Systems and methods for determining immune activation status - Patents.com

Abstract

A system or method for detecting an immune system activation state in a patient may include a sample preparation system configured to isolate white blood cells from a patient sample using a spindle configured to be attached to a test tube, a cytometry module configured to determine a biophysical property of the white blood cells of the sample, and an analysis module configured to analyze the biophysical property to determine a probability associated with the patient having sepsis.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 17 / 575,388, filed January 13, 2022, which is incorporated by reference in its entirety.

[0002] The present invention relates generally to the health field, and more specifically to novel and useful systems and methods in the health field. [Brief description of the drawings]

[0003] [Figure 1] FIG. 1 is a schematic diagram of an example of an apparatus. [Diagram 2] FIG. 1 is a schematic diagram of an example method. [Diagram 3] FIG. 1 is a schematic diagram of an example of a blood sample (eg, before processing) on ​​a sample holder. [Figure 4] FIG. 1 is a schematic diagram of an example of lysis and quenching of a blood sample. [Diagram 5] FIG. 1 is a schematic diagram of an example of washing a lysed blood sample. [Figure 6] FIG. 1 is a schematic diagram of an example of suspending washed white blood cells. [Figure 7] FIG. 1 is a schematic diagram of an example of suspending white blood cells to achieve a target temperature after suspension. [Figure 8] FIG. 1 is a schematic diagram of an exemplary microfluidic channel. [Figure 9] 1 is a schematic diagram of an exemplary imaging system. [Figure 10] 1 is a flow chart of an example method. [Figure 11] 1 is a flow chart of an example of an analysis performed on white blood cells. [Figure 12] FIG. 1 is a schematic diagram of an example of determining a biophysical parameter and / or immune activation status of a patient. [Figure 13] FIG. 1 is a schematic diagram of an example of a sample preparation module. [Figure 14] FIG. 1 is a schematic diagram of an example of a system. [Figure 15] FIG. 1 is a schematic diagram of an example of a sample holder interfacing with a spindle. [Figure 16A] 16A and 16B are schematic diagrams of an exemplary assembled cartridge and its exemplary microfluidic channels, respectively. [Figure 16B] 16A and 16B are schematic diagrams of an exemplary assembled cartridge and its exemplary microfluidic channels, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] The following description of preferred embodiments of the invention is not intended to limit the invention to those preferred embodiments, but rather to enable any person skilled in the art to make and use the invention.

[0005] 1. Overview As shown in FIG. 1, the system (10) may include a sample preparation module, a measurement module, a computing system, and / or any suitable components.

[0006] 2, the method (20) may include preparing a sample and measuring the sample. The method optionally includes pre-conditioning the system (or portions thereof), determining the immune activation status of the patient, providing an intervention to the patient (e.g., based on the immune activation status), and / or any suitable steps.

[0007] The system and / or method may function to prepare a sample for measurement and / or analysis, measure characteristics of the sample, determine a patient's immune activation state, and / or otherwise. In a specific example, the system and / or method may be used to triage patients (e.g., diagnose, determine the likelihood that a patient is experiencing a condition, such as a sepsis-related illness, etc.) in an emergency department (e.g., in a hospital), urgent care, and / or a physician's office. Examples of immune activation states include sepsis (e.g., septicemia), sepsis-related illness, anemia, bleeding illness, systemic inflammatory response syndrome (SIRS), blood cell health, organ health, cancer illness (e.g., metastatic cancer), inflammation, cytokine release (e.g., cytokine storm), autoimmune disorders (e.g., rheumatic disorders such as arthritis, lupus, etc.), graft-versus-host disease, and / or any suitable immune activation state or other health condition. The immune activation state may be caused by one or more pathogens (e.g., a class of pathogens such as bacteria, viruses, fungi, chemicals, etc., a specific pathogen, etc.) and / or other causes of the immune activation state and / or may include a diagnosis thereof (e.g., the probability that the immune activation state is caused). The immune activation state may be a binary state (e.g., yes or no as to whether the patient is positive for a given condition), a severity index (e.g., "healthy", "no symptoms", "mild", "moderate", "severe", "severe", "acute", "life-threatening", etc.), a numerical value, a probability and / or likelihood that the patient has the condition, and / or other representation of the immune activation state.

[0008] advantage. Variations in the technology may confer several benefits and / or advantages.

[0009] First, the variations of the technology may improve the reproducibility, repeatability, and / or reliability of blood sample preparation for measurement and / or analysis. The improved reproducibility and / or reliability may be across different practitioners, different medical practices, different geographical areas, different times (e.g., hours, days, weeks, months, years, etc.), different systems, and / or under any suitable conditions. For example, standardizing sample preparation procedures (e.g., using the same or same type of sample preparation module), determining optimal timing (e.g., process duration, relative timing, amount of time between processes, total process time, etc.), and / or determining preferred conditions (e.g., humidity, temperature, reagent concentration, reagent addition rate, reagent identity, etc.) may enable technical advantages of improved reproducibility and / or reliability. For example, the system and / or method may achieve a reproducibility of 1.0 score units (e.g., the score may range from about 0 to 10, 0 to 100, and / or have any suitable range), and the score may be related to immune activation status. In another illustrative example, the system and / or method may achieve a repeatability of at least 10% (e.g., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, values ​​therebetween, etc.) in measuring biophysical parameters of samples in different conditions. However, the system and / or method may achieve any suitable repeatability and / or reliability.

[0010] Second, variations on the technology can reduce the occurrence of user error (improving the reliability, repeatability, reproducibility, validity of results, etc.). For example, automating most or all of the steps and / or sample interactions can reduce user interaction with the sample (which can also be beneficial to reduce the risk of user exposure to patient blood samples).

[0011] However, variations on the techniques may impart any other suitable benefits and / or advantages.

[0012] As used herein, "substantially" or other similar terms (e.g., "about," "approximately," etc.) may be within a given error threshold or tolerance of a metric, component, or other reference (e.g., within 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, etc. of the reference) or may be otherwise interpreted.

[0013] 3. System. The system may include a sample preparation module, a measurement module, a computing system, and / or any suitable components, as shown in Figure 1. The sample preparation module, the measurement module, the computing system, and / or other components may be integrated into a common housing (e.g., an enclosure, unit, etc.), into separate housings and / or units (e.g., as shown in the example of Figure 14), two or more components may share a common housing, and / or the components may be integrated or connected in any manner.

[0014] The system is preferably capable of functioning to prepare a sample (e.g., a blood sample) for measurement, measure the sample, analyze the sample (e.g., determine a patient's health status, determine a patient's immune activation status, etc.), and / or otherwise function.

[0015] The sample preparation module (100) (e.g., sample preparation system, sample preparation subsystem, blood sample system, etc.) preferably functions to prepare a patient sample to be measured. The sample preparation module may additionally and / or alternatively function to store the sample (e.g., prepare the blood sample for storage, modify the blood sample to increase shelf life, etc.) and / or function otherwise. Sample (15) preferably refers to a blood sample associated with a patient (e.g., arterially drawn blood, venipuncture drawn blood, peripheral blood sample, finger stick blood sample, venous blood, capillary blood, red blood cells, white blood cells, platelets, serum, plasma, etc.), but may additionally or alternatively include a mucus sample, a urine sample, a saliva sample, a sputum sample, feces, semen and / or other bodily fluids, etc. The sample can be provided in a centrifuge tube (e.g., a microcentrifuge tube), a test tube, a vacutainer tube, a vial, a well plate (e.g., a 24-well plate, a 48-well plate, a 96-well plate, a 384-well plate, a standard well plate, a deep well plate, etc.), and / or any suitable container or receptacle. The sample container (17) can be made of glass, plastic, metal, and / or any suitable material. The sample container can have a diameter (e.g., maximum diameter, minimum diameter, average diameter, diameter near the center, opening diameter, etc.) of about 10 mm to 50 mm (e.g., 15 mm, 16 mm, 17 mm, 20 mm, 21 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, etc.), a diameter less than about 10 mm, or a diameter greater than about 50 mm.

[0016] The sample preparation module is preferably autonomous (e.g., functions without user interaction, robotic, etc.), but may be semi-autonomous (e.g., requires user engagement to move between one or more steps) and / or manual (e.g., requires user input to perform one or more steps, such as adding a reagent). For example, a user may load a sample into the sample preparation module, which may then perform the steps of preparation without further user interaction. In another example, a user may perform a blood draw for a patient, and the sample preparation module may automatically load and process the blood sample from the drawn blood. However, the sample preparation module may be otherwise automated.

[0017] Inputs to the sample preparation module include sample, lysing solutions, quenching solutions, buffers, washes, fixatives, stains, and / or any suitable solutions or inputs. Outputs of the sample preparation module may include prepared sample (16), waste (e.g., by-products from lysis, quenching, washing, etc., such as cell fragments, proteins, plasma, salts, residual reagents, etc.), and / or any suitable output. The output sample (e.g., residual material not degraded or removed from the sample) is preferably in substantially the same activation state as before it was isolated and / or prepared (e.g., the biophysical properties of the processed sample are the same, within a threshold, or less than about 15%, 10%, 5%, 2%, 1%, 0.5%, 0.1%, values ​​in between, less than <0.1%, etc., relative to the biophysical properties of the same species prior to the processing step). The biophysical properties include cell subpopulations, relative amounts of cell subpopulations, cell mechanical properties, cell size, cell shape, cell deformability, cell response to forces (e.g., stress, strain, compression, expansion, etc.), trajectories (e.g., in a microfluidic chip), and / or any suitable properties. However, the output sample may be in a different activation state than the original sample and / or may have any suitable state. By way of illustration, the input sample may be a blood sample associated with a patient and the output of the sample preparation module may be a white blood cell sample (e.g., red blood cells, platelets, etc. may be removed from the blood sample). In this specific example, processing the blood sample does not substantially change the activation state of the white blood cells (e.g., their biophysical properties). However, the sample preparation system may receive any suitable input and / or output.

[0018] For example, as shown in FIG. 13, the sample preparation module may include a sample holder, reagent ports, spindles, a temperature control system, sensors, manifolds, reagent reservoirs, motors (e.g., stepper motors, centrifuges, etc.), and / or any suitable components.

[0019] The sample holder (110) preferably functions to support a sample within the sample preparation system. The sample holder can include (e.g., be) a test tube holder (e.g., a test tube rack, a ring having a diameter matching the center diameter of a sample container, etc.), a clamp (e.g., a finger clamp, a ring clamp, etc.), tongs, and / or any suitable sample holder. The sample (e.g., a sample container) can be suspended within the sample holder, rest on a surface (e.g., a curved surface matching the curvature of the sample container), and / or the sample can otherwise be held by the sample holder. The sample holder is preferably connected to a motor (e.g., so that the sample holder can be translated relative to other components of the system). The sample holder is preferably located below the spindle (e.g., centered below the spindle), but can have any suitable orientation with respect to the spindle. The sample holder can optionally be connected to a mixer and / or agitation mechanism (e.g., a magnetic stirrer, shaker, etc.).

[0020] The sample holder may include an automated sampler, which may function to automatically load, change, unload, and / or otherwise automatically modify samples. For example, the automated sampler may include a sample carousel (e.g., configured to hold multiple patient samples for a single patient, multiple patients, etc., where the samples may include labels, locations, holders, or other identifiers to identify and coordinate sample transfer, processes, etc.), robotics (e.g., configured to transfer samples between the carousel and the sample holder), and / or any suitable automated sampler.

[0021] In some variations, the sample holder may be the same as (eg, incorporated into) the spindle and / or reagent port (eg, as shown in FIG. 15).

