System and method for detecting separated gas in a sample chamber

Abstract

The present invention provides a flow cytometer device, which includes: (a) a flow cell; (b) a fluid path having a second end coupled to a first end of the flow cell; (c) a probe coupled to the first end of the fluid path; (d) at least one sensor configured to detect one or more characteristics of the fluid in the fluid path and positioned between the probe and the first end of the flow cell; (e) a processor in communication with the at least one sensor; and (f) a non-transitory computer-readable medium storing instructions executable to cause the processor to perform functions including: (i) receiving, via the processor, the one or more characteristics of the fluid in the fluid path detected by the at least one sensor, and (ii) determining, based on the one or more detected characteristics of the fluid in the fluid path, the presence of separated gas in the fluid in the fluid path.

Description

[0001] Cross - reference to Related Applications

[0002] This application claims priority to U.S. Application Serial No. 16 / 544,842, filed on August 19, 2020, with the title "System and Method for Separation Gas Detection Between Samples", the content of which is incorporated herein by reference in its entirety. Background Art

[0003] High - throughput flow cytometer systems use a pump system to fill a sample line with a continuous flow of discrete sample particle suspensions that are aspirated from the wells of a microplate and separated from each other by air gaps. The entire sample flow is continuously conveyed to the flow cytometer, whereby data from all samples in the microplate are acquired and stored in a single data file. During data acquisition, high - precision time parameters are also recorded. Time gaps in particle detection are created by air gaps in the data stream, which, when plotted in combination with the time parameters, 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. However, in many cases, these time distributions are not sufficient to accurately identify individual sample wells, and misidentifications sometimes occur. Summary of the Invention

[0004] The present invention discloses methods and systems for detecting bubbles in a continuous fluid flow stream of samples separated by bubbles. In one example, the scattered waveform output of a scatter detector of a flow cytometer is used to detect bubbles.

[0005] Some embodiments of the present disclosure provide a method for: during a period when a flowing stream including a plurality of samples passes through a flow cytometer, generating a voltage output signal by a scatter detector, wherein each of the plurality of samples is separated by a separation gas; sampling the voltage output signal; and recording a timestamp and a voltage value of each sampled voltage of the voltage output signal greater than a separation gap threshold. The method may further include the steps of: before the generating step, moving a plurality of samples including particles into the flowing stream; inserting the separation gas between adjacent samples among the plurality of samples to separate the samples from each other in the flowing stream, and the flowing stream thus constitutes a flowing stream of gas-separated samples; guiding the flowing stream of fluid-separated samples including the separated samples and the separation fluid to and through the flow cytometer; and when the fluid flowing stream passes through the flow cytometer, continuously operating the flow cytometer to concentrate the flowing stream of gas separation and detect scattered light by the scatter detector. In other embodiments, the method may include the steps of: before the moving step, obtaining a plurality of samples from a plate having a plurality of sample wells, wherein each of the plurality of samples is obtained from a corresponding well of the plurality of wells.

[0006] Embodiments of the present disclosure further include a non-transitory computer-readable medium storing instructions that, when executed, cause a processor to perform the methods described herein.

[0007] Other embodiments of the present disclosure include a system comprising: a flow cytometer including a scatter detector; a processor in communication with an output of the scatter detector; and a non-transitory computer-readable medium storing instructions that, when executed, cause the processor to perform the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A is a schematic diagram of a flow cytometer device.

[0009] Figure 1B is Figure 1A a cross-sectional schematic view of a section adjacent to a sample in a conduit of the flow cytometer device of

[0010] Figure 2 An exemplary graph showing a sample event waveform output from a forward scatter detector is shown.

[0011] Figure 3 An exemplary graph showing a bubble gap waveform output from a forward scatter detector is shown.

[0012] Figure 4An exemplary histogram showing sample event data for pre-plate prime air bubbles obtained from a flow cytometer, the first row A of a sample plate with inter-row shaking, and the first two wells of row B of the sample plate, plotted from separated gas timing output data obtained from the forward scatter detector of the flow cytometer.

[0013] Figure 5 is Figure 4 an enlarged view of a portion of.

[0014] Figure 6 is Figure 4 an enlarged view of a portion of.

[0015] Figure 7 An exemplary histogram showing sample event data for a complete 96-well plate obtained from a flow cytometer, plotted from separated gas timing output data obtained from the forward scatter detector of the flow cytometer.

[0016] Figure 8 An exemplary histogram showing sample event data for processed FSC-A output obtained from a flow cytometer, plotted from separated gas timing output data obtained from the forward scatter detector of the flow cytometer.

[0017] Figure 9 An exemplary histogram showing sample event data for processed SSC-A output obtained from a flow cytometer, plotted from separated gas timing output data obtained from the side scatter detector of the flow cytometer.

[0018] Figure 10 A schematic diagram showing an alternative arrangement of a flow cytometer device according to one exemplary embodiment.

[0019] Figure 11 A flowchart showing a method for detecting separated gas in a fluid flow stream according to one exemplary embodiment.

[0020] Figure 12 A schematic diagram showing another alternative arrangement of a flow cytometer device according to one exemplary embodiment.

[0021] Figure 13 A flowchart showing a method for forming a sample fluid flow stream with gas separation according to one exemplary embodiment. Detailed Description

[0022] Unless the context clearly requires otherwise, the words "comprising", "including", and the like in the specification and claims shall be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, the sense of "including but not limited to". The use of the singular or plural words also correspondingly includes the plural or singular.

[0023] The description of embodiments / examples of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. While specific embodiments and examples of the present disclosure are described herein for purposes of illustration, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure.

[0024] All embodiments of any aspect of the present invention may be used in combination unless the context clearly dictates otherwise.

[0025] For the purposes of the present invention, as used herein, the term "sample" refers to any quantity of liquid that may contain particles of interest or labeled particles detectable by a particle analyzer. More specifically, a sample may include a fluid solution or suspension containing particles of interest or labeled particles, which particles will be detected and / or analyzed using the methods and / or devices disclosed herein. The particles of interest in the sample may be labeled, such as with a fluorescent label. The particles of interest may also be conjugated to beads, receptors, or other useful proteins or polypeptides, or may simply exist as free particles, such as particles naturally present in a cell lysate, purified particles from a cell lysate, particles from a tissue culture, etc. The sample may include organic or inorganic chemicals for reacting with the particles of interest. When the particles of interest are biological materials, a drug may be added to the sample to cause a reaction or response in the biological material particles. When the sample is in a sample source well, a chemical, drug, or other additive may be added to and mixed with the sample, or after the sample is aspirated by an autosampler, a chemical, drug, or other additive may be added to the sample in the fluid flow stream.

[0026] As used herein, the term "biological material" refers to any organic material obtained from a living organism, whether alive or dead. The term "biological material" also refers to any synthetic biological material, such as synthetic oligonucleotides, synthetic polypeptides, etc. A synthetic biological material may be a synthetic version of a naturally occurring biological material, or a non-naturally occurring biological material made from parts of a naturally occurring biological material, such as a fusion protein, or two biological materials that have been joined together, such as an oligonucleotide, such as DNA or RNA covalently or non-covalently conjugated to a peptide, which oligonucleotides are not normally conjugated to peptides in nature.

[0027] As used herein, the term "oligonucleotide" refers to any oligonucleotide, including double-stranded and single-stranded DNA, RNA, PNA (peptide nucleic acid), and any nucleic acid sequence, whether natural or synthetic, derivatized or underivatized.

[0028] As used herein, "peptide" refers to all types of peptides and conjugated peptides, including: peptides, proteins, polypeptides, protein sequences, amino acid sequences, denatured proteins, antigens, oncogenes, and partial oncogenes.

[0029] As used herein, the term "organism" refers not only to animals, plants, bacteria, viruses, etc., but also to cell cultures, replicated oligonucleotides, etc., made of organic materials obtained from animals, plants, bacteria, viruses, etc.

[0030] As used herein, the term "drug" refers to any type of substance that is commonly regarded as a drug. A drug can be a substance that acts on the central nervous system of an individual, such as an anesthetic, hallucinogenic, barbiturate, or psychotropic drug. For the purposes of the present invention, a drug can also be a substance that kills or inactivates pathogenic infectious organisms. Additionally, a drug can be a substance that affects the activity or function of specific cells or body organs. A drug can be an organic or inorganic chemical substance, a biological material, etc.

[0031] As used herein, an "aliquot" is a small sample taken from a well via a probe of a flow cytometer.

[0032] As used herein, the term "duct" refers to a device such as a tube, channel, etc. through which a fluid flow passes. A duct can be composed of several individual devices, such as multiple connected or joined pipe fittings or a single-piece pipe fitting, alone or in combination with a channel or other different devices. In various embodiments, the duct can include any tube that can be used with a peristaltic pump having compression characteristics that allow the peristaltic pump to move samples separated from aliquots of separated gas or labeled particles through the tube at a rate of at least 6 samples per minute without causing adjacent samples to mix with each other.

[0033] As used herein, "labeled particle" can include control particles, beads, or microbeads, and further refers to one or more particles that can be detected by a flow cytometer system (e.g., the systems described in U.S. Patent No. 6,878,556 and W02010005617), which can aspirate an aliquot of a sample suspected of containing particles of interest to be analyzed from a sample container.

