Tracking sample volume in flow cytometry

Abstract

A method of performing a flow cytometry experiment includes determining a volume of sample held in a container prior to streaming the sample through an interrogation location. The method includes monitoring the volume of the sample held in the container while running the flow cytometry experiment. The method includes determining whether the volume of the sample held in the container satisfies a threshold volume for performing the flow cytometry experiment, and generating an alert when the volume of the sample held in the container does not satisfy the threshold volume for performing the flow cytometry experiment.

Description

TRACKING SAMPLE VOLUME IN FLOW CYTOMETRYCROSS REFERENCE TO RELATED APPLICATION

[0001] This application is being filed as a PCT International patent application and claims priority to and the benefit of U.S. Provisional Patent Application Number 63 / 590,027 filed on October 13, 2023, the subject matter of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] In flow cytometry, particles are arranged in a sample stream, and typically pass one-by-one through one or more excitation light beams with which the particles interact. Light scattered or emitted by the particles upon interaction with the one or more excitation beams is collected and analyzed to characterize and differentiate the particles. In a sorting flow cytometer, particles may be extracted out of the sample stream after having been characterized by their interaction with the one or more excitation beams, and thereby sorted into different groups.SUMMARY

[0003] In general terms, the present disclosure relates to performing a flow cytometry7experiment. In one possible configuration, a volume of sample is monitored while running the flow cytometry experiment, and an alert is generated when the volume of sample does not satisfy a threshold volume for performing the flow cytometry experiment. Various aspects are described in this disclosure, which include, but are not limited to, the following aspects.

[0004] One aspect relates to a method of performing a flow cytometry experiment, the method comprising: determining a volume of sample held in a container prior to streaming the sample through an interrogation location; monitoring the volume of the sample held in the container while running the flow cytometry experiment; determining whether the volume of the sample held in the container satisfies a threshold volume for performing the flow cytometry’ experiment; and generating an alert when the volume of the sample held in the container does not satisfy the threshold volume for performing the flow cytometry' experiment.

[0005] Another aspect relates to a system for performing a flow cytometry’ experiment, the system comprising: a sample station configured to receive a container having a sample; a pump configured to pump the sample from the container forstreaming the sample through an interrogation location; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: receive experimental parameters for performing the flow cytometry experiment; and calculate a threshold volume for the sample for performing the flow cytometry experiment based on the experimental parameters.

[0006] Another aspect relates to a system for performing a flow cytometry experiment, the system comprising: a sample station configured to receive a container having a sample; a pump configured to pump the sample from the container for streaming the sample through an interrogation location; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: determine a volume of the sample held in the container prior to streaming the sample through the interrogation location; monitor the volume of the sample held in the container while running the flow cytometry experiment; determine whether the volume of the sample held in the container satisfies a threshold volume for performing the flow cytometry experiment; and generate an alert when the volume of the sample held in the container does not satisfy the threshold volume for performing the flow cytometry experiment.

[0007] A variety of additional aspects will be set forth in the description that follows. The aspects can relate to individual features and to combination of features. It is to be understood that both the foregoing general description and the following detailed description are exemplary' and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.DESCRIPTION OF THE FIGURES

[0008] The following drawing figures, which form a part of this application, are illustrative of the described technology and are not meant to limit the scope of the disclosure in any manner.

[0009] FIG. 1 illustrates an example of a system for performing a flow cytometry’ experiment, the system including a flow cytometer and a workstation.

[0010] FIG. 2 schematically illustrates an example of the system of FIG 1.

[0011] FIG. 3 is an isometric view of the sample station of the flow cytometer ofFIG. 1.

[0012] FIG. 4 shows a holder in a sample loading position inside the sample station of the flow cytometer of FIG. 1.

[0013] FIG. 5 shows the holder in a standby position inside the sample station of the flow cytometer of FIG. 1.

[0014] FIG. 6 shows the holder in a sample acquisition position inside the sample station of the flow cytometer of FIG. 1.

[0015] FIG. 7 schematically illustrates a fluidic system of the flow cytometer of FIG 1.

[0016] FIG. 8 schematically illustrates an example of a method of performing a flow cytometry experiment that can be performed by the system of FIG. 1.

[0017] FIG. 9 schematically illustrates an example of a method of determining a threshold volume of sample for performing a flow cytometry experiment that can be performed by the system of FIG. 1.

[0018] FIG. 10 schematically illustrates another example of a method of performing a flow cytometry experiment that can be performed by the system of FIG.1.

[0019] FIG. 11 illustrates an example of a graphical user interface that can be displayed on a display monitor of the workstation of FIG. 1.

[0020] FIG. 12 illustrates an example of a graphical user interface that can be displayed on the display monitor of the workstation of FIG. 1, the graphical user interface allowing a user of the system to enter a volume value of sample held in a container.

[0021] FIG. 13 illustrates an example of a graphical user interface that can be displayed on the display monitor of the workstation of FIG. 1, the graphical user interface allowing the user of the system to run the flow cytometry experiment after the volume value is entered.

