Device and method for determining particle concentration in a sample

Abstract

The present invention provides a cartridge for determining the concentration of target cells in a sample, the cartridge comprising a separation portion and a detection portion. The separation portion comprises a first surface and a second surface defining a separation chamber. The separation portion can accommodate a density medium having a density greater than a density of a first portion of the sample and less than a density of a second portion of the sample (which comprises the target cells). The separation chamber can be fluidically connected to an inlet reservoir so that the sample can reach the separation chamber from the inlet reservoir during rotation. The detection portion comprises a detection surface forming a boundary of the detection chamber. The detection surface is not parallel to the first surface so that the target cell impacts on the detection surface when entering the detection chamber. The detection surface is configured to capture the target cell.

Description

[0001] Cross-references to related patent applications

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 669,357, filed on May 9, 2018, entitled “Device and Method for Bacterial Assay,” and U.S. Provisional Application Serial No. 62 / 785,752, filed on December 28, 2018, entitled “Devices and Method for Determining Particle Concentration in a Sample,” the disclosure of each of which is incorporated herein by reference in its entirety. Background Art

[0003] Embodiments described herein relate to devices and methods for enumerating target cells in biological samples having known or suspected microbial content. Specifically, embodiments described herein relate to cartridges and methods that include centrifugally plating cells onto a detection surface, separating cells based on size and / or density, and enumerating particles based on signals generated from captured cells.

[0004] Bloodstream infection or sepsis is a microbial infection in the bloodstream that accounts for up to 1-2% of hospitalizations. It causes more than 200,000 deaths per year in the United States. Treatment pathways for sepsis rely primarily on brute force and often begin before a definitive diagnosis is made. More information about the nature of the infection can give healthcare providers greater ability to develop targeted treatment plans. Alternatively, healthcare practitioners or researchers may wish to analyze the nature of the infection by using known assays that may have varying efficiencies depending on the concentration of cells in a sample. For example, when testing to determine whether a target bacterium is susceptible to treatment with a known antibiotic (i.e., antibiotic susceptibility testing or AST), it is often required that the bacteria in the sample be within an expected concentration range. For example, if the concentration of bacteria in the sample is below the expected range, the results of the AST may falsely identify the bacteria as susceptible. Conversely, if the concentration of bacteria in the sample is above the expected range, the results of the AST may falsely identify the bacteria as resistant to the antibiotic. Therefore, some known tests involve first enumerating the bacteria in the sample.

[0005] Current methods for bacterial enumeration include quantitative culture, most probable number analysis (MPN), direct hemocytometer counts, and direct photometric measurement of optical density. In sepsis samples, the primary method used to enumerate bacteria is quantitative culture, which involves plating serial dilutions of cells on culture agar followed by an incubation period of 16 hours or longer, depending on the strain identity or strain mixture identity. After the incubation period, cells are counted as colony forming units (CFU) by the formation of colonies on the plate. Using hemocytometer counts or optical density measurements may yield actionable information more quickly, but the results of the known methods may be obscured by the heterogeneous nature of blood samples. Hemocytometer counts are the direct counting of cells on a fixed depth slide containing a grid or by imaging with a microscope reticle. This technique may not work if other cells are present, which may be hidden in the case of red blood cells (RBCs) or white blood cells (WBCs) or have a similar apparent size in brightfield imaging (platelets). Optical density measures light scattering that is usually correlated to cell density, however, this technique results in variable correlation curves with respect to sample matrix and strain identity, and is also prone to error due to the presence of other non-analyte cells that may scatter light. In other words, the presence of particulate contaminants often hinders the measurement of the number of target analyte particles. For example, measuring the concentration of bacteria suspended in whole blood using known devices and methods is problematic due to the presence of similarly sized particles, such as platelets and apoptotic fragments of blood cells. Particulate contamination of whole blood is even more severe in the case of measurements made using blood culture media, where degradation of blood cells and coagulation of proteins can produce large numbers of bacteria-sized particles that may greatly exceed the concentration of actual bacteria. Other prior art particle counting methods, such as flow cytometry or absorption spectroscopy, are also interfered with by contaminating particles.

[0006] Furthermore, known methods such as MPN may involve serial dilution of samples and statistical formulas to determine the original number and may take 24 to 48 hours to determine the results.

[0007] Additionally, some known methods for enumerating bacteria can vary depending on the strain of bacteria being analyzed (e.g., whether the bacteria are Gram-positive or Gram-negative). Currently, strain identification in the blood laboratory is accomplished through advanced microbiology work performed by trained technicians using selective media and microscopy. Gram staining is used to identify the Gram identity of bacteria. Selective media (agar plates containing growth media that only include certain nutrients; certain classes of bacteria may or may not grow in the presence of certain missing or included nutrients) are often used to narrow down a particular bacterial family or genus. In an advanced research setting, strain identification is accomplished through 16S-rRNA sequencing or through the use of MALD-TOF. Each of these techniques requires a minimum of 16 hours, where the cell culture plate must be grown enough to isolate a certain number of bacteria that can be stained for imaging or plated onto multiple growth medium plates.

[0008] Therefore, there is a need for improved devices and methods for determining the concentration of target cells (e.g., bacteria) within a sample. In particular, there is a need for improved devices and methods for enumerating bacteria that can provide accurate results over a wide dynamic range of bacterial concentrations. Summary of the invention

[0009] Devices, systems, kits and methods for determining the concentration of target cells (e.g., bacteria) in a sample are described herein. In some embodiments, a system, kit or method for determining the concentration of target cells in a sample includes a cartridge configured to be removably coupled to a rotating element that rotates the cartridge around an axis of rotation to capture target cells in the sample. The cartridge includes an inlet portion that defines an inlet reservoir configured to accommodate a sample, a separation portion and a detection portion. The separation portion includes a first surface and a second surface that define a separation chamber. The separation portion may include a density medium having a density greater than the density of the first portion of the sample and less than the density of the second portion of the sample. The second portion of the sample includes target cells. The separation chamber is configured to be fluidically coupled to the inlet reservoir so that when the cartridge is rotated, at least the second portion of the sample can be transported from the inlet reservoir to the separation chamber. The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidically coupled to the separation chamber so that at least the target cell can pass through the density medium and enter the detection chamber. The detection surface is non-parallel to at least one of the first surface or the second surface such that the target cells impinge on the surface upon entering the detection chamber. The detection surface is configured to capture a plurality of target cells.

[0010] The cartridge disclosed herein may include an inlet portion defining an inlet reservoir configured to accommodate a sample; a separation portion including a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidically coupled to the inlet reservoir so that when the cartridge is rotated, at least a portion of the sample can be transported from the inlet reservoir to the separation chamber; and a detection portion defining a detection chamber, the detection portion including a detection surface forming a boundary of the detection chamber, the detection chamber being fluidically coupled to the separation chamber so that at least a plurality of target cells can pass through the separation chamber and enter the detection chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, and the detection surface being configured to capture a plurality of target cells.

[0011] In one aspect, a kit is provided, the kit comprising a cartridge configured to be removably coupled to an instrument, the instrument configured to rotate the cartridge around a rotation axis and detect a plurality of target cells in a sample, the cartridge comprising: an inlet portion defining an inlet reservoir configured to accommodate the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber configured to be fluidically coupled to the inlet reservoir so that when the cartridge is rotated, at least a portion of the sample can be transported from the inlet reservoir to the separation chamber; and a detection portion defining a detection chamber, the detection portion comprising a detection surface forming a boundary of the detection chamber, the detection chamber being fluidically coupled to the separation chamber so that at least a plurality of target cells can pass through the separation chamber and enter the detection chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, the detection surface being configured to capture a plurality of target cells; a density of 1.01 g / cm 3 and 1.13 g / cm 3 ; and a stain that is formulated to bind and enhance the detection of multiple target cells. Such a kit can be used to detect the concentration of multiple target cells in a sample. In some embodiments, the kit also includes a diluent. In some embodiments, the detection angle between the detection surface and the first surface of the separation portion is between about 1 degree and about 8 degrees; and the detection surface includes a surface modification to enhance the adhesion of multiple target cells. In some embodiments, the first surface of the separation portion and the detection surface are integrally constructed; and the detection angle is about 2 degrees. In some embodiments, the detection surface includes a chemical modification to enhance the adhesion of multiple target cells. In certain embodiments, the surface modification includes a coating that includes a charged polymer. In some embodiments, the density medium is stored in a prefilled sealed container separated from the cartridge and has a volume less than fifty percent of the volume of the cartridge. In some embodiments, the density medium includes any one of a defoamer, a wetting agent, a dispersant, or an emulsifier. In some embodiments, the diluent is stored with a prefilled container separated from the cartridge, and the diluent includes any one of a defoamer, a wetting agent, a dispersant, or an emulsifier.