[0034] For the purposes of the present invention, the term "particle" as used herein refers to small objects that may be present in a sample and detected using a flow cytometer device, including but not limited to biological particles such as molecules, cells, proteins, protein aggregates, cell components such as cell nuclei, and mitochondria, organisms including microorganisms and viruses, microspheres, microbeads, and synthetic particles such as compounds and chemical aggregates, etc.

[0035] For the purposes of the present invention, the term "sample" refers to a fluid solution or suspension that may contain particles of interest.

[0036] For the purposes of the present invention, the term "well" as used herein refers to any structure that contains an aliquot of a sample, control, or labeled particle to be analyzed.

[0037] For the purposes of the present invention, the terms "plate", "microplate", and "microtiter plate" refer to structures that contain aliquots of a sample, control, or labeled particle to be analyzed.

[0038] For the purposes of the present invention, the term "about" means + / - 5% of the parameter.

[0039] For the purposes of the present invention, the term "detector" refers to any detector capable of detecting scattered light, including photomultiplier tubes (PMTs) and single photon avalanche diodes (SPADs).

[0040] For the purposes of the present invention, the term "separating gas" refers to any gas such as air, an inert gas, or a fluid, etc., that can be used to form gas bubbles or immiscible fluids between adjacent samples or between a sample and a buffer fluid. An immiscible fluid is a fluid that is substantially non-miscible with and does not contaminate the sample.

[0041] For the purposes of the present invention, the term "adjacent samples" refers to two samples in a fluid flow stream that are separated from each other only by a separating gas such as a gas bubble.

[0042] For the purposes of the present invention, the term "flow cytometer" includes any flow cytometer device, including but not limited to those described in U.S. Patent Nos. 5,895,764; 5,824,269; 5,395,588; 4,661,913, the entire contents and disclosures of which are incorporated herein by reference. In a flow cytometer, samples can be sorted particle by particle using known methods.

[0043] As used herein, "coupled" means directly and indirectly associated. For example, member A can be directly associated with member B, or can be indirectly associated therewith, for example, via another member C. It will be understood that not all relationships between the various disclosed elements must be presented. Thus, couplings other than those depicted in the block diagrams can also exist.

[0044] Unless otherwise specified, the terms "first", "second", etc. are used herein only as labels and are not intended to impose an order, position, or ranking requirement on the items to which these terms refer. Additionally, the reference to a "second" item, for example, does not require or preclude the presence of a "first" or lower-numbered item and / or a "third" or higher-numbered item, for example.

[0045] As used herein, a system, device, apparatus, structure, article, element, component, or hardware that is "configured to" perform a particular function is capable of performing the particular function without any change, rather than simply having the potential to perform the particular function after further modification. In other words, a system, device, structure, article, element, component, or hardware that is "configured to" perform a particular function is specifically selected, created, implemented, utilized, programmed, and / or designed to perform the particular function. As used herein, "configured to" represents an existing characteristic of a system, device, structure, article, element, component, or hardware that enables the system, device, structure, article, element, component, or hardware to perform the specified function without further modification. For the purposes of this disclosure, a system, device, structure, article, element, component, or hardware described as "configured to" perform a particular function may alternatively or additionally be described as "adapted to" and / or "operable to" perform the function.

[0046] The present disclosure describes a novel system and method for detecting bubbles in a continuous sample fluid stream separated by bubbles using the scattered waveform output of a flow cytometer detector.

[0047] Figure 1A An exemplary flow cytometer device 100 used in conjunction with the present invention is shown. The flow cytometer device 100 includes a conventional autosampler 102 having an adjustable arm 101 with a hollow probe 106 mounted thereon. As the arm 104 moves back and forth ( Figure 1A left and right in []) and left and right ( Figure 1A in and out of the plane of []), the probe 106 descends into a single source well 108 of a microtiter plate 110 to obtain a sample including particles (which may be labeled with a fluorescent tag ( Figure 1A not shown in [])) that will be analyzed using the flow cytometer device 100. Between obtaining sample material from each source well 108, the probe 106 is allowed to aspirate an aliquot of a separating fluid, such as air, thereby forming separating bubbles between successive samples in the fluid flow stream.

[0048] Once a sample is picked up by the probe 106, it is introduced into a fluid flow stream, and the peristaltic pump 112 forces the sample through a conduit 114 that extends from the autosampler 102 through the peristaltic pump 112 and into a flow cytometer 116 that includes a flow cell 118 and a laser interrogation device 120. As the fluid flow stream passes through the flow cytometer, the flow cell 118 can be continuously operated to focus the fluid flow stream and analyze the particles in each of a plurality of samples. The laser interrogation device 120 examines individual samples flowing out of the flow cell 118 at a laser interrogation point 122.

[0049] Figure 1B A series of samples 130, 132, and 134 separated from each other by separation bubbles 136 and 138 in the conduit 114 are shown, forming a bubble-separated fluid flow stream. In Figure 1B it, sample 130 is adjacent to sample 132, and sample 132 is adjacent to sample 134. When samples 130, 132, and 134 pass through the laser interrogation point 122, the particles in the samples are detected by the flow cytometer 116. Forward scattered light is detected by a forward scatter detector 124. Fluorescence emitted by labeled particles in the flow cell is detected by a fluorescence detector 126. Side scattered light is detected by a side scatter detector 128. In contrast, when bubbles 136 and 138 pass through the laser interrogation point 122, no particles are sensed. Thus, a plot of the sensed fluorescence data points of a series of samples analyzed using the flow cytometer versus time will form distinct groups, each group aligned with the time when a sample containing particles passes through the laser interrogation point. Such a plot can be generated from the outputs of the forward scatter detector 124, the fluorescence detector 126, and / or the side scatter detector 128.

[0050] When analyzing and using the output data of a flow cytometer, it is important to correctly identify the sample well from which each sample was taken. In some high-throughput flow cytometer system methods, a sampling protocol including parameters such as probe dip time (the duration of the probe in the well), probe rise time (the amount of time the probe pauses outside the well to aspirate air), multi-well shaking and flushing steps, sampling order, and the height and spacing between event peaks is used to split a data file of an entire microtiter plate into individual well data. However, even with these elements, well identification errors can still occur.

[0051] In one embodiment of the present invention, detection of the separation bubble gap is used to accurately identify individual sample wells. In some cases, this can be used as a supplement or alternative to parameters that have already been used. During a period when a flow stream having a plurality of samples separated by a separation gas passes through a flow cytometer, the separation bubble gap is identified by analyzing the voltage output signal generated by a scatter detector (such as a forward scatter detector 124 or a side scatter detector 128) of the flow cytometer. When the samples (each expected to contain particles of interest) in the flow stream travel through the flow cell of the flow cytometer, each event triggered by a particle generates a fairly consistent scatter waveform pattern having a duration between about 4 μs and 10 μs and a peak-to-peak detector voltage output between about 1.4 volts and 1.6 volts. Figure 2 Shows a sample event waveform obtained via an oscilloscope connected to the forward scatter detector output.

[0052] The separation bubble gap after the sample travels through the flow cell of the flow cytometer also generates a fairly consistent scatter waveform pattern. The waveform pattern is shown in Figure 3 and has a characteristic of having a duration between 50 μs and 90 μs and a peak-to-peak detector voltage output between about 4.2 volts and 4.8 volts, the duration being about seven to nine times greater than the sample event waveform, and the peak-to-peak detector voltage output being three times greater than the sample event waveform. When the bubble passes through the flow cell, it acts as a mirror, reflecting most of the excitation laser to the forward scatter detector. This intensity of the scattered light causes the detector to output a signal with the maximum voltage, as shown by the waveform.

[0053] Using these waveforms, a processor integrated as part of or in communication with the flow cytometer analyzes the voltage output over time to match one of the following signal patterns: background (no event measured), measured event, or bubble measured based on the above waveforms. Then, these patterns can be used to identify each source well in the data stream.

[0054] Specifically, a method for detecting a separation gas in a fluid flow stream includes: (a) during a period when a flow stream including a plurality of samples (each sample separated by a separation gas) passes through a flow cytometer, generating a scatter voltage output signal indicative of the intensity of scattered light with a scatter detector, (b) sampling the scatter voltage output signal, and (c) recording the timestamp and voltage value of each sampled voltage of the scatter voltage output signal that is greater than a separation gap threshold. In one example, each of the plurality of samples is suspected of containing particles of interest. The method may also include comparing each sampled voltage of the scatter voltage output signal with the separation gap threshold.

[0055] In operation, the processor samples the voltage output signal of the scatter detector and records the voltage values greater than the separation gap threshold. In some instances, each sampled voltage of the voltage output signal is compared with the separation gap threshold. In some instances, the separation gap threshold has a value that is at least twice greater than the maximum voltage output of the plurality of samples, which may depend on the type of flow cytometer and the electronics of the forward scatter detector. In conjunction with the experimental data given below, the maximum voltage that can be detected by the forward scatter detector is 5V, and a separation gap threshold of 3.9V was selected (corresponding to (800 / 1023)*5V). This threshold is more than twice the maximum expected sample output of 1.6V from the forward scatter detector. Additionally, the voltage output signal is sampled at a frequency. In some instances, the sampling frequency is between 5 kHz and 500 kHz. In another instance, a sampling frequency of up to approximately 10 MHz is used.