[0022] FIG. 14 that illustrates an example of a graphical user interface that can be displayed on the display monitor of the workstation of FIG. 1, the graphical user interface displaying a threshold volume of sample determined for performing a flow cytometry experiment.

[0023] FIG. 15 schematically illustrates an example of a computing system for implementing aspects of the flow cytometry system of FIG. 1.DETAILED DESCRIPTION

[0024] Various embodiments will be described in detail with reference to the drawings, wherein like reference numerals represent like parts and assemblies throughout the several views. Reference to various embodiments does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments for the appended claims.

[0025] FIG. 1 illustrates an example of a system 10 that can be used to perform a flow cytometry' experiment. The system 10 includes a flow cytometer 100 and a workstation 200. In general, the flow cytometer 100 is an analytical instrument that detects physical and chemical properties of samples of particles or cells. In some examples, the flow cytometer 100 is designed to capture robust and high quality data for characterizing biologically relevant nanoparticles. The flow cytometer 100 is a single instrument that provides simultaneous assessment of nanoparticle size, concentration, and cargo to understand biological mechanisms of action and nanoparticle origins. The flow cytometer 100 can collect data from millions of particles or cells in a matter of minutes for display in a variety of formats on a display monitor 204 of the workstation 200.

[0026] The flow cytometer 100 includes a housing 101 having a sample station 104 which receives a container 106 containing a sample of cells and / or particles. In some examples, the container 106 contains a sample of nanoparticles such as extracellular vesicles (EVs). A user of the system 10 can manually load the container 106 into the sample station 104. Once loaded in the sample station 104, the flow cytometer 100 can acquire the sample from the container 106 to perform the flow cytometry experiment. In some examples, the container 106 is a sample tube such as a 1.5-mL or 2-rnL microtube, and / or has a diameter of 12 mm and a height of 75 mm.

[0027] The flow cytometer 100 can further include a sheath fluid container 107 for holding sheath fluid that is mixed with the sample during the flow cytometry experiment. For example, the sheath fluid is pumped into the flow cytometer 100 causing a laminar flow, and the sample are injected into the center of the stream, at a higher pressure. Hydrodynamic focusing causes the particles to align, single file in the direction of the laminar flow. The sheath fluid container 107 is connected to the flow cytometer 100 via tubing 109.

[0028] The flow cytometer 100 can further include a waste container 108 for collecting waste fluid that is produced from running the flow cytometry’ experiment. The waste container 108 is connected to the flow cytometer 100 via tubing 109.

[0029] The workstation 200 connects to the flow cytometer 100 to receive data from the flow cytometer 100 for display on the display monitor 204. The workstation 200 includes one or more user input devices such as a mouse 206 and a keyboard 208 allowing a user of the system 10 to enter data and information, control the flow cytometer 100, and alter the display of the data on the display monitor 204. As will be described in more detail, in some examples, the user can enter a volume of the sample in the container 106 on a graphical user interface displayed on the display monitor 204 using the mouse 206 and / or the keyboard 208. Thereafter, the system 10 can track the volume of the sample while running a flow cytometry experiment on the sample. In some examples, the system 10 determines a nanoparticle count using the volume of the sample entered by the user. In further examples, the flow cytometer can include a sensor that can be used to detect and monitor the volume of the sample in the container 106 while running a flow cytometry experiment on the sample.

[0030] The workstation 200 further includes a computing device 202. In some examples, the workstation 200 utilizes the computing device 202 to process raw data received from the flow cytometer 100. Alternatively, or additionally, the flow cytometer 100 can also include a computing device that processes the data collected from the flow cytometry experiment such that the workstation 200 receives processed data for display on the display monitor 204.

[0031] FIG. 2 schematically illustrates an example of the system 10 that includes three main subsystems: a fluidic system 110, an optical system 120, and an electronic system 130. The fluidic system 110 includes a nozzle 1 12 which receives a sample containing particles or cells suspended in a fluid. The nozzle 112 creates and ejects a fluid stream 114 of the sample arranged in single file in the laminar flow of the sheath fluid. Each particle passes through one or more light beams produced by a light source 102. The point at which a particle intersects with a light beam inside the flow' cytometer 100 is known as an interrogation location 116.

[0032] The optical system 120 includes the light source 102, optical elements 122, and detectors 124. At the interrogation location 116, light from the light source 102 hits a particle and scatters. The optical elements 122 direct the scattered light toward thedetectors 124. The detectors 124 may include a forw ard scatter (FSC) detector to measure scatter along the path of the light source 102, a side scatter (SSC) detector to measure scatter at a ninety -degree angle relative to the light source 102, and / or one or more fluorescence detectors (e.g., FL1, FL2, and FL3) to measure the emitted fluorescence intensity7of different wavelengths of light.

