present disclosure provides various improvements for high-throughput flow cytometry systems. Summary of the Invention [Means for solving the problem]

[0004] In a first aspect, the present disclosure provides a flow cytometry system, the flow cytometry system comprising: (a) a flow cell; (b) a fluid path in fluid communication with the flow cell; (c) a probe in fluid communication with the fluid path, the probe configured to inject aliquots of a plurality of samples and a separation gas between successive ones of the plurality of samples into the fluid path; (d) two or more lasers, the two or more lasers positioned such that an illumination spot of each of the two or more lasers is directly over the flow cell; (e) two or more side scatter detection modules in communication with the two or more lasers; (f) a processor in communication with the two or more side scatter detection modules; and (g) a flow cytometer system, wherein when the flow cytometer system is used, (i) two or more side scatter detection modules. and a non-transitory computer-readable medium having stored thereon instructions executable to cause a processor to perform functions including (i) detecting a first sample of the plurality of samples in the fluid pathway at a first timestamp via a first side scatter detection module of the above side scatter detection modules; (ii) detecting a first sample of the plurality of samples in the fluid pathway at a second timestamp after the first timestamp via a second side scatter detection module of the two or more side scatter detection modules; and (iii) determining a time delta between a first laser of the two or more lasers and a second laser of the two or more lasers based on a difference between the plurality of first timestamps and the plurality of second timestamps.

[0005] In a second aspect, the present disclosure provides a flow cytometry system including: (a) a flow cell; (b) a fluid pathway in fluid communication with the flow cell; (c) a probe having a first end and a second end opposite the first end, the second end of the probe in fluid communication with the fluid pathway; and (d) a color washing module disposed adjacent to the probe, the color washing module including a top opening, a bottom opening in fluid communication with the top opening via a common shaft, a first side opening, and a second side opening in fluid communication with the first side opening via the common shaft, the probe being configured such that the first end of the probe is positioned outside the color washing module from a first position, the first end of the probe being positioned between the first side opening and the second side opening. The probe is configured to transition from a second position disposed inside the color cleaning module to a third position disposed outside the color cleaning module, and the first end of the probe is configured to move fluid between the first side opening and the second side opening when the probe is in the second position, thereby cleaning the probe, the first end of the probe is configured to introduce a plurality of samples from a plurality of sample wells into the fluid pathway when the probe is in the third position, thereby forming fluid flow streams in the fluid pathway, and the first end of the probe is configured to introduce an aliquot of separation gas between successive ones of the plurality of samples in the fluid flow stream to configure the fluid flow streams as separation-gas-separated fluid flow streams.

[0006] In a third aspect, the present disclosure provides a flow cell module for a flow cytometry device, the flow cell module including: (a) a flow cell; (b) an upper manifold arranged upstream of the flow cell; (c) a lower manifold coupled to the upper manifold and movable relative to the upper manifold; (d) a sample injection needle having a first end and a second end opposite the first end; (e) a needle adjustment member fixedly coupled to the second end of the sample injection needle and movably disposed on the lower manifold, the needle adjustment member having an internal thread on at least a portion thereof such that rotation of the needle adjustment member adjusts the position of the first end of the sample injection needle relative to the flow cell; and (f) a fluid link connection screw coupled to the needle adjustment member, the internal thread of the fluid link connection screw being configured to receive a fluid path.

[0007] In a fourth aspect, the present disclosure provides a system comprising: (a) the flow cytometry system of the first aspect; (b) the flow cytometry system of the second aspect; and (c) the flow cell module of the third aspect.

[0008] The features, functions, and advantages that have been discussed can be achieved independently in various examples or can be combined in still other examples, further details of which can be seen with reference to the following description and figures.

[0009] The novel features believed characteristic of the illustrative examples are set forth in the appended claims. However, the illustrative examples, as well as the preferred mode of use, further objects and explanations thereof, will best be understood by reference to the following detailed description of illustrative examples of the present disclosure when read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of an example flow cytometry system. [Figure 2]1 is a table and corresponding histogram generated using data acquired from two or more side scatter detection modules of a flow cytometry system. [Figure 3] Figure 1 illustrates per-laser data correlation on the field-programmable gate array (FPGA) level using a global source trigger with data acquired from two or more side scatter detection modules of a flow cytometry system. [Figure 4] FIG. 10 illustrates a search time window for adapting to fluid flow changes using data acquired from two or more side scatter detection modules of a flow cytometry system. [Figure 5] FIG. 1 illustrates steps in a data correlation algorithm that correlates data acquired from two or more side scatter detection modules of a flow cytometry system. [Figure 6] FIG. 10 illustrates another step in a data correlation algorithm that correlates data acquired from two or more side scatter detection modules of a flow cytometry system. [Figure 7] FIG. 10 illustrates another step in a data correlation algorithm that correlates data acquired from two or more side scatter detection modules of a flow cytometry system. [Figure 8A] FIG. 1 shows a collar cleaning module with the probe in a first position. [Figure 8B] 8B shows the collar cleaning module of FIG. 8A with the probe in a second position. [Figure 8C] 8B shows the collar cleaning module of FIG. 8A with the probe in a third position. [Figure 9A] FIG. 1 shows a collar cleaning module with the probe in a first position. [Figure 9B] 9B shows the collar cleaning module of FIG. 9A with the probe in a second position. [Figure 9C] FIG. 9B shows the collar cleaning module of FIG. 9A with the probe in a third position. [Figure 10]FIG. 1 is a cross-sectional view of an example flow cell module of a flow cytometry system. [Figure 11] FIG. 11 is a perspective view of the flow cell module of FIG. 10. [Figure 12] FIG. 11 is a perspective view of a set screw of the flow cell module of FIG. 10. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the following description, numerous specific details are set forth to provide a thorough understanding of the disclosed concepts, although the practice may be without some or all of these details. In other instances, details of known devices and / or processes have been omitted to avoid unnecessarily obscuring the disclosure. While some concepts have been described in conjunction with specific examples, it will be understood that these examples are not intended to be limiting. All examples of any aspect of the invention can be used in combination unless the context clearly dictates otherwise.

[0012] Unless otherwise indicated, terms such as "first," "second," etc. are used herein merely as labels and are not intended to impose any order, position, or hierarchical requirements on the items to which they refer, and reference to, for example, a "second" item does not require or exclude the presence of, for example, a "first" or lower-numbered item, and / or, for example, a "third" or higher-numbered item.

[0013] References herein to "one embodiment" or "one example" mean that one or more features, structures, or characteristics described in connection with that example are included in at least one implementation. The phrases "one embodiment" or "one example" in various places throughout the specification may or may not refer to the same example.