[0022] The reagent ports (120, 120') may function to introduce reagents to the sample, remove reagents from the sample (e.g., aspirate the sample), mix the sample, and / or otherwise function. The reagent ports preferably have a smaller diameter than the sample container (e.g., so that the reagent port can fit inside the sample container), but may have a larger and / or equal diameter than the diameter of the sample container. The reagent ports may include needles, tubes, manifolds, tubing, and / or any suitable port may be used.

[0023] During operation, the reagent port is preferably inserted into the sample vessel. The reagent port is fully inserted to a threshold height (e.g., above the sample, relative to the sample holder, relative to the top of the sample holder, relative to the bottom of the sample holder, etc.), to a minimum depth (e.g., as high as the inlet of the vessel), submerged into the sample (e.g., to a threshold depth such that the sample covers one or more orifices), and / or to any suitable depth. However, the reagent port may additionally or alternatively remain outside the sample vessel (e.g., where different reagent ports may be used to introduce different reagents) and / or may be aligned differently relative to the sample. When the system is not in operation, the reagent port is preferably positioned above the sample holder (e.g., such that the port only needs to translate in one axis to enter the sample), but may have any suitable arrangement (e.g., requires translation in two or three axes to access the sample).

[0024] The reagent ports preferably include a set of orifices (e.g., openings, holes, etc.) at an end (e.g., the end that is inserted into a sample container), which can allow reagents to be added and / or removed from the sample. The orifices can share a common manifold (e.g., the manifold can split and / or connect to different endpoints) and / or can connect to different manifolds (e.g., each manifold connects to a different reagent or endpoint). Illustratively, the orifice can be used to introduce lysis reagents, quenching reagents, wash reagents, buffers, water, fixatives, vacuum (e.g., connected to a vacuum pump (179) to generate a vacuum pressure that is <10 mTorr, 10 mTorr, 20 mTorr, 50 mTorr, 100 mTorr, 150 mTorr, 200 mTorr, 300 mTorr, 400 mTorr, 500 mTorr, 600 mTorr, 700 mTorr, 710 mTorr, 720 mTorr, 750 mTorr, 755 mTorr, 760 mTorr, values ​​or ranges therebetween, etc.), and / or any suitable reagent.

[0025] In some embodiments, the reagents introduced using the orifices may depend on the motor position and / or the sample holder position (e.g., height). For example, the orifices may be open and / or closed depending on the position, valves or other components may function depending on the sample position (e.g., via interlocks, switches, etc.), the orifices may form closed fluid paths depending on the position (e.g., the tubes may be aligned and / or the orifices may depend on the motor and / or sample holder position). For example, as shown in FIG. 4, at a first height, the reagent ports may introduce lysis and / or quenching reagents. At a second height, as shown in FIG. 5, the reagent ports may introduce wash reagents and vacuum (e.g., concurrently, contemporaneously, simultaneously, sequentially, according to a predetermined pattern, etc.). At a third height, the reagent ports may be used to introduce buffer (e.g., to dilute the sample), for example, as shown in FIG. 6. However, the reagent ports may additionally and / or alternatively perform these operations at the same height (eg, at a given time) and / or under any suitable conditions.

[0026] The reagent ports may include multiple through manifolds (e.g., separate fluid pathways as shown in FIG. 3), with each internal manifold being able to connect to a separate endpoint (e.g., waste collection, vacuum, different reagents, etc.). However, the endpoints (or pathways to the endpoints) may be split (e.g., include valves, distributors, connectors, etc. that allow multiple endpoints to use a single manifold or reagent port) and / or configured differently.

[0027] In some variations, the system may include separate reagent ports, for example, different reagent ports may be used to introduce each reagent, a subset of reagents (e.g., reagents introduced before centrifugation, reagents introduced during centrifugation, reagents introduced after centrifugation), and / or any suitable reagents.

[0028] The spindle (130) preferably functions to centrifuge the sample, which is useful for separating species of the sample (e.g., based on solubility, density, mass, etc.). For example, a blood sample can be centrifuged (e.g., after hemolysis) to enrich for white blood cells and remove red blood cell fragments.

[0029] The spindle is preferably the same as the reagent port (eg, a cooperatively integrated component), but may be separate from the reagent port.

[0030] The sample is preferably centrifuged at a relative centrifugal force of about 500-5000 g, but can be centrifuged below 500 g and / or above 5000 g. For example, the sample can be centrifuged at about 8000 rpm (e.g., in a sample vessel of about 15 mm diameter, such as 8000±1000 rpm).

[0031] The spindle preferably includes a seal (135) (e.g., adhesive, gasket, chemical, thread, mechanical sealant, etc.) that functions to connect and / or seal the spindle and the sample (e.g., to allow a vacuum to be pulled, to prevent the sample from ejecting the sample container, etc.). The seal is preferably reversible, but may be irreversible. For example, a gasket (e.g., a rubber gasket, an o-ring, etc.) may be expanded and / or contracted to seal and unseal the connection, a snug fit (so that it can be overcome by opposing forces), a threaded seal, and / or any suitable seal may be used.

[0032] The reagent reservoirs (170) (e.g., bottles, containers, etc.) preferably function to store reagents to be dispensed and / or used for sample preparation. The reagent reservoirs may be located within a housing (e.g., sample preparation system housing), outside a housing (e.g., outside the sample preparation system housing), and / or in any suitable location. The reagent reservoirs are preferably coupled to the sample preparation system (e.g., reagent ports, spindles, samples, etc.) through a manifold (e.g., tubing), but may be directly fed (e.g., gravity coupled) and / or otherwise coupled to the sample preparation system. The reservoirs may be closed and / or open to the external environment. The reservoirs may be made of glass, plastic, metal, and / or any suitably suitable material.

[0033] The reagents in the reservoirs are preferably pre-made (e.g., including a mixture of reagents for a given processing step, separate reservoirs for each process, etc.). However, additionally or alternatively, separate reservoirs can exist for each component of the reagent and / or reaction process, and / or the reservoirs can otherwise contain any suitable reagents. In a first specific example, the lysis reservoir can contain a prepared lysis solution (172). In a second specific example, the first reservoir can contain formic acid (e.g., for lysis), and the second reservoir can contain a buffer (e.g., the formic acid and buffer can be mixed (e.g., in a mixing chamber, in a manifold, etc.) and / or dispensed (e.g., to achieve a target concentration)), and the lysis solution can be formed in situ, in a manifold, and / or at any suitable location.

[0034] For example (e.g., as shown in FIG. 13), the sample preparation system may include a lysis reagent reservoir (e.g., containing a lysis reagent (172)), a quench reagent reservoir (e.g., containing a quench reagent (174)), a wash reservoir (e.g., including or containing a wash solution (176)), and / or a buffer reservoir (e.g., including or containing a buffer reagent (178)). However, the sample preparation system may include any suitable reservoirs.

[0035] The manifold (160) functions to transfer one or more reagents between the endpoints. The manifold may operate under positive and / or negative pressure. The manifold preferably includes (e.g., is made of) chemically compatible materials (e.g., plastics, metals, glass, etc. that do not react with reagents that intentionally or accidentally contact the material), but may include weakly-compatible (e.g., compatible for a threshold duration of contact, compatible for a threshold concentration of reagent) and / or incompatible materials. Each reservoir may have a separate manifold, which may meet (e.g., with connectors, valves, distributors, etc.), the reservoirs may share a manifold (e.g., one or more manifolds may be closed to allow selective reagent uptake), and / or the manifold may be configured differently. In a specific example, each reservoir may include a manifold (e.g., tubing) configured to uptake a reagent from the reservoir. In this particular example, the manifolds may be connected or combined (e.g., using splitters, valves, multi-port connectors, etc.) and reagents from a single manifold may be added at one time (e.g., via reagent ports, spindles, etc.) (e.g., a single manifold is fluidly coupled to the sample at one time). However, the manifolds may be configured differently.

[0036] The motor (140) preferably functions to move (e.g., rotate, move, translate, rotate, etc.) the sample. The motor may move the sample directly (e.g., directly connected to the sample) and / or indirectly (e.g., via an intermediate component). The motor may be connected to the sample, the sample holder, the spindle, the manifold, the sensor, the wash tank, and / or any suitable component. Exemplary motors may include brushed motors (e.g., DC brushed motors, AC brushed motors, etc.), brushless motors (e.g., brushless DC motors, brushless AC motors, etc.), switched reluctance motors, universal motors, induction motors, hysteresis motors, pancake motors, stepper motors, and / or any suitable motor.

[0037] The sample preparation system may include multiple motors. For example, a stepper motor may be used to set the height of the sample (e.g., adjust, translate, etc. the height of the sample, sample holder, spindle, etc.) and a DC motor may be used to centrifuge the sample (e.g., rotate the sample via the spindle). However, any suitable motor may be used for any suitable purpose.

[0038] The temperature control system (150) of the sample preparation system preferably functions to maintain the temperature of the sample in the sample preparation system at or near a target sample temperature, which is preferably about 25° C. (e.g., 25±1° C., 24-26° C., 20-30° C., 25±0.5° C., 21-29° C., 22.5-27.5° C., etc.), but can be less than about 25° C. (e.g., 0° C., 5° C., 10° C., 15° C., 20° C., values ​​therebetween, etc.) and / or greater than about 25° C. (e.g., 30° C., 35° C., 37° C., 38° C., 39° C., 40° C., 45° C., values ​​therebetween, etc.). The temperature control system may include a heater (e.g., radiative heater, convection heater, conductive heater, resistive heater, gas heater, etc.) and / or a cooler (e.g., air conditioner, Peltier cooler, heat sink, thermoelectric cooler, absorption cooler, compression cooler, etc.). The temperature control system may be in direct contact with the component (e.g., touching the sample container, touching the manifold, touching the spindle, integrated into the spindle, integrated into the sample holder, integrated into the sample container, etc.), in indirect contact with the component (e.g., including an air gap between the heater and / or cooler and the component, including a thermally conductive material between the heater and / or cooler, etc.), isolated from the component (e.g., to prevent heating or cooling of one or more components, such as by having thermally isolating material between the component and the heater and / or cooling unit), and / or otherwise positioned.

[0039] The temperature can be actively controlled (e.g., using a positive feedback loop, a negative feedback loop, based on sensor readings, using PI control, PD control, PID control, etc.) and / or passively controlled.

[0040] In a first illustrative example, the environment within the sample preparation system may be maintained at approximately the target sample temperature. In a second illustrative example, the sample may be maintained at the target sample temperature. In a variation of the second illustrative example, the sample and reagents (e.g., reservoir, manifold, etc.) may be maintained at the target sample temperature. In a third illustrative example, the temperature of one or more components and / or reagents may be controlled to modify the sample temperature to maintain approximately the target sample temperature. For example (as shown in FIG. 7), when the temperature of the sample is approximately 20° C., the temperature of the reagents (e.g., lysis reagent, quench reagent, buffer, diluent, etc.) may be set to approximately 27° C. such that the final sample temperature (e.g., after mixing) is 25° C. The specific temperature of each component may be measured, empirically determined, heuristically determined, determined according to an equation (e.g., thermal mass balance), and / or otherwise determined. The temperature of the reagent may be set in the reservoir, in the manifold, in the reagent port, in the spindle, and / or at any suitable location within the sample preparation system. However, the temperature of any suitable component may be controlled in any manner.

[0041] The sensor (180) may function to measure one or more aspects (e.g., parameters, characteristics, etc.) of a sample within the sample preparation system. Exemplary embodiments include mass, volume, temperature, height (e.g., height relative to other components, total height within the sample container, etc.), color, density, solubility, activation state, pressure, and / or any suitable embodiment. The embodiments may be measured continuously, intermittently, at predetermined times, at predetermined frequencies, at specific times during operation of the system, and / or at any suitable timing. Sensors may be incorporated into components (e.g., spindles, manifolds, reagent ports, sample containers, sample holders, motors, etc.), incorporated into other components (e.g., spindles, manifolds, reagent ports, sample containers, sample holders, motors, etc.), and / or otherwise located inside or outside the sample preparation system.