[0012] In another aspect, a method for detecting the concentration of multiple target cells in a sample is provided. The method includes delivering the sample to an inlet reservoir of a cartridge, the cartridge also including a separation portion and a detection portion, the separation portion including a first surface and a second surface defining a separation chamber, the separation chamber configured to be fluidically coupled to the inlet reservoir, the detection portion defining a detection chamber and including a detection surface forming a boundary of the detection chamber, the detection chamber fluidly coupled to the separation chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, the detection surface configured to capture multiple target cells; coupling the cartridge to a rotating element of an instrument; and starting the instrument so that the instrument rotates the cartridge at a certain rotation speed, sufficient to cause a) at least a portion of the sample to be delivered from the inlet reservoir through the separation chamber and into the detection chamber; and b) multiple target cells to be captured on the detection surface; receiving a signal indicating the number of multiple target cells captured on the detection surface via a detector of the instrument; and determining based on the signal that the concentration of multiple target cells in the sample is within a lower limit of 10^3 colony forming units (CFU) per milliliter (mL) and an upper limit of 10^9 CFU per milliliter.

[0013] In another aspect, a method for detecting the concentration of a plurality of target cells in a sample is provided, the method comprising delivering the sample to an inlet reservoir of a cartridge. The cartridge comprises a separation portion and a detection portion. The separation portion comprises a first surface and a second surface defining a separation chamber, the separation chamber being fluidically coupled to the inlet reservoir. The detection portion comprises a detection surface forming a boundary of the detection chamber. The detection chamber is fluidically coupled to the separation chamber, and the detection surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture a plurality of target cells. The cartridge is coupled to a rotating element of an instrument. The instrument is then activated so that the instrument: (1) rotates the cartridge at a rotational speed, thereby causing at least a portion of the sample to be delivered from the inlet reservoir through the separation chamber and into the detection chamber, and the target cells to be captured on the detection surface; (2) receives a signal indicating the number of target cells captured on the detection surface via a detector of the instrument; and (3) determines the concentration of the plurality of target cells in the sample based on the signal.

[0014] In another aspect, a method for detecting the concentration of a plurality of target cells in a sample is provided. The method includes transferring a density medium from a container into a cartridge, the cartridge including an inlet portion, a separation portion, and a detection portion, the inlet portion defining an inlet reservoir, the separation portion including a first surface and a second surface defining a separation chamber, the separation chamber being fluidly connected to the inlet reservoir, the detection portion including a detection surface forming a detection chamber boundary, the detection chamber being fluidly connected to the separation chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, the detection surface being configured to capture a plurality of target cells; and performing the transfer so that the density medium is contained within the separation chamber, the density of the density medium having a density greater than a density of a first portion of the sample and less than a density of a second portion of the sample, the second portion of the sample including a plurality of target cells; delivering the sample to the inlet reservoir; rotating the cartridge within an instrument at a rotational speed and for a rotational duration sufficient to cause a) the second portion of the sample to pass from the inlet reservoir through the density medium in the separation chamber and into the detection chamber; and b) the plurality of target cells to be captured on the detection surface; receiving a signal indicating the number of the plurality of target cells captured on the detection surface via a detector of the instrument; and determining the concentration of the plurality of target cells in the sample based on the signal.

[0015] In another aspect, a method for determining the sensitivity of a plurality of target cells to a treatment regimen is provided. The method includes delivering a first portion of a sample containing a plurality of target cells to a cartridge, the cartridge including a separation portion and a detection portion, the separation portion including a first surface and a second surface defining a separation chamber, the detection portion including a detection surface forming a boundary defining a detection chamber, the detection chamber being fluidly coupled to the separation chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, the detection surface being configured to capture a plurality of target cells; activating a first instrument so that the first instrument rotates the cartridge at a rotational speed sufficient to cause a) the first portion of the sample to be delivered through the separation chamber and into the detection chamber and b) the plurality of target cells to be captured on the detection surface; receiving a signal indicating the number of the plurality of target cells captured on the detection surface via a detector of the first instrument; and determining the concentration of the plurality of target cells in the first portion of the sample based on the signal; processing a second portion of the sample based on the concentration of the plurality of target cells in the first portion of the sample; after processing, delivering the second portion of the sample to a reaction chamber; and activating a second instrument to perform a sensitivity test on the second portion of the sample to determine the sensitivity of the plurality of target cells to the treatment regimen. In some embodiments, the method processing includes diluting the second portion of the sample until the concentration of the plurality of target cells in the second portion of the sample is within a predetermined range.

[0016] In the above methods and some embodiments herein, the concentration of multiple target cells is within a lower limit of 10^3 colony forming units (CFU) per milliliter (mL) and an upper limit of 10^9 CFU per milliliter. The concentration can be determined by an instrument. In some embodiments, the lower limit is about 10^5 CFU per milliliter. In some embodiments, the portion of the sample that is transported from the inlet reservoir through the separation chamber and into the detection chamber is the second portion; and the cartridge contains a density medium in the separation chamber, the density of the density medium is greater than the density of the first portion of the sample and less than the density of the second portion of the sample, and after rotating the cartridge, the first portion of the sample is maintained in the separation chamber. In some embodiments, the portion of the sample that is transported from the inlet reservoir through the separation chamber and into the detection chamber is the second portion, and the method further includes delivering the density medium to the cartridge before the sample is transported to the inlet reservoir, the density of the density medium is greater than the density of the first portion of the sample and less than the density of the second portion of the sample, and after rotating the cartridge, the first portion of the sample is maintained in the separation chamber. In some embodiments, the method further comprises delivering a diluent to the cartridge, at least one of the diluent or the density medium comprising any one of a defoamer, a wetting agent, a dispersant, or an emulsifier. In some embodiments, delivering the diluent comprises mixing the diluent with the sample and delivering the diluent and the sample to the inlet reservoir of the cartridge in the same operation. In some embodiments, the diluent is delivered to the cartridge together with the density medium, at least one of the diluent or the density medium comprising any one of a defoamer, a wetting agent, a dispersant, or an emulsifier. In some embodiments, the density of the density medium is between 1.05 g / cm 3 and 1.09 g / cm 3 In some embodiments, at least one of the diluent or the density medium comprises a poloxamer. In some embodiments, the sample is a body fluid. In certain embodiments, the sample is any one of a blood sample or a urine sample. In specific embodiments, the blood sample is a cultured blood sample, and the plurality of target cells are a plurality of bacterial cells. In certain embodiments, the plurality of bacterial cells comprises Enterobacteriaceae, Pseudomonas, Acinetobacter, Staphylococcus, Streptococcus, or EnterococcusIn some embodiments, the method further comprises processing the original cultured blood sample to produce a cultured blood sample before the cultured blood sample is delivered to the inlet reservoir of the cartridge. In some embodiments, the method further comprises mixing a stain with the sample, the stain being formulated to bind and enhance the detection of multiple target cells. In certain embodiments, the stain is a fluorescent stain formulated for staining multiple target cells. In some embodiments, the instrument rotates the cartridge at a rotational speed between about 3000 rpm and 15,000 rpm for a period of at least 1 minute. In certain embodiments, the rotational speed is between about 6000 rpm and 7000 rpm. In some embodiments, the container and the cartridge are packaged together in a test kit.