[0014] As used herein, a system, apparatus, device, structure, article, element, part, or hardware that is "configured to" perform a particular function is in fact capable of performing the particular function without any modification, rather than merely having the potential to perform the particular function after further modification. In other words, a system, apparatus, structure, article, element, part, or hardware that is "configured to" perform a particular function is specifically selected, created, implemented, utilized, programmed, and / or designed for the purpose of performing the particular function. As used herein, "configured to" refers to an existing characteristic of a system, apparatus, structure, article, element, part, or hardware that enables the system, apparatus, structure, article, element, part, or hardware to perform a particular function without further modification. For purposes of this disclosure, a system, apparatus, structure, article, element, part, or hardware described as "configured to" perform a particular function can additionally or alternatively be described as "adapted" and / or "operable" to perform that function.

[0015] As used herein, "coupled" means directly as well as indirectly associated. For example, member A may be directly associated with member B, or may be indirectly associated, for example, through another member C. It will be understood that not all relationships between the various disclosed elements are necessarily represented.

[0016] Example methods and systems are described herein. It should be understood that the words "example," "exemplary," and "illustrative" are used herein to mean "serving as an example, instance, or illustration." Any example or feature described herein as being "example," "exemplary," or "illustrative" should not necessarily be construed as preferred or advantageous over other examples or features. The examples described herein are not meant to be limiting. It will be readily understood that the aspects of the present disclosure as outlined and illustrated in the figures herein can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0017] Unless the context clearly requires otherwise, throughout the description and claims, the terms "comprise," "comprising," and the like, are to be construed in an inclusive sense, i.e., "including, but not limited to," rather than in an exclusive or exhaustive sense. Words using the singular or plural also include the plural and the singular, respectively.

[0018] In FIG. 1 referenced above, solid lines connecting various elements and / or components may represent mechanical, electrical, fluid, optical, electromagnetic, and other couplings and / or combinations thereof, if any. As used herein, "coupled" means directly as well as indirectly associated. For example, component A may be directly associated with component B, or may be indirectly associated, for example, via another component C. It will be understood that not all relationships between the various disclosed elements are necessarily represented. Thus, other couplings may exist than those shown in the block diagram. Dashed lines connecting blocks representing various elements and / or components, if any, represent couplings similar in function and purpose to those represented by solid lines, but the couplings represented by dashed lines may either be provided in preference or may pertain to alternative examples of the present disclosure. Similarly, elements and / or components represented by dashed lines, if any, represent alternative examples of the present disclosure. One or more elements shown with solid and / or dashed lines may be omitted from a particular example without departing from the scope of the present disclosure. Environmental elements, if any, are represented by dotted lines. Hypothetical (imaginary) elements may be depicted for clarity. Those skilled in the art will appreciate that some of the features depicted in Figure 1 can be combined in various ways without necessarily including other features described in Figure 1, other figures, and / or the accompanying disclosure, even if such combinations are not explicitly depicted herein. Similarly, additional features, not limited to the examples presented, can be combined with some or all of the features shown and described herein.

[0019] For purposes of the present invention, the term "particle" as used herein refers to small objects having a physical size between 1 nm and 1 mm, including, but not limited to, molecules, cells, proteins, protein aggregates, microorganisms, viruses, microspheres, microbeads, cellular components such as nuclei, mitochondria, compounds, and chemical aggregates.

[0020] As used herein, the term "sample" refers to any volume of liquid that may contain particles of interest or marker particles that are detectable by a particle analysis device. More specifically, a sample can include a fluid solution or suspension containing particles of interest or marker particles to be detected and / or analyzed using the methods and / or devices disclosed herein. Particles of interest in a sample can be tagged, for example, with a fluorescent tag. Particles of interest can be bound to beads, receptors, or other useful proteins or polypeptides, or can simply exist as free particles, such as those found naturally in cell lysates, particles purified from cell lysates, particles from tissue culture, etc. A sample can include either organic or inorganic chemicals used to induce a reaction with the particles of interest. When the particles of interest are biomaterials, agents can be added to the sample to induce a reaction or response in the biomaterial particles. Chemicals, agents, or other additives can be added to the sample and mixed with the sample while it is in the sample source well, or chemicals, agents, or other additives can be added to the sample in the fluid flow stream after the sample is captured by the autosampler.

[0021] For purposes of the present invention, the term "well" as used herein can include any container for containing a sample, such as a chamber, dish, tube, bottle, vial, reservoir trough, or well on a microtiter plate.

[0022] As used herein, "microplate" and "plate" refer to a structure capable of holding one or more aliquots of sample or marker particles to be analyzed.

[0023] As used herein, the term "fluid pathway" or "conduit" refers to a device such as a tube, channel, etc. through which a fluid stream flows. A fluid pathway can be made up of several separate devices, such as multiple connected or joined pieces of tubing or a single piece of tubing, alone or in combination with channels or other different devices.

[0024] By the terms "about," "approximately," or "substantially" with respect to a quantity or measurement described herein, it is meant that the stated characteristic, parameter, or value need not be achieved exactly, but rather that deviations or variations, including, for example, tolerances, measurement errors, limits of measurement accuracy, and other factors known to those of ordinary skill in the art, may occur in an amount that does not interfere with the effect that the characteristic is intended to provide. For example, in one embodiment, the term "about" can refer to ±5% of a given value.

[0025] Various other features of the example systems discussed above, as well as methods for using these systems, are also described below with reference to the accompanying drawings. Illustrative, non-exhaustive examples of subject matter according to the present disclosure, which may or may not be claimed, are provided below.

[0026] Referring to the figures, FIG. 1 illustrates an example flow cytometry system 100. As shown in FIG. 1, flow cytometry system 100 includes a flow cell 102 and a fluid path 104 in fluid communication with flow cell 102. Flow cytometry system 100 further includes a probe 106 in fluid communication with fluid path 104. The probe is configured to inject aliquots of a plurality of samples and a separation gas between successive ones of the plurality of samples into fluid path 104. Flow cytometry system 100 further includes two or more lasers 108A-108D, which are positioned such that the illumination spots of each of the two or more lasers 108A-108D are directly over flow cell 102. Flow cytometry system 100 further includes two or more side scatter detection modules 110A-110D in communication with the two or more lasers 108A-108D. Although FIG. 1 shows the number of two or more lasers and the number of two or more side scatter detection modules as being four, this number may be two, three, four, or five, as non-limiting examples.

[0027] In operation, the probe 106 can, for example, collect a sample 111 from a sample well 115 in a well plate 117 and then advance the sample 111 into the fluid path 104. A pump 119 can then propel a fluid flow stream containing the sample 111 from the well 115 through the fluid path 104 to the flow cell 102. In one such embodiment, the flow cell 102 is in fluid communication with the probe 106 via the fluid path 104, and the flow cytometry system 100 is configured to focus the fluid flow stream delivered by the fluid path 104 from the probe 106 and selectively analyze particles in each of the multiple samples 111 as the fluid flow stream passes through the flow cell 102.