[0042] Exemplary sensors include optical sensors (e.g., cameras, lasers, interferometers, etc.), acoustic sensors, pressure sensors, rulers, ultrasonic sensors, depth sensors (e.g., RADAR, LIDAR, SONAR, depth cameras, stereo cameras, etc.), spectrometers, scanners (185) (e.g., to scan the identity of a patient associated with a sample, to scan a cartridge lot, to scan a reagent tag, to facilitate or accelerate data entry, to ensure accurate data entry), and / or any suitable sensor may be used.

[0043] The sample preparation system may optionally include a wash unit (190). The wash unit preferably functions to wash the sample preparation system (e.g., spindle, reagent ports, etc.). For example, after a sample is prepared, the wash unit may be used to wash the sample preparation system (e.g., its spindle, reagent ports, etc.) before another sample is prepared. However, the wash unit may be used at any suitable time. For example, as shown in FIG. 13, the wash unit may be translated (and / or rotated) out of the way of the sample being loaded and then positioned at the sample position after the sample has been prepared.

[0044] The washing unit may be, for example, an empty sample container, an empty container, a filled container, and / or any suitable container. The washing unit may be filled (e.g., via a reagent port, spindle, manually, etc.) with a washing reagent (e.g., buffer, detergent, soap, etc.) and processed in the same manner as a sample, may be processed for one or more washing steps (e.g., including agitation, washing solution addition, buffer addition, centrifugation, spinning, mixing, aspiration, etc.), and / or may be processed differently to wash the sample preparation system and / or its components. The washing unit is preferably integrated into the sample preparation system, but may be separate from the sample preparation system (e.g., manually inserted by a user, etc., before and / or after preparing a sample). The sample preparation system is preferably washed automatically, but may be washed manually, semi-automatically, and / or with any suitable responsiveness. The sample preparation system is preferably cleaned after each use, but may be cleaned after a predetermined number of uses, before each use, at a predetermined frequency, based on the need for cleaning (e.g., how dirty the sample preparation system is), according to a cleaning schedule, and / or at any suitable time.

[0045] The measurement module (200) preferably functions to measure one or more sample parameters (e.g., sample characteristics). The measurement module preferably operates on prepared samples (e.g., samples prepared by a sample preparation system), but may operate on unprepared samples and / or any suitable sample. Samples may be transferred automatically (e.g., using a robotic arm, using an automated pipette, etc.), manually (e.g., by a user), and / or otherwise transferred from the sample preparation system to the measurement module.

[0046] Sample properties may include individual properties (e.g., properties of individual cells), aggregate properties (e.g., combinations of properties of individual cells as described by a distribution, mean property, median property, modal property, 95th percentile value, etc.), bulk properties (e.g., properties of the entire sample such as density, light absorption, etc.), and / or any suitable property.

[0047] Exemplary sample characteristics may include structural parameters, trajectory parameters, patient parameters, positional parameters (e.g., cell position relative to the image frame, cell position relative to the channel, cell position relative to the outlet, cell position relative to the inlet, cell position relative to the stagnation point, etc.), and / or any parameter. Examples of structural parameters include shape (e.g., ellipticity, helicity, ellipticity, circularity, curvature, skewness, etc.), aspect ratio (e.g., ratio of longest dimension to shortest dimension, ratio of length to width, etc.), size (e.g., lateral extent, longitudinal extent, depth, height, width, length, volume, surface area, etc.), constituent structures (e.g., location of cell membranes, cell shape, cell wall structure, etc.), constituent morphology (e.g., cell morphology, cell shape, particle shape, etc.), internal structure (e.g., shape, morphology, size of the nucleus and / or other organelles of the cell, etc.), and / or any other structural parameter. Examples of patient parameters include complete particle counts, complete blood counts, complete white blood cell counts, complete neutrophil counts, complete monocyte counts, complete lymphocyte counts, complete basophil counts, complete eosinophil counts, complete red blood cell counts, complete platelet counts, component counts (e.g., number of cells and / or cell segments, number of particles and / or particle segments, etc.), presenting symptoms (e.g., patient temperature, blood pressure, weight, blood oxygenation, etc.), cell density, cell culture results, hydration, and / or any other sample parameters.

[0048] A trajectory parameter can be a parameter related to and / or determined from the trajectory of a cell through a microfluidic cartridge (e.g., a deformation region, a focusing region, etc. of a microfluidic cartridge). A trajectory can be a series of discrete positions of a cell (e.g., a center of gravity, a center of mass, a reference point, an average position of a cell, etc.), a continuous path of a cell, and / or corresponds to any motion of a cell as it passes through a fluid stream (e.g., a deformation region of a microfluidic cartridge). Examples of trajectory parameters include a direction of motion of objects and / or features, a velocity of motion of objects and / or features (e.g., average velocity, instantaneous velocity, etc.), an acceleration of motion of objects and / or features, oscillations in the motion of objects and / or features (e.g., oscillation amplitude, oscillation frequency, oscillation phase, oscillation modulation, oscillation damping, etc., as shown in FIG. 12), a viscoelastic inertial response (VEIR), deviations in particle stream trajectories (e.g., from a linear path, an expected path, etc.), and / or any other trajectory parameter. The trajectory parameters may be determined based on differences, sums, amplitudes, maxima, minima, averages, and / or other characteristics of one or more positions of the trajectory. In variations in which the trajectory parameters include vibrations, the vibrations may correspond to vibrations of a reference point (e.g., a center of gravity, an extremum, etc.), a reference axis (e.g., one or more dimensions such as length, width, depth, etc.), one or more reference surfaces (e.g., an object boundary, an internal boundary of the object, such as corresponding to a sub-organ boundary, etc.), a reference volume (e.g., a volume of the object, an internal structure of the object, etc.), and / or other parts of the object. The vibrations preferably occur along a reference axis perpendicular to the direction of motion of the object. However, the reference axis may be parallel to the direction of motion and / or have any orientation relative to the direction of motion. The vibration amplitude is preferably on a micron size scale (e.g., 1-10 μm, 10-100 μm, etc.), but may additionally or alternatively be on a nanometer scale (e.g., 1-100 nm, 100 nm to 1 μm, etc.) and / or any suitable distance.

[0049] However, any suitable sample property may be measured.

[0050] The measurement module is preferably housed in an enclosure (e.g., a unit, enclosure, etc., as shown in FIG. 14, etc.) separate from the sample preparation module. However, the measurement module and the sample preparation module may be housed in the same housing. The measurement module is preferably located within a threshold distance of the sample preparation module (e.g., on the same table, in the same room, within 1 m, 2 m, 5 m, 10 m, 20 m, 50 m, etc.), which may ensure that the temperature of the sample does not change significantly between sample preparation and sample measurement, facilitate rapid sample measurement, and / or provide any suitable technical advantage.

[0051] In illustrative examples, the measurement module may be a cytometry module (e.g., a flow cytometry module) where a sample (e.g., a prepared sample) may be loaded into a cytometry cartridge (210) (e.g., a microfluidic device, a microfluidic channel, a flow path, etc.). The measurement module may include an imaging system (220), a motor (230), a temperature control system (240), a vibration isolator (250), a pressure system (260), and / or any suitable components. However, the measurement module may additionally or alternatively include an atomic force microscope module, an optical probe module, and / or any suitable modules and / or components.

[0052] A cartridge (e.g., a cytometry cartridge) may function to receive a sample, sort the sample, apply a stimulus to the sample, and / or otherwise function. The cartridge is preferably a single-use cartridge (e.g., to reduce the risk of contamination between different samples), but may be a multi-use cartridge. The cartridge may be made of plastic, glass, metal, and / or any suitable material.

[0053] For example, as shown in FIG. 16A, the cartridge may include an inlet (211), an inlet filter (212), a microfluidic channel (213), an outlet filter (216), an outlet (217), and / or any suitable components. For example, as shown in FIG. 8 and / or FIG. 16B, a sample may enter the cartridge through an inlet (211), pass through an inlet filter (212) (e.g., to remove particle clumps, minimize cross contamination within a measurement module, etc.), enter a focusing region (214) (e.g., of a microfluidic flow path including multiple sharp bends or turns, which may function to sort the sample into individual components, focus the components into a path of the microfluidic device, etc.), enter a deformation region (215) (e.g., an elongational flow region including a first stream including sample constituents and a second stream intersecting the first stream, which may function to apply stress, strain, pressure, force, etc. to the sample), and exit through an outlet (e.g., via an outlet filter). However, the cartridge may be arranged in other manners. The intersections (e.g., at the deformation regions) may be three-way intersections (e.g., "T" junctions, "Y" junctions, etc.), two-way intersections (e.g., "L" junctions, "I" junctions, "V" junctions, Venturi junctions, etc.), four-way intersections (e.g., "x" junctions, "t" junctions, "K" junctions, etc.), and / or any suitable shape.

[0054] The cartridge is preferably loaded into a cartridge holder configured to hold the cartridge (e.g., within a threshold tolerance of 0.0001", 0.0005", 0.001", 0.005", 0.01", 0.05", etc., without the cartridge shifting or moving within the holder).

[0055] In a specific example, the cartridge may be any of the following: U.S. Application No. 16 / 374,663, filed April 3, 2019, and entitled "SYSTEM AND METHOD FOR DEFORMING AND ANALYZING PARTICLES," U.S. Application No. 15 / 868,025, filed January 11, 2018, and entitled "METHOD AND DEVICE FOR HIGH-THROUGHPUT CELL DEFORMABILITY MEASUREMENTS," U.S. Application No. 16 / 676,352, filed November 6, 2019, and entitled "METHOD AND DEVICE FOR HIGH-THROUGHPUT SOLUTION EXCHANGE FOR CELL AND PARTICLE SUSPENSIONS," U.S. Application No. 16 / 676,352, filed October 18, 2013, and entitled "SYSTEM AND METHOD FOR DEFORMING, IMAGING AND ANALYZING PARTICLES," each of which is incorporated by reference in its entirety. No. 9,464,977, filed April 3, 2017, and entitled "SYSTEM AND METHOD FOR DEFORMING PARTICLES," and may include and / or be any suitable microfluidic system disclosed in U.S. Patent No. 10,252,260, filed April 3, 2017, and entitled "SYSTEM AND METHOD FOR DEFORMING PARTICLES."

[0056] The imaging system preferably functions to acquire images of the sample (eg, images of cells in the sample), which images can be analyzed to determine sample properties. The imaging system is preferably positioned around the cartridge (e.g., above, below, next to, optionally optically connected via mirrors, optical fibers, free space coupling, optical material, immersion oil, etc., as shown in the example of FIG. 9, etc.), however, the imaging system may be positioned in other ways.

[0057] The imaging system may include an illumination system (222), imaging optics (225), an image sensor (228), and / or any suitable components. The illumination system is preferably a light emitting diode (e.g., an LED, which may be beneficial for introducing a relatively small amount of heat into the measurement module), but may additionally or alternatively include an incandescent lamp, a fluorescent lamp, a black light, a halogen lamp, and / or any suitable light source. The light source is preferably positioned for Kohler illumination of the sample (e.g., to generate uniform illumination of the sample without generating an image of the illumination source in the resulting image, such as by including a collector lens and / or a field lens, a field diaphragm, a condenser diaphragm, a condenser lens, etc.), but may additionally or alternatively be positioned for critical illumination and / or any suitable illumination. The light source may be positioned above, below, and / or otherwise relative to the sample.