[0033] Generally, FSC intensity is proportional to the size or diameter of cells due to light diffraction around the cell. FSC can be used to discriminate cells by relative size. SSC, on the other hand, is produced from light refracted or reflected by internal structures of the cells, and provides information about internal complexity or granularity of the cells. SSC is proportional to nanoparticle size. By adding fluorescent labelling to a sample, different fluorescent signals / channels (e.g.. green, orange, and red) can be analyzed for functional characteristics of a cell or nanoparticle. For example, since T-cells present CD3 binding sites, a sample containing T-cells may be “stained” with anti-CD3 antibodies conjugated with a fluorescent molecule. As these cells pass through the interrogation location 116, the laser light excites the fluorescent tag, or fluorochrome, to emit photons at a wavelength detectable by a fluorescence detector. The detectors 124 may therefore simultaneously measure a number of parameters and enable categorization of particles by their function based on detected w avelengths of light.

[0034] The electronic system 130 includes a waveform acquisition device 140 and a waveform analysis device 150. The waveform acquisition device 140 is communicatively coupled with the detectors 124 and is configured to receive analog waveform data 126 generated by the detectors 124. The w aveform acquisition device 140 includes an analog-to-digital converter (ADC) 142 configured to digitize the waveform data.

[0035] The waveform analysis device 150 is configured to receive the digital w aveform data and display it for a user of the system 10. In some embodiments, the waveform analysis device 150 includes the computing device 202 communicatively coupled with the flow cytometer 100, and the flow cytometer 100 may include the fluidic system 110, optical system 120, and the waveform acquisition device 140. In other embodiments, the waveform analysis device 150 is integrated with the flow cytometer 100.

[0036] As further shown in FIG. 2, the waveform analysis device 150 includes a graphics processing unit (GPU) 152. The GPU 152 can be used to process the digitized waveform data received from the waveform acquisition device 140.

[0037] The example of the system 10 shown in FIG. 2 includes elements which are shown and described for purposes of discussion and it will be appreciated that numerous variations in components and functions are possible. The optical elements 122 may include a series of filters, dichroic mirrors, and / or beam splitters to select out different wavelengths of light and provide the wavelength to an appropriate detector. The detectors 124 may include, for example, photomultiplier tubes (PMTs) or avalanche photodiodes (APDs).

[0038] FIG. 3 is an isometric view of an example of the sample station 104 of the flow cytometer 100. The sample station 104 includes a holder 160 for holding the container 106. The holder 160 can hold containers having a diameter of 12 mm and a height of 75 mm. In some examples, the holder 160 can hold sample tube such as a 1.5- mL or 2-mL microtube. In some examples, the holder 160 can be a well in a microplate.

[0039] The sample station 104 includes a probe 162 to draw and transfer the sample from the container 106 into the fluid stream 114 inside the flow cytometer 100. The sample station 104 can include a wash station and mixer 164 that can be used when acquiring the sample to mix the sample for a default time (e.g., 1 second), and to clean the probe 162 and a sample fluid path 172, 174 (shown in FIG. 7) when the flow cytometer 100 performs a backflush.

[0040] FIG. 4 shows the holder 160 in a sample loading position 161. FIG. 5 shows the holder 160 in a standby position 163. FIG. 6 shows the holder 160 in a sample acquisition position 165. Referring now to FIGS. 4-6, the holder 160 is connected to an arm 166 that is controlled by the flow cytometer 100 to move the holder 160 from the sample loading position 161 into the standby position 163 once the container 106 has been loaded. As shown by the arrows in FIGS. 4 and 5, the standby position 163 is positioned inward in the sample station 104 relative to the sample loading position 161. Thereafter, the arm 166 is controlled by the flow cytometer 100 to move the holder 160 from the standby position 163 to the sample acquisition position 165. As shown by the arrow- in FIG. 6, the sample acquisition position 165 is positioned upward relative to the standby position 163 in the sample station 104. While in the sample acquisitionposition 165, the probe 162 is used to draw and transfer the sample from the container 106 into the fluid stream 114 (see FIG. 2) inside the flow cytometer 100.

[0041] FIG. 7 schematically illustrates the fluidic system 110 of the flow cytometer 100. The fluidic system 110 includes a pump 170 that pumps the sample from the container 106 via the probe 162 to the fluid stream 114 that includes the sheath fluid from the sheath fluid container 107. In some examples, the pump 170 provides positive pressure displacement for loading the sample from the container 106. In some examples, the pump 170 is a piston pump. In other examples, the pump 170 is a peristaltic pump. Additional types of pumps are possible.

[0042] As further shown in FIG. 7, the fluid stream 114 enters a transparent receptacle 115 such as a cuvette where the interrogation location 116 is located. After passing through the interrogation location 116, the fluid stream 1 14 is collected in the waste container 108.

[0043] In some example embodiments, the flow cytometer 100 includes a sensor 168 that can be used by the flow cytometer 100 to measure a volume of the sample held in the container 106 prior to streaming the sample through the interrogation location 116. In some further example embodiments, the sensor 168 can be used to monitor the volume of the sample held in the container 106 while running the flow cytometry experiment.