[0028] As discussed above, flow cytometry system 100 further includes two or more lasers 108A-108D, positioned such that the illumination spot of each of the two or more lasers 108A-108D is directly on flow cell 102. The two or more lasers 108A-108D are configured to inspect individual samples flowing from flow cell 102, as discussed in further detail below. As sample 111 passes through the illumination spots of the two or more lasers 108A-108D, particles within sample 111 are sensed by various components of flow cytometry system 100. Forward scattered light is detected by one or more forward scatter detectors 121. Fluorescence emitted from tagged particles within flow cell 102 is detected by one or more fluorescence detectors 123. In one example, one or more fluorescence detectors 123 include one or more photomultiplier tube detectors. Side scattered light is detected by two or more side scatter detectors 110A-110D. In contrast, particles are not sensed when the separation gas 113 passes through the illumination spots of two or more lasers 108A-108D. Thus, for a series of samples analyzed using a flow cytometer, a graph of sensed fluorescence data points versus time will form distinct groups, each time a particle-containing sample passes through the illumination spots of two or more lasers 108A-108D. Such a graph can be generated by the outputs of both one or more forward scatter detectors 121, one or more fluorescence detectors 123, and / or one or more side scatter detectors 110A-110D.

[0029] In one example, pump 119 includes a peristaltic pump. In one embodiment, such a peristaltic pump can be operated to reduce pulsating flow, thereby improving sample properties in flow cytometry system 100. In another embodiment, pump 119 includes a gear pump. In yet another example, pump 119 includes a syringe pump. While pump 119 is shown in front of flow cell 102 in FIG. 1 , in another embodiment, pump 119 can be positioned downstream of flow cell 102. Furthermore, additional pumps can be added to flow cytometry system 100 to perform various functions. For example, a combination of one or more peristaltic pumps, one or more gear pumps, and / or one or more syringe pumps can be used to transport a sample through fluid pathway 104.

[0030] In one embodiment, the fluid pathway 104 can be made of elastomeric tubing such as nitrile (NBR), Hypalon, Viton, silicone, polyvinyl chloride ("PVC"), ethylene propylene diene monomer ("EPDM"), EPDM+polypropylene, polyurethane, or natural rubber, among other possibilities. One example of such tubing can be polyvinyl chloride (PVC) tubing having an inner diameter of about 0.01 inch to 0.03 inch and a wall thickness of about 0.01 inch to 0.03 inch. In one embodiment, a preferred tubing for the fluid pathway 104 can be PVC tubing having an inner diameter of about 0.02 inch and a wall thickness of about 0.02 inch.

[0031] 1, fluid path 104 includes a fluid flow stream in which a series of samples 111 are each separated by an aliquot of separation gas 113, such as, by way of non-limiting example, air bubbles. The separation gas 113 can be formed by having probe 106 aspirate air (or other gas) between aspirating sample material from each of sample wells 115. In this manner, probe 106 is used to introduce aliquots of separation gas 113 between successive ones of samples 111 in the fluid flow stream, configuring the fluid flow stream in fluid path 104 as a separation-gas-separated fluid flow stream.

[0032] 1 , flow cytometry system 100 can include a processor 112, data storage 114, and a controller 126, which can all be part of a control system 118. Processor 112 can operate as one or more general-purpose or special-purpose hardware processors (e.g., digital signal processors, application-specific integrated circuits, etc.). Processor 112 can be configured to execute non-transitory computer-readable medium 120 and manipulate data 122, both of which are stored in data storage 114. Processor 112 can also directly or indirectly interact with other components of flow cytometry system 100, such as, by way of non-limiting example, a communication link 124.

[0033] The data storage 114 can be one or more types of hardware memory. For example, the data storage 114 can include or take the form of one or more computer-readable storage media that can be read or accessed by the processor 112. The one or more computer-readable storage media can include volatile and / or non-volatile storage components, such as optical, magnetic, organic, or another type of memory or storage, and these storage components can be integrated in whole or in part with the processor 112. In some implementations, the data storage 114 can be a single physical device. In other implementations, the data storage 114 can be implemented using two or more physical devices, which can communicate with each other via wired or wireless communications. As previously mentioned, the data storage 114 can include a non-transitory computer-readable medium 120 and data 122. The data 122 can be any type of data from the flow cytometry system 100, such as configuration data, sensor data, and / or diagnostic data, among other possibilities.

[0034] Controller 126 may include one or more electrical circuits, units of digital logic, computer chips, and / or microprocessors configured to interface between any combination of the various components of flow cytometry system 100 (perhaps among other tasks). In some implementations, controller 126 may be an embedded device specially constructed to perform specific operations on one or more subsystems of flow cytometry system 100.

[0035] Control system 118 can monitor and physically change the operating conditions of flow cytometry system 100. In doing so, control system 118 can act as a link between portions of flow cytometry system 100. In some cases, control system 118 can act as an interface between flow cytometry system 100 and another computing device. Additionally, control system 118 can act as an interface between flow cytometry system 100 and a user.

[0036] In some implementations, the control system 118 of the flow cytometry system 100 can also include a communications link 124 configured to transmit and / or receive information. The communications link 124 can transmit data indicative of the status of various components of the flow cytometry system 100. For example, information from two or more side scatter detection modules 110A-110D can be transmitted to a separate device via the communications link 124. Other diagnostic information indicative of the integrity or health of various components of the two or more side scatter detection modules 110A-110D can be transmitted to an external communications device via the communications link 124.

[0037] In some implementations, flow cytometry system 100 can receive information over communication link 124, which is then processed by processor 112. The received information can represent data accessible by processor 112 during execution of instructions stored on non-transitory computer-readable medium 120. Additionally, the received information can alter aspects of controller 126, which can affect operating parameters of various components of flow cytometry system 100. In some cases, the received information can represent a query requesting specific information (e.g., one or more operational states of components of flow cytometry system 100). Processor 112 can then transmit the specific information over communication link 124.

[0038] In some cases, the communication link 124 can include a wired connection. Accordingly, the flow cytometry system 100 can include one or more ports for interfacing the communication link 124 to an external device. In addition to or instead of a wired connection, the communication link 124 can include a wireless connection. Some example wireless connections can utilize cellular connections such as CDMA, EVDO, GSM / GPRS, or 4G communications such as WiMAX or LTE. Alternatively, or in addition, the wireless connection can utilize a Wi-Fi® connection to transmit data to a wireless local area network (WLAN). In some implementations, the wireless connection can also communicate via an infrared link, Bluetooth®, or a near-field communication (NFC) device.