[0058] The imaging optics may include a microscope objective, a condenser, a lens, a mirror, a polarizer, a wave plate, and / or any suitable components. The imaging optics may be fixed (e.g., set in a fixed location relative to the sample) and / or movable (e.g., movable relative to the sample so that the optics can move and / or the sample can be moved to achieve a target focal plane). For example, the sample and / or the optics may be translated (e.g., using a motor) to set the focus of the imaging optics on the sample (e.g., on the deformation region of the sample). In this example, a set of optical guides (e.g., on the cartridge) may be used to facilitate focusing and / or alignment of the sample to the imaging system (e.g., the imaging optics).

[0059] The image sensor is preferably a high speed camera (e.g., a camera capable of capturing at least 1000 fps, 2000 fps, 5000 fps, 10,000 fps, 20,000 fps, 50,000 fps, 100,000 fps, 200,000 fps, 500,000 fps, 1,000,000 fps, values ​​in between, >1,000,000 fps, etc.), but may be a low speed camera (e.g., a camera capable of capturing up to 1000 fps, 120 fps, 100 fps, 60 fps, 50 fps, 30 fps, 24 fps, 20 fps, 12 fps, values ​​in between, <12 fps, etc.) and / or any suitable camera. The camera is preferably a visible camera (e.g., CCD, CMOS, etc., capable of detecting radiation at least between 400-800 nm), but may additionally or alternatively be sensitive to infrared (e.g., near infrared, mid infrared, far infrared, etc.), ultraviolet, x-ray, microwave, radio wave, and / or any suitable radiation. The image sensor may be positioned above, below, and / or at any suitable position or orientation relative to the sample (e.g., cartridge).

[0060] In an illustrative example, the imaging system may be (e.g., may be configured as) a microscope (e.g., an upright microscope, an inverted microscope, a confocal microscope, etc.), however, any suitable imaging system may be used.

[0061] The temperature control system may function to pre-heat the cartridge (e.g., heat and / or cool the cartridge to achieve a target temperature prior to addition of the sample), maintain the temperature of the cartridge, maintain the temperature of the sample, maintain the temperature of the measurement module, and / or otherwise. The temperature control system may be local (e.g., to components of the measurement module) and / or global (e.g., effects of the entire measurement module, impact, etc.). For example, the temperature control system may be incorporated into the cartridge holder, the cartridge, the imaging system, the vibration isolation system, and / or any suitable component. The temperature control system may be radiative, non-radiative, convective, conductive, combinations thereof, and / or any suitable temperature control system.

[0062] The temperature control system (e.g., of the measurement module) can include and / or be the same as and / or different from the temperature control system as described above for the sample preparation system. For example, the measurement module temperature control system can have a higher temperature tolerance (e.g., achieve, maintain, etc. a more accurate, precise temperature, etc.). For example, the temperature of the sample preparation system can be maintained with a tolerance of about ±1° C. and the temperature of the measurement module can be maintained with a tolerance of about ±0.3° C. However, the temperature tolerance of the measurement module can be the same as and / or less than the temperature tolerance of the sample preparation system.

[0063] The vibration isolator preferably functions to dampen and / or minimize the effect of cartridge motion on cartridge alignment. The vibration isolator is preferably connected to the cartridge (e.g., via a cartridge holder), but may be built into the cartridge, built into the cartridge holder, connected to the imaging system, connected to the housing of the measurement module, and / or otherwise connected and / or aligned. Exemplary vibration isolators include honeycomb structures, springs, pneumatic isolators, sheets or pads of flexible material (e.g., rubber, elastomer, cork, foam, laminates, etc.), tuned mass dampeners, rope isolators, active vibration isolators (including, e.g., sensors such as piezoelectric accelerometers, force sensors, MEMs accelerometers, geophones, proximity sensors, interferometers, etc., actuators such as linear motors, pneumatic actuators, piezoelectric motors, controllers, etc.), and / or any suitable vibration isolator may be used. The vibration isolators may attenuate low frequency vibrations (e.g., <0.1 Hz, <1 Hz, <5 Hz, <10 Hz, etc.), medium frequency vibrations (e.g., 10-10000 Hz), high frequency vibrations (e.g., >10000 Hz), combinations thereof, and / or any suitable frequency vibrations. The vibration isolators may be horizontal isolators, vertical isolators, six degree of freedom isolators, three degree of freedom isolators, and / or isolate vibrations in any suitable axis or direction.

[0064] The motor preferably functions to translate the sample (e.g., cartridge) relative to the imaging system (e.g., change portions of the cartridge, sample, etc. within the field of view, focal plane, etc. of the imaging system). However, the motor may function differently. The motor is preferably coupled (e.g., connected, mechanically coupled, capable of translating, etc.) to the cartridge (e.g., cartridge holder), but may be coupled to the imaging system (e.g., light source, optical components, image sensor, etc.) and / or any suitable components. The motor may be continuously operable (eg, to set the position to an arbitrary position) and / or discretely operable (eg, to set the sample position to one or more discretized positions).

[0065] The sample may be stationary, translated, and / or otherwise moved during the measurement. For example, the sample may be moved at predetermined times according to a movement schedule to randomly correct for vibrations (e.g., to isolate systematic movement errors, vibrations, etc.) and / or otherwise moved. In a specific example, after loading, the sample may be aligned (e.g., moved) such that the deformation region of the cartridge is within the field of view of the imaging system. In a second specific example, the sample may be focused (e.g., by translating the sample along the optical axis of the imaging system). In a third specific example, the sample may be translated such that the imaging system acquires multiple images of the sample at a position upstream of the deformation region before (or after) measuring an image of the sample (e.g., cells in the deformation region). Images from the third specific example may be used to determine (e.g., measure, calculate, estimate, etc.) the flow velocity of the sample (e.g., cells in the sample, fluid velocity in the sample, etc.) and / or any suitable sample characteristics (e.g., cell mechanics in the absence of a deformation force, cell number, etc.). After the sample characteristics are determined, the sample may be translated so that the deformation region is within the field of view of the imaging system. However, the sample characteristics (e.g., flow rate) may be determined otherwise. These specific examples are non-limiting and non-exclusive examples, and the sample may be translated according to one or more of these examples in combination.

[0066] The pressure system preferably functions to urge the sample through the cartridge (e.g., through the microfluidic channels). The pressure system can be a positive pressure (positive displacement, such as pushing the sample out) system, a negative pressure (negative displacement, such as pulling the sample out) system, and / or any suitable combination thereof and / or type of displacement system. The pressure system is preferably connected to the cartridge (e.g., its inlet, outlet, etc.), but can additionally or alternatively be connected to any suitable components. Exemplary pressure systems include the use of vacuum pumps, peristaltic pumps, syringe pumps, microfluidic pumps, microfluidic precision pumps, and / or any suitable pump. The pressure system preferably generates a pressure differential (e.g., between the inlet and outlet of the cartridge) that is about 90 psi (e.g., 90±1, 90±3, 90±5, 90±10, etc.), but may generate a pressure differential of about 50-150 psi, less than 50 psi, and / or more than 150 psi. The pressure differential depends on the target flow rate, sample temperature, sample viscosity, sample pH, sample zeta potential, average sample particle size, channel size (eg, nano, micro, milli, etc. channels), and / or on any suitable characteristic.

[0067] The computing system (300) may function to control the operation of the sample preparation system, control the operation of the measurement module, process (e.g., analyze) data (e.g., images) acquired by the measurement module, determine scores (e.g., leukocyte structure index (LSI), sepsis, diagnostic score, probability of diagnosis, etc.), and / or function in other ways. The computing system may be local (e.g., a dedicated computer as shown in FIG. 14 ), remote (e.g., cloud computing, server, database, etc.), and / or distributed in any manner (e.g., the sample preparation system may include a computing system, the measurement module may include a computing system, and a dedicated processing computing system). The computing system may include a processor, a microprocessor, a computer processing unit, a graphics processing unit, and / or any suitable processor. In a specific example, the computing system may include GPU-accelerated computation, which may be beneficial for quickly and / or efficiently handling (e.g., processing) large data sets (such as those generated by image sets). In some variations, the computing system may include an analysis module (e.g., operable to determine, detect, estimate, predict, calculate, etc., the immune activation state of a patient based on biophysical characteristics, etc.), which may include an image analyzer, a quality detector, and / or any suitable components.

[0068] The computing system may include an image analyzer (320), which may function to determine characteristics of the sample from a set of images, particularly but not limited to mechanical properties and / or trajectory parameters. The image analyzer may include a segmentation module (e.g., which may function to segment the image into a foreground, such as cells, and a background), a feature module (e.g., which may function to identify or determine image features, such as cell centroids, cell boundaries, cell edges, cell membranes, etc.), a positioning module (e.g., which determines the location of features within the image), a processing module (e.g., which may function to determine sample characteristics based on the location, shape of features, evolution of feature shape, evolution of location, etc.), and / or any suitable components. The image analyzer may include machine learning algorithms, stereo algorithms (e.g., optical flow), classifiers, and / or any suitable algorithms and / or equations. The image analyzer may process individual images (e.g., frame-level analysis, such as determining cell characteristics in a given frame), sets of images (e.g., event-level analysis, such as tracking cells moving through a deformation region, field of view, etc.), population analysis (e.g., to process the sample as a whole), and / or any suitable analysis.

[0069] The image analyzer may include a quality detector (325) that may function to determine the quality (e.g., score, ranking, use or not, etc.) of an image, which may be used to determine whether an image should be analyzed or included in the analysis. The quality may be frame-level, event-level, population-level, and / or any suitable level of quality. For example, low quality images may be excluded from the set of analyzed images, images within a threshold number of frames may be excluded from the set of analyzed images, images containing the same features (e.g., the same cells) may be excluded from the set of analyzed images, and / or may be otherwise used to modify which images are analyzed. In specific examples, the quality may be determined based on the number of cells in an image, overlap of cells in an image (e.g., low quality when cells are touching and / or overlapping, high quality when cells are separated), distance between cells, and / or the quality may be derived from the cells in any suitable manner.

[0070] The computing system may include a scoring module (340) that may be operable to use sample characteristics (e.g., as determined by the image analyzer) to determine a diagnostic score (e.g., LSI, probability of diagnosis, diagnosis, etc.) associated with the sample (and / or patient). The scoring module may include one or more equations, machine learning algorithms (e.g., neural networks, convolutional neural networks, recurrent neural networks, etc.), classifiers, lookup tables, statistical regression, Bayesian regression, and / or any suitable method for determining a score. Inputs to the scoring module may include sample characteristics, patient information (e.g., patient demographic information, symptomatic patients, etc.), one or more images (e.g., average image, representative image, full image, images of cells at or near a particular location on the cartridge, etc.), ambient conditions (e.g., temperature, humidity, pressure, etc.), flow rate, sample temperature, and / or any suitable input. The output of the scoring module may be a score (e.g., a numeric score, a number between 0-1, 0-10, 1-10, 0-100, 1-100, etc., etc.), a classification (e.g., "low-probability of sepsis", "medium probability of sepsis", "high probability of sepsis", "further testing recommended", "other diagnostic tests recommended", "sepsis treatment recommended", etc.), color-coded information (e.g., green to indicate low probability of sepsis, low probability of a dangerous immune activation state, etc., yellow to indicate that further testing is needed, the result was inconclusive, etc., red to indicate high probability of sepsis, high immune activation, etc.), and / or any suitable information.

[0071] A computing system (e.g., an image analyzer, a scoring module, etc.) can be trained using a set of training data (e.g., labeled training data, unlabeled training data, etc.). The set of training data preferably includes data related to patients diagnosed with a high immune activation state (e.g., having sepsis, having metastatic cancer, having cancer, etc.) and data related to patients diagnosed with a low immune activation state (e.g., not having sepsis, not having metastatic cancer, not having cancer, etc.). However, the training data set may include any suitable data.

[0072] In some embodiments, the computing system (e.g., the image analyzer and / or scoring module, alone or in combination) may perform any suitable operations and / or methods disclosed in U.S. Patent Application No. 17 / 401,627, entitled "SYSTEM AND METHOD FOR IMMUNE ACTIVITY DETERMINATION," filed August 13, 2021, and incorporated by reference in its entirety. However, the computing system may be operated otherwise.