[0056] The analysis software algorithm executed by the processor can consist of two parts, initial time correlation and bubble gap event timing, to delineate a single microplate well from a continuous flow cytometer data stream. The bubble gap event timing algorithm can be used in conjunction with other well identification parameters, such as those described above.

[0057] When collecting scatter data, a timestamp is recorded at the time when each sampled voltage above the threshold occurs. Thus, the flow cytometer system or a processor integrated therein or communicating therewith can also include a clock. This timestamp will be used to correlate the detected pattern with the data stream from the flow cytometer. The flow cytometer system can also include or communicate with a memory in which the sampled voltage values above the threshold and the timestamps are recorded.

[0058] Additionally, at the start of a microplate sampling run, a start time calibration sequence can be performed before sampling the first microplate well. In such an instance, the method can further include: before the generating step, moving a plurality of samples including particles into a flow stream; inserting a separation gas between adjacent samples among the plurality of samples to separate the samples from each other in the flow stream, which thereby constitutes a gas-separated sample flow stream; guiding the fluid-separated sample flow stream including the separated samples and a separation fluid to and through the flow cytometer; and continuously operating the flow cytometer as the fluid flow stream passes through the flow cytometer to focus the gas-separated flow stream and detect scattered light by the scatter detector. In such an instance, the method can further include: before the moving step, obtaining a plurality of samples from a plate having a plurality of sample wells, where each of the plurality of samples is obtained from a corresponding well of the plurality of wells.

[0059] In one particular instance, three separate bubble gaps are introduced, each separated by dipping in deionized water for one second followed by eight seconds of deionized water. When sample event data acquisition begins from the flow cytometer, the bubble gap detector microprocessor starts with a timestamp of zero. Using this calibration sequence, the timestamp output (separated gas timing data) of the bubble gap detector can be correlated with the flow cytometer sample event data timing to synchronize the start of the plate sampling run. In operation, separated gas timing data is generated from the captured scatter voltage signal and corresponding timestamp, which is applied when the output of the scatter detector exceeds a set voltage threshold. Based on the timing, this separated gas timing data is synchronized with the sample event data from the flow cytometer. The separated gas timing data is plotted using a sample event versus time histogram for well identification. Thus, the bubble detection pattern timing output can be used to delineate the bubble gaps between wells where microbubbles, debris, insufficient sample, sample preparation errors, or artifacts make it difficult to do so using only event counts over time.

[0060] In one instance, the scatter detector includes a forward scatter detector, as described in more detail below in conjunction with Figures 4 to 8 In another instance, the scatter detector includes a side scatter detector, as described in more detail below in conjunction with Figure 9 In an embodiment where the scatter detector includes a forward scatter detector, the method may further include: generating a side scatter voltage output signal indicative of the intensity of side scatter light with a side scatter detector during a period in which a flow stream including a plurality of samples (each sample separated by separated gas) passes through the flow cytometer; generating a fluorescence voltage output signal indicative of the intensity of emitted fluorescence light with a fluorescence detector during a period in which a flow stream including a plurality of samples (each sample separated by separated gas) passes through the flow cytometer; and generating sample event data based at least in part on the forward scatter voltage output signal, the side scatter voltage output signal, and the fluorescence voltage output signal.

[0061] The exemplary bubble detector of the present invention was experimentally tested by first measuring the forward scatter flow cytometer waveforms of bubbles and samples and determining a method for differentiating between the two waveforms. In Figures 4 to 7 The resulting separated gas timing data is shown, plotted using a sample detection data histogram. The flow cytometer generates sample detection data based on the outputs of the forward scatter detector, the side scatter detector, and the fluorescence detector. The number of wells identified by the well identification algorithm and the total number of wells in the sample plate are shown at the top of the histogram. In these plots, the tall vertical lines on the time histogram are associated with bubbles passing through the flow cell, and the short vertical lines are associated with the number of events in the sample. In Figure 4Detection of separated bubbles can be seen particularly well in the pre-plate startup sequence shown, which also shows the detector output from sampling the first row A of the well plate, followed by shaking the microplate to resuspend any particles in the sample, inter-row shaking, and the detector output from sampling the first two wells of row B. Figure 5 yes Figure 4 A close-up view of a portion of a well plate, specifically, the detector output from sampling the first row A of the well plate, followed by microplate shaking, and the detector output from sampling the first two wells of row B. Figure 6 Too Figure 4 A close-up view of a portion of the detector, particularly the detector output from sampling the first six wells of row A of the well plate. Figure 7 is a histogram of the detector output from a full 96-well plate sampled. In some cases, the control software used to operate the flow cytometer device can allow the user to program a customized sampling protocol, which can include, for example, a set of probe washes and / or microplate shaking sequences performed after a certain number of wells have been sampled. In the illustrated example, the 96-well microplate is sampled row by row, and the microplate is shaken after each row.

[0062] exist Figures 4 to 7 In each of, each door marked with letters and numbers corresponds to the corresponding hole of the orifice plate identified by the method of the present disclosure. Compared with the aforementioned method, this new method of detecting bubble gaps using forward scattering output limits hole identification errors. The forward scattering waveform analysis used in conjunction with the sampling protocol described herein can allow accurate identification and verification of sampling protocol features, such as row or column plate shaking and probe flushing, and bubble gaps between samples, regardless of the detection of the sample itself. In the forward scattering waveform analysis, the sample is depicted using the detected bubble sequence rather than the low event count sequence between the samples. This can eliminate possible errors that may occur in the case of low event count sequences in the sample, which may be due to, for example, sample preparation errors, incorrectly distributed samples, samples with very few particles of interest (e.g., toxicity assays), insufficient resuspension of samples, and blockage of fluid sample tubes. In addition, forward scattering waveform analysis can provide real-time feedback on the consistency of bubble gaps in the flow cell, which can be used to detect blockage of the fluid path from the sample probe to the flow cell.

[0063] Figure 8 An exemplary histogram of sample event data of processed FSC-A output acquired from a flow cytometer is shown, plotted with separated gas timing output data acquired from the forward scatter detector of the flow cytometer. Figure 8In the illustrated example, the flow cytometer detector itself (not an external device) converts the voltage output PMT from forward scatter into a processed FSC-A digital output value for each event. The boundary between the separated gas and the liquid sample will cause one or more events with FSC-A values to be at the upper limit of detection (e.g., the highest value generated by the flow cytometer), thereby forming a gate around the highest FSC-A event value, as Figure 8 shown. The flow cytometer also records a timestamp for each event recorded. Using this information, based on timing, the separated gas timing data is synchronized with the sample event data from the flow cytometer. The sample events for well identification are used to plot the off-gas timing data against a time histogram. Thus, the bubble detection pattern timing output can be used to depict the bubble gaps between wells, where microbubbles, debris, insufficient sample, sample preparation errors, or remnants make it difficult to do this using only event counts over time.

[0064] Figure 9 An exemplary histogram of sample event data showing the processed SSC-A output obtained from a flow cytometer, plotted with the separated gas timing output data obtained from the side scatter detector of the flow cytometer. In Figure 9 the illustrated example, the flow cytometer detector itself (not an external device) converts the voltage output PMT from side scatter into a processed SSC-A digital output value for each event. The flow cytometer also records a timestamp for each event recorded. Using this information, based on timing, the separated gas timing data is synchronized with the sample event data from the flow cytometer. The sample events for well identification are used to plot the off-gas timing data against a time histogram.

[0065] Figure 10 Another exemplary flow cytometer device 200 used in conjunction with the present invention is shown. In particular, as Figure 10 shown, the flow cytometer device 200 includes a flow cell 202 having a first end 204 and a second end 206. The flow cytometer device 200 also includes a fluid path 208 having a first end 210 and a second end 212. As Figure 10 shown, the second end 212 of the fluid path 208 is coupled to the first end 204 of the flow cell 202. The flow cytometer device 200 also includes a probe 214 coupled to the first end 210 of the fluid path 208. As Figure 10 shown, the flow cytometer device 200 also includes at least one sensor 216 positioned between the probe 214 and the first end 204 of the flow cell 202. The sensor 216 is configured to detect one or more characteristics of the fluid in the fluid path 208. Although Figure 10A single sensor 216 is shown, but additional sensors may be positioned between the probe 214 and the first end 204 of the flow cell 202. Additionally, one or more additional sensors may also be positioned downstream of the flow cell 202.

[0066] Exemplary probe 214 may include a 0.01-inch ID, 1 / 16-inch OD stainless steel needle compatible with HPLC ferrule fittings. In one embodiment, to reduce sample residue between pores, probe 214 may have a tapered tip. In another embodiment, silicone or other hydrophobic agents may be coated on the tip of sampling probe 214 to help minimize sample carryover. In alternative embodiments, the entire probe 214 may be made of hydrophobic material to reduce carryover. Suitable hydrophobic materials for coating or fabricating the entire hydrophobic probe include: (polytetrafluoroethylene (PTFE)), (polyvinylidene fluoride), (ethylene-tetrafluoroethylene copolymer), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer resin (PFA), tetrafluoroethylene-hexafluoropropylene copolymer (EFP), polyetheretherketone (PEEK), etc.