[0017] In another aspect, a device is provided, the device comprising a cartridge configured to be removably coupled to a rotating element, the rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of target cells within a sample, the cartridge comprising an inlet portion, the inlet portion defining an inlet reservoir configured to hold the sample; a separation portion, the separation portion comprising a first surface and a second surface defining a separation chamber, the separation portion being configured to hold a density medium, the density of the density medium being greater than a density of a first portion of the sample and less than a density of a second portion of the sample, the second portion comprising a plurality of target cells, the separation chamber being configured to be fluidically coupled to the inlet reservoir so that when the cartridge is When the cartridge rotates, at least a second portion of the sample can reach the separation chamber from the inlet reservoir; and a detection portion comprising a detection surface, the detection surface forming a boundary of the detection chamber, the detection chamber being fluidly connected to the separation chamber so that at least a plurality of target cells can pass through the density medium and enter the detection chamber, the detection surface being non-parallel to at least one of the first surface or the second surface so that a plurality of target cells impact on the detection surface when entering the detection chamber, the detection surface being configured to capture a plurality of target cells; and a diluent, wherein at least one of the diluent or the density medium comprises any one of a defoaming agent, a wetting agent, a dispersant or an emulsifier.

[0018] In another aspect, a device is provided, comprising a cartridge configured to be removably coupled to a rotating element, the rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of target cells within a sample, the cartridge comprising an inlet portion defining an inlet reservoir configured to hold the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidically coupled to the inlet reservoir such that when the cartridge is rotated, at least a portion of the sample can be transported from the inlet reservoir to the separation chamber; and a detection portion defining a detection chamber, the detection portion comprising a detection surface forming a boundary of the detection chamber, the detection chamber being fluidically coupled to the separation chamber such that at least a plurality of target cells can be transported through the separation chamber and into the detection chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, the detection surface comprising a surface modification to enhance adhesion of the plurality of target cells, and a ratio of a volume of the separation chamber to a volume of the detection chamber of at least about 2.0.

[0019] In yet another aspect, a device is provided, comprising a cartridge configured to be removably coupled to a rotating element, the rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of target cells within a sample, the cartridge comprising an inlet portion defining an inlet reservoir configured to hold the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation portion being configured to hold a density medium having a density greater than a density of a first portion of the sample and less than a density of a second portion of the sample, the second portion comprising a plurality of target cells, the separation chamber being configured to be fluidically coupled to the inlet reservoir such that when the cartridge is rotated, at least the second portion of the sample can enter the separation chamber from the inlet reservoir; and a detection portion, the detection portion comprising a detection surface forming a boundary of the detection chamber, the detection chamber being fluidically coupled to the separation chamber such that at least a plurality of target cells can pass through the density medium and enter the detection chamber, the detection surface being non-parallel to at least one of the first surface or the second surface such that a plurality of target cells impact on the detection surface when entering the detection chamber, the detection surface being configured to capture a plurality of target cells.

[0020] In another aspect, a device is provided, comprising a cartridge configured to be removably coupled to a rotating element, the rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of target cells within a sample, the cartridge comprising an inlet portion defining an inlet reservoir configured to hold the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidically coupled to the inlet reservoir such that when the cartridge is rotated, at least a portion of the sample can be transported from the inlet reservoir to the separation chamber; and a detection portion defining a detection chamber, the detection portion comprising a detection surface forming a boundary of the detection chamber, the detection chamber being fluidically coupled to the separation chamber such that at least a plurality of target cells can be transported through the separation chamber and into the detection chamber, the detection surface and the first surface of the separation portion forming a non-zero detection angle, the detection surface being configured to capture a plurality of target cells, and the ratio of the volume of the separation chamber to the volume of the detection chamber being at least about 2.0.

[0021] In some embodiments of the devices described above and herein, the first surface of the separation portion and the detection surface form a detection angle between about 1 degree and about 8 degrees. In certain embodiments, the detection angle is about 2 degrees. In some embodiments, the transition zone between the first surface of the separation portion and the detection surface defines a maximum radius of curvature of less than 0.1 mm. In some embodiments, the distance between the first surface of the separation portion and the second surface of the separation portion defines a thickness of the separation chamber, which is less than about 0.6 mm. In certain embodiments, the thickness of the separation chamber is less than about 0.3 mm. In some embodiments, the first surface of the separation portion and the detection surface are integrally constructed. In some embodiments, the cartridge defines a radial axis that intersects the axis of rotation; the first surface of the separation portion and the detection surface are integrally constructed; and the detection angle is along the radial axis and within a cross-sectional plane defined by the radial axis and the axis of rotation. In some embodiments, the inlet portion defines an opening through which a sample can be delivered to the inlet reservoir; and the detection surface is angled in a downward direction relative to the opening. In some embodiments, the ratio of the volume of the separation chamber to the volume of the detection chamber is at least about 2.0. In some embodiments, the ratio of the volume of the separation chamber to the volume of the detection chamber is between about 2.0 and 5.0. In certain embodiments, the ratio of the volume of the separation chamber to the volume of the detection chamber is at least about 2.5. In some embodiments, the apparatus further comprises a density medium having a density of 1.01 g / cm 3 and 1.13 g / cm 3 In some embodiments, the density of the density medium is between 1.05 g / cm 3 and 1.09 g / cm 3In some embodiments, the separation portion defines a density medium reservoir, and the density medium is contained in the density medium reservoir. In some embodiments, at least one of the diluent or the density medium includes a poloxamer. In certain embodiments, the poloxamer comprises poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). In some embodiments, the device further comprises a stain, the stain being formulated to bind and enhance detection of multiple target cells. In some embodiments, the detection surface comprises a surface modification to enhance adhesion of multiple target cells. In certain embodiments, the surface modification comprises a coating comprising a charged polymer. In specific embodiments, the coating is a (-NH 3 + ) of poly-L-lysine. In some embodiments, the cartridge is configured to concentrate a plurality of target cells captured on the detection surface.

[0022] In some embodiments, the cartridge of the above-described apparatus is a centrifuge cartridge that includes a density medium and / or one or more non-plastic components within the cartridge. In some embodiments, the cartridge can be used to separate target particles (e.g., cells) initially contained in a biological sample from contaminants of varying density and size. This is accomplished by centrifuging the biological sample through a density medium in the cartridge. The density medium has a higher density than the biological sample and a lower density than the target particles. In some embodiments, the density medium may be contained in the imaging cartridge prior to adding the biological sample and will occupy an area of ​​the angled imaging surface of the cartridge. In some embodiments, the target particles are captured by the angled imaging surface, while the contaminant particles are either retained in the biological sample (for small or low density contaminants) or centrifuged through the density medium and away from the angled imaging surface (for larger and high density contaminants). The number of target particles per given imaging area will be related to the original concentration of target particles suspended in the biological sample. In some embodiments, the specificity of imaging target particles (as opposed to contaminants) may be improved by adding a dye that specifically labels the target particles.

[0023] The cartridge or the method for determining the concentration of target cells can be used as part of a biological test. For example, the method for determining the sensitivity of target cells to a treatment regimen may include delivering a first portion of a sample containing target cells to a cartridge. The first instrument is then activated to rotate the cartridge at a certain rotation speed sufficient to allow: A) the first portion of the sample to be transported through the separation chamber and into the detection chamber, and B) the target cells to be captured on the detection surface within the cartridge. The first instrument then receives a signal indicating the number of target cells captured on the detection surface via a detector, and determines the concentration of target cells in the first portion of the sample based on the signal. The second portion of the sample is then processed based on the concentration of multiple target cells in the first portion of the sample. For example, in certain cases, the second portion of the sample may be diluted. The method may also include delivering the second portion of the sample to a reaction chamber after the second portion of the sample is processed. The second instrument is then activated to perform a sensitivity test on the second portion of the sample to determine the sensitivity of the target cells to the treatment regimen.