[0073] 4. Method. As shown in Figure 2, the method may include preparing a sample and measuring the sample. The method optionally includes pre-treating the system (or portions thereof), determining the immune activation status of the patient, providing an intervention to the patient (e.g., based on the immune activation status), and / or any suitable steps.

[0074] The method is preferably performed by a system (e.g., as described above), but may be performed by any suitable system. In some embodiments of the method (and / or steps thereof), timing may be beneficial to achieve accurate, reproducible, reliable, valid, and / or other quality results (e.g., diagnosis). For example, duration of steps, amount of time between steps (e.g., maximum amount of time, minimum amount of time, etc.), total amount of time the method is performed, amount of time to prepare the sample, amount of time to measure the sample, amount of time to analyze the measurement of the sample, and / or any suitable timing may enable technical advantages of the method. In some embodiments of the method (and / or steps thereof), temperature (e.g., temperature of the sample, system, system components, etc.) may be beneficial to achieve accurate, reproducible, reliable, valid, and / or other quality results (e.g., diagnosis). For example, maintaining the temperature of the sample at a target temperature during sample preparation, during sample storage (e.g., before, during, and / or after sample preparation or measurement), during sample measurement, and / or any suitable time may help ensure that the measurement data is of sufficient quality. These embodiments may be related, combined, separate, and / or otherwise related or unrelated, but the method may in some circumstances provide results (e.g., of sufficient quality) without (or with less strict control over) timing, temperature, and / or other appropriate characteristics.

[0075] The method preferably minimizes user input and / or feedback, which is beneficial to ensure that the method is performed consistently for different samples. For example, the steps of the method may be performed automatically, may include checks to ensure that the steps are performed properly, and / or may be performed otherwise. However, the method and / or its steps may be performed with input and / or feedback from a user. In a specific example of the method, a user may load a sample into a sample preparation module (e.g., for sample preparation) and / or transfer a prepared sample to a measurement module (e.g., for measurement), and other method steps in this example may be performed without user intervention (e.g., the method may be performed upon closing the sample preparation module, upon closing the measurement module, etc.). However, a sample may be automatically added (e.g., using a robotic arm, an autosampler, an autopipette, etc.), may be automatically transferred (e.g., using a robotic arm, an autosampler, an autopipette, etc.), and a user may initiate one or more method steps and / or perform the method steps otherwise.

[0076] Pre-treating the system (S100) preferably functions to prepare the system to receive a sample. Pre-treating the system may be particularly beneficial to ensure that the system is within target conditions for operation. Pre-treating the system may include pre-treating a sample preparation module, pre-treating a measurement module, and / or pre-treating any suitable components. Pre-treating the system may be performed concurrently (e.g., contemporaneously, simultaneously, etc.) with preparing the sample, before preparing the sample, and / or after preparing the sample (e.g., before measuring the sample).

[0077] Pre-treating the system may include loading a cartridge (e.g., into the measurement module), heating (and / or cooling) the cartridge to a desired temperature, cleaning the sample preparation system (e.g., treating buffers, treating cleaning solutions, etc.), cleaning the measurement module, processing a calibration sample (e.g., a sample having particles of known characteristics, a sample having fixed cells, etc.), pre-treating the sample (e.g., diluting, heating, cooling, adding anticoagulant, fixing, etc.), setting environmental conditions of the sample preparation module (e.g., temperature, pressure, humidity, etc.), setting environmental conditions of the measurement module (e.g., temperature, pressure, humidity, etc.), cooling the imaging system, and / or any suitable steps. In a specific example, the cartridge may be loaded into the measurement module before the sample can be introduced into the sample preparation system, which may be beneficial to allow for alignment of the cartridge with the imaging system, pre-heating of the cartridge (e.g., to a target temperature, to a stabilized temperature, etc.), and / or otherwise beneficial. However, pre-conditioning the system may include any suitable steps.

[0078] Preparing the sample (S200) preferably functions to prepare the sample for measurement. The sample is preferably prepared using a sample preparation module, but may be prepared by a user and / or by any suitable system. The sample is preferably prepared before the sample is measured. However, the sample may be prepared at the same time (e.g., the sample may be measured while it is being prepared) and / or after the sample is measured (e.g., the sample may be post-processed after measurement, the processing may be used to measure sample properties, etc.).

[0079] Preferably, about 0.1-10 mL of sample is prepared (e.g., transferred to the sample preparation system). For example, about 1 mL (e.g., 1 mL ± 10%) of sample can be transferred. In this example, if more or less sample is provided to the sample preparation system (e.g., less than 900 μL, more than 1.1 mL, such as resulting from user error, pipette error, etc.), a new sample can be loaded (e.g., the user can instruct the sample to be discarded and a new sample to be loaded), the process can be adjusted to account for the volume (e.g., increasing and / or decreasing reagents, times, speeds, etc. based on the volume loaded), the sample can be treated as normal (e.g., including a flag indicating that results derived therefrom may not be conclusive as a result), and / or the process can be modified differently and / or remain the same.

[0080] Preparing the sample may include lysing the sample (S220), quenching the sample (S240), washing the sample (S260), suspending the sample (S280), and / or any suitable process.

[0081] Lysing the sample preferably functions to break down red blood cells (e.g., erythrocytes) and / or other cells (e.g., cells other than white blood cells, cells not being measured, etc.) from the sample (e.g., haemolysis, hemolysis, etc.). Lysing the sample preferably does not change the activation state and / or any properties of the species being measured (e.g., cells, white blood cells, leukocytes, etc.), which may be beneficial to ensure that the measurement is representative of the state of the sample. However, lysing the sample may change the activation state and / or properties of the species being measured (e.g., by a known amount, in a known manner, to effect a predetermined change, to remove one or more subpopulations of white blood cells, to remove a subpopulation of a species, etc.).

[0082] Lysing the sample preferably involves adding a lysis solution to the sample and mixing the sample (e.g., stirring, agitating, etc.), but may additionally or alternatively involve heating the sample, sonicating the sample, irradiating the sample, and / or any suitable process.

[0083] The lysis solution may contain a lysis reagent (e.g., cell membrane disruptors that may function to lyse species, such as formic acid, ammonium chloride, CTAB, diethylene glycol, saponin, detergents, etc.), an anticoagulant (e.g., that function to prevent clotting, clumping, etc., such as sodium EDTA, heparin, warfarin, sodium citrate, acid-citrate-dextrose, citrates, oxalates, batroxobin, hementin, vitamin E, alcohols, coumarins, etc.), a detergent (e.g., that function to modify the surface tension of the lysis solution, such as phosphatidylcholine, sophorolipids, rhamnolipids, lecithin, bile salts, etc.), a fixative (e.g., that function to preserve the species and / or subspecies of the sample, such as formaldehyde, paraformaldehyde, glutaraldehyde, phenol, etc.), a solvent (e.g., , water, saline, seawater, potassium bicarbonate, buffered saline, phosphate buffered saline (PBS), Tris buffered saline (TBS), borate buffered saline, Tris-NaCl-Tween buffer (TNT), phosphate buffered Tween (PBT), HEPES, sodium acetate, cacodylate, citrate, Sorensen phosphate buffer, "Good's" buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate citrate buffer, Tris buffer, barbital buffer, etc., buffers, stains (e.g., contrast agents, fluorescent dyes, phosphorescent dyes, etc.), and / or any suitable components. The lysis solution preferably includes mild reagents, but may include harsh reagents, corrosive reagents, and / or any suitable reagents. In a specific example, the lysis solution may include a 1-5% (e.g., by weight, volume, composition, etc.) formic acid solution (e.g., in water, in a buffer, etc.). Specific example variations may include saponin (e.g., 0.1%-1% saponin concentration by weight, volume, composition, etc.) and / or may be saponin-free (e.g., consisting essentially of a solution with no saponin or other terpenoids), however, any suitable dissolution solution may be used.

[0084] Dissolution of the sample can use (e.g., in one or more aliquots or additives) about 0.1 ml to 100 ml of dissolution solution (e.g., 0.1 ml, 0.2 ml, 0.5 ml, 1 ml, 2 ml, 5 ml, 10 ml, 20 ml, 50 ml, 100 ml, any value or range therebetween, etc.).

[0085] The sample is preferably lysed over a lysis time. The lysis time may depend on the amount of sample, the sample temperature, the lysis solution (e.g., concentration, reagents, components, etc.), the type of sample, the quench solution, the quench mechanism, the measurement module (e.g., the type of measurement to be performed), the target sample (e.g., the characteristics of the target sample), and / or may be determined in other ways. The lysis time is preferably about 5 seconds to 60 seconds (e.g., 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, values ​​therebetween, etc.). If the lysis time is too long, the white blood cells (or other species being measured) may change their activation state, be lysed, and / or be otherwise altered or affected. If the lysis time is too short, the red blood cells may be incompletely lysed (e.g., and therefore may be difficult to separate) and / or the sample may be otherwise incompletely processed. However, the lysis time may be less than 5 seconds and / or more than 60 seconds. In a specific example, the dissolution time can be about 10 seconds (e.g., 9-11 seconds, 6-12 seconds, 9.5-11.5 seconds, 5-15 seconds, 6-14 seconds, 8-12 seconds, 9.5-10.5 seconds, 5-30 seconds, etc.), but the dissolution time can be any suitable time.

[0086] The sample may be dissolved at ambient temperature (e.g., room temperature, environmental temperature, 15-25°C, etc.), elevated temperature (e.g., about 25°C, 30°C, 35°C, 40°C, 45°C, etc.), reduced temperature (e.g., about 0°C, 10°C, 15°C, etc.), and / or any suitable temperature.

[0087] Quenching the sample preferably functions to quench (e.g., pause, stop, slow, etc.) the dissolution reaction. The sample is preferably quenched before the species to be measured are dissolved or otherwise affected by the dissolution process. However, the sample may be dissolved after the species to be measured are affected. For example, the sample may be quenched shortly after the dissolution time has elapsed (e.g., within up to 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, etc.). However, the sample may be quenched concurrently with dissolution (e.g., quenching may occur via diffusion rather than mixing) and / or at any suitable time.

[0088] Quenching the sample preferably does not change the activation state and / or any properties of the species being measured (e.g., cells, white blood cells, leukocytes, etc.), which can be beneficial to ensure that the measurement is representative of the sample state. However, quenching the sample can change the activation state and / or properties of the species being measured (e.g., by a known amount, in a known manner, to effect a predetermined change, to remove one or more subpopulations of white blood cells, to remove a subpopulation of a species, etc.).

[0089] Quenching the sample preferably includes adding a quenching solution to the sample and mixing the sample (e.g., stirring, agitating, etc.), but may additionally or alternatively include heating the sample (e.g., burning off the lysis reagent, evaporating the lysis agent, etc.), cooling the sample, sonicating the sample, irradiating the sample, and / or any suitable process. The quenching solution may depend on the sample (e.g., volume, concentration, identity, species, etc.), the lysis solution (e.g., concentration, lysis reagent, etc.), temperature, humidity, pressure, and / or any suitable sample or environmental characteristics. Quenching the sample may use about 0.1 ml to 100 ml of quenching solution (e.g., 0.1 ml, 0.2 ml, 0.5 ml, 1 ml, 2 ml, 5 ml, 10 ml, 20 ml, 50 ml, 100 ml, values ​​or ranges therebetween, etc.) (e.g., in one or more aliquots or additives).