[0039] During operation, control system 118 can communicate with other systems of flow cytometry system 100 via wired or wireless connections, and can be configured to communicate with one or more users of the system. As one possible example, control system 118 can receive input (e.g., from two or more side scatter detection modules 110A-110D of flow cytometry system 100) indicating a change in the operating state of flow cytometry system 100. The input to control system 118 can be received via communication link 124. Based on this input, control system 118 can perform actions that cause flow cytometry system 100 to perform one or more tasks.

[0040] The operations of control system 118 may be performed by processor 112. Alternatively, these operations may be performed by controller 126, or a combination of processor 112 and controller 126. In some implementations, control system 118 may be partially or entirely located on a device other than flow cytometry system 100, and thus may at least partially control flow cytometry system 100 remotely. Remote communications may be performed at least in part using communications link 124.

[0041] As described above, flow cytometry system 100 includes processor 112 in communication with two or more side scatter detection modules 110A-110D and non-transitory computer-readable medium 120 having instructions stored thereon executable to cause processor 112 to perform several functions. In particular, these functions may include (i) detecting a first sample of multiple samples 111 in fluid path 104 at a first timestamp via a first side scatter detection module 110A of the two or more side scatter detection modules, (ii) detecting a first sample of multiple samples 111 in fluid path 104 at a second timestamp that follows the first timestamp via a second side scatter detection module 110B of the two or more side scatter detection modules, and (iii) determining a time delta between a first laser 108A of the two or more lasers and a second laser 108B of the two or more lasers based on a difference between the first plurality of timestamps and the second plurality of timestamps. In one example, the time delta ranges from about 50 μsec to about 200 μsec between any two adjacent lasers of the two or more lasers 108A-108D.

[0042] In one example, the time delta is determined based on a standard deviation of the differences between the first plurality of timestamps and the second plurality of timestamps. In another example, the time delta is determined based on an average of the differences between the first plurality of timestamps and the second plurality of timestamps.

[0043] In one example, a fiber optic cable that collects scattered light signals from a flow cell 102 illuminated by a given one of two or more lasers 108A-108D connects to a given one of two or more side scatter detection modules 110A-110D.

[0044] In one example, the flow cytometry system 100 includes a plurality of photomultiplier tube detectors, a plurality of photodiode detectors, a filter, an analog-to-digital converter, and a field programmable gate array (FPGA). In one such example, the filter includes one or more of a bandpass filter, a longpass filter, and a dichroic filter.

[0045] In one particular example, the plurality of photomultiplier tube detectors and the plurality of photodiode detectors include two lasers (one blue and one red), eight photomultiplier tube detectors (five blue and three red), and three photodiode detectors (one blue forward scatter detector, one blue side scatter detector, and one red side scatter detector). In another example, the plurality of photomultiplier tube detectors and the plurality of photodiode detectors include three lasers (one violet, one blue, and one red), 16 photomultiplier tube detectors (eight violet, five blue, and three red), and four photodiode detectors (one blue forward scatter detector, one violet side scatter detector, one blue side scatter detector, and one red side scatter detector). In another example, the plurality of photomultiplier tube detectors and the plurality of photodiode detectors include four lasers (one violet, one blue, one yellow, and one red), 22 photomultiplier tube detectors (eight violet, five blue, six yellow, and three red), and five photodiode detectors (one blue forward scatter detector, one violet side scatter detector, one blue side scatter detector, one yellow side scatter detector, and one red side scatter detector). These example component counts are given for illustrative purposes only, and other numbers of these features are possible.

[0046] In one example, the number of the two or more lasers 108A-108D is equal to the number of the two or more side scatter detection modules 110A-110D. In one example, the number of the two or more lasers 108A-108D and the number of the two or more side scatter detection modules 110A-110D is 2. In one such example, the first laser 108A is configured to emit only light having red wavelengths, and the second laser 108B is configured to emit only light having blue wavelengths.

[0047] In another example, the number of the two or more lasers 108A-108D and the number of the two or more side scatter detection modules 110A-110D is three. In one such example, the third laser 108C is configured to emit light having only violet wavelengths. In one such example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including (i) detecting a first sample of the plurality of samples 111 in the fluid path 104 via the third side scatter detection module 110C of the two or more side scatter detection modules at a third timestamp that is after the first timestamp, and (ii) determining a second time delta between the first laser 108A of the two or more lasers and the third laser 108C of the two or more lasers based on a difference between the plurality of first timestamps and the plurality of third timestamps.

[0048] In another example, the number of the two or more lasers 108A-108D and the number of the two or more side scatter detection modules 110A-110D is four. In one such example, the fourth laser 108D is configured to emit only light having a yellow wavelength. In one such example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including: (i) detecting a first sample of the plurality of samples 111 in the fluid path 104 via the fourth side scatter detection module 110D of the two or more side scatter detection modules at a fourth timestamp that follows the first timestamp, and determining a third time delta between the first laser 108A of the two or more lasers and the fourth laser 108D of the two or more lasers based on a difference between the plurality of first timestamps and the plurality of fourth timestamps.

[0049] In another example, the number of the two or more lasers 108A-108D and the number of the two or more side scatter detection modules 110A-110D is five. In one such example, the fifth laser is configured to emit light having only ultraviolet wavelengths. In one such example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including: (i) detecting a first sample of the plurality of samples 111 in the fluid path 104 via a fifth side scatter detection module of the two or more side scatter detection modules at a fifth timestamp after the first timestamp, and determining a fourth time delta between the first laser 108A of the two or more lasers and the fifth laser of the two or more lasers based on a difference between the plurality of first timestamps and the plurality of fifth timestamps.

[0050] In another example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including correlating, using a time delta, data corresponding to the first sample detected by the first side scatter detection module 110A with data corresponding to the first sample detected by the second side scatter detection module 110B to create single event data for the first sample.

[0051] In another example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including (i) determining the presence of a separation gas 113 in the fluid in the fluid path 104 based on one or more characteristics of the fluid in the fluid path 104, (ii) generating separation gas timing data including the detected one or more characteristics of the fluid in the fluid path 104 and a corresponding timestamp, and (iii) identifying each sample well of the plurality of sample wells 115 based at least in part on the separation gas timing data.

[0052] In another example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including correlating the first timestamp with a second timestamp to determine a detection event for a first sample of the plurality of samples. In one example, correlating the first timestamp with the second timestamp includes (i) accumulating data from the first side scatter detection module 110A and the second side scatter detection module 110B in separate queues for each side scatter detection module at predetermined time intervals, and (ii) combining the data from the first side scatter detection module 110A and the second side scatter detection module 110B if the first timestamp and the second timestamp are within a threshold time difference.