[0067] As Figure 10 shown, the flow cytometer device 200 may include one or more processors 218, a data memory 219, and one or more controllers 221, which together may be part of a control system 223. One or more processors 218 may operate as one or more general-purpose hardware processors or dedicated hardware processors (e.g., digital signal processors, application-specific integrated circuits, etc.). One or more processors 218 may be configured to execute a non-transitory computer-readable medium 220 and manipulate data 225, both of which are stored in the data memory 219. One or more processors 218 may also interact directly or indirectly with other components of the flow cytometer device 200, such as, by way of non-limiting example, the sensor 216 and / or the communication link 227.

[0068] The data memory 219 can be one or more types of hardware memories. For example, the data memory 219 can include or take the form of one or more computer-readable storage media that can be read or accessed by one or more processors 218. 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 memories, which can be integrated with the one or more processors 218, either wholly or in part. In some embodiments, the data memory 219 can be a single physical device. In other embodiments, two or more physical devices can be used to implement the data memory 219, and the physical devices can communicate with each other via wired or wireless communication. As previously described, the data memory 219 can include the non-transitory computer-readable medium 220 and the data 225. The data 225 can be any type of data from the flow cytometer device 200, such as configuration data, sensor data, and / or diagnostic data, among other possibilities.

[0069] The controller 221 can include one or more circuits, digital logic units, computer chips, and / or microprocessors that are configured to interface (possibly among other tasks) between any combination of the various components of the flow cytometer device 200. In some embodiments, the controller 221 can be a dedicated embedded device that is used to perform specific operations with one or more subsystems of the flow cytometer device 200.

[0070] The control system 223 can monitor and physically change the operating conditions of the flow cytometer device 200. In doing so, the control system 223 can act as a link between parts of the flow cytometer device 200. In some cases, the control system 223 can act as an interface between the flow cytometer device 200 and another computing device. Additionally, the control system 223 can act as an interface between the flow cytometer device 200 and the user.

[0071] In some embodiments, the control system 223 of the flow cytometer device 200 can also include one or more communication links 227 that are configured to send and / or receive information. The one or more communication links 227 can transmit data indicating the status of the various components of the flow cytometer device 200. For example, the information read by the sensor 216 can be transmitted via the one or more communication links 227 to a separate device. Other diagnostic information indicating the integrity or health of the various components of the flow cytometer device 200 can be transmitted via the one or more communication links 227 to an external communication device.

[0072] In some embodiments, the flow cytometer device 200 may receive information at one or more communication links 227, which is then processed by one or more processors 218. The received information may indicate data accessible to one or more processors 218 during the execution of instructions stored by the non-transitory computer-readable medium 220. Additionally, the received information may alter aspects of one or more controllers 221, which may affect the operating parameters of various components of the flow cytometer device 200. In some cases, the received information may indicate a query requesting specific information (e.g., the operating status of one or more components of the flow cytometer device 200). One or more processors 218 may then transmit the specific information back outside of the one or more communication links 227.

[0073] In some cases, one or more communication links 227 may include a wired connection. Thus, the flow cytometer device 200 may include one or more ports for docking the one or more communication links 227 to an external device. In addition to or as an alternative to the wired connection, one or more communication links 227 may include a wireless connection. Some exemplary wireless connections may utilize cellular connections such as CDMA, EVDO, GSM / GPRS, or 4G telecommunications such as WiMAX or LTE. Alternatively or additionally, the wireless connection may utilize a Wi-Fi connection to transmit data to a wireless local area network (WLAN). In some embodiments, the wireless connection may also communicate via an infrared link, Bluetooth, or a near field communication (NFC) device.

[0074] During operation, the control system 223 may communicate with other systems of the flow cytometer device 200 via a wired or wireless connection and may further be configured to communicate with one or more users of the system. As a possible example, the control system 223 may receive an input indicating a change in the operating status of the flow cytometer device 200 (e.g., from a sensor 216 of the flow cytometer device 200). Input to the control system 223 may be received via one or more communication links 227. Based on this input, the control system 223 may perform operations to cause the flow cytometer device 200 to perform one or more tasks.

[0075] The operations of the control system 223 may be performed by one or more processors 218. Alternatively, these operations may be performed by the controller 221 or a combination of one or more processors 218 and the controller 221. In some embodiments, the control system 223 may reside partially or entirely on a device different from the flow cytometer device 200 and may thus at least partially remotely control the flow cytometer device 200. Remote communication may be performed at least in part using one or more communication links 227.

[0076] As described above, the flow cytometer device 200 includes a processor 218 in communication with a sensor 216, and a non-transitory computer-readable medium 220 storing instructions executable to cause the processor 218 to perform functions. In particular, these functions can include: (i) receiving, via the processor 218, one or more characteristics of the fluid in the fluid path 208 detected by the sensor 216, and (ii) determining the presence of separated gas in the fluid in the fluid path 208 based on the one or more characteristics of the fluid in the detected fluid path 208.

[0077] The processor 218 can transmit the separated gas detection data to a processed data channel that is combined with other detector data channels, such as, by way of non-limiting example, a forward scatter detector 124, a fluorescence detector 126, and / or a side scatter detector 128. In this way, the separated gas presence data can be processed and integrated with sampling protocol information as part of a well identification algorithm. The processor 218 can be configured to correct a known time offset between flow cell event triggering and sensor triggering. The time offset can be adaptive based on the sample flow rate. Additionally, this time offset accounts for the position of the sensor 216 leading the other sensors in the data stream.

[0078] In one instance, the sensor 216 is directly coupled to the fluid path 208. In another instance, the sensor 216 is positioned adjacent to the fluid path 208 without physically contacting the fluid path 208. The sensor 216 can be positioned between about 0.10 inches and about 48 inches from the first end 204 of the flow cell 202, between about 0.10 inches and about 24 inches from the first end 204 of the flow cell 202, between about 0.10 inches and about 12 inches from the first end 204 of the flow cell 202, between about 0.10 inches and about 6 inches from the first end 204 of the flow cell 202, between about 0.10 inches and about 4 inches from the first end 204 of the flow cell 202, or between about 0.10 inches and about 2 inches from the first end 204 of the flow cell 202. Positioning the sensor 216 closer to the flow cell 202 can provide improved accuracy for detecting separated gas in the fluid path 208. As discussed above, although Figure 10 a single sensor 216 is shown, one or more additional sensors can be positioned between the probe 214 and the first end 204 of the flow cell 202. Additionally, one or more additional sensors can also be positioned downstream of the flow cell 202.

[0079] As Figure 10 shown, the flow cytometer device 200 can also include a pump 222 in fluid communication with the fluid path 208. Although in Figure 10The middle pump 222 is shown before the flow cell 202, but in another embodiment, the pump 222 can be positioned downstream of the flow cell 202. In a particular instance, the pump 222 includes a peristaltic pump. An exemplary peristaltic pump is the Gilson Minipuls3, but other exemplary peristaltic pumps can also be used. In one embodiment, such a peristaltic pump can be operated in a manner that reduces pulsatile flow, thereby improving the sample characteristics in the flow cytometer device 200. In another embodiment, the pump 222 includes an injection pump. Additionally, additional pumps can be added to the flow cytometer device 200 to perform various functions. For example, a combination of one or more peristaltic pumps and one or more injection pumps can be used to transport the sample through the fluid path 208.

[0080] In one embodiment, the fluid path 208 can be made of elastomeric tubing, such as nitrile (NBR), chlorosulfonated polyethylene, fluororubber, silicone, polyvinyl chloride (“PVC”), ethylene propylene diene monomer (“EPDM”), EPDM + polypropylene, polyurethane, or natural rubber, among other possibilities. An example of such tubing can be polyvinyl chloride (PVC) tubing having an inner diameter of about 0.01 inches to 0.03 inches and a wall thickness of about 0.01 inches to 0.03 inches. In one embodiment, the preferred tubing for the fluid path can be PVC tubing having an inner diameter of about 0.02 inches and a wall thickness of about 0.02 inches.

[0081] In one embodiment, the flow cytometer device 200 can include an autosampler 224 coupled to the probe 214. A particular non - limiting example of an autosampler 224 that can be used with the flow cytometer device 200 is the Gilson 215 Liquid Handler. In one embodiment, the autosampler 224 can include an adjustable arm 226. As the adjustable arm 226 of the autosampler 224 moves left - right and up - down, the probe 214 descends into a single sample well 228 of the microplate 230 to obtain a sample that has been labeled with labeled particles, which will be analyzed using the flow cytometer device 100. Further, the probe 214 is coupled to the first end 210 of the fluid path 208, and the pump 222 is in fluid communication with the fluid path 208.

[0082] In operation, for example, the probe 214 can obtain a sample 229 from a sample well 228 in the microtiter plate 230 and then advance the sample 229 into the fluid path 208. The pump 222 can then drive a fluid flow stream 234 including the sample 229 from the well 228 through the fluid path 208 to the flow cell 202. In this embodiment, the sensor 216 is in fluid communication with the autosampler 224 via the fluid path 208, and the flow cytometer device 200 is configured to focus the fluid flow stream 234 delivered from the autosampler 224 through the fluid path 208 and selectively analyze particles in each of a plurality of samples 229 as the fluid flow stream 234 passes by the sensor 216 and the flow cell 202. In one embodiment, the flow cytometer device 200 further includes a laser interrogation device 236 positioned downstream of the flow cell 202. The laser interrogation device 236 is configured to examine individual samples flowing out of the flow cell 202 at a laser interrogation point.