[0090] The quenching solution is preferably a basic solution (e.g., a buffer with a pH greater than 7, a solution with a pH greater than that of the dissolution solution, etc.), but may be an acidic solution and / or a neutral solution. For example, the quenching solution may include a quenching agent such as a carbonate (e.g., carbonates of sodium, lithium, potassium, rubidium, cesium, beryllium, magnesium, calcium, strontium, barium, aluminum, etc.), a bicarbonate (e.g., bicarbonate of sodium, lithium, potassium, rubidium, cesium, beryllium, magnesium, calcium, strontium, barium, aluminum, etc.), an acetate (e.g., acetate of sodium, lithium, potassium, rubidium, cesium, beryllium, magnesium, calcium, strontium, barium, aluminum, etc.), a salt (e.g., sodium chloride, sodium sulfate, etc.), and / or any suitable quenching species. The quenching agent may be dissolved in water, saline, a buffer, a solvent (e.g., alcohol, ether, glycol, solvent mixture, etc.), and / or any suitable solvent. As an illustrative example, the quench solution may include a 1-10% sodium bicarbonate solution (e.g., in water, in a buffer, etc.) The quench solution may accurately neutralize the lysate, may over-neutralize the lysate (e.g., the remaining solution may have residual quenching agent), and / or may under-neutralize the lysate (e.g., the remaining solution may have residual lysing reagent).

[0091] The sample is preferably quenched (e.g., mixed, in the presence of a quenching agent, etc.) for a quench time. The quench time may depend on the amount of sample, the sample temperature, the lysis solution (e.g., concentration, reagents, components, etc.), the quench solution (e.g., concentration, reagents, components, etc.), the type of sample, the quench mechanism, the measurement module (e.g., the type of measurement being performed), the target sample (e.g., the characteristics of the target sample), and / or may be determined otherwise. The quench time is preferably about 5 seconds to 60 seconds (e.g., 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, values ​​therebetween, etc.). If the quench time is too long, the white blood cells (or other species being measured) may change their activation state, be lysed, and / or be otherwise altered or affected. If the quenching time is too short, the lysis solution or its reagents may be insufficiently quenched, which allows lysis to continue and / or the sample may otherwise be incompletely processed. However, the quenching time may be less than 5 seconds and / or more than 60 seconds. In specific examples, the quenching time may be about 10 seconds (e.g., 9-11 seconds, 6-12 seconds, 9.5-11.5 seconds, 5-15 seconds, 6-14 seconds, 8-12 seconds, 9.5-10.5 seconds, 5-30 seconds, etc.). However, the quench time can be any suitable time.

[0092] The sample may be quenched at ambient temperature (e.g., room temperature, ambient temperature, 15-25° C., etc.), elevated temperature (e.g., about 25° C., 30° C., 35° C., 40° C., 45° C., etc.), reduced temperature (e.g., about 0° C., 10° C., 15° C., etc.), and / or any suitable temperature.

[0093] Washing the sample may preferably function to remove particle debris from the sample, to isolate the species of the sample being measured from by-products or other sample components, to remove lysis and / or quench by-products, and / or otherwise. The sample is preferably washed within a threshold time after the sample is quenched. For example, the sample may be washed immediately (e.g., within 1 second) after the quench time has elapsed. In another example, the sample may be washed within a threshold time (e.g., 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, etc.) that the quench time has elapsed. However, the sample may be washed after the sample is quenched (e.g., a delay may be introduced between quenching the sample and washing the sample) and / or the sample may be washed otherwise.

[0094] Washing the sample may include introducing a wash solution (S262), agitating the sample (S264), collecting the sample (S266), aspirating the sample (S268), decanting the sample, and / or any suitable steps and / or processes.

[0095] The wash fluid is preferably a buffer, but may be water, saline, a solvent (e.g., ether, alcohol, etc.), and / or any suitable solvent and / or solution. The wash fluid may be added all at once, in aliquots, dropwise (e.g., slowly), and / or in any suitable manner. In some variations, the wash fluid may be added concurrently (e.g., contemporaneously, simultaneously, etc.) with aspirating the sample. However, washing the sample may include alternating addition of wash fluid, agitating (e.g., mixing) the sample, and aspirating the sample, and / or the sample may be washed in any manner. Washing the sample may use about 1 ml to 100 ml (e.g., 1 ml, 2 ml, 5 ml, 10 ml, 20 ml, 50 ml, 100 ml, values ​​or ranges therebetween, etc.) of wash fluid (e.g., in one or more aliquots or additions).

[0096] Collecting the sample preferably serves to separate the prepared sample into samples to be measured (e.g., sample subspecies) and sample components to be discarded (e.g., waste, by-products, degradants, residual reagents, etc.). Sample components to be measured are preferably retained (e.g., collected), but may be used in any manner. Sample components to be discarded are preferably discarded (e.g., to a waste process), but may additionally or alternatively be measured (e.g., using a separate measurement module, to obtain auxiliary data for analysis, etc.) and / or used in any manner.

[0097] Collecting the sample preferably includes centrifuging the sample. The sample may be centrifuged using a microcentrifuge, a low-speed centrifuge, a high-speed centrifuge, an ultracentrifuge, and / or any suitable centrifuge. For example, a spindle may be used to centrifuge the sample. The centrifuge may be used as a fractionation centrifuge, a differential centrifugation, a density gradient centrifugation, and / or in any suitable centrifugation process. The sample may be centrifuged at a speed that sediments the components to be measured, does not sediment the components to be discarded, does not substantially change the activation state of the components, does not damage the components, and / or any suitable speed based on the centrifugation duration (e.g., the time required to sediment the components). For example, the sample may be centrifuged at a speed that generates a relative centrifugal force of about 500-5000 g (e.g., a speed of about 1000-20000 RPM). In this example, the sediment (e.g., pellet) preferably contains the sample components to be measured (e.g., white blood cells) and the supernatant contains the sample components to be discarded (e.g., lysed red blood cells). However, the pellet may contain the sample components to be discarded, the supernatant may contain the sample components to be measured, the sample may be separated into multiple fractions (e.g., red blood cells, buffy coat, plasma, etc.), and / or the sample may be separated in any suitable manner. However, the sample may be centrifuged at a speed generating a relative centrifugal force of less than 500 g and / or more than 5000 g. However, collecting the sample may additionally or alternatively include settling the sample (and / or components), sedimenting the sample (and / or components), and / or any suitable process.

[0098] After centrifuging the sample, the supernatant is preferably removed. The supernatant is typically discarded, but may be stored, processed, measured, and / or otherwise handled. The supernatant may be removed by decanting, suction, flicking, harvesting, and / or using any suitable technique.

[0099] The total wash time is preferably about 5 seconds to 120 seconds (e.g., 5 seconds, 10 seconds, 15 seconds, 17 seconds, 20 seconds, 23 seconds, 30 seconds, 35 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds, etc.). If the wash time is too short, waste materials may be insufficiently removed, which may result in contamination of the data set, slow data collection, and / or otherwise be detrimental to sample processing and / or measurement. If the wash time is too long, the measured sample may be lost (e.g., retention may be low) and / or other problems may occur (e.g., the wash process may damage or alter the measured sample).

[0100] In a specific example of washing the quenched sample, a wash solution can be added to the sample, the sample can be centrifuged, and the supernatant can be aspirated. These steps can be performed concurrently (e.g., contemporaneously, simultaneously, etc.), sequentially, iteratively (e.g., add solution, centrifuge, aspirate, repeat), and / or in any suitable order and / or manner.

[0101] The washed sample (e.g., components to be measured) may be dry, wet (e.g., wet pellet, containing capture solution, etc.), contain solution (e.g., water, buffer, saline, etc.), and / or be otherwise arranged. For example, the washed sample may contain about 300 μl (e.g., 50-1000 μl) of buffer (e.g., residual buffer from the wash solution).

[0102] The sample may be washed at ambient temperature (e.g., room temperature, environmental temperature, 15-25°C, etc.), elevated temperature (e.g., about 25°C, 30°C, 35°C, 40°C, 45°C, etc.), reduced temperature (e.g., about 0°C, 10°C, 15°C, etc.), and / or any suitable temperature.

[0103] After the sample has been quenched and / or washed, the sample is generally stable (e.g., the activation state of the sample does not change substantially, the properties of the sample remain approximately the same, etc.). For example, the sample may be stable on a time scale of minutes to hours (e.g., the sample may be used after these steps for any suitable length of time up to about 24 hours before or after other processes are performed), which may be beneficial to reduce the urgency of steps to be performed, may allow other preparatory steps to be performed, and / or may be otherwise beneficial. However, the sample may additionally or alternatively be unstable (e.g., need to be measured within a threshold time, need to be further processed within a threshold time, etc.) and / or have any suitable stability.

[0104] Suspending the washed sample (e.g., to preserve pellets, sample components, etc.) serves to suspend the washed sample in a solution (e.g., a buffer) to facilitate measurement of the sample. The suspension solution may be referred to as a preparation solution. However, the prepared solution may additionally or alternatively include any suitable solution and / or material from other processes during the preparation of the sample for measurement and / or any suitable sample. The washed sample is preferably suspended in a buffer (e.g., PBS buffer), but may be suspended in water, saline, and / or any suitable solution.

[0105] The washed sample is preferably suspended to a target concentration. The target concentration is preferably about 4000-11000 cells per microliter of solution. However, the target concentration can be less than 4000 cells / μL and / or more than 11000 cells / μL. The suspended sample preferably has a volume that is about 1 mL (e.g., 900 μL-1.1 mL), but can have a volume less than 1 mL or more than 1 mL.

[0106] The suspension sample is preferably suspended at a target temperature. The target temperature is preferably about the same as the measurement temperature, but may be different from the measurement temperature. If the washed sample is at a temperature different from the target temperature, the suspension solution may be heated and / or cooled so that the mixture (e.g., the resulting suspension solution) is at the target temperature. For example, as shown in FIG. 7, the washed sample (e.g., concentrated cells) may be at ambient temperature (e.g., about 20° C.). The sample temperature may be measured (e.g., using a sensor, using a temperature probe, etc.), inferred (e.g., from ambient temperature, from the temperature of the reagent reservoir, etc.), and / or otherwise determined. In this example, the target temperature may be about 25° C. To achieve this target temperature, the buffer added to the washed sample may be heated to about 27° C. and added to the washed sample. The temperature of the suspension solution may be empirically determined, heuristically determined, determined based on a thermal mass balance (e.g., taking into account the heat capacity, mass, volume, temperature, etc. of the solution, target suspension, container, manifold, etc.), and / or determined in any manner. The suspension solution may be heated in the reservoir, in the manifold, in the spindle, in the reagent port, and / or in any suitable location, however, the prepared solution may additionally or alternatively be heated and / or cooled to achieve a target temperature and / or the prepared sample may be at any suitable temperature (e.g., a temperature at which the sample does not decompose).

[0107] The suspension solution is preferably transferred to a measurement module. For example, the suspension is preferably loaded into a cartridge in the measurement module (S310). The cartridge is preferably at a target temperature (e.g., the measurement temperature as obtained from (S100)), but can be at ambient temperature (e.g., heated or cooled to the measurement temperature) and / or any suitable temperature. The suspension solution can be transferred by a user, by a robot, by an automaton, and / or otherwise. The amount of sample transferred (and ultimately measured) is preferably 0.1-10 mL, but can be less than 0.1 mL and / or more than 10 mL. For example, about 1 mL (e.g., 1 mL ± 10%) of the suspension sample can be measured. In this example, if more or less sample (e.g., the volume changes by more than a threshold amount) is transferred (e.g., more than 1.1 mL or less than 0.9 mL), the measurement can be interrupted and resumed (e.g., by adding new sample) and / or the measurement can be otherwise affected.

[0108] After transferring the suspension solution, the sample preparation system can be optionally washed. For example, blank solutions, buffers, water, solvents, and / or any suitable materials can be loaded into the sample container, the wash container, and / or any suitable container, and then subjected to a washing process similar to that performed on the sample. The sample preparation system washing can function to minimize and / or prevent cross-contamination between samples, remove particulate residues, prevent the accumulation of particulate residues, and / or otherwise.