[0044] In some examples, the sensor 168 is an optical sensor that can measure the volume of the sample held in the container 106 such as by detecting a location of a meniscus of the sample in the container 106. In such examples, the container 106 is made of a transparent material such as polystyrene, and the optical sensor emits a light beam that can be used to detect the volume of the sample held in the container 106.

[0045] In some examples, the optical sensor is a through-beam sensor that includes an emitter positioned on one side of the container 106, and a receiver positioned on an opposite side of the container 106. The emitter emits the light beam and the receiver detects changes in the light beam due to a presence or an absence of the sample in the container 106.

[0046] In other examples, the optical sensor is a reflective sensor that include the emitter and the receiver positioned on the same side of the container 106. The emitter emits a light beam that is reflected off the container 106, and the receiver detects changes in the light beam reflected from the container 106 due to a presence or anabsence of the sample in the container 106. Additional types of optical sensors that can be used to detect and / or monitor the volume of the sample in the container 106 are possible.

[0047] Referring to FIGS. 3-6, the sensor 168 can be positioned inside the sample station 104. In such examples, the sensor 168 is positioned relative to the container 106 to measure the volume of sample when the holder 160 is in the sample loading position 161, the standby position 163. and / or the sample acquisition position 165. In some examples, the sensor 168 moves inside the sample station 104 when the holder 160 moves between the sample loading position 161, the standby position 163, and the sample acquisition position 165.

[0048] In further examples, gravimetric techniques can be used for determining the sample volume. Such techniques can include measuring the mass of the sample and the container 106 before and after sample volume is displaced from the container 106 to determine the mass of the sample. The mass of the sample can be used to measure the sample volume based on a known density of the sample. For example, the sample held in the container is mostly water, which has a density of about Ig / mL. The displaced volume subtracted by the dead volume of the fluidic systeml 10 equals the sample volume interrogated by the flow cytometer 100.

[0049] In examples where gravimetric techniques are used, the sensor 168 can be connected to the arm 166 to measure a total mass of the sample and the container 106. In such examples, the flow cytometer 100 can determine the volume of the sample when the container 106 has a know n mass, such that any additional mass above the know n mass of the container 106 is due to the presence or absence of the sample. As discussed above, the sample has a known density such that the volume of the sample can be determined from the mass of the sample.

[0050] Additional types of techniques and sensors for detecting the volume of the sample in the container 106 are possible, and the flow' cytometer 100 is not limited to the types of techniques and sensors described above. Further, the sensor 168 is optional such that in some examples the flow cytometer 100 does not include the sensor 168. In such examples, the flow cytometer 100 monitors the sample volume in the container 106 without using the sensor 168.

[0051] FIG. 8 schematically illustrates an example of a method 800 of performing a flow cytometry experiment. In some example embodiments, the method 800 can beperformed by the system 10. As shown in FIG. 8, the method 800 includes an operation 802 of prompting a user to enter a sample volume. In some examples, the sample volume is a volume value expressed in microliters (pL) or in another unit of measurement. Operation 802 can include displaying a graphical user interface on the display monitor 204 that includes a message prompt requesting the user to enter the sample volume. In some examples, operation 802 includes prompting the user to enter the sample volume once the container 106 is detected inside the sample station 104.

[0052] The method 800 includes an operation 804 of receiving the sample volume entered by the user. In some examples, the sample volume is received via the user entering the sample volume in a data field included on the graphical user interface displayed on the display monitor 204 such as by using the mouse 206 and / or the keyboard 208 of the workstation 200.

[0053] The method 800 includes an operation 806 of monitoring the sample volume in the container 106 while running the flow cytometry experiment. In some examples, operation 806 includes determining a volume of the sample removed from the container 106 and subtracting the volume of the sample removed from the container 106 from the sample volume received in operation 804. In some examples, the volume of the sample removed from the container 106 is determined based on the parameters of the flow cytometry experiment. In such examples, the method 800 includes monitoring the sample volume in the container 106 without using data collected from the sensor 168 such that the sensor is optional.

[0054] The method 800 includes an operation 808 of determining whether the sample volume in the container 106 is sufficient for running the flow cytometry experiment. In some examples, operation 808 includes comparing the sample volume monitored in operation 806 with a threshold volume for performing the flow cytometry experiment. In some examples, the threshold volume is determined based on the parameters of the flow cy tometry7experiment.

[0055] When the method 800 determines that the sample volume in the container 106 is sufficient for running the flow cytometry experiment (i.e., "Yes” in operation 808), the method 800 can return to operation 806 to continue monitoring the sample volume in the container 106 while running the flow cytometry7experiment.

[0056] Alternatively, when the method 800 determines that the sample volume in the container 106 is not sufficient for running the flow cytometry experiment (i.e.,“No’' in operation 808), the method 800 proceeds to an operation 810 of generating an alert. As an illustrative example, the alert generated in operation 810 can be displayed on the display monitor 204. In some examples, the alert generated in operation 810 is displayed as a warning that the flow cytometry experiment cannot be completed based on the current sample volume in the container 106. In some examples, the alert includes an audible alarm.