[0024] A system for determining the concentration of target cells in a sample is also provided, comprising a centrifugal cartridge, a motor, a connection for rotating the cartridge via the motor, and a controller for controlling the centrifugal operation. Here, the system may include imaging hardware for imaging in or through the cartridge, including any software or firmware therein.

[0025] A method of manufacturing a cartridge is also provided, the method comprising delivering a density medium into the cartridge.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a top view of a portion of a cartridge according to an embodiment.

[0028] FIG. 2 is a cross-sectional view of the cartridge shown in FIG. 1 taken along line XX in FIG. 1 .

[0029] 3A and 3B show top views of a portion of the cartridge shown in FIG. 1 containing a sample before rotation ( FIG. 3A ) and after rotation ( FIG. 3B ).

[0030] 4A and 4B show cross-sectional views of a portion of a cartridge according to an embodiment containing a sample and a density medium at the beginning of a rotation cycle ( FIG. 4A ) and after rotation ( FIG. 4B ).

[0031] 5A is a top view of a portion of a cartridge according to an embodiment.

[0032] FIG. 5B is a cross-sectional view of the cartridge shown in FIG. 5A taken along line BB in FIG. 5A .

[0033] 5C is a cross-sectional schematic diagram of a cartridge according to an embodiment.

[0034] FIG. 6 is a schematic cross-sectional view of a cartridge according to an embodiment.

[0035] FIG. 7 is a schematic cross-sectional view of a cartridge according to an embodiment.

[0036] FIG. 8A is an enlarged view of a portion of the cartridge shown in FIG. 7 identified by area Z in FIG. 7 .

[0037] 8B is an enlarged view of a portion of a cartridge showing an area similar to the area identified by area Z in FIG. 7 , according to an embodiment.

[0038] 9 and 10 are perspective views of a cartridge according to an embodiment.

[0039] FIG. 11 is a cross-sectional view of a portion of the cartridge shown in FIGS. 9 and 10 , taken along line XX in FIG. 9 .

[0040] 12 and 13 are perspective views of the top member of the cartridge shown in FIGS. 9 and 10 , illustrating the outer surface ( FIG. 12 ) and the inner surface ( FIG. 13 ).

[0041] 14 and 15 are top perspective views of the bottom member of the cartridge shown in FIGS. 9 and 10 .

[0042] 16 is a bottom perspective view of the bottom member of the cartridge shown in FIGS. 9 and 10 .

[0043] 17 is a top view of the bottom member of the cartridge shown in FIGS. 9 and 10 , showing the separation portion and the imaging portion.

[0044] 18 is a schematic diagram of the top member of the cartridge shown in FIGS. 9 and 10, illustrating the surface treatment on the detection surface.

[0045] 19 and 20 are graphs showing experimental data associated with surface treatment on a detection surface according to an embodiment.

[0046] 21 is a graph illustrating experimental data associated with a density medium within a cartridge, according to an embodiment.

[0047] FIG. 22 is a graph showing experimental data associated with stains used in a method, according to an embodiment.

[0048] FIG. 23 is a flow chart of a method for detecting target cell concentration in a sample according to an embodiment.

[0049] 24A and 24B are schematic diagrams illustrating a method of detecting a target cell concentration in a sample according to an embodiment.

[0050] 25 is a flow chart of a method for determining the sensitivity of a target cell to a therapeutic regimen, according to an embodiment.

[0051] 26A and 26B are graphs showing experimental data of cell concentration associated with cartridges and methods according to various embodiments.

[0052] 27 is a graph showing experimental data of particle concentration associated with cartridges and methods according to various embodiments.

[0053] 28 is a graph showing experimental data of bacterial cell concentration associated with cartridges and methods according to various embodiments.

[0054] 29A and 29B are graphs showing experimental data of bacterial cell concentration associated with cartridges and methods according to various embodiments.

[0055] 30A-30C are diagrams showing bilirubin associated with cartridges and methods according to various embodiments. Fig. 30A ), lipids (Figure 30B), and hemoglobin (Figure 30C).

[0056] 31 is a perspective view of a cartridge according to an embodiment.

[0057] 32A and 32B are schematic diagrams of cartridges according to various embodiments. DETAILED DESCRIPTION

[0058] Described herein are devices and methods for determining the concentration of target cells (e.g., bacteria) within a sample. In some embodiments, the devices described herein are used for rapid automatic or semi-automatic quantification of particles such as bacteria or cells in biological samples such as blood or urine. The embodiments described herein can provide accurate results over a wide dynamic range of cell concentrations. Specifically, in some embodiments, the device and / or method can determine the cell concentration with a detection limit of 1000 (i.e., 10^3) ​​colony forming units (CFU) per milliliter (mL). In some embodiments, the device and / or method can determine the cell concentration of the sample within the range of 10^3 CFU / mL and 10^9 CFU / mL. In addition, the embodiments described herein can produce accurate particle or cell concentration results in samples with a wide range of contaminants. Therefore, the embodiments described herein are applicable to a variety of samples, including cultured blood samples.

[0059] In some embodiments, a system or method for determining the concentration of target cells in a sample includes a cartridge configured to be removably coupled to a rotating element, the rotating element rotating the cartridge around a rotation axis to capture target cells in the sample. The cartridge includes an inlet portion defining an inlet reservoir, a separation portion, and a detection portion configured to accommodate the sample. The separation portion includes a first surface and a second surface defining a separation chamber. The separation portion may include a density medium having a density greater than the density of the first portion of the sample and less than the density of the second portion of the sample. The second portion of the sample includes target cells. The separation chamber is configured to be fluidically coupled to the inlet reservoir so that when the cartridge rotates, at least the second portion of the sample can be transported from the inlet reservoir to the separation chamber. The detection portion includes a detection surface forming a boundary of the detection chamber. The detection chamber is fluidically coupled to the separation chamber so that at least the target cell can pass through the density medium and enter the detection chamber during rotation. The detection surface is not parallel to at least one of the first surface or the second surface so that the target cell impacts on the surface when passing through the density medium into the detection chamber. The detection surface is configured to capture multiple target cells.

[0060] In some embodiments, a method includes centrifuging a sample within a cartridge to separate target particles (e.g., cells) originally contained in the sample from contaminants of varying density and size. In some embodiments, the sample is centrifuged through a density medium or density gradient within the cartridge that has a density greater than that of the sample but less than that of individual target particles. The density medium may be contained within the cartridge prior to adding the sample and may occupy an area of ​​an angled detection (or imaging) surface. In this manner, target particles may be captured by the angled detection surface while contaminant particles are either retained in the sample in the separation chamber (for small concentrations or low density contaminants) or centrifuged through the density medium (for larger and high density contaminants). The number of target particles within the known detection (or imaging) area may be related to the original concentration of target particles suspended in the sample.

[0061] In some embodiments, the cartridge or kit includes a density medium for separating target particles from contaminants. The density medium may be composed of a material having a mass density of 1.01 g / cm 3 and 1.13 g / cm 3 In some embodiments, for example, when the target particles are bacteria, the mass density of the density medium is 1.03 g / cm 3 and 1.09 g / cm 3 In other embodiments, the mass density of the density medium is between 1.06 g / cm 3 and 1.08 g / cm 3 between.

[0062] In some embodiments, the cartridge includes a defined density medium reservoir in which the density medium is contained prior to and during use. The density medium reservoir ensures that a desired amount of density medium is within the cartridge to ensure desired performance. During centrifugation, the sample will form a distinct layer that contacts the density medium and rotates inwardly from the density medium. During use, mixing of the sample and the density medium is limited in the measurement technique to limit the possibility of introducing contaminants into the detection (or imaging) surface.