[0109] Measuring the sample (S300) preferably functions to obtain data related to one or more biophysical properties of the sample (and / or the patient associated therewith). The measurement of the sample is preferably performed using a measurement module, but can be performed using any suitable system and / or component. The data can be images (e.g., one or more images of each cell), image features, temperature, pressure, flow rate, and / or any suitable data. The measured sample is preferably a prepared sample (e.g., a suspended sample, a washed sample, etc.), but can be an as-collected sample, a lysed sample, a quenched sample, an unwashed sample, and / or any suitable sample. In a first specific example, the measurement sample can include white blood cells (e.g., essentially composed of white blood cells, consisting of white blood cells, including white blood cells, etc.). In a second specific example, the measurement sample can include one or more white blood cell subpopulations (e.g., neutrophils, eosinophils, basophils, monocytes, lymphocytes, etc.) and can exclude one or more other white blood cell subpopulations. However, the measurement sample may include any suitable cells, species, and / or components.

[0110] The sample is preferably measured (e.g., as shown in FIG. 10 ) within a threshold time of preparing the sample (e.g., the measurement is initiated within the threshold time, the measurement is completed within the threshold time, a portion of the measurement is performed within the threshold time, etc.). The threshold time is preferably on the order of 1 minute (e.g., 30 seconds, 1 minute, 90 seconds, 2 minutes, 2.5 minutes, 3 minutes, 4 minutes, 5 minutes, values ​​or ranges therebetween, etc.), but can be shorter (e.g., the threshold time can be less than 30 seconds, less than 20 seconds, less than 10 seconds, less than 5 seconds, etc.) and / or longer (e.g., on the order of several minutes, on the order of an hour, on the order of a day, etc.). Having a threshold time of about 1 minute (e.g., on the order of 1 minute) can be beneficial to limit temperature changes (e.g., after suspending, heating, etc. the prepared sample), to limit the amount of change (e.g., degradation) of the sample, and / or can be otherwise beneficial (e.g., a larger threshold time may experience, be subject to, etc. some of these undesirable effects).

[0111] During the measurement of the sample, the sample temperature is preferably about 25°C (e.g., 25°C±1°C, 25°C±0.5°C, 25°C±0.3°C, 25°C±0.1°C, etc.). The measurement temperature may additionally or alternatively be about 15-45°C, less than 15°C, and / or greater than 45°C. Significant deviations from the target measurement temperature (e.g., deviations from the target temperature by more than about 1°C) may invalidate and / or reduce the accuracy of the results from the measurement. Thus, the sample temperature (and / or cartridge temperature) is preferably measured throughout the sample measurement. If the sample temperature deviates from the target measurement temperature by more than a threshold amount, the measurement may be paused until the temperature returns to the target measurement temperature, data following the deviation may be excluded from analysis, the method may be terminated (e.g., analysis may be initiated if sufficient data is present), the method may be resumed (e.g., a new sample may be loaded for measurement, a new sample may be prepared, etc.), a temperature correction may be applied to the data, and / or the method may include any appropriate response.

[0112] Measuring the sample may include aligning the microfluidic cartridge with the imaging sensor (S320). Aligning the microfluidic cartridge may function to set the microfluidic cartridge in a focal plane of the imaging system, to set a predetermined portion of the microfluidic cartridge (e.g., a deformation region, a linear region, etc.) in the field of view of the imaging system, and / or otherwise. The microfluidic cartridge may be aligned using an alignment guide (e.g., an alignment pattern on the microfluidic cartridge), using a motor (e.g., to translate, rotate, etc. the microfluidic cartridge and / or the imaging system), and / or otherwise aligned.

[0113] Measuring the sample preferably includes acquiring a plurality of images of the sample measured in a flow cytometer (e.g., deformation cytometry) (S360). The plurality of images preferably includes a plurality of images of cells (or other sample components) in a deformation region, an elongation region, a focusing region, a linear region, and / or any suitable region of the flow path (e.g., of a cartridge). However, measuring the sample may additionally or alternatively include performing atomic force microscopy using a force sensor (e.g., an optical force sensor) and / or perform any suitable data or measurement of the sample.

[0114] The number of cells measured (e.g., per instance of the method, per sample, etc.) is preferably about 10,000 to 100,000 cells. For example, the average number of cells may be about 55,000 with a standard deviation of about 35,000. In this specific example, about 5000 cells / sec may be measured. However, the method may measure about 1000 cells / sec to 10,000 cells / sec, less than 1000 cells / sec, and / or more than 10,000 cells / sec. If the number of cells measured is less than about 10,000, the method may be restarted and continued (e.g., more cells from the same sample may be added to the measurement) until the number of cells exceeds 10,000, the confidence of the result may be reported (e.g., a flag may be issued based on the number of cells measured), and / or any suitable result may be generated. If more than 100,000 cells are measured, the data may be downsampled (e.g., to select a target number of cells, randomly, based on the probability that a given cell is an outlier, etc.), all measurements may be used, the method may be repeated (e.g., with the same sample, another sample from the same patient, etc.), the analysis may proceed unchanged, and / or the analysis or method may be modified in any manner. In general, fewer cells may not provide sufficient data for analysis, and additional cells may result in overfitting and / or slow data analysis, but fewer than 10,000 or more than 100,000 cells may be measured. However, any suitable number of cells may be measured.

[0115] For each measured cell, preferably at least about 10 images (e.g., 10, 11, 12, 14, 15, 16, 17, 20, 25 values ​​or ranges therebetween, >25, etc.) of the cell are measured (within the deformation, elongational flow, etc. area of ​​the cytometer). Having at least 10 images is often sufficient to ensure that an event-level (e.g., how the cell unfolds in response to force) analysis can be performed on the cell. If there are fewer than 10 images of a cell, the cell can be excluded from the event-level analysis, excluded from the population analysis, excluded from the set of measured cells (e.g., rejected data), included in the frame-level analysis, included in the event-level analysis, flagged (e.g., to indicate a potential source of unreliable data), and / or otherwise used and / or excluded. However, fewer than 10 images can be used.

[0116] Measuring the sample may include determining a flow rate of the sample through the cartridge (S340). The flow rate may be determined using multiple images, using a flow meter, based on timing (e.g., how long it takes for a measured amount of sample to flow through the cartridge), using a separate flow channel (e.g., a separate channel in a microfluidic pathway), based on applied pressure or force (e.g., using an equation to calculate the flow rate taking into account the cartridge dimensions, geometry, etc.), based on thermal changes, using a Coriolis mass flow meter, using acoustics, using an electrochemical sensor, and / or in any manner. In an illustrative example, the cartridge may be translated such that a set of flow images (e.g., images of one or more cells in a linear region of the cartridge) are acquired, the set of flow images may be segmented to identify cells in the flow images, and the flow rate of the sample through the cartridge is calculated based on frame rate, cell size, cartridge (e.g., fluidic channel) size, field of view, and / or any suitable information. However, the flow rate may be determined in any manner.

[0117] The flow rate is preferably determined prior to measuring the sample (e.g., prior to acquiring images of cells in the deformation region), but may be determined at any suitable time. By determining (e.g., measuring) the flow rate, the flow rate can be adjusted (if necessary) to achieve a target flow rate. The target flow rate is preferably about 0.1-10 m / s (e.g., 0.1 m / s, 0.2 m / s, 0.4 m / s, 1 m / s, 2 m / s, 4 m / s, 10 m / s, values ​​or ranges therebetween, etc.), but can be any suitable flow rate (e.g., less than 0.1 m / s, greater than 10 m / s). The flow rate may depend on the sample concentration, sample viscosity, applied force, frame rate of the imaging sensor, sample temperature, and / or any suitable characteristic. The flow rate can be adjusted, for example, by changing the sample concentration, by changing the sample viscosity, by changing the applied pressure (e.g., using positive and / or negative pressure or force to generate flow), and / or by otherwise adjusting the flow rate.

[0118] The step of determining the patient's health status (S400) preferably functions to determine the patient's health status. The patient's health status is preferably determined using a computing system (e.g., its image analyzer, scoring module, etc.), but may be performed using any suitable components. The patient's health status may include an index (e.g., a white blood cell structure index, a normalized index such as 0-1, an unnormalized index such as 0-10, 0-100, 0.1-10, 0.1-100, 1-10, 1-100, etc.), a diagnosis, a classification, a probability of a diagnosis applicable to the patient, and / or any suitable information.

[0119] The health state can be determined from a model, an equation, a lookup table, a machine learning algorithm (e.g., trained to output a score, trained to output an index, trained to output a disease state, etc.), and / or can be determined otherwise. Examples of models that can be used include logistic regression (e.g., linear logistic regression, non-linear logistic regression, etc.), decision trees, Bayesian classifiers, nearest neighbor techniques, support vector machines, decision forests (e.g., random forests), neural networks, gradient boosting, and / or any model can be used. The model can be a predefined model, a general model, a model selected based on auxiliary information, and / or any other suitable model.

[0120] Inputs to the model may include one or more biophysical characteristics; one or more parameters; auxiliary information such as age, maturity (e.g., infant, toddler, child, adolescent, adult, etc.), weight, height, race, sex, temperature, body mass index, percentage of body fat, pre-existing conditions, duration of symptoms, onset of symptoms, travel, blood oxygenation level, blood pressure, source of infection; treatment parameters (e.g., hospitalization, treatment administered, etc.); collection parameters (e.g., temperature, other compounds found in the sample, etc.); images (e.g., from a set of images); images of cells and / or analyses thereof under different lighting conditions (e.g., different intensities of light, different wavelengths of light, etc.); different event-level information; frame-level analysis; population analysis; and / or any input. In illustrative examples, the inputs may include trajectory parameters (e.g., oscillation amplitude, average oscillation amplitude, oscillation amplitude distribution, etc., for each cell, each cell with a threshold, etc.), cell size (e.g., thickness), and / or cell enumeration (e.g., number of neutrophils, number of white blood cells, etc., e.g., total number of cells, total number of cells with threshold trajectory parameters, etc.).

[0121] The output of the model may include an index (e.g., a health status index, a Leukocyte Structure Index (LSI), a value between 0-1, 0-10, etc., a score, a number of components having parameters and / or combinations of such parameters above a threshold such as a total number of components above a threshold, a fraction of the total number of components above a threshold, a probability, etc.), a health state, a severity of the health state, a probability of the health state, and / or any output.

[0122] In an illustrative example, the health status can be determined based on the index. If the index exceeds a threshold (e.g., 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 0.95, 0.99, 1, etc.), the health status can indicate that the patient has the condition (or has at least a 50% probability, etc.). If the index is below the threshold (e.g., 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 0.85, 0.99, 1, etc.), the health status can indicate that the patient does not have the condition (or has a high probability that the patient does not have the disease, such as at least a 50% probability that the patient does not have the disease). However, an index below the threshold may indicate disease, an index above the threshold may indicate the absence of the condition, and / or the index may be used in any manner. The threshold may be determined based on a training data set (e.g., in a manner similar to how the model is generated, in a manner similar to how the inputs to the model are selected), may be determined in advance, may be determined according to an equation (e.g., an equation based on and / or taking into account auxiliary sample information), and / or may be determined otherwise.

[0123] In some variations, a single index may be determined (e.g., population parameters for each cell subpopulation may be aggregated into a single calculation). In other variations, multiple indexes may be determined for a patient and / or sample. For example, a neutrophil index and a monocyte index may be determined. Each index may have the same or different thresholds. For example, a monocyte index above a threshold of 0.75 may indicate disease, and a neutrophil index above a threshold of 0.9 may indicate disease. When multiple indexes are used, the health status may be determined based on votes, favorable indexes, weighted averages of index results, equations relating the indexes and / or thresholds, and / or may be determined otherwise.