[0057] In some examples, the method 800 includes an operation 812 of automatically stopping acquisition of the sample from the container 106 when the method 800 determines in operation 808 that the sample volume in the container 106 is not sufficient for running the flow cytometry experiment. Operation 812 can be performed before the sample volume in the container runs out (i.e.. before it reaches zero). For example, the flow cytometer 100 can control the pump 170 to pump the sample from the container 106 for streaming the sample through the interrogation location 116. Additionally, the flow cytometer 100 can control the pump 170 to stop the pumping of the sample from the container 106 when the volume of the sample held in the container 106 does not satisfy the threshold volume for performing the flow cytometry experiment (i.e., “No” in operation 808).

[0058] Advantageously, operation 812 provides a technical effect by preventing erroneous results from being collected by the flow cytometer 100 for display on the display monitor 204. Also, operation 812 provides a further technical effect by conserving energy and resources such as sheath fluid that would otherwise be wasted once the sample volume in the container 106 runs out. Additionally, operation 812 provides a further technical effect by preventing the need to re-prime the flow cytometer 100 with the sample, which can avoid undesirable down time.

[0059] FIG. 9 schematically illustrates an example of a method 900 of determining a threshold volume of sample for performing a flow cytometry experiment. In some example embodiments, the method 900 can be performed by the system 10.

[0060] The method 900 includes an operation 902 of prompting a user to enter one or more experimental parameters for the flow cytometry experiment. In some examples, operation 902 includes displaying a graphical user interface on the display monitor 204 that includes one or more data fields for the user to enter the experimental parameters. In some examples, operation 902 includes prompting the user to enter experimental parameters that include a start gain, an end gain, an increment of gain,and a time or quantity of events to record when the user wants to perform a series of tests as part of a gain titration experiment (sometimes called "gain tra on"). Operation 902 includes prompting the user to enter the experimental parameters before the container 106 is prepared to include the sample, and before the container 106 with the sample is placed inside the sample station 104 (see FIGS. 3-6).

[0061] The method 900 includes an operation 904 of receiving the experimental parameters for performing the flow cytometry experiment. In some examples, operation 904 includes receiving the experimental parameters via the user entering the experimental parameters into one or more data fields in the graphical user interface displayed on the display monitor 204 such as by using the mouse 206 and / or the keyboard 208 of the workstation 200.

[0062] The method 900 includes an operation 906 of calculating a threshold volume of sample for performing the flow cytometry experiment based on the experimental parameters received in operation 904. For example, operation 906 can include calculating the threshold volume of sample for performing a gaintration experiment based on a start gain, an end gain, an increment of gain, and a volume, a time, or a quantity of events to record.

[0063] The method 900 can include an operation 908 of displaying the threshold volume. In some examples, the threshold volume is displayed on a graphical user interface generated on the display monitor 204. Once the threshold volume is displayed on the display monitor 204, the user can load a volume of the sample into the container 106 that is equal to or greater than the threshold volume before loading the container 106 into the sample station 104.

[0064] In some examples, the method 900 can further include an operation 910 of determining the sample volume inside the container 106 once the container 106 is loaded inside the sample station 104. As an illustrative example, operation 910 can include using data received from the sensor 168 to detect the sample volume inside the container 106.

[0065] The method 900 can further include an operation 912 of determining whether the sample volume in the container 106 detected in operation 910 is sufficient for running the flow cytometry experiment. In some examples, operation 912 includes comparing the sample volume determined in operation 910 with the threshold volume calculated in operation 906.

[0066] When the method 900 determines that the sample volume in the container 106 is sufficient for running the flow cytometry experiment (i.e., “Yes’" in operation 912), the method 900 can proceed to operation 912 of running the flow cytometry experiment.

[0067] Alternatively, when the method 900 determines that the sample volume in the container 106 is not sufficient for running the flow cytometry experiment (i.e.,i£No” in operation 912), the method 900 proceeds to an operation 914 of generating an alert. As an illustrative example, the alert generated in operation 914 can be displayed on the display monitor 204. In some examples, the alert generated in operation 914 is displayed as a warning that the flow cytometry experiment cannot be completed based on the current sample volume in the container 106. In some examples, the alert includes an audible alarm. In some examples, when the user incorporates a sample volume in the container 106 that is less than the threshold volume, the alert requests either the user increase the sample volume or change to the experimental parameters to accommodate the sample volume in the container 106.

[0068] FIG. 10 schematically illustrates another example of a method 1000 of performing a flow cytometry experiment. In some example embodiments, the method 1000 can be performed by the system 10. The method 1000 includes an operation 1002 of determining a volume of sample held in the container 106 prior to streaming the sample through an interrogation location. In some examples, the operation 1002 of determining the volume of the sample held in the container 106 prior to streaming the sample through the interrogation location is based on a volume value entered by a user of the system 10. For example, the user can enter the volume value into a graphical user interface displayed on the display monitor 204 using the mouse 206 and the keyboard 208 of the workstation 200. In such examples, operation 1002 is performed without using the sensor 168.