[0053] Each flow cytometry system 100 may experience some small variations in flow rate due to tubing wear associated with peristaltic or gear pumps, tubing condition, pump output variations from machine to machine, and other conditions. Using two or more side scatter detection modules 110A-110D as described above allows flow cytometry system 100 to adaptively measure the time delta between laser spot positions, allowing it to automatically adapt to fluid flow variations. In addition to the initial time delta calibration, flow cytometry system 100 uses two or more side scatter detection modules 110A-110D to correlate sample data across multiple laser spot positions.

[0054] A time delta calculation is used to synchronize events from lasers that are physically separated on the fluid path 104. Typical time differences between lasers in a flow cytometry system 100 can be approximately 60 μs to 95 μs, but can vary due to differences in flow flow and small differences in mechanical construction and laser point angle. The following algorithm is used to determine the actual time offset between lasers 108A-108D in a flow cytometry system 100 under real-world conditions:

[0055] The calculation uses only the side scatter channel and works best with sparse event streams (low concentrations). The side scatter channel is configured in "threshold mode" so that it is self-triggered. For each event, each laser will produce a separate peak with a timestamp unique to that laser.

[0056] For purposes of explanation, the lowest numbered board will be referred to as board 0. When the algorithm begins, data is collected for 4096 peaks for all installed boards. The data is then sorted by timestamp. The timestamp delta for each board relative to board 0 is then calculated and stored in a table. A histogram is formed for each board, and the time delays corresponding to the peaks in the histogram are used to calculate the mean and standard deviation of the time delay for each board. The number of peaks for each board as well as the standard deviation are further used to indicate the quality of the measurement.

[0057] Below is a description of the algorithm used to calculate the above time delta, according to one non-limiting example.

[0058] System Settings 1. Set all side scatter channels to threshold mode with time offset = 0 and local peak search disabled.

[0059] 2. Set the channel list to VLSSC, BLSSC, YLSSC, RLSSC so that only the side scatter channels for the installed lasers are enabled.

[0060] 3. Configure the system for 32-byte packets.

[0061] 4. Change the network transfer size to smaller packets (4096).

[0062] process Referring to Figure 2, a table is created with three entries: (1) board ID, (2) timestamp, and (3) timestamp delta.

[0063] Next, start the data stream. While streaming, each board is polled in turn, and each peak received is added to a table in the order it was received, along with its board ID and timestamp. Data is collected until the table is filled. Currently, this is set to 4096 peaks, but other numbers of peaks are possible.

[0064] Once the table is filled, (1) sort the table by timestamp, (2) determine the lowest numbered board in the system and call it board 0, and (3) for each event, calculate the time delta relative to board 0 and create a delta-time histogram for each of the boards (currently 1024 points). Then, for each entry for board 0, look for subsequent peaks from other boards, (1) calculate and store the timestamp delta between this and the previous board 0 timestamp, and (2) add the calculated delta to the histogram for that board.

[0065] Using the histogram shown in Figure 2, for each board (except board 0, which in this case corresponds to the violet laser), the delta time for the board corresponding to the peak in the histogram table is obtained. For each non-zero board, the standard deviation is used as a marker of the quality of the delta time. The standard deviation and mean are calculated over a window of + / - 4 μsec. Furthermore, the data can be qualified by requesting a minimum table size / 8 (512) points for each board. The standard deviation and mean delta values ​​can be returned for each board relative to the second laser board.

[0066] Figure 3 shows the data correlation per laser on the field programmable gate array (FPGA) level using a global source trigger.

[0067] Figure 4 shows the search time window to accommodate fluid flow fluctuations. Note that if a second peak is triggered within the sample window, it will be rejected due to coincidence.

[0068] Data Correlation Algorithm Data packets received from a single FPGA are in chronological order. However, transmission between FPGAs is not synchronized. Packets are sent based on network traffic and the buffering capacity on the FPGA board.

[0069] Data from different FPGAs may not arrive in chronological order at the network host, so it is necessary to time-align the separate FPGA packets to ensure peaks are combined into events before being transferred to the ForeCyt analysis software in an ordered format known as FCS data.

[0070] An FCS event consists of peak data from all lasers in the system corresponding to a given timestamp. To create an FCS event, a sorting process is performed. This is achieved by simply holding the data in separate queues for each FPGA for a given time interval (currently 1.00 seconds), then combining packets from different FPGAs with matching timestamps before forwarding them to ForeCyt as an FCS event.

[0071] The data arriving from each FPGA is stored in a separate queue. Note that packets from the FPGA are guaranteed to be incrementally ordered in time. Therefore, each packet in the queue is implicitly ordered by this mechanism, as shown in Figure 5.

[0072] Each queue will receive data from the FPGA as it becomes available. Once enough time has passed to ensure data is available in each queue, a pass is made to peek at the head of each queue and find the lowest timestamp. This timestamp, T, will be used to time-correlate groups of packets. Packets will only be dequeued if their timestamp tolerance is within 10 time steps of T; otherwise, they will remain in the queue. Dequeued packets are then added to a group of correlated packets that will be used to form events, as shown in Figure 6.

[0073] Each group of correlated packets is filtered and formatted into an FCS event, which is then added to a list of FCS events that can be sent to ForeCyt, as shown in Figure 7.

[0074] In one example, there are two software filters that a user can apply to a selected channel before qualifying an FCS event. These software filters specify minimum altitude values ​​for the specified channel that must be met for the event to be accepted. Additionally, the software filters can include a data integrity filter that checks for incomplete events. For example, if data is missing from any valid FPGA (laser), the event is discarded.

[0075] 8A-9C, the probe 106 of the flow cytometry system 100 includes a first end 107 and a second end 109 opposite the first end 107. The second end 109 of the probe 106 is in fluid communication with the fluid path 104. The flow cytometry system 100 may further include a collar washing module 128 disposed adjacent to the probe 106. As shown in FIGS. 8A-9C, the collar washing module 128 includes a top opening 130, a bottom opening 132 in fluid communication with the top opening 130 via a common shaft 134, a first side opening 136, and a second side opening 138 in fluid communication with the first side opening 136 via the common shaft 134.

[0076] 8A-9C, the probe is configured to transition from a first position (FIGS. 8A, 9A) in which the first end 107 of the probe 106 is disposed outside the collar cleaning module 128, to a second position (FIGS. 8B, 9B) in which the first end 107 of the probe 106 is disposed inside the collar cleaning module 128 between the first side opening 136 and the second side opening 138, to a third position (FIGS. 8C, 9C) in which the first end 107 of the probe 106 is disposed outside the collar cleaning module 128. In one example, as shown in FIGS. 8C and 9C, the first end 107 of the probe 106 extends through the bottom opening 132 in the third position. In use, in the third position shown in Figures 8C and 9C, the first end 107 of the probe 106 extends through the bottom opening 132 into the sample well 115 so that the sample 111 can be aspirated and delivered to the flow cell 102 for analysis.