[0124] Determining the health condition preferably includes determining biophysical properties (e.g., parameters such as structural parameters, orbital parameters, patient parameters, location parameters, etc.) from the measurement data (S430). The biophysical properties are preferably used to determine the health condition, but may be otherwise used.

[0125] The set of biophysical properties preferably corresponds to population parameters (e.g., parameters representative of a sample), but may also correspond and / or be related to event parameters (e.g., parameters representative of an image, such as parameters determined based on features or objects in an image, parameters representative of components, such as parameters determined based on multiple images of components, features, objects, etc.), frame parameters (e.g., parameters associated with a single image), and / or any suitable criteria. The population parameters may be determined from the event parameters, frame parameters, and / or from any suitable parameters, and / or may be determined otherwise. The population parameters may be an average of multiple parameters, a characteristic parameter of a parameter (e.g., maximum, minimum, mean, median, mode, etc.), and / or may be otherwise related to the parameter. In specific examples, determining one or more parameters of the set of parameters may include averaging the parameters determined for each event (e.g., traversing the image, device, or component through the deformation region, etc.), averaging parameters that exceed a threshold (e.g., averaging parameters greater than the 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, 90th, 95th percentile, averaging parameters less than the 5th, 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, 90th, 95th percentile, etc.), averaging a subset of the parameters (e.g., parameters associated with the same feature type, such as the same cell type), averaging extreme parameters (e.g., maximum, minimum, etc.), determining correlations between parameters, determining the variance and / or standard deviation of the set (or subset) of event parameters, using equations relating event parameters to population parameters, determining regressions (e.g., linear regression, nonlinear regression, etc.) between distributions of event and / or frame parameters using weighted averages of event parameters, and / or may be otherwise determined.

[0126] In an illustrative example, determining the structural parameters may include determining boundaries of the features (e.g., inner boundaries, outer boundaries, etc.) and determining the structural parameters based on the boundaries. In a first variation of this illustrative example, the structural parameters may be determined based on a known geometry of the detection system and a sample geometry (e.g., the size of the features may be determined based on the system geometry and known camera parameters such as focal length, optical sensor size, etc.). In a second variation of this illustrative example, the structural parameters may be determined by determining a disparity map between two images containing the same features. In a specific example, the aspect ratio of the cells in each frame and / or the position along the deformation region may be determined based on the cell dimensions extracted from the respective frames.

[0127] In an illustrative example, determining the trajectory parameters includes determining a centroid location of a feature (e.g., object, component) in each image of the multiple images, tracking changes in the location of the centroid between the multiple images, measuring an amplitude of the vibration (e.g., using one or more peaks or cycles of the vibration), and calculating the parameters based on the amplitude of the vibration. In a first variation of this illustrative example, measuring the amplitude can exclude measuring the amplitude of the first, second, and / or any suitable peak of the vibration, which can increase the reproducibility of the measurement. In a second variation of this illustrative example, measuring the amplitude can include measuring the amplitude of the third, fourth, fifth, and / or any peak of the vibration and averaging the amplitudes to determine the fitting parameters. In a third variation of this illustrative example, measuring the amplitude may include measuring the amplitude of the third, fourth, fifth and / or any peak of the vibration and fitting the vibration to a predetermined equation (e.g., an oscillation function such as a trigonometric function, a damped oscillation, an exponential function, etc.) to determine the trajectory parameters (e.g., fit parameters such as amplitude, offset, phase, frequency, decay, damping, drive, etc.). In a fourth variation of this illustrative example, the trajectory parameters may include one or more Fourier coefficients from a Fourier decomposition of the trajectory (e.g., a Fourier cosine transform, a Fourier sine transform, a Fourier transform, etc.). In a fifth variation of this specific example, the trajectory parameters may be determined based on changes in structural parameters between images containing the same feature. In a specific example, the VEIR of a cell passing through a deformation region may be determined based on the cell vibration amplitude extracted from the frames of the respective time series. In a sixth variation of this illustrative example, the orbital parameters may be determined based on the amplitudes of the primary, secondary, tertiary, quaternary, quinary, secular, septenary, octonary, nonary, denarius, and / or other cycles of oscillation, and / or combinations of the above (e.g., excluding some cycles, such as primary and / or secondary cycles, including a subset of cycles, such as tertiary, quaternary, and quinary cycles, etc.), but the orbital parameters may be determined in other ways.

[0128] Determining the biophysical properties may include detecting overlapping cells in the image. If overlapping cells are detected, the cells can be excluded from the analysis (e.g., frames in which the cells overlap can be excluded, events containing one or more overlapping cells can be excluded, etc.), the cells can be separated (e.g., using a machine learning algorithm), corrections can be applied based on the overlap (e.g., the degree of overlap), and / or the overlapping cells can be treated differently.

[0129] In a specific example (e.g., as shown in FIG. 11 and / or FIG. 12), determining the patient's health status may include receiving an image of cells (e.g., white blood cells) undergoing deformation, extracting cellular features from the image (e.g., dimensions and location within the flow field) for each cell (S420), determining trajectory parameters (e.g., vibration amplitude) and / or structural parameters (e.g., aspect ratio) for each cell based on the cellular features, calculating an index based on the trajectory parameter and / or structural parameter values ​​(S435), and determining the patient's health status based on the index (S440). The method may optionally include separating the cell data according to cell subpopulations (e.g., monocytes, lymphocytes, neutrophils), and the index may be calculated based on parameters of a subset of the cell subpopulations (e.g., monocytes and neutrophils).

[0130] In a second specific example, determining the patient's health status can be performed as disclosed in U.S. patent application Ser. No. 17 / 401,627, filed August 13, 2021, entitled "SYSTEM AND METHOD FOR IMMUNE ACTIVITY DETERMINATION," and incorporated by reference in its entirety.

[0131] However, the patient's health status may be determined differently.

[0132] Providing an intervention (S500) may function to provide an intervention to the patient. The intervention is preferably dependent on a health state (e.g., immune activation state), but may be dependent on biophysical characteristics, patient data, and / or any suitable data and / or information. Providing an intervention is preferably performed automatically (e.g., a computing system generates a suggestion based on the health state), but can be performed manually and / or in any manner. Example interventions include tracking the progression of a patient's disease state over time, determining treatment parameters (e.g., estimated length of hospital stay, estimated triage requirements, etc.), initiating secondary analyses (e.g., innate immune activation assays such as light microscopy to assay structural features, cytological assays, immunofluorescent labeling of CD11b, CD18, CD64, CD66b, chemical change assays using flow cytometry, etc., transcriptomic signature analyses, etc.), suggesting an antibiotic regimen, suggesting an antiviral regimen, suggesting a chemotherapy regimen, informing a health care provider of a patient's health status, indicating the urgency of a health status, informing a patient of a health status, displaying a health status, and / or other appropriate interventions.

[0133] The method of the preferred embodiment and variations thereof may be embodied and / or performed at least in part as a machine configured to receive a computer-readable medium storing computer-readable instructions. The computer-readable medium may be stored on any suitable computer-readable medium, such as a RAM, a ROM, a flash memory, an EEPROM, an optical device (CD or DVD), a hard drive, a floppy drive, or any suitable device. The computer-executable component is preferably a general or application-specific processor, although any suitable dedicated hardware or hardware / firmware combination device may alternatively or additionally execute the instructions.

[0134] Embodiments of the systems and / or methods may include any combination and permutation of the various system components and various method processes, and one or more example methods and / or processes described herein may be performed asynchronously (e.g., serially), simultaneously (e.g., in parallel), or in any other suitable order by and / or using one or more example systems, elements, and / or entities described herein.

[0135] As those skilled in the art will recognize from the foregoing detailed description and drawings and the appended claims, modifications and variations can be made to the preferred embodiments of the invention without departing from the scope of the invention, which is defined in the following claims.

Claims

1. A system for detecting immune system activation status in a patient, the system comprising:

1. A sample preparation system comprising: lysing red blood cells from said patient's blood sample in a lysis reaction; quenching the lytic reaction before leukocytes from the blood sample are damaged; Separating the white blood cells from the lysed red blood cells, and a sample preparation system configured to maintain the temperature of the blood sample at 24-26°C; 1. A cytometer configured to determine a biophysical property of the white blood cells of the blood sample, the cytometer comprising: an inlet configured to receive the separated white blood cells; a focusing region fluidly connected to the inlet through a filter, the focusing region configured to align the white blood cells; a deformation region fluidly connected to the focusing region, the deformation region configured to deform the white blood cells; and a cytometer comprising a microfluidic channel including an outlet fluidly connected to the deformation region through a second filter; a processor configured to detect an immune system activation state in the patient based on the biophysical characteristic; A system comprising:

2. The system described in claim 1, wherein the cytometer further comprises a thermal system that maintains the microfluidic channel at a temperature of 24.5°C to 25.5°C.

3. The system described in claim 1, wherein the cytometer further comprises an imaging system configured to acquire images of the deformation region, and wherein the frame rate of the image sensor of the imaging system is at least 50,000 frames per second.

4. The system described in claim 3, wherein the imaging system is further configured to acquire images of white blood cells in a channel upstream of the deformation region, and the images of the white blood cells in the channel are used to determine the flow rate of the blood sample through the microfluidic channel.

5. The system described in claim 1, wherein lysing the red blood cells does not change the activation state of the white blood cells.

6. The system described in claim 1, wherein the flow of the blood sample within the cytometer module is established using compressed air at a pressure of 91±3 PSI.

7. The system of claim 1, wherein the biophysical properties include at least one of trajectory parameters including oscillation of the center of mass of the white blood cells, cell count, or white blood cell subpopulation.

8. The system of claim 1, wherein the processor uses a machine learning algorithm trained to detect immune system activation status in the patient based on the biophysical characteristics, and wherein the system further includes an analysis module processor including a graphics processing unit accelerated computation.

9. A system for detecting an immune system activation state in a patient, the system comprising:

1. A sample preparation system comprising: lysing red blood cells from said patient's blood sample in a lysis reaction; quenching the lytic reaction before leukocytes from the blood sample are damaged; Separating the white blood cells from the lysed red blood cells, and a sample preparation system configured to maintain the temperature of the blood sample at 24-26°C; a cytometer configured to determine a biophysical property of the white blood cells of the blood sample, the biophysical property further comprising at least one of a cell count or a white blood cell subpopulation; a processor configured to detect the immune system activation state in the patient based on the biophysical characteristics, the biophysical characteristics including trajectory parameters, the trajectory parameters including oscillations of center of mass of cells of the white blood cells; and A system comprising:

10. The cytometer an inlet configured to receive the separated white blood cells; a focusing region fluidly connected to the inlet through a filter, the focusing region configured to align the white blood cells; a deformation region fluidly connected to the focusing region, the deformation region configured to deform the white blood cells; an outlet fluidly connected to the deformation region through a second filter; 10. The system of claim 9, comprising a microfluidic channel comprising:

11. A system as described in claim 9 or 10, wherein the cytometer further comprises a thermal system that maintains the microfluidic channel at a temperature of 24.5°C to 25.5°C.

12. A system as described in claim 9 or 10, wherein the cytometer further comprises an imaging system configured to acquire images of the deformation region, and wherein the frame rate of the image sensor of the imaging system is at least 50,000 frames per second.

13. The system described in claim 12, wherein the imaging system is further configured to acquire images of white blood cells in a channel upstream of the deformation region, and the images of the white blood cells in the channel are used to determine the flow rate of the blood sample through the microfluidic channel.

14. The system described in claim 9 or 10, wherein lysing the red blood cells does not change the activation state of the white blood cells.

15. The system described in claim 9 or 10, wherein the processor uses a machine learning algorithm trained to detect the immune system activation state in the patient based on the biophysical characteristics.

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