[0069] Alternatively, the operation 1002 of determining the volume of the sample held in the container 106 prior to streaming the sample through the interrogation location is based on a measurement value detected by the sensor 168. For example, once the container 106 is loaded inside the sample station 104, the flow cytometer 100 can use the sensor 168 to measure the volume of the sample held in the container 106.

[0070] The method 1000 includes an operation 1004 of pumping the sample from the container 106 for streaming the sample through the interrogation location 116. Forexample, the flow cytometer 100 can perform operation 1004 by controlling the pump 170 to pump the sample from the container 106 to the interrogation location 116 (see FIG. 7).

[0071] The method 1000 includes an operation 1006 of monitoring the volume of the sample held in the container 106 while running the flow cytometry experiment (i.e., while the pump 170 is being controlled to pump the sample through the interrogation location 116). In some examples, operation 1006 includes determining a volume of the sample removed from the container 106, and subtracting the volume of the sample removed from the container 106 from the volume of the sample determined in operation 1002. In some examples, determining the volume of the sample removed from the container 106 is calculated based on the experimental parameters of the flow cytometry experiment. In such examples, operation 1006 is performed without using the sensor 168.

[0072] In alternative examples, the operation 1006 of monitoring the volume of the sample held in the container 106 while running the flow cytometry experiment is based on measurement values detected by the sensor 168. For example, the sensor 168 can be used to monitor the volume of the sample in the container 106 while the flow cytometer 100 is acquiring the sample examples from the container 106 in the sample acquisition position 165.

[0073] The method 1000 includes an operation 1008 of determining whether the volume of the sample held in the container 106 satisfies a threshold volume for performing the flow cytometry experiment. Operation 1008 can include comparing the volume of the sample monitored in operation 1006 with the threshold volume for performing the flow cytometry’ experiment. The threshold volume can be determined by receiving experimental parameters for performing the flow cytometry experiment, and calculating the threshold volume for performing the flow cytometry experiment based on the experimental parameters. As an illustrative example, the experimental parameters can include a start gain, an end gain, an increment of gain, and a time or quantity of events to record.

[0074] When the method 1000 determines that the volume of the sample held in the container 106 is sufficient for running the flow cytometry' experiment (i.e., “Yes” in operation 1008), the method 1000 returns to operation 1006 to continue monitoring thevolume of the sample held in the container 106 while running the flow cytometry experiment.

[0075] When the method 1000 determines that the volume of the sample held in the container 106 is not sufficient for running the flow cy tometry experiment (i.e., “No” in operation 1008), the method 1000 proceeds to an operation 1010 of generating an alert. As an illustrative example, the alert generated in operation 810 can be displayed on the display monitor 204. In some examples, the alert generated in operation 810 is displayed as a warning that the flow cytometry experiment cannot be completed based on the current volume of the sample held in the container 106. In some examples, the alert includes an audible alarm.

[0076] The method 1000 can include an operation 1012 of stopping the pumping of the sample from the container 106 when the method 1000 determines in operation 1008 that the volume of the sample held in the container 106 does not satisfy the threshold volume for performing the flow cytometry experiment. Operation 1012 can be performed before the volume of the sample held in the container 106 runs out (i.e.. before it reaches zero). For example, the flow cytometer 100 can control the pump 170 to stop the pumping of the sample from the container 106 when the volume of the sample held in the container 106 does not satisfy the threshold volume for performing the flow cytometry experiment (i.e., “No” in operation 1008).

[0077] FIG. 11 illustrates an example of a graphical user interface 1100 that can be displayed on the display monitor 204 of the workstation 200. Using the mouse 206, a user of the system 10 can select an “Advanced” drop down menu 1102 and select a “load sample setting” option 1104.

[0078] FIG. 12 illustrates an example of a graphical user interface 1200 that can be displayed on the display monitor 204 of the workstation 200 when the “load sample setting” option 1104 is selected on the graphical user interface 1100 of FIG. 11. The graphical user interface 1200 allows a user of the sy stem 10 to enter one or more load sample settings. The graphical user interface 1200 includes a data field 1202 that allows the user of the system 10 to enter a volume value of sample held in the container 106. The user can use the mouse 206 to select up and down arrows 1204 to increase or decrease the volume value entered in the data field 1202. Alternatively, the user can use the keyboard 208 to type the volume value in the data field 1202.

[0079] The graphical user interface 1200 can further include a continuous load sample option 1206 that can be selected or deselected by the user. When selected, the continuous load sample option 1206 causes the flow cytometer 100 to remove aliquots of the specified volume from the sample in the container 106 until one or more acquisition limits are met. Examples of the acquisition limits include volume, time, or events. Once the volume value of sample is entered in the data field 1202 and the continuous load sample option 1206 is selected or deselected as desired by the user, the user can select the '‘Ok” option 1208 to submit the load sample settings, or can select the “Cancel” option 1210 to cancel the load sample settings.