[0077] A fluid is configured to travel between the first side opening 136 and the second side opening 138 when the probe 106 is in the second position, thereby cleaning the probe 106. In particular, the fluid can clean the exterior surface of the probe 106 via the liquid in the collar cleaning module 128 and further clean the fluid path 104 by wicking the liquid from the collar cleaning module 128 through the first end 107 of the probe 106. The fluid can be, by way of non-limiting example, a buffer solution or a decontamination solution. In one example, as shown in FIGS. 8B and 9B , the fluid is configured to enter the first side opening 136 and exit the second side opening 138 when the probe 106 is in the second position, thereby cleaning the probe 106. In another example, the fluid is configured to enter the second side opening 138 and exit the first side opening 136 when the probe 106 is in the second position, thereby cleaning the probe 106.

[0078] The first end 107 of the probe 106 is configured to introduce a plurality of samples 111 (shown in FIG. 1 ) from a plurality of sample wells 115 into the fluid path 104 when the probe 106 is in the third position, thereby forming fluid flow streams in the fluid path 104. The first end 107 of the probe 106 is further configured to introduce aliquots of a separation gas 113 between successive ones of the plurality of samples 111 in the fluid flow stream to configure the fluid flow streams as separation-gas-separated fluid flow streams. In one example, when the probe 106 is in the first position, aliquots of a separation gas 113 are introduced between successive ones of the plurality of samples 111.

[0079] In one example, as shown in Figure 8A, in the first position, the first end 107 of the probe 106 extends through the bottom opening 132 of the collar cleaning module 128. In another example, as shown in Figure 9A, in the first position, the first end 107 of the probe 106 extends through the top opening 130 of the collar cleaning module 128 but above the first side opening 136.

[0080] In one example, the probe 106 is configured to transition between the second and third positions between each of the plurality of sample wells 115. In another example, the probe 106 is configured to transition between the first and second positions multiple times before transitioning to the third position. In one such example, fluid is configured to move between the first and second side openings 136 and 138 each time the probe 106 is in the second position, thereby washing the probe 106 multiple times between successive sample collections. In one example, the number of times the probe 106 enters the second position to be washed between successive sample collections can be selected by a user.

[0081] In one example, the flow cytometry system 100 further includes a pump 119 configured to draw fluid through the color wash module 128 and out a second side opening 138 of the color wash module 128 into a waste container. The color wash module 128 described herein can help reduce sample carryover from one well to another by washing the probe 106 and the interior surfaces of the tubing. Additionally, as mentioned above, the color wash module 128 has the ability to backflush the tubing with sheath fluid from the flow cell by reversing the pump 119, while the backflush fluid in the sample tubing goes from the probe tip to the color wash module 128 and will be aspirated into the waste tank (which will help keep the sample tubing well / clean without clogging issues).

[0082] In one example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including (i) detecting a clog in the fluid pathway 104, and (ii) pumping fluid through the collar cleaning module 128 via the pump 119 to the second end 109 of the probe 106 and into a waste container to remove the clog from the fluid pathway 104.

[0083] In one example, such clogs are detected via one or more air bubble sensors 142 disposed on the fluid path 104. The one or more air bubble sensors 142 can be used to look for unwanted air bubble bursts and to calibrate for volumetric sampling. The one or more air bubble sensors 142 can be configured to record the number of samples and the number of air gaps. If the number of samples and the number of air gaps detected by the one or more air bubble sensors 142 indicate the absence of air gap and / or sample movement, the flow cytometry system 100 can determine that a clog exists in the fluid path 104 and can alert the user and / or automatically initiate a clog removal procedure.

[0084] In one example, the non-transitory computer-readable medium 120 further causes the processor 112 to perform functions including (i) determining the presence of a separation gas 113 in the fluid in the fluid path 104 via one or more bubble sensors 142 disposed on the fluid path 104, (ii) generating separation gas timing data including one or more detected characteristics of the fluid in the fluid path 104 and a corresponding timestamp, and (iii) identifying each sample well of the plurality of sample wells based at least in part on the separation gas timing data.

[0085] 10-12, the flow cytometry system 100 may further include a flow cell module 144. The flow cell module 144 includes the flow cell 102 described above. As shown in FIGS. 10-12, the flow cell module 144 further includes an upper manifold 146 disposed upstream of the flow cell 102. The flow cell module 144 further includes a lower manifold 148 coupled to the upper manifold 146 and movable relative to the upper manifold 146. The flow cell module 144 further includes a sample injection needle 150 having a first end 152 and a second end 154 opposite the first end 152. The flow cell module 144 further includes a needle adjustment member 156 fixedly coupled to the second end 154 of the sample injection needle 150 and movably disposed in the lower manifold 148. At least a portion of the needle adjustment member 156 is internally threaded such that rotation of the needle adjustment member 156 adjusts the position of the first end 152 of the sample injection needle 150 relative to the flow cell 102. In one example, rotating the needle adjustment member 156 in and out of the lower manifold 148 can adjust the first end 152 of the sample injection needle 150 by + / - 10 mm.

[0086] The flow cell module 144 further includes a fluid link connection screw 158 coupled to the needle adjustment member 156. An interior of the fluid link connection screw 158 is configured to receive the fluid pathway 104. In one example, an exterior of at least a portion of the fluid link connection screw 158 includes threads complementary to the threads of the needle adjustment member 156.

[0087] 10, the first end of the flow cell 102 positioned adjacent the first end 152 of the sample injection needle 150 is conical in shape. Such an arrangement can help ensure that the fluid flow through the flow cell is laminar.

[0088] 10-11 , the lower manifold 148 is movable relative to the upper manifold 146 via four adjustment screws 160A-160D configured to move the lower manifold 148 in the x and y directions relative to the upper manifold 146. Because the sample injection needle 150 is fixed relative to the lower manifold 148, such movement further adjusts the position of the first end 152 of the sample injection needle relative to the flow cell 102 in the x and y directions. In contrast, rotation of the needle adjustment member 156 adjusts the position of the first end 152 of the sample injection needle 150 relative to the flow cell 102 in the Z direction.

[0089] 10-11, the lower manifold 148 includes an input port 162 for the sheathed wire. In one example, the upper manifold 146, the lower manifold 148, and the sample injection needle 150 each include stainless steel. In another example, the sample injection needle 150 includes a one-piece design. The sample injection needle 150 can be bonded or welded to the needle adjustment member 156. Rotating the needle adjustment member 156 in the Z direction also rotates the sample injection needle 150 in the Z direction.