[0083] As Figure 10 shown, the fluid flow stream 234 can include a series of samples 229, each separated by a separation gas 238, such as, by way of non-limiting example, a bubble. The separation gas 238 can be formed by allowing the probe 214 to aspirate air (or other gas) between sample materials from each sample well 228. In this way, the autosampler 224 and the pump 222 cooperate to introduce aliquots of the separation gas 238 between successive samples 229 in the fluid flow stream 234 so as to configure the fluid flow stream 234 as a separation gas-separated fluid flow stream.

[0084] The sensor 216 can take various forms. In one example, the sensor 216 includes an ultrasonic sensor. In such an example, one or more characteristics of the fluid in the fluid path 208 detected by the sensor 216 include the density of the fluid. The non-transitory computer-readable medium 220 can store first density data corresponding to a range of density values for the sample and can also store second density data corresponding to a range of density values for air. The processor 218 can compare the density measured by the ultrasonic sensor with the stored first density data and second density data to determine whether separation gas is present in the fluid in the fluid path 208.

[0085] In another example, sensor 216 includes an ultrasonic sensor. In such an example, one or more characteristics of the fluid in fluid path 208 detected by sensor 216 include the reflection of light passing through the fluid path 208. In particular, the optical sensor measures the light transmission through the conduit of fluid path 208 and looks for differences between air reflections and sample reflections. As such, the non-transitory computer-readable medium 220 can store first reflection data corresponding to a range of reflection values for the sample and can also store second reflection data corresponding to a range of reflection values for air. The processor 218 can compare the reflection measured by the optical wave sensor with the stored first and second reflection data to determine whether a separated gas is present in the fluid in fluid path 208.

[0086] In another example, sensor 216 includes an image sensor. In such an example, one or more characteristics of the fluid in fluid path 208 detected by sensor 216 include an image of the fluid in fluid path 208 captured by the image sensor. The image sensor can be a camera, such as a charge-coupled device or a complementary metal-oxide semiconductor (CMOS) sensor. The image sensor uses the camera and image processing to record the sample and extract information from the fluid flow stream 234. In particular, the non-transitory computer-readable medium 220 can store first image data corresponding to a known image of the sample and can also store second image data corresponding to a known image of the separated gas. The processor 218 can compare the image data from the image sensor with the stored first and second image data to determine whether a separated gas is present in the fluid in fluid path 208.

[0087] In yet another example, sensor 216 includes a mass flow sensor. In such an example, one or more characteristics of the fluid in fluid path 208 detected by sensor 216 include the temperature of the fluid in fluid path 208. In particular, one end of the mass flow sensor can be configured to heat the sample, and the mass flow sensor determines how much the sample has cooled when it reaches the other end of the mass flow sensor. Since the sample will heat and cool at a different rate than air, the temperature difference can indicate whether there is a separated gas in the fluid path. In particular, non-transitory computer-readable medium 220 can store first temperature data corresponding to a known temperature drop range between opposite ends of the mass flow sensor when the sample is heated at one end of the mass flow sensor. Non-transitory computer-readable medium 220 can also store second temperature data corresponding to a known temperature drop range between opposite ends of the mass flow sensor when the separated gas is heated at one end of the mass flow sensor. Processor 218 can compare the temperature drop detected from the mass flow sensor with the stored first and second temperature data to determine whether there is a separated gas in the fluid in fluid path 208.

[0088] In one embodiment, non-transitory computer-readable medium 220 causes processor 218 to further perform functions including: (i) generating separated gas timing data including one or more characteristics of the fluid in detected fluid path 208 and corresponding timestamps, and (ii) identifying a corresponding sample well among a plurality of sample wells 228 at least in part based on the separated gas timing data, as discussed in further detail above.

[0089] In another embodiment, non-transitory computer-readable medium 220 causes processor 218 to further perform functions including: (i) generating a scattered voltage output signal indicative of the intensity of scattered light with a scatter detector during a period when the fluid passes through flow cytometer device 200, and (ii) sampling the scattered voltage output signal, wherein it is further determined at least in part based on the sampled scattered voltage output signal that there is a separated gas in the fluid in fluid path 208, as discussed in further detail above.

[0090] In another embodiment, non-transitory computer-readable medium 220 causes processor 218 to further perform functions including: recording a timestamp and the voltage value of each sampled voltage of the scattered voltage output signal that is greater than a separated gas threshold, wherein the separated gas threshold has a constant value, as discussed in further detail above.

[0091] In another embodiment, the non-transitory computer-readable medium 220 causes the processor 218 to further perform functions, the functions including: (i) determining a first timestamp at which a sample begins in the fluid path 208 based on one or more characteristics of the fluid in the detected fluid path 208; (ii) determining a second timestamp at which the sample ends in the fluid path 208 based on one or more characteristics of the fluid in the detected fluid path; and (iii) determining the volume of the sample based at least in part on the first timestamp, the second timestamp, the flow rate of the fluid in the fluid path 208, and the diameter of the fluid path.

[0092] In another embodiment, the non-transitory computer-readable medium 220 causes the processor 218 to further perform functions, the functions including: determining that there are labeled particles present in the fluid in the fluid path 208 based on one or more characteristics of the fluid in the detected fluid path 208. In one example, the presence of labeled particles in the fluid in the fluid path 208 can be indicated by physical or chemical characteristics, such as the fluorescence intensity of the sample. The presence of labeled particles in the fluid in the fluid path 208 can be used to determine the boundaries of the sample in the fluid path 208. Additionally, the labeled particle data can provide a comparison of the relative fluorescence of the fluorescently labeled particles to the processed cells as a consistency parameter for samples obtained from wells within one or more plates of a flowing sample stream in an experiment recorded in a single data file. Thus, data obtained from multiple plates can be normalized to the labeled particles, allowing for direct comparison of results across large experimental data sets.

[0093] In another embodiment, the non-transitory computer-readable medium 220 causes the processor 218 to further perform functions, the functions including: (i) determining a count of the number of separated bubbles detected in the fluid in the fluid path 208 based on one or more characteristics of the fluid in the detected fluid path 208 over a period of time, and determining that the flow cytometer device 200 is blocked if the count is below a minimum count tolerance. In one embodiment, the count tolerance can be at least fifteen separated bubbles detected per second. Other count tolerances are possible.

[0094] Figure 11 is a flow chart of a method for detecting separated gas in a fluid flow stream according to an exemplary embodiment. The embodiment can be performed by the flow cytometer device 200 as described above with respect to Figure 10 the flow cytometer device 200 described. The method 300 can include one or more operations, functions, or actions as shown in one or more of blocks 302 - 312. Although these blocks are shown in a sequential order, these blocks can also be performed in parallel, and / or in an order different from that described herein. Additionally, multiple blocks can be combined into fewer blocks, split into additional blocks, and / or deleted based on the goal to be achieved.

[0095] In addition, for the method 300 and other processes and operations disclosed herein, block diagrams illustrate possible embodiments. In this regard, each block may represent a module, segment, or portion of program code, which includes one or more instructions executable by a processor or computing device for implementing a particular logical operation. The program code may be stored on any type of computer-readable medium, such as a storage device including, for example, a magnetic disk or a hard disk drive. The computer-readable medium may include a non-transitory computer-readable medium, such as a computer-readable medium that stores data for a short time, such as register memory, processor cache, and random access memory (RAM). For example, the computer-readable medium may also include secondary or persistent long-term memory, such as read-only memory (ROM), optical disk or magnetic disk, and compact disc read-only memory (CD-ROM). The computer-readable medium may also be any other volatile or non-volatile storage system. The computer-readable medium may be considered, for example, a computer-readable storage medium or a tangible storage device.

[0096] In addition, Figure 11 each block in may represent a circuit wired to perform a particular logical operation.

[0097] At block 302, method 300 includes: obtaining a plurality of samples from a plate having a plurality of sample wells, wherein each sample of the plurality of samples is obtained from a corresponding well of the plurality of sample wells. At block 304, method 300 includes moving the plurality of samples into a fluid flow stream in a fluid path. At block 306, method 300 includes inserting a separation gas between adjacent samples of the plurality of samples to separate the plurality of samples from each other in the fluid flow stream, and the fluid flow stream thus constitutes a sample fluid flow stream separated by the separation gas. At block 308, method 300 will include guiding the gas-separated sample fluid flow stream including the separated samples and the separation fluid to and through at least one sensor and a flow cell of a flow cytometer, wherein at least one sensor is positioned upstream of the flow cell. At block 310, method 300 includes detecting one or more characteristics of the fluid flow stream in the fluid path via at least one sensor. At block 312, method 300 includes determining the presence of a separation gas in the fluid flow stream in the fluid path based on the one or more characteristics of the fluid flow stream detected in the fluid path.

[0098] In one example, as discussed above with respect to Figure 10 , the plurality of samples are obtained from a plate having a plurality of sample wells via an autosampler coupled to a probe. In addition, as discussed above with respect to Figure 10 , at least one sensor 216 may include one or more of an ultrasonic sensor, an optical sensor, an image sensor, and a mass flow sensor.

[0099] In another embodiment, method 300 further includes: (i) generating separation gas timing data including one or more characteristics of a fluid flow stream detected in a fluid path and corresponding timestamps, and (ii) identifying a corresponding sample well among a plurality of sample wells based at least in part on the separation gas timing data.