[0063] In some embodiments, the detection (or imaging) surface of the cartridge may be configured to capture multiple target cells. For example, in some embodiments, the detection surface may include a surface treatment or modification to enhance the adhesion of target particles (or cells) to the detection surface. In particular, such surface modification may enhance particle capture by having a detection surface with a shallow angle (relative to the wall of the separation chamber). Such a surface modification may prevent target particles from traveling along the surface to form a pellet at the distal end of the cartridge. In some embodiments, the adhesion force of the detection surface may be greater than the effective centrifugal force caused by the rotation of the cartridge. Therefore, the shallower the angle of the detection surface, the greater the adhesion force must be to prevent the migration of target particles for a given rotation rate.

[0064] In some embodiments, the detection surface can include a coating having a charged polymer. For example, in some embodiments, the detection surface can include poly-L-lysine having a positive charge.

[0065] In some embodiments, the cartridge may be configured to limit undesirable mixing of the sample (including target particles) and the density medium. Specifically, in some embodiments, the depth of certain chambers (or channels) may be selected to minimize mixing. For example, in some embodiments, the cartridge may include a separation chamber having a depth (or thickness) of less than 0.60 mm, less than 0.40 mm, or less than 0.30 mm. In other embodiments, the thickness of the separation chamber may be less than 0.3 mm, or less than 0.2 mm, or less than 0.1 mm, thereby enabling imaging. The layers inside the sample and the fluid contained in the cartridge may also be stabilized by constructing the cartridge so that the angled detection (or imaging) channel and the detection (or imaging) surface are angled downward. This downward angle allows the density medium to be naturally retained in the distal region of the cartridge (including the angled imaging channel) under natural gravity.

[0066] In some embodiments, a device or test kit includes a cartridge configured to be removably connected to a rotating element that rotates the cartridge around an axis of rotation to capture target cells in a sample. The cartridge includes an inlet portion that defines an inlet reservoir, a separation portion, and a detection portion for accommodating the sample. The separation portion includes a first surface and a second surface that define a separation chamber (or channel). The separation chamber can be fluidically connected to the inlet reservoir so that at least a portion of the sample can be transported from the inlet reservoir to the separation chamber when the cartridge rotates. The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidically connected to the separation chamber so that at least the target cell can pass through the separation chamber and enter the detection chamber. The detection surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture target cells and may include a coating, a positively charged treatment, etc. The ratio of the volume of the separation chamber to the volume of the detection chamber is at least about 2.0. By maintaining the volume ratio within a desired range, the ratio of sample to density medium within the cartridge can be maintained within a desired range to ensure that the detection (or imaging) volume is properly filled with the density medium.

[0067] In some embodiments, the ratio of the volume of the separation chamber to the volume of the detection chamber is between about 2.0 and 5.0. In certain embodiments, the ratio of the volume of the separation chamber to the volume of the detection chamber is about 2.5. In some embodiments, a non-zero detection angle can affect the effectiveness of the detection surface in capturing target cells. In some embodiments, the detection angle between the detection surface and the first surface of the separation portion is between about 1 degree and about 8 degrees. In some embodiments, the first surface of the separation portion and the detection surface are integrally constructed, and the detection angle is about 2 degrees.

[0068] In some embodiments, the methods, cartridges, and / or kits described herein may include and / or employ stains formulated to bind and enhance detection of target cells. The use of stains may improve the specificity of detecting (e.g., imaging) target particles in samples containing contaminants. In some embodiments, stains may include DNA dyes such as Syto, DAPI, or Hoechst 33258, which are used to label DNA within target cells (e.g., bacterial particles) while leaving platelets (which do not contain DNA) unstained. In some embodiments, unbound dyes (small molecules) will remain in the biological sample and will not migrate to the detection (e.g., imaging) surface. In addition to differences in labeling, target particles may also be identified by size or shape for residual contaminants.

[0069] In some embodiments, a kit for performing a method for determining the concentration of target cells in a sample includes a cartridge, a density medium, and a stain. The cartridge may be removably coupled to an instrument configured to rotate the cartridge around an axis of rotation and detect target cells in the sample. The cartridge includes an inlet portion defining an inlet reservoir, a separation portion, and a detection portion configured to accommodate a sample. The separation portion includes a first surface and a second surface defining a separation chamber. The separation chamber may be fluidically coupled to the inlet reservoir so that at least a portion of the sample may reach the separation chamber from the inlet reservoir when the cartridge rotates. The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidically coupled to the separation chamber so that at least the target cell may pass through the separation chamber and enter the detection chamber. The detection (e.g., imaging) surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture target cells. The density medium may be either pre-loaded into the cartridge or included in a separate container for use in any of the methods described herein. The density of the density medium is 1.01 g / cm 3 and 1.13 g / cm 3 The stain is formulated to bind and enhance detection of multiple target cells. In some embodiments, the kit further comprises a diluent for mixing with the sample prior to and / or during use.

[0070] In some embodiments, a method for determining the concentration of target cells in a sample may include imaging a detection surface that captures target cells. In some embodiments, a method for detecting the concentration of multiple target cells in a sample includes delivering the sample to an inlet reservoir of a cartridge. The cartridge includes a separation portion and a detection portion. The separation portion includes a first surface and a second surface that define a separation chamber, and the separation chamber can be fluidically connected to the inlet reservoir. The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidically connected to the separation chamber, and the detection surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture multiple target cells. The cartridge is connected to a rotating element of an instrument. The instrument is then activated so that the instrument: (1) rotates the cartridge at a certain rotational speed, thereby causing at least a portion of the sample to be delivered from the inlet reservoir to the separation chamber, and the target cells to be delivered to the detection chamber and captured on the detection chamber; (2) receives a signal indicating the number of target cells captured on the detection surface via a detector of the instrument; and (3) determines the concentration of target cells in the sample based on the signal.

[0071] In some embodiments, the concentration of target cells determined by the instrument is within a lower limit of 10^3 CFU / mL and an upper limit of 10^9 CFU / mL.

[0072] In some embodiments, the sample portion transported from the inlet reservoir through the separation chamber and into the detection chamber is the second part, and the method may further include transporting the density medium to the cartridge before the sample is transported to the inlet reservoir. The density of the density medium is greater than the density of the first part of the sample and less than the density of the second part of the sample. After the cartridge is rotated, the first part of the sample is maintained in the separation chamber. The density medium can be any density medium composition and can have any property (e.g., density value) described herein. For example, in some embodiments, the density medium may be included in a test kit packaged with the cartridge. In such an embodiment, a predetermined amount of density medium (e.g., an amount sufficient to fill the cartridge to a desired level without overfilling) may be packaged in a container separated from the cartridge. In some embodiments, the volume of the density medium may be less than fifty percent of the volume of the cartridge. By packaging the density medium and the cartridge separately, the test kit can extend the shelf life. In addition, if some tests require a density medium and other determinations require a second different density medium, packaging the density medium and the cartridge separately can allow the same cartridge to be used in different test kits (each test kit comes with different reagents, density media, etc.).

[0073] However, in other embodiments, the method may be performed with a cartridge pre-filled with the density medium.

[0074] In some embodiments, the method further comprises delivering a diluent to the cartridge before delivering the sample to the inlet reservoir. In some embodiments, the diluent may be included as part of the density medium delivered to the cartridge. In other embodiments, the diluent may be added to the cartridge in an operation separate from the addition of the density medium. At least one of the diluent or the density medium comprises any one of a defoamer, a wetting agent, a dispersant, or an emulsifier. As used herein, a dispersant may be any substance that improves particle separation (e.g., prevents agglomeration or agglomeration). The dispersant may be or include a surfactant. For example, in some embodiments, the diluent (or density medium) may include a poloxamer that may act as a surfactant to improve the separation of particles (e.g., target cells, waste particles, etc.). In some embodiments, the diluent may be included in a kit packaged with the cartridge. In such an embodiment, a predetermined amount of diluent (e.g., an amount sufficient to fill the cartridge to a desired level without overfilling) may be packaged in a container separate from the cartridge. By packaging the diluent and the cartridge separately, the kit may extend the shelf life. Additionally, if some assays require one diluent and other assays require a second, different diluent, packaging the diluent and cartridge separately allows the same cartridge to be used in different kits (each with different reagents, density media, etc.).