[0090] 12, the lower manifold 148 includes a set screw 164 configured to lock the position of the sample injection needle 150 by preventing further rotation of the needle adjustment member 156. When the set screw 164 locks the needle adjustment member 156, it also locks the position of the sample injection needle 150. As shown in FIG. 12, in one example, the set screw 164 includes a nylon or brass ball 166 configured to directly contact the threads of the needle adjustment member 156 to prevent damage to the threads while preventing further rotation of the needle adjustment member 156. Other soft or hard set screw mechanisms are possible.

[0091] In use, the ability to adjust the x, y, and z positions of the first end 152 of the sample injection needle 150 can be useful in the above-described flow cytometry system 100. In particular, the x-y positioning of the sample injection needle 150 helps ensure that multiple samples 111 are centered through the flow cell 102 and into the fluidic path 104, thereby ensuring that two or more lasers 108A-108D illuminate the samples so that two or more side scatter detection modules 110A-110D can detect the multiple samples 111. The z-positioning of the sample injection needle 150 helps ensure laminar flow through the flow cell 102, which is important for the separated gas-separated fluid flow stream of the flow cytometry system 100. If the z-position of the sample injection needle 150 is incorrect, air may become entrained in the sample injection needle 150 or by the inner wall surface of the chamber surrounding the sample injection needle 150, causing a shift in the position of the sample within the fluidic path 104 and thereby misaligning the sample through the flow cell 102. Because flow cytometry system 100 intentionally introduces separation gas between samples, it is useful to ensure layered vias by adjusting the Z position of sample injection needle 150. In one example, data 122 from control system 118 can be used to determine whether XY or Z adjustment of sample injection needle 150 is required.

[0092] It should be understood that the configurations described herein are for example purposes only. Thus, those skilled in the art will recognize that other configurations and other elements (e.g., machines, interfaces, functions, sequences, and groupings of functions) can be substituted, and that some elements can be omitted entirely, depending on the desired results. Furthermore, many of the described elements are functional entities that can be implemented as separate or distributed components, or in conjunction with other components, in any suitable combination and location, or can be combined with other structural elements described as independent structures.

[0093] While various aspects and examples have been disclosed herein, other aspects and examples will be apparent to those skilled in the art. The various aspects and examples disclosed herein are for illustrative purposes only and are not intended to be limiting, with the true scope being indicated by the following claims, along with the full scope of equivalents to which such claims are entitled. It should also be understood that the terms used herein are for the purpose of describing particular examples only and are not intended to be limiting. [Explanation of symbols]

[0094] 100 Flow Cytometry System 102 flow cell 104 Fluid Path 106 Probe 107 First end 108A First Laser 108B Second Laser 108C Third Laser 108D The Fourth Laser 109 Second end 110A First Side Scatter Detection Module 110B Second Side Scatter Detection Module 110C Third Side Scatter Detection Module 110D Fourth Side Scatter Detection Module 111 samples 112 processors 113 Separation Gas 114 Data Storage 115 sample wells 117-well plate 118 Control System 119 Pump 120 Non-transitory computer-readable medium 121 Forward Scatter Detector 122 Data 123 Fluorescence Detector 124 communication links 126 Controller 128 Color Cleaning Module 130 Top opening 132 Bottom opening 134 Common Shaft 136 First side opening 138 Second Side Opening 142 Air bubble sensor 144 Flow Cell Module 146 Upper manifold 148 Lower manifold 150 Sample injection needle 152 first end 154 Second End 156 Needle adjustment member 158 Fluid link connecting screw 160A~160D Adjustment screw 162 input ports 164 Set screw 166 balls

Claims

1. 1. A flow cytometry system, comprising: A flow cell; a fluid pathway in fluid communication with the flow cell; a probe in fluid communication with the fluid path, the probe configured to inject aliquots of a plurality of samples and a separation gas between successive ones of the plurality of samples into the fluid path; two or more lasers positioned such that the illumination spot of each of the two or more lasers is directly on the flow cell; two or more side scatter detection modules in communication with the two or more lasers; a processor in communication with the two or more side scatter detection modules; When using the flow cytometer system, detecting a first sample of the plurality of samples in the fluid path at a first timestamp via a first side scatter detection module of the two or more side scatter detection modules; detecting the first sample of the plurality of samples in the fluid path at a second timestamp after the first timestamp via a second side scatter detection module of the two or more side scatter detection modules; and determining a time delta between a first one of the two or more lasers and a second one of the two or more lasers based on a difference between a plurality of first timestamps and a plurality of second timestamps; a non-transitory computer-readable medium having stored thereon instructions executable to cause the processor to perform functions including: A flow cytometry system comprising:

2. 2. The flow cytometry system of claim 1, wherein the number of said two or more lasers is equal to the number of said two or more side scatter detection modules.

3. 3. The flow cytometry system of claim 2, wherein the number of the two or more lasers and the number of the two or more side scatter detection modules comprises 2, 3, 4, or 5.

4. The flow cytometry system of claim 1 , wherein the time delta is determined based on a standard deviation of the differences between the plurality of first timestamps and the plurality of second timestamps.

5. The flow cytometry system of claim 1 , wherein the time delta is determined based on an average of the differences between the plurality of first timestamps and the plurality of second timestamps.

6. 6. The flow cytometry system of claim 1, wherein a fiber optic cable that collects scattered light signals from the flow cell illuminated by a given one of the two or more lasers connects to a given one of the two or more side scatter detection modules.

7. a plurality of photomultiplier tube detectors, a plurality of photodiode detectors, a filter, an analog-to-digital converter, and a field-programmable gate array; 7. The flow cytometry system of claim 1, further comprising:

8. 8. The flow cytometry system of claim 7, wherein the filter comprises one or more of a bandpass filter, a longpass filter, and a dichroic filter.

9. 9. The flow cytometry system of claim 7 or 8, wherein the plurality of photomultiplier tube detectors and the plurality of photodiode detectors include one of: (i) 8 photomultiplier tube detectors and 3 photodiode detectors; (ii) 16 photomultiplier tube detectors and 4 photodiode detectors; or (iii) 22 photomultiplier tube detectors and 5 photodiode detectors.

10. 10. The flow cytometry system of claim 1, wherein the time delta ranges from about 50 μsec to about 200 μsec between any two adjacent lasers of the two or more lasers.

11. 11. The flow cytometry system of claim 1, wherein the first laser is configured to emit light having only red wavelengths and the second laser is configured to emit light having only blue wavelengths.