[0100] In yet another example, method 300 further includes: (i) generating a scattered voltage output signal indicative of an intensity of scattered light with a scatter detector during a period when a fluid flow stream passes through a flow cytometer, (ii) sampling the scattered voltage output signal, and (iii) recording a timestamp and a voltage value for each sampled voltage of the scattered voltage output signal that is greater than a separation gap threshold, where the separation gap threshold has a constant value.

[0101] Figure 12 Another exemplary flow cytometer device 400 for use in connection with the present invention is shown. In particular, as Figure 12 shown, flow cytometer device 400 includes a flow cell 402 having a first end 404 and a second end 406. Flow cytometer device 400 further includes a fluid path 408 having a first end 410 and a second end 412. As Figure 12 shown, the second end 412 of fluid path 408 is coupled to the first end 404 of flow cell 402. Flow cytometer device 400 further includes a probe 414 coupled to the first end 410 of fluid path 408. Probe 414 may be configured similar to probe 214 described above with respect to Figure 10 As Figure 12 shown, flow cytometer device 400 further includes a sensor 416A positioned between probe 414 and the first end 404 of flow cell 402. Sensor 416A is configured to detect one or more characteristics of a fluid in fluid path 408. Sensor 416A may take various forms as discussed above with respect to Figure 10 discussed.

[0102] As Figure 12 shown, flow cytometer device 400 further includes a valve 417 positioned between sensor 416A and flow cell 402. Valve 417 is configured to move between a first position (shown as position A in Figure 12 ) and a second position (shown as position B in Figure 12 ), in the first position, the fluid flow stream in fluid path 408 is directed to the first end 404 of flow cell 402, and in the second position, the fluid flow stream in fluid path 408 is directed to a waste port 415. In one example, valve 417 includes a shear valve. In another example, valve 417 includes a rotary valve. Other valves are possible.

[0103] As Figure 12As shown, the flow cytometer device 400 further includes an auxiliary sheath fluidic pathway 431 in fluid communication with valve 417, a first sheath port 433 in fluid communication with flow cell 402, and a second sheath port 435 in fluid communication with flow cell 402. Each of the auxiliary sheath fluidic pathway 431, the first sheath port 433, and the second sheath port 435 provides sheath fluid to various components of the flow cytometer device 400, as discussed in further detail below.

[0104] As Figure 12 As shown, the flow cytometer device 400 may include one or more processors 418, a data memory 419, and one or more controllers 421, which together may be part of a control system 423. The one or more processors 418 may operate as one or more general-purpose hardware processors or as dedicated hardware processors (e.g., digital signal processors, application specific integrated circuits, etc.). The one or more processors 418 may be configured to execute a non-transitory computer-readable medium 420 and manipulate data 425, both of which are stored in the data memory 419. The one or more processors 418 may also interact directly or indirectly with other components of the flow cytometer device 400, such as, by way of non-limiting example, sensors 416A, valves 417, and / or communication link 427.

[0105] The data memory 419 may be one or more types of hardware memory. For example, the data memory 419 may include or take the form of one or more computer-readable storage media that may be read or accessed by the one or more processors 418. The one or more computer-readable storage media may include volatile and / or non-volatile storage components, such as optical, magnetic, organic, or another type of memory or memories, which may be integrated, in whole or in part, with the one or more processors 418. In some embodiments, the data memory 419 may be a single physical device. In other embodiments, two or more physical devices may be used to implement the data memory 419, which may communicate with each other via wired or wireless communication. As previously described, the data memory 419 may include the non-transitory computer-readable medium 420 and data 425. The data 425 may be any type of data from the flow cytometer device 400, such as configuration data, sensor data, and / or diagnostic data, among other possibilities.

[0106] The controller 421 may include one or more circuits, digital logic units, computer chips, and / or microprocessors configured to interface (possibly among other tasks) between any combination of the various components of the flow cytometer device 400. In some embodiments, the controller 421 may be a dedicated embedded device for performing specific operations with one or more subsystems of the flow cytometer device 400.

[0107] The control system 423 may monitor and physically alter the operating conditions of the flow cytometer device 400. In doing so, the control system 423 may act as a link between parts of the flow cytometer device 400. In some cases, the control system 423 may act as an interface between the flow cytometer device 400 and another computing device. Additionally, the control system 423 may act as an interface between the flow cytometer device 400 and a user.

[0108] In some embodiments, the control system 423 of the flow cytometer device 400 may also include one or more communication links 427 configured to send and / or receive information. The one or more communication links 427 may transmit data indicative of the status of the various components of the flow cytometer device 400. For example, the information read by the sensor 416A may be transmitted via the one or more communication links 427 to the valve 417, as a non-limiting example. Other diagnostic information indicative of the integrity or health of the various components of the flow cytometer device 400 may be transmitted via the one or more communication links 427 to an external communication device.

[0109] In some embodiments, the flow cytometer device 400 may receive information at one or more communication links 427, which is then processed by one or more processors 418. The received information may indicate data accessible to the one or more processors 418 during the execution of instructions stored on the non-transitory computer-readable medium 420. Additionally, the received information may alter aspects of the one or more controllers 421 that may affect the operating parameters of the various components of the flow cytometer device 400. In some cases, the received information may indicate a query requesting specific information (e.g., the operating status of one or more components of the flow cytometer device 400). The one or more processors 418 may then transmit the specific information back outside of the one or more communication links 427.

[0110] In some cases, one or more communication links 427 may include a wired connection. Thus, the flow cytometer device 400 may include one or more ports for docking one or more communication links 427 to an external device. In addition to or as an alternative to the wired connection, one or more communication links 427 may include a wireless connection. Some exemplary wireless connections may utilize cellular connections such as CDMA, EVDO, GSM / GPRS, or 4G telecommunications such as WiMAX or LTE. Alternatively or additionally, the wireless connection may utilize a Wi-Fi connection to transmit data to a wireless local area network (WLAN). In some embodiments, the wireless connection may also communicate via an infrared link, Bluetooth, or a near field communication (NFC) device.

[0111] During operation, the control system 423 may communicate with other systems of the flow cytometer device 400 via a wired or wireless connection and may further be configured to communicate with one or more users of the system. As a possible example, the control system 423 may receive an input indicating one or more characteristics of the fluid in the fluid path 408 (e.g., from a sensor 416A of the flow cytometer device 400). Inputs to the control system 423 may be received via one or more communication links 427. Based on this input, the control system 423 may perform operations to cause the flow cytometer device 400 to perform one or more tasks.

[0112] The operations of the control system 423 may be executed by one or more processors 418. Alternatively, these operations may be executed by the controller 421 or a combination of one or more processors 418 and the controller 421. In some embodiments, the control system 423 may reside partially or entirely on a device different from the flow cytometer device 400 and may thus at least partially remotely control the flow cytometer device 400. Remote communication may be performed using at least partially one or more communication links 427.

[0113] As described above, the flow cytometer device 400 includes a processor 418 that communicates with a sensor 416A and a valve 417, and a non-transitory computer-readable medium 420 storing instructions executable to cause the processor 418 to perform functions. In particular, these functions may include: (i) receiving, via the processor 418, one or more characteristics of the fluid in the fluid path 408 detected by the sensor 416A, and (ii) adjusting the valve 417 from a first position A to a second position B based on the one or more characteristics of the fluid detected in the fluid path 408. Although Figure 12 only the sensor 416A communicating with the control system 423 is shown, each of the sensors 416A - 416L may communicate with the control system 423 in the manner described with respect to the sensor 416A.

[0114] In one embodiment, the flow cytometer device 400 can include an autosampler 424 coupled to a probe 414. A particular non-limiting example of an autosampler 424 that can be used with the flow cytometer device 400 is a Gilson 215 liquid handler. In one embodiment, the autosampler 424 can include an adjustable arm 426. As the adjustable arm 426 of the autosampler 424 moves left and right and up and down, the probe 414 descends into a single sample well 428 of a microplate 430 to obtain a sample that has been labeled with labeled particles, and the labeled particles will be analyzed using the flow cytometer device 400. Further, the probe 414 is coupled to a first end 410 of a fluid path 408.

[0115] In operation, for example, the probe 414 can obtain a sample 429 from the sample well 428 in the microplate 430 and then advance the sample 429 into the fluid path 408. A vacuum pressure at a waste outlet 422 downstream of the flow cell 402 can then pull a fluid flow stream 434 that includes the sample 429 from the well 428 through the fluid path 408 to the flow cell 402. In such an embodiment, the sensor 416A is in fluid communication with the autosampler 424 via the fluid path 408, and the flow cytometer device 400 is configured to focus the fluid flow stream 434 conveyed from the autosampler 424 through the fluid path 408 and selectively analyze the particles in each of a plurality of samples 429 as the fluid flow stream 434 passes by the sensor 416A and the flow cell 402.