[0080] FIG. 13 illustrates an example of a graphical user interface 1300 that can be displayed on the display monitor 204 of the workstation 200 when the “Ok” option 1208 is selected on the graphical user interface 1200 of FIG. 12. The graphical user interface 1300 allows the user of the system 10 to run the flow cytometry experiment after the volume value is entered using the graphical user interface 1200 of FIG. 12. The graphical user interface 1300 includes a “Run” option 1302 that causes the flow cytometer 100 to continually cycle until a manual stop. Each additional cycle will reuse the 3 pL air gap and 34 pL dead volume.

[0081] The graphical user interface 1300 can further include a “Record” option 1304 that will follow stop limits. The stop limits can include a volume of sample interrogated, a time, or a quantity of events. The “Record” option 1304 automatically executes acquisition of the sample, and stops based on the stop limits. In contrast, the “Run” option 1302 is a manual acquisition, and is stopped manually by the user. Both the “Run” option 1302 and the “Record” option 1304 generate a Flow Cytometry Standard (FCS) file when stopped.

[0082] FIG. 14 that illustrates an example of a graphical user interface 1400 that can be displayed on the display monitor 204 of the workstation 200. As shown in FIG. 14, the graphical user interface displays a threshold volume 1402 of sample determined for performing a flow cytometry experiment. As described above, the threshold volume 1402 can be calculated by the system 10 based on one or more experimental parameters entered by the user such as a start gain, an end gain, an increment of gain, and a time or quantity of events to record.

[0083] FIG. 15 schematically illustrates an example of a computing device 1500 for implementing aspects of the system 10, including functions of the flow cytometer 100and the workstation 200. Examples of the computing device 1500 include a server computer, a desktop computer, a laptop computer, a tablet computer, a mobile computing device (such as a smartphone), or other devices configured to process digital instructions.

[0084] The computing device 1500 includes one or more processing devices 1502. Examples of the one or more processing devices 1502 include central processing units (CPUs), digital signal processors, field-programmable gate arrays, and other types of electronic computing circuits. The one or more processing devices 1502 can be part of a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to perform the functionalities described herein.

[0085] The computing device 1500 further includes a system memory 1504, and a system bus 1506 that couples various system components including the system memory 1504 to the one or more processing devices 1502. The system bus 1506 is one of any number of types of bus structures including a memory bus, or a memory controller; a peripheral bus; and a local bus using any of a variety of bus architectures.

[0086] The system memory 1504 can include a read only memory (ROM) 1508 and a random access memory7(RAM) 1510. A basic input / output system (BIOS) 1512 containing the basic routines that act to transfer information within computing device 1500, such as during start up, can be stored in the system memory 1504. The GPU 152 can use the RAM 1510 for loading and subsequently analyzing the waveform data (e.g., stored in a raw waveform data file, which can include digitalized raw waveform data).

[0087] The computing device 1500 can also include one or more secondary storage devices 1514 such as a hard disk drive for storing digital data. The one or more secondary storage devices 1514 are connected to the system bus 1506 by a secondary storage interface 1516. The one or more secondary storage devices 1514 and associated computer readable media provide nonvolatile storage of computer readable instructions (including application programs and program modules), data structures, and other data for the computing device 1500. Although the example described herein employs a hard disk drive as a secondary storage device, other types of computer readable storage media are used in other embodiments. Examples of these other types of computer readable storage media include the ROM 1508 and / or the RAM 1510. Some examplesinclude non-transitoiy media. Additionally, such computer readable storage media can include local storage or cloud-based storage.

[0088] The computing device 1500 typically includes at least some form of computer readable media. Computer readable media includes any available media that can be accessed by the computing device 1500. By way of example, computer readable media include computer readable storage media and computer readable communication media.

[0089] Computer readable storage media includes volatile and nonvolatile, removable and non-removable media implemented in any device configured to store information such as computer readable instructions, data structures, program modules or other data. Computer readable storage media includes, but is not limited to, random access memory, read only memory, electrically erasable programmable read only memory, flash memory or other memory technology, or any other medium that can be used to store the desired information and that can be accessed by the computing device 1500.

[0090] Computer readable communication media can embody computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term “modulated data signal'’ refers to a signal that has one or more characteristics set in a manner as to encode information in the signal. For example, computer readable communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency, infrared, and other wireless media. Combinations of any of the above are also included within the scope of computer readable media.

[0091] A number of program modules can be stored in secondary storage device 1514 or the system memory 1504, including an operating system 1518, application programs 1520, program modules 1522 (such as the softw are engines), and program data 1524. The computing device 1500 can utilize any suitable operating system, such as Microsoft Windows™, Google Chrome™. Apple OS, and any other operating system suitable for a computing device.

[0092] A user provides inputs to the computing device 1500 through one or more input devices 1526. Examples of input devices 1526 include the mouse 206, the keyboard 208, a microphone 1532, and a touch sensor 1534 (such as a touchpad ortouch sensitive display). Additional types of the input devices 1526 are contemplated. The input devices 1526 are often connected to the one or more processing devices 1502 through an input / output interface 1536 that is coupled to the system bus 1506. These input devices 1526 can be connected by any number of input / output interfaces, such as a parallel port, serial port, game port, or a universal serial bus. Wireless communication between input devices and the input / output interface 1536 is possible as well, and includes infrared, BLUETOOTH® wireless technology, 802. l la / b / g / n. cellular, or other radio frequency communication systems in some possible embodiments.