[0075] In some embodiments, the diluent can be mixed with the sample, and the mixture of the sample and the diluent can then be delivered to the inlet reservoir of the cartridge. Similarly, in some embodiments, the diluent and the sample can be delivered to the inlet reservoir in the same operation.

[0076] However, in other embodiments, the method may be performed with a cartridge pre-filled with diluent.

[0077] In some embodiments, the signal can be an optical signal received by an optical detector of an imaging subsystem of the instrument. Such imaging techniques may include, for example, identifying and counting particles of interest based on light generated from a detection portion of the cartridge. Such imaging techniques may include bright field, dark field, and / or may be enhanced by any of the stains described herein.

[0078] In some embodiments, all or part of the measurement technique can be accomplished by automated steps (i.e., steps that do not require further human intervention after the instrument is activated). In some embodiments, a system for determining particle concentration can automatically identify target particles (e.g., to distinguish target cells from contaminants) through a recognition algorithm and calculate the original concentration of target particles in a biological sample.

[0079] In some embodiments, a method for detecting the concentration of target cells in a sample includes rotating a cartridge in an instrument. The cartridge includes an inlet portion, a separation portion, and a detection portion. The inlet portion defines an inlet reservoir that holds the sample before rotation. The separation portion includes a first surface and a second surface that define a separation chamber, the separation chamber containing a density medium having a density greater than the density of the first portion of the sample and less than the density of the second portion of the sample. The second portion of the sample contains target cells. During rotation, the separation chamber is fluidically coupled to the inlet reservoir. The detection portion includes a detection surface that forms a boundary of a detection chamber that is fluidically coupled to the separation chamber. The detection surface and the first surface of the separation portion form a non-zero detection angle, and the detection surface is configured to capture a plurality of target cells. The rotation is performed at a certain rotation speed and within a certain rotation duration, so as to be sufficient to cause: A) the second portion of the sample to pass from the inlet reservoir through the density medium in the separation chamber and then enter the detection chamber, and B) the target cells to be captured on the detection surface. The method further includes receiving a signal via a detector of the instrument, the signal indicating the number of target cells captured on the detection surface. The concentration of target cells in the sample is then determined based on the signal.

[0080] In some embodiments, a method for detecting the concentration of target cells in a sample includes delivering a density medium from a container to a cartridge. The cartridge includes an inlet portion, a separation portion, and a detection portion. The inlet portion defines an inlet reservoir. The separation portion includes a first surface and a second surface defining a separation chamber. The density of the density medium is greater than the density of the first portion of the sample and less than the density of the second portion of the sample, and is delivered to the separation chamber. The second portion of the sample contains target cells. The separation chamber is fluidically connected to the inlet reservoir. The detection portion includes a detection surface, which forms a boundary of a detection chamber that is fluidically connected to the separation chamber. The detection surface and the first surface of the separation portion form a non-zero detection angle, and the detection surface is configured to capture multiple target cells. The sample is delivered to the inlet reservoir. The cartridge is then rotated in the instrument at a certain rotation speed and within a certain rotation duration, so as to be sufficient to cause: A) the second portion of the sample to pass from the inlet reservoir through the density medium in the separation chamber and then enter the detection chamber, and B) the target cells are captured on the detection surface. The method further includes receiving a signal via a detector of the instrument, which indicates the number of target cells captured on the detection surface. The concentration of target cells in the sample is then determined based on the signal.

[0081] In some embodiments, the container (including the density medium) and the cartridge are packaged together in a kit.

[0082] In some embodiments, the method further comprises delivering a diluent to the cartridge. At least one of the diluent or density medium comprises any one of a defoamer, a wetting agent, a dispersant, or an emulsifier. In some embodiments, at least one of the diluent or density medium comprises a poloxamer. In some embodiments, delivering the diluent comprises mixing the diluent with the sample in the same operation and delivering the diluent and the sample to the inlet reservoir of the cartridge.

[0083] In some embodiments, any of the methods, cartridges, and kits for detecting target cell concentrations in a sample can be used as part of an assay to determine the sensitivity of target cells to a treatment regimen. For example, in some embodiments, any of the methods, cartridges, and kits for detecting target cell concentrations can be used as part of an antibiotic sensitivity test (AST). For example, in some embodiments, a method includes delivering a first portion of a sample into a cartridge. The cartridge includes a separation portion and a detection portion. The separation portion includes a first surface and a second surface defining a separation chamber. The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidically coupled to the separation chamber, and the detection surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture a plurality of target cells. The cartridge is coupled to a first instrument. The first instrument is activated so that the instrument: (1) rotates the cartridge at a rotational speed sufficient to deliver the first portion of the sample through the separation chamber and into the detection chamber and capture the target cells on the detection surface; (2) receives a signal indicating the number of target cells captured on the detection surface via a detector of the instrument, and (3) determines the concentration of target cells in the sample based on the signal. The method further includes processing a second portion of the sample based on the concentration of target cells in the first portion of the sample. The second portion of the sample is then delivered to a reaction chamber. The method then includes activating a second instrument to perform a sensitivity test on the second portion of the sample to determine the sensitivity of the target cells to the treatment regimen.

[0084] As used herein, the singular forms "a", "an", and "the" include plural references unless otherwise specified. Thus, for example, the term "component" is intended to mean a single component or a combination of components, and "material" is intended to mean one or more materials or a combination thereof.

[0085] As used herein, the terms "about" and "approximately" when used in conjunction with a reference numerical designation refer to the reference numerical designation plus or minus ten percent of the stated value. For example, about 0.5 would include 0.45 and 0.55, about 10 would include 9 to 11, and about 1000 would include 900 to 1100.

[0086] As used herein, unless the context clearly indicates otherwise, terms referring to multiple components or portions thereof are intended to refer to a first component or a first portion thereof and / or a second component or a second portion thereof. Thus, for example, the term "reagent" is intended to mean a "first reagent" and / or a "second reagent."

[0087] As used herein, a "set" may refer to multiple features or a single feature with multiple parts. For example, when referring to a set of walls, the set of walls may be considered to be one wall with different parts, or the set of walls may be considered to be multiple walls.

[0088] The specific words selected to describe one or more embodiments are not intended to limit the present invention. For example, spatial relative terms (such as "below ... ", "below", "below", "above", "above", "near side", "distal side", etc.) can be used to describe the relationship between an element or feature and another element or feature as illustrated in the figure. These spatial relative terms are intended to cover different positions (i.e., translational displacements) and orientations (i.e., rotational displacements) of the device in use or operation in addition to the positions and orientations shown in the drawings. For example, if the device in the drawings is flipped, the elements described as "below" or "below ... " of other elements or features will be "above" or "above ... " of other elements or features. Therefore, the term "below" can cover the position and orientation above and below. The device can be oriented in other ways (e.g., rotated 90 degrees or in other orientations), and the spatial relative descriptors used in this article are interpreted accordingly. Similarly, the description of the movement along (translation) and around (rotation) various axes includes various spatial device positions and orientations.

[0089] Similarly, unless the context indicates otherwise, geometric terms such as "parallel," "perpendicular," "circular," or "square" are not intended to require absolute mathematical precision. Rather, such geometric terms allow for variations due to manufacturing or equivalent functions. For example, if an element is described as "circular" or "substantially circular," the description still encompasses components that are not exactly circular (e.g., slightly oblong or polygonal components).