[0116] As Figure 12 shown, the fluid flow stream 434 can include a series of samples 429, each sample separated by a separation gas 438, such as a bubble as a non-limiting example. The separation gas 438 can be formed by allowing the probe 414 to aspirate air (or other gas) between the sample materials from each sample well 428. In this way, the vacuum pressure at the autosampler 424 and the waste outlet 422 cooperate to introduce aliquots of the separation gas 438 between consecutive samples 429 in the fluid flow stream 434 so as to configure the fluid flow stream 434 as a separation gas-separated fluid flow stream. The separation gas-separated fluid flow stream advances to the flow cell 402 for sample measurement. As Figure 12 shown, the flow cytometer device 400 can include a first sheath port 433 and a second sheath port 435 in fluid communication with the flow cell 402. The first sheath port 433 and the second sheath port 435 provide sheath fluid to the sample 429 to wrap the sample 429 with the sheath fluid.

[0117] The presence of separation gas 438 between samples 429 helps reduce carryover between samples, allowing the system to track sample identification (e.g., which sample reservoir 428 the sample 429 came from) and enabling faster sampling. However, it is advantageous to reduce the size of the separation gas 438 between samples 429. This reduction in the size of the separation gas 438 can provide numerous advantages. First, it can improve the information of the flow cytometer device 400 regarding well identification (e.g., knowing which sample well 428 the sample 429 came from when the sample 429 is being measured in the flow cell 402). Additionally, reducing the size of the separation gas 438 between samples 429 can improve the consistency of aspirated sampling. In particular, as air passes through the flow cell 402, the fluid in the sample line moves faster and affects the volume of the aspirated sample. Thus, reducing the amount of air present in the flow cell 402 can reduce these potential problems. Furthermore, reducing the size of the separation gas 438 between samples 429 can improve the stability of the core flow passing through the flow cell 402. When air exits the injection needle in the flow cell 402, there are pressure transients that dynamically change the size of the core flow and the measurements taken on that flow.

[0118] As discussed above, valve 417 is configured to move between a first position (shown as position A in Figure 12 ) and a second position (shown as position B in Figure 12 ). In the first position, the fluid flow stream in fluid path 408 is directed to the first end 404 of the flow cell 402, and in the second position, the fluid flow stream in fluid path 408 is directed to the waste port 415. When the valve 417 is in the second position, at least some of the separation gas 438 in the gas-separated sample fluid flow stream 434 is replaced by sheath fluid from the auxiliary sheath fluid path 431, thereby reducing the gap size between adjacent samples 429 in the gas-separated sample fluid flow stream 434.

[0119] As discussed above, the flow cytometer device 400 includes a sensor 416A positioned between the probe 414 and the first end 404 of the flow cell 402, and the sensor is configured to detect one or more characteristics of the fluid in the fluid path 408. In one example, the sensor 416A includes a first sensor positioned between the probe 414 and the valve 417, and the flow cytometer device 400 further includes a second sensor 416B positioned between the first sensor 416A and the valve 417. The first sensor 416A and the second sensor 416B can be used to detect the position and velocity of the separation gas 438, and can be used to control the timing of the valve 417 to replace a portion of the separation gas 438 with sheath fluid from the auxiliary sheath fluid path 431.

[0120] As Figure 12As shown, the flow cytometer device 400 may further include a third sensor 416C positioned between the valve 417 and the first end 404 of the flow cell 402. The third sensor 416C can be used to detect the separated gas 438 in the sample fluid flow stream 434 downstream of the valve 417 for use as feedback in the process of replacing a portion of the air gap with sheath fluid via the shear valve, as discussed in further detail below. This process of replacing a portion of the air gap with sheath fluid via the shear valve may be referred to herein as the air gap replacement algorithm. In addition, the third sensor 416C provides an updated position of the sample to the flow cytometer device 400 system.

[0121] As Figure 12 shown, the flow cytometer device 400 may further include a fourth sensor 416D positioned between the third sensor 416C and the first end 404 of the flow cell 402, and a fifth sensor 416E positioned in fluid communication with the waste port 415. In this configuration, the fourth sensor 416D acts in series with the third sensor 416C to detect residual separated gas and serve as feedback for the air gap replacement algorithm. In addition, the fourth sensor 416D helps to provide better certainty regarding the velocity of the sample 429 for maintaining accurate pore identification. The fifth sensor 416E adds a measurement of the sample volume that does not enter the flow cell 402 for minimizing wasted sample volume.

[0122] As Figure 12 shown, the flow cytometer device 400 may further include a sixth sensor 416F positioned downstream of the flow cell 402. The sixth sensor 416F can add the ability to track the sample that has passed through the flow cell 402 as verification to assist in tracking pore identification after the addition of sheath fluid from the first sheath port 433 and the second sheath port 435.

[0123] As Figure 12 shown, the flow cytometer device 400 may further include a seventh sensor 416G positioned in fluid communication with the auxiliary sheath fluid path 431, and an eighth sensor 416H also positioned in fluid communication with the auxiliary sheath fluid path 431. This arrangement can add the ability to monitor the volume and velocity of an unwanted additional separated gas gap from the sheath fluid, which may be mistaken for the intentional separated gas 438, thereby improving pore identification.

[0124] As Figure 12As shown, the flow cytometer device 400 may also include a ninth sensor 416I in fluid communication with the first sheath port 433, and a tenth sensor 416J in fluid communication with the second sheath port 435. This arrangement may add the ability to monitor air bubbles that enter the flow cell 402, which may disrupt sample measurements in the flow cell 402. Additionally, the ninth sensor 416I and the tenth sensor 416J may provide information about unwanted air gaps to improve well identification.

[0125] like Figure 12 As shown, the flow cytometer device 400 may further include an eleventh sensor 416K in fluid communication with the first sheath port 433 and positioned between the ninth sensor 416I and the flow cell 402, and a twelfth sensor 416L in fluid communication with the second sheath port 435 and positioned between the tenth sensor 416J and the flow cell 402. This arrangement improves the measurements of the ninth sensor 416I and the tenth sensor 416J by adding velocity information about the air gap that may not be needed.

[0126] In various embodiments, in the flow cytometer device 400, any combination of sensors 416A-416L can be used. For example, the flow cytometer device 400 includes only the first sensor 416A. In another example, the flow cytometer device 400 includes only the first sensor 416A and the third sensor 416C. In another example, the flow cytometer device 400 includes only the first sensor 416A, the second sensor 416B, and the third sensor 416C. These are non-limiting examples, and in the flow cytometer device 400 described herein, any number and combination of sensors 416A-416L can be used.

[0127] It may be preferred that the air gap size and sample volume sucked be controlled by limiting the pulling pressure (e.g., negative pressure or vacuum) applied to the fluid path 408 at the probe 414 using the timing of the valve 417. This technique can use the first sensor 416A, the second sensor 416B, the third sensor 416C, and the fourth sensor 416D to control the timing as described above, but without the air gap replacement algorithm described above. In this embodiment, the auxiliary sheath fluid path 431 is used to maintain the sample line flow, which minimizes the pressure transients in the flow cell 402. Maintaining the sample line flow means vacuum suction of the auxiliary sheath fluid instead of air or sample so that the flow is not interrupted. In other embodiments, it may be preferred that the sheath fluid is degassed to prevent bubbles from being generated in the fluid flow stream 434 leading to the flow cell 402.

[0128] Figure 13 is a flow chart of a method for forming a sample fluid flow stream for gas separation according to an exemplary embodiment.Figure 12 Performed by the flow cytometer device 400 described above. The method 500 may include one or more operations, functions, or actions as shown in one or more of the blocks 502-512. Although these blocks are shown in sequential order, these blocks may also be performed in parallel and / or in a different order than described herein. Additionally, multiple blocks may be combined into fewer blocks, split into additional blocks, and / or deleted based on the goal to be achieved.

[0129] Additionally, for the method 500 and other processes and operations disclosed herein, the block diagrams illustrate possible implementations. In this regard, each block may represent a module, segment, or portion of program code that includes one or more instructions executable by a processor or computing device to implement a particular logical operation. The program code may be stored on any type of computer-readable medium, such as a storage device including a magnetic disk or hard drive. The computer-readable medium may include non-transitory computer-readable media, such as computer-readable media that stores data for a short time, such as register memory, processor cache, and random access memory (RAM). For example, the computer-readable medium may also include secondary or permanent long-term memory, such as read-only memory (ROM), optical disk or magnetic disk, compact disc read-only memory (CD-ROM). The computer-readable medium may also be any other volatile or non-volatile storage system. The computer-readable medium may be considered, for example, a computer-readable storage medium or a tangible storage device.

[0130] Additionally, Figure 13 each of the in may represent circuitry wired to perform a particular logical operation.

[0131] In block 502, the method 500 includes: obtaining a plurality of samples from a plate having a plurality of sample wells, wherein each of the plurality of samples is obtained from a corresponding one of the plurality of sample wells. In block 504, the method 500 includes moving the plurality of samples into a fluid flow stream in a fluid path. In block 506, the method 500 includes inserting a separation gas between adjacent samples among the plurality of samples to separate the plurality of samples from each other in the fluid flow stream, which thereby constitutes a sample fluid flow stream separated by the separation gas. In block 508, the method 500 will include guiding the sample fluid flow stream separated by the separation gas of the separated samples and the separation fluid to and through a valve, at least one sensor, and a flow cell, wherein the valve and the at least one sensor are positioned upstream of the flow cell. In block 510, the method 500 includes detecting one or more characteristics of the fluid flow stream in the fluid path via the at least one sensor. In block 512, the method 500 includes adjusting the valve from a first position to a second position based on the detected one or more characteristics of the fluid flow stream in the fluid path, in the first position, the fluid flow stream is guided to the flow cell, and in the second position, the fluid flow stream is guided to a waste port.