[0093] The display monitor 204 can include a liquid crystal display device, a touch sensitive display device, and the like. The display monitor 204 connects to the sy stem bus 1506 via an interface, such as a video adapter 1540. In addition to the display monitor 204, the computing device 1500 can include various other peripheral devices, such as speakers or a printer.

[0094] When used in a local area networking environment or a wide area networking environment (such as the Internet), the computing device 1500 is typically connected to a network 1544 through a network interface 1542. such as an Ethernet interface. Other possible embodiments use other communication devices. For example, some embodiments of the computing device 1500 include a modem for communicating across the network 1544.

[0095] The computing device 1500 is an example of programmable electronics, which may include one or more such computing devices. When multiple computing devices are included, such computing devices can be coupled together with a suitable data communication network so as to collectively perform the various functions, methods, or operations disclosed herein.

[0096] The various embodiments described above are provided by way of illustration only and should not be construed to be limiting in any way. Various modifications can be made to the embodiments described above without departing from the true spirit and scope of the disclosure.

Claims

What is claimed is:

1. A method of performing a flow cytometry experiment, the method comprising: determining a volume of sample held in a container prior to streaming the sample through an interrogation location; monitoring the volume of the sample held in the container while running the flow cytometry experiment; determining whether the volume of the sample held in the container satisfies a threshold volume for performing the flow cytometry experiment; and generating an alert when the volume of the sample held in the container does not satisfy the threshold volume for performing the flow cytometry experiment.

2. The method of claim 1, further comprising: pumping the sample from the container for streaming the sample through the interrogation location; and stopping the pumping of the sample from the container when the volume of the sample does not satisfy the threshold volume for performing the flow cytometry experiment.

3. The method of claim 1 or 2, wherein determining the volume of the sample held in the container prior to streaming the sample through the interrogation location is based on a volume value entered by a user of the system.

4. The method of any of claims 1-3, wherein monitoring the volume of the sample held in the container includes: determining a volume of the sample removed from the container; and subtracting the volume of the sample removed from the container from the volume of the sample held in the container prior to streaming the sample through the interrogation location.

5. The method of claim 1 or 2, wherein determining the volume of the sample held in the container prior to streaming the sample particle through the interrogation location is based on a measurement value detected by a sensor.

6. The method of any of claims 1-3 or 5, wherein monitoring the volume of the sample held in the container is based on measurement values detected by a sensor.

7. The method of any of claims 1-6, further comprising: receiving experimental parameters for performing the flow cytometry experiment, wherein the experimental parameters include a start gain, an end gain, an increment of gain, and a time or quantity of events to record; and calculating the threshold volume for performing the flow cytometry experiment based on the experimental parameters.

8. A system for performing a flow cytometry experiment, the system comprising: a sample station configured to receive a container having a sample; a pump configured to pump the sample from the container for streaming the sample through an interrogation location; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: determine a volume of the sample held in the container prior to streaming the sample through the interrogation location; monitor the volume of the sample held in the container while running the flow cytometry experiment; determine whether the volume of the sample held in the container satisfies a threshold volume for performing the flow cytometry experiment; and generate an alert when the volume of the sample held in the container does not satisfy the threshold volume for performing the flow cytometry experiment.

9. The system of claim 8. wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: pump the sample from the container for streaming the sample through the interrogation location; andstop pumping the sample from the container when the volume of the sample held in the container does not satisfy the threshold volume for performing the flow cytometry experiment.

10. The system of claim 8 or 9, wherein determine the volume of the sample held in the container prior to streaming the sample through the interrogation location is based on a volume value entered by a user of the system.

11. The system of any of claims 8-10, wherein monitor the volume of the sample held in the container includes: determine a volume of the sample removed from the container; and subtract the volume of the sample removed from the container from the volume of the sample held in the container prior to streaming the sample through the interrogation location.

12. The system of claim 8 or 9, wherein determine the volume of the sample held in the container prior to streaming the sample particle through the interrogation location is based on a measurement value detected by a sensor in the sample station.

13. The system of claim 12, wherein monitor the volume of the sample held in the container includes: determine a volume of the sample removed from the container; and subtract the volume of the sample removed from the container from the volume of the sample held in the container prior to streaming the sample through the interrogation location.

14. The system of claim 12, wherein monitor the volume of the sample held in the container is based on measurement values detected by the sensor in the sample station.

15. The system of any of claims 8-14, wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to:receive experimental parameters for performing the flow cytometry experiment, wherein the experimental parameters include a start gain, an end gain, an increment of gain, and a time or quantity of events to record; and calculate the threshold volume for performing the flow cytometry experiment based on the experimental parameters.

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