[0090] As used in this specification, the term "reagent" includes any substance used in combination with any method described herein. For example, a reagent may include a composition, a washing solution, an anticoagulant solution, a dye, a staining solution, etc., for diluting a sample. A reagent may include a mixture of one or more components. A reagent may include such components, regardless of their state of matter (e.g., solid, liquid, or gas). In addition, a reagent may include multiple components that may be included in a substance in a mixed state, an unmixed state, and / or a partially mixed state. A reagent may include an active ingredient and an inert ingredient. Therefore, as used herein, a reagent may include an inactive and / or inert ingredient, such as water, a colorant, etc.

[0091] FIG. 1 shows a top view of a portion of an imaging cartridge 101. The structure shown here can be an internal structure surrounded by an imaging cartridge 101. The imaging cartridge 101 includes a closed inlet reservoir 102 in which a clinical sample can be deposited. The inlet reservoir 102 is in fluid communication with a valve 103, which in turn is in fluid communication with a separation channel 104. The separation channel (also referred to herein as a separation chamber) can have any suitable thickness or channel depth. In some embodiments, the thickness of the separation channel will be between 0.40 mm and 0.02 mm. In other embodiments, the thickness of the separation channel will be between 0.05 mm and 0.20 mm. The separation chamber is continuous with the imaging channel 105. Optionally, the imaging channel 105 terminates in a waste chamber 106. The cartridge 101 further includes a hub 107 around which the cartridge rotates. Optionally, the cartridge 101 may include one or more media reservoirs 108 having a greater depth than the separation channel 105 or the imaging channel 106. It should be understood that portions of the imaging cartridge (not shown) may include mirrored copies of the structures 102-106 and 108 shown in this figure, which are symmetrical about a plane that bisects the hub 107.

[0092] FIG. 2 shows a cross-sectional view of a portion of the imaging cartridge 101 taken along line XX in FIG. 1 . The cartridge may be constructed of two parts: a bottom 201 and a top 202. The bottom 201 and the top 202 may be joined by any process described herein, such as ultrasonic welding or laser welding, to form an integral detection (or imaging) cartridge 101. The imaging cartridge 101 includes a closed inlet reservoir 102 in which a clinical sample may be deposited. The closed inlet reservoir 102 may be connected to the outside through an inlet hole 203 and may have a greater depth than a channel located further distally (e.g., a separation channel or a detection channel). The inlet reservoir 102 is in fluid communication with a valve 103, which may include a plurality of channel depths that are smaller than the inlet reservoir 102. The valve may include a first depth, followed by a second depth greater than the first depth, and then a third depth equal to the first depth. This structure is used to restrict the flow of liquid through the valve 103 unless sufficient pressure is applied to the fluid (e.g., during centrifugation of the cartridge). The valve 103 is in fluid communication with the separation channel 104 .

[0093] The separation chamber 104 is continuous with the angled detection (or imaging) channel 105, which is continuous at a vertical angle 204 relative to the separation chamber 104. Suitable values ​​for the angle 204 may include 0.25 degrees to 20 degrees, or preferably 0.5 degrees to 10 degrees. Optionally, the imaging channel 105 terminates in a waste chamber 106, which may not share the same angle 204 as the angled imaging channel 105. The separation channel 104 and the angled imaging channel 105 may have the same depth, measured perpendicular to the top and bottom closed surfaces. In some embodiments, the cartridge (and any cartridge described herein) may include a balancing feature so that the center of mass of the cartridge is within the hub 107.

[0094] FIG3 shows a top view of a portion of the imaging cartridge 101 in two different states of action during intended operation. The imaging cartridge 101 initially contains a fluid density medium 301. The density medium 301 may fill the waste chamber 106, the angled imaging chamber 105, and a portion of the separation channel 104 and the medium reservoir 108. The density medium 301 may be any suitable medium of the type described herein that has a density greater than the density of a first portion of the sample and less than the density of a second portion of the sample. Specifically, the density of the density medium 301 (and any density medium described herein) may be greater than the density of certain contaminants within the sample (e.g., low-density platelets, unbound dye, etc.). The density of the density medium 301 (and any density medium described herein) may be less than the density of certain particles in the sample (e.g., target cells, blood cells, high-density platelets, etc.). In this manner, as described below, during use, target cells can be transported through the density medium 301 and into the imaging channel 105 while other (eg, lighter) contaminants are prevented from entering the detection region.

[0095] A clinical sample 302 may be introduced into the inlet chamber 102 through an inlet hole 203 (as described in FIG. 2 ). The clinical sample 302 (and any sample described herein) may contain particles 303, such as bacteria or other cells. The clinical sample 302 may be held in the inlet reservoir for a period of time through the valve 103. During this period of time, reagents may be introduced into the clinical sample 302 for reaction. For example, in some embodiments, a membrane-permeable DNA dye, such as Syto 83, DAPI, or Hoechst 33258, may be introduced to fluorescently label cells containing DNA. After centrifugation (i.e., the cartridge rotates), the clinical sample 302 will enter the valve 103 and the separation chamber 104, and will form a distinct layer from the density medium inwardly relative to the center of rotation, and contact the density medium fluid. During centrifugation, the clinical sample 301 may also occupy a portion of the medium reservoir 108. A portion of the clinical sample 301 may also remain in the inlet reservoir 102.

[0096] FIG4A and FIG4B illustrate cross-sections of the distal end of the imaging cartridge 101 at the beginning (FIG4A) and end (FIG4B) of a rotation cycle (also referred to as "spin"). As shown, the upper surface of the imaging channel 105 includes a detection (or imaging) surface 401. In the initial state, the clinical sample 302 forms a layer from the center of rotation of the density medium 301 inward. The clinical sample contains target particles 303 (e.g., bacteria or cells), low-density contaminants 403, and high-density contaminants 404 (e.g., red blood cells, white blood cells, crystals, or large protein aggregates). It should be understood that the "low-density contaminants" 403 may be replaced by objects of higher density that are substantially smaller than the particles 303 and therefore do not migrate through the clinical sample 302 or the density medium 301 in a timely manner during the rotation. The low-density contaminants 403 may include unbound dyes, cell fragments, proteins, or other matrix components in the clinical sample 302. The effective centrifugal force causes the target particles 303 and high density contaminants 404 to migrate in a radially outward direction 402 (through the density medium).

[0097] 4B, upon completion of the rotation, the target particles 303 will migrate until they contact the imaging surface 401. However, low density contaminants 403 will not migrate (or pass through) the density medium 301, and therefore will remain away from the imaging chamber 105. High density contaminants 404 will migrate faster and more vigorously than the target particles 303, and therefore will reside in the waste chamber 106 after the rotation is completed. By making the upper surface of the waste chamber 106 coplanar with or lower than the bottom surface of the separation chamber 104, the capture of all particles 303 by the imaging surface 401 is facilitated. For example, Figure 6 The cartridge 2100 shown in shows a coplanar arrangement of the lower separation portion surface 2142 and the upper waste portion surface 2181 (aligned with plane P). This arrangement ensures that particles 303 initially dispersed in the clinical sample 302 will contact the imaging surface 401 assuming the effective centrifugal force is much higher than the natural gravitational force.

[0098] In some embodiments, the detection (or imaging) surface 401 (and any detection surface described herein) can be configured to capture target particles 303. For example, in some embodiments, the detection (or imaging) surface 401 is treated with a surface modification 407 that attracts or binds to the particles 303. Moreover, the surface modification can be selected so that high density contaminants 404 do not easily bind to the detection surface 401. For example, in some embodiments, the chemical modification 407 can include a charged coating that includes chemical components that maintain a positive or negative charge at the pH of the density medium. When the surface is used to capture bacteria, the charge should be positive. Examples of suitable coatings are polymers that are positively charged at neutral + / - 3 pH, such as poly-L-lysine or polyethyleneimine, which will bind negatively charged particles, such as bacteria. The surface modification can be selected to minimize interference or disruption of the imaging technique. Furthermore, by making the top outer surface 408 of the imaging chamber 105 and the bottom outer surface 409 of the imaging chamber 105 parallel to the imaging surface 401 , imaging of the target particles 303 may be facilitated.