[0132] In one example, when the valve is in the second position, at least a portion of the separated gas in the sample fluid flow of the gas separation is replaced by a fluid such as a sheath fluid, thereby reducing the gap size between adjacent samples in the sample fluid flow of the gas separation.

[0133] In one example, as described above with respect to Figure 12 discussed, multiple samples are obtained from a plate having multiple sample wells via an autosampler coupled to a probe.

[0134] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting, with the true scope being indicated by the following claims.

Claims

1. A flow cytometer device, comprising: A flow cell having a first end and a second end; A fluid path having a first end and a second end, wherein the second end of the fluid path is coupled to the first end of the flow cell; A probe coupled to the first end of the fluid path; At least one sensor positioned between the probe and the first end of the flow cell, wherein the at least one sensor is configured to detect one or more characteristics of the fluid in the fluid path; A processor in communication with the at least one sensor; And A non-transitory computer-readable medium storing instructions that can be executed to cause the processor to perform functions when using the flow cytometer device, the functions including: Receiving, via the processor, the one or more characteristics of the fluid in the fluid path detected by the at least one sensor; and Based on the one or more detected characteristics of the fluid in the fluid path, Determining that separated gas is present in the fluid in the fluid path; Determining a count of separated bubbles detected in the fluid in the fluid path based on the one or more characteristics of the fluid in the fluid path detected over a period of time; and Determining that the flow cytometer device is blocked if the count is below a minimum count tolerance.

2. The flow cytometer device according to claim 1, wherein the at least one sensor is coupled to the fluid path.

3. The flow cytometer device according to claim 1, wherein the at least one sensor is positioned between 0.10 inches and 48 inches from the first end of the flow cell.

4. The flow cytometer device according to claim 1, wherein the at least one sensor comprises an ultrasonic sensor, and wherein the one or more characteristics of the fluid in the fluid path detected by the at least one sensor include the density of the fluid.

5. The flow cytometer device according to claim 1, wherein the at least one sensor comprises an optical sensor, and wherein the one or more characteristics of the fluid in the fluid path detected by the at least one sensor include the reflection of light through the fluid path.

6. The flow cytometer device according to claim 1, wherein the at least one sensor comprises an image sensor, and wherein the one or more characteristics of the fluid in the fluid path detected by the at least one sensor include an image of the fluid in the fluid path captured by the image sensor.

7. The flow cytometer device according to claim 1, wherein the at least one sensor comprises a mass flow sensor, and wherein the one or more characteristics of the fluid in the fluid path detected by the at least one sensor include the temperature of the fluid in the fluid path.

8. The flow cytometer device according to claim 1, further comprising: A pump in fluid communication with the fluid path.

9. The flow cytometer device according to claim 1, further comprising: An automatic sampler coupled to the probe, wherein the automatic sampler is configured to insert a plurality of samples containing particles from a plurality of respective sample wells into a fluid flow stream in the fluid path.

10. The flow cytometer device according to claim 9, wherein the automatic sampler includes an adjustable arm.

11. The flow cytometer device according to claim 9, wherein the at least one sensor is in fluid communication with the automatic sampler via the fluid path, and wherein the flow cytometer device is configured to concentrate the fluid flow stream delivered from the automatic sampler through the fluid path and selectively analyze the particles in each of the plurality of samples as the fluid flow stream passes through the at least one sensor and the flow cell.

12. The flow cytometer device according to claim 9, wherein the automatic sampler and the pump cooperate to introduce aliquots of a separation gas between consecutive samples in the fluid flow stream to configure the fluid flow stream as a separation gas-separated fluid flow stream.

13. The flow cytometer device according to claim 1, further comprising: A laser interrogation device positioned downstream of the flow cell, wherein the laser interrogation device is configured to examine a single sample flowing out of the flow cell at a laser interrogation point.

14. The flow cytometer device according to claim 1, wherein the non-transitory computer-readable medium causes the processor to further perform functions including: Generating separation gas timing data, the separation gas timing data including one or more characteristics of the fluid in the detected fluid path and corresponding timestamps; and Identifying a corresponding sample well among the plurality of sample wells at least in part based on the separation gas timing data.

15. The flow cytometer device according to claim 1, wherein the non-transitory computer-readable medium causes the processor to further perform functions including: Generating a scattered voltage output signal indicative of the intensity of scattered light by a scatter detector during a period when the fluid passes through the flow cytometer device; and Sampling the scattered voltage output signal, wherein the presence of the separation gas in the fluid in the fluid path is further determined at least in part based on the sampled scattered voltage output signal.

16. The flow cytometer device according to claim 15, wherein the non-transitory computer-readable medium causes the processor to further perform functions including: Recording a timestamp and a voltage value of each sampled voltage of the scattered voltage output signal greater than a separation gap threshold, wherein the separation gap threshold has a constant value.

17. The flow cytometer device according to claim 1, wherein the non-transitory computer-readable medium causes the processor to further perform functions including: Determining a first timestamp of the start of a sample in the fluid path based on one or more characteristics of the fluid in the detected fluid path; Determine a second timestamp indicating the end of a sample in the fluid path based on one or more characteristics of the fluid in the detected fluid path; And Determine the volume of the sample at least in part based on the first timestamp, the second timestamp, the flow rate of the fluid in the fluid path, and the diameter of the fluid path.

18. The flow cytometer device according to claim 1, wherein the non-transitory computer-readable medium causes the processor to further perform functions including the following: Determine that there are labeled particles in the fluid in the fluid path based on one or more characteristics of the fluid in the detected fluid path.

19. The flow cytometer device according to claim 1, further comprising: A valve positioned between the probe and the first end of the flow cell, wherein the valve is configured to move between a first position and a second position. In the first position, the fluid in the fluid path is directed to the first end of the flow cell. In the second position, the separation gas in the fluid in the fluid path is directed to a waste port in fluid communication with the valve.

20. The flow cytometer device according to claim 19, wherein the valve comprises a shear valve.

21. The flow cytometer device according to claim 19, further comprising: An auxiliary sheath fluid path in fluid communication with the valve.

22. The flow cytometer device according to claim 19, further comprising: One or more sheath ports in fluid communication with the flow cell.

23. The flow cytometer device according to claim 19, wherein the at least one sensor comprises a first sensor positioned between the probe and the valve, and the flow cytometer device further comprises one or more second sensors positioned between the first sensor and the valve.

24. The flow cytometer device according to claim 19, further comprising: A third sensor positioned between the valve and the first end of the flow cell.

25. The flow cytometer device according to claim 24, further comprising one or more of the following items: A fourth sensor positioned between the third sensor and the first end of the flow cell; A fifth sensor positioned in fluid communication with the waste port; A sixth sensor positioned downstream of the flow cell; A seventh sensor positioned in fluid communication with an auxiliary sheath fluid path that is in fluid communication with the valve; An eighth sensor positioned in fluid communication with an auxiliary sheath fluid path that is in fluid communication with the valve; A ninth sensor in fluid communication with a first sheath port that is in fluid communication with the flow cell; A tenth sensor in fluid communication with a second sheath port that is in fluid communication with the flow cell; An eleventh sensor, the eleventh sensor being in fluid communication with the first sheath port and positioned between the ninth sensor and the flow cell; and A twelfth sensor, the twelfth sensor being in fluid communication with the second sheath port and positioned between the tenth sensor and the flow cell.

26. A method of detecting separated gas in a fluid flow stream by a flow cytometer device according to any one of claims 1 to 25, comprising: Obtaining a plurality of samples from a plate having a plurality of sample wells, wherein each of the plurality of samples is obtained from a respective one of the plurality of sample wells; Moving the plurality of samples into a fluid flow stream in a fluid path; Inserting separated gas between adjacent samples of the plurality of samples to separate the plurality of samples from each other in the fluid flow stream, the fluid flow stream thereby constituting a gas-separated sample fluid flow stream; Directing and guiding the gas-separated sample fluid flow stream comprising the separated samples and separated fluid to and through at least one sensor and a flow cell of the flow cytometer, wherein the at least one sensor is positioned upstream of the flow cell; Detecting one or more characteristics of the fluid flow stream in the fluid path via the at least one sensor; and Based on the one or more characteristics of the fluid flow stream detected in the fluid path, determining the presence of the separated gas in the fluid flow stream in the fluid path.

27. The method of claim 26, wherein the plurality of samples are obtained from the plate having the plurality of sample wells via an autosampler coupled to a probe.

28. The method of claim 26, further comprising: Generating separated gas timing data, the separated gas timing data comprising one or more characteristics of the fluid flow stream detected in the fluid path and corresponding timestamps; and Identifying the respective sample wells of the plurality of sample wells at least in part based on the separated gas timing data.

29. The method of claim 26, further comprising: Generating a scattered voltage output signal indicative of the intensity of scattered light by a scatter detector during a period when the fluid flow stream passes through the flow cytometer; Sampling the scattered voltage output signal; and Recording timestamps and voltage values of each sampled voltage of the scattered voltage output signal that is greater than a separation gap threshold, wherein the separation gap threshold has a constant value.

30. The method of claim 26, wherein the at least one sensor comprises one or more of an ultrasonic sensor, an optical sensor, an image sensor, and a mass flow sensor.

Images