12. The non-transitory computer-readable medium comprises: detecting the first sample of the plurality of samples in the fluid path at a third timestamp after the first timestamp via a third side scatter detection module of the two or more side scatter detection modules; and determining a second time delta between the first one of the two or more lasers and a third one of the two or more lasers based on a difference between a plurality of first timestamps and a plurality of third timestamps; 12. The flow cytometry system of claim 11, further causing the processor to perform functions including:

13. 13. The flow cytometry system of claim 12, wherein the third laser is configured to emit light having only violet wavelengths.

14. The non-transitory computer-readable medium comprises: detecting the first sample of the plurality of samples in the fluid path at a fourth timestamp after the first timestamp via a fourth side scatter detection module of the two or more side scatter detection modules; and determining a third time delta between a first one of the two or more lasers and a fourth one of the two or more lasers based on a difference between the first plurality of timestamps and the fourth plurality of timestamps; The flow cytometry system of claim 12 or 13, further comprising causing the processor to perform functions including:

15. 15. The flow cytometry system of claim 14, wherein the fourth laser is configured to emit light having only a yellow wavelength.

16. The non-transitory computer-readable medium comprises: using the time delta to correlate data corresponding to the first sample detected by the first side scatter detection module with data corresponding to the first sample detected by the second side scatter detection module to create single event data for the first sample.

16. The flow cytometry system of claim 1, further comprising:

17. The non-transitory computer-readable medium comprises: determining the presence of a separation gas in the fluid in the fluid path based on one or more characteristics of the fluid in the fluid path; generating separated gas timing data including the detected one or more properties of the fluid in the fluid path and corresponding timestamps; and identifying each sample well of a plurality of sample wells based at least in part on the separation gas timing data; 17. The flow cytometry system of claim 1, further comprising:

18. The non-transitory computer-readable medium comprises: correlating the first timestamp with the second timestamp to determine a detection event for the first sample of the plurality of samples; 18. The flow cytometry system of claim 1, further causing the processor to perform functions including:

19. Correlating the first timestamp with the second timestamp comprises: accumulating data from the first side scatter detection module and the second side scatter detection module in separate queues for each side scatter detection module at predetermined time intervals; and combining data from the first side scatter detection module and the second side scatter detection module if the first timestamp and the second timestamp are within a threshold time difference; 20. The flow cytometry system of claim 18, comprising:

20. A flow cell; a fluid pathway in fluid communication with the flow cell; a probe having a first end and a second end opposite the first end, the second end of the probe in fluid communication with the fluid path; a collar cleaning module disposed adjacent to the probe, the collar cleaning module including a top opening, a bottom opening in fluid communication with the top opening via a common shaft, a first side opening, and a second side opening in fluid communication with the first side opening via the common shaft; Including, the probe is configured to transition from a first position in which the first end of the probe is disposed outside the color cleaning module, to a second position in which the first end of the probe is disposed inside the color cleaning module between the first side opening and the second side opening, to a third position in which the first end of the probe is disposed outside the color cleaning module; when the probe is in the second position, fluid is configured to travel between the first side opening and the second side opening, thereby washing the probe; the first end of the probe is configured to introduce a plurality of samples from a plurality of sample wells into the fluid pathway when the probe is in the third position, thereby creating a fluid flow stream in the fluid pathway; a flow cytometry system, wherein the first end of the probe is configured to introduce an aliquot of a separation gas between successive ones of the plurality of samples in the fluid flow stream to configure the fluid flow stream as a separation-gas-separated fluid flow stream.

21. 21. The flow cytometry system of claim 20, wherein the fluid comprises a buffer.

22. 21. The flow cytometry system of claim 20, wherein the fluid comprises a decontamination solution.

23. 23. The flow cytometry system of claim 20, wherein the probe is configured to transition between the second position and the third position between each of the plurality of sample wells.

24. a pump configured to draw the fluid through the collar cleaning module and out the second side opening of the collar cleaning module to a waste container; 24. The flow cytometry system of any one of claims 20 to 23, further comprising:

25. a processor; When using the flow cytometry system, detecting a blockage in the fluid path; and pumping fluid through the collar cleaning module via a pump to the second end of the probe and to a waste container to remove the clog from the fluid path; a non-transitory computer-readable medium storing instructions executable to cause the processor to perform functions including:

25. The flow cytometry system of claim 24, further comprising:

26. 26. The flow cytometry system of claim 25, wherein the clog is detected via one or more air bubble sensors disposed on the fluid path.

27. a processor; When using the flow cytometry system, determining the presence of the separation gas in the fluid in the fluid path via one or more air bubble sensors disposed on the fluid path; generating separated gas timing data including one or more detected properties of the fluid in the fluid path and corresponding timestamps; and identifying each sample well of the plurality of sample wells based at least in part on the separation gas timing data; a non-transitory computer-readable medium storing instructions executable to cause the processor to perform functions including:

27. The flow cytometry system of any one of claims 20 to 26, further comprising:

28. 1. A flow cell module for a flow cytometry device, comprising: A flow cell; an upper manifold positioned upstream of the flow cell; a lower manifold coupled to the upper manifold and movable relative to the upper manifold; a sample injection needle having a first end and a second end opposite the first end; a needle adjustment member fixedly coupled to the second end of the sample injection needle and movably disposed on the lower manifold, at least a portion of the needle adjustment member being internally threaded such that rotation of the needle adjustment member adjusts the position of the first end of the sample injection needle relative to the flow cell; a fluid link connection screw coupled to the needle adjustment member, an interior of the fluid link connection screw configured to receive a fluid path; a flow cell module comprising:

29. 30. The flow cell module of claim 28, wherein the lower manifold includes a set screw configured to lock the position of the sample injection needle by preventing further rotation of the needle adjustment member.

30. 30. The flow cell module of claim 28 or 29, wherein a first end of the flow cell positioned adjacent to the first end of the sample injection needle is conical in shape.

31. 31. A flow cell module according to any one of claims 28 to 30, wherein the lower manifold is movable relative to the upper manifold via four adjustment screws configured to move the lower manifold relative to the upper manifold in xy directions.

32. 32. The flow cell module of any one of claims 28 to 31, wherein the rotation of the needle adjustment member adjusts the position of the first end of the sample injection needle relative to the flow cell in a Z direction.

33. 33. A flow cell module according to any one of claims 28 to 32, wherein the lower manifold includes an input port for a sheath line.

34. 34. The flow cell module of any one of claims 28 to 33, wherein the upper manifold, the lower manifold, and the sample injection needle each comprise stainless steel.

35. 35. A flow cell module according to any one of claims 28 to 34, wherein the sample injection needle comprises a one-piece design.

36. A flow cytometry system according to any one of claims 1 to 19; A flow cytometry system according to any one of claims 20 to 27; A flow cell module according to any one of claims 28 to 35; A system including:

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