[0099] In use, the cartridge 101 (and any cartridge described herein) can be used in an optical system 410 that can receive a signal associated with the amount of target particles 303 captured on the detection surface 401. Referring to FIG. 4B, the optical system can be configured at the same angle 204 as the imaging chamber 105 relative to an axis 411 that is perpendicular to the imaging surface 401. In other embodiments, this angle may not be included in the optical system 410, resulting in a distorted but potentially acceptable image. The imaging surface 401 can be illuminated by light 413 emitted by a light source 412 to facilitate imaging by the optical system 410. The light 412 can have a wavelength that enables fluorescent imaging of the particles 303. The light 412 can also be configured to provide non-fluorescent brightfield or darkfield illumination of the imaging surface 401. The light source can be placed coaxially with the optical system 410, or can be pointed in any direction that illuminates the particles 303. The light source 412 can be integrated into the optical system 410, such as with an epifluorescence system. The optical system 410 can capture images of the particles 303 bound to the imaging surface 401, thereby enabling quantification of the particles 303. The number of particles 303 in a given image of the imaging surface 401 will be related to the original concentration of particles 303 suspended in the clinical sample 302.

[0100] In some embodiments, to improve analytical accuracy, a method may include capturing multiple images from different regions of the angled imaging channel 105 to obtain an average particle count for each image. One method of achieving this goal is to slowly rotate the cartridge 101 about the hub 107 so that different portions of the angled imaging channel 105 are located in the imaging path of the optical system 410. In such an embodiment, the imaging surface 401 would include a curved surface rather than a flat surface to maintain a constant cross-sectional angle 204 at each radial cross section of the cartridge as the cartridge is rotated. The curvature increases as the angle 204 increases, thereby reducing the area in which any given image can remain in focus (some areas will be closer to the optical system and others will be farther away)

[0101] As shown, the detection (or imaging) surface 401 and the surface defining the separation channel 104 define a detection angle 204. The detection angle 204 is along the radial axis of the cartridge 101, i.e., the axis intersecting the rotation axis (or center point) of the cartridge in the radial direction. Specifically, the detection angle 204 is within the cross-sectional plane defined by the rotation axis and the radial axis, as shown in the cross-sectional views of Figures 2, 4A, and 4B. In addition, the detection angle 204 of the cartridge 101 is in a downward direction (relative to the opening 102 and / or the direction of gravity). In other words, the detection angle 204 causes the waste chamber 106 to be below the separation channel 104 relative to the opening 102. In this way, the imaging surface 401 is on the upper (or top) surface of the top 202 that forms the cartridge. This arrangement can provide advantageous imaging from the top (compared to bottom imaging systems). Such an arrangement may also provide improved liquid stability within the channel, which may facilitate including a chamber (or channel) of greater depth (or thickness) than a cartridge having an upward detection angle. Including a surface treatment to enhance capture of target particles 303 may minimize the effects of gravity settling after rotation is complete and during imaging.

[0102] However, in other embodiments, the detection angle may be in an upward direction (relative to the opening and / or the direction of gravity). Such an arrangement may limit any negative effects of gravity. Figures 5A and 5B show an imaging cartridge 101', which is an alternative embodiment of the cartridge 101 shown in Figures 1 to 4B. In this embodiment, the imaging cartridge 101' surrounds a sample inlet chamber 102', a valve 103', a separation channel 104', an angled imaging chamber 105', and a waste chamber 106'. In this embodiment, the imaging surface 401' is on the bottom 201', which has the advantage of retaining any particles on the imaging surface with the help of natural gravity. In this embodiment, the thickness of the separation chamber 104' is greater than the thickness of the waste chamber 106', and the bottom of the imaging chamber 105' is at a certain angle to connect the separation chamber 104' to the waste chamber 106'. In some embodiments, the imaging cartridge 101' rotates around an axis of rotation 501 outside the cartridge 101'. The cartridge 101' can be held in rotation by a rotor 502, which holds, locks or constrains the cartridge in place during rotation. The rotor 502 can be connected to the cartridge 101' through a protrusion 503 on the cartridge 101', which interlocks with a protrusion 504 on the rotor 502. In other embodiments, the cartridge can be attached to the rotor or rotating hub by any suitable mechanism. During rotation, the cartridge 101' will be held in place by the reaction force from the protrusion 504 on the rotor 502. The contacting surfaces of the protrusions 503 and 504 will preferably be vertical relative to the axis of rotation 501. As described herein, this embodiment may include a density medium and a chemical modification of the imaging surface 401. This embodiment can also be evaluated by the optical system and illuminator described in conjunction with Figures 4A and 4B.

[0103] FIG. 5C is a schematic cross-sectional view of a cartridge 1100 according to an embodiment. Like cartridge 101', cartridge 1100 is also characterized by having an upward detection angle that causes target particles TP to be captured on the bottom surface of cartridge 1100. Specifically, cartridge 1100 includes at least a detection portion 1160 and a waste portion 1180. Detection portion 1160 includes a first (or upper) surface 1161 and a second (or bottom) surface 1162 that define a detection chamber. In use, as shown by arrow R, rotation of cartridge 1100 causes the sample to flow, transport and / or otherwise move from the separation portion to the detection portion 1160 in a radial direction. In some embodiments, cartridge 1100 may include a density medium of the type described herein. Centrifugation of the sample causes target particles TP to contact and be captured by second surface 1162. In some embodiments, second surface 1162 may be configured to enhance adhesion of target particles TP thereto. As shown in FIG. 5C , larger and / or denser contaminants are transported to the waste portion 1180 and therefore do not cause undesirable interference with the detection (ie, counting and / or imaging) of the target particles TP captured on the second surface 1162 .

[0104] In some embodiments, the size and geometry of the portions of any cartridge described herein may be selected to optimize collection performance and detection for specific target particles and / or specific samples. For example, in some embodiments, a higher detection angle may increase certain particle capture characteristics (i.e., due to greater impact of target particles), but may also result in greater instability during and after rotation of the cartridge. Specifically, in some embodiments, a separation chamber and / or detection chamber having a depth of 125 μm (0.125 mm) exhibits suitable mixing stability at a detection angle of 5 degrees. In contrast, in other embodiments, a separation chamber and / or detection chamber having a depth of 250 μm (0.25 mm) exhibits undesirable mixing stability at a detection angle of 5 degrees. Additionally, in some embodiments, making the upper surface of the waste chamber coplanar with or lower than the bottom surface of the separation chamber may improve the capture efficiency of the detection surface. For example, FIG. 6 is a cross-sectional schematic diagram of a cartridge 2100 according to an embodiment. The cartridge 2100 is composed of a first (or upper) member 2101 and a second (or lower) member 2102 to form a separation section 2140, a detection (or imaging) section 2160, and a waste (or collection) section 2180. The separation section 2140 defines a separation chamber (or channel) defined by a first surface 2141 (of the first member 2101) and a second surface 2142 (of the second member 2102). The separation chamber has a depth D1 (also referred to herein as thickness) perpendicular to the first surface 2141 and the second surface 2142. The depth D1 is also perpendicular to the radial direction R.

[0105] The detection portion 2160 defines a detection chamber (or channel) 2163 defined by a first surface 2161 (of the first member 2101) and a second surface 2162 (of the second member 2102). The first surface 2161 (also referred to as a detection surface or imaging surface) is not parallel to at least one of the first surface 2141 or the second surface 2142, thereby allowing target particles (or cells) to impact on the detection surface 2161, as described in detail herein. Similarly, the detection surface 2161 and the first surface 2141 of the separation portion 2160 form a non-zero detection angle θ. The detection angle θ is along the radial axis of the cartridge 2100 (the radial direction is indicated by the arrow R), i.e., the axis intersecting the rotation axis (or center point) of the cartridge in the radial direction. Specifically, the detection angle θ is within a cross-sectional plane defined by the rotation axis and the radial axis. In addition, the detection angle θ is in a downward direction. In this manner, the detection surface 2161 is on the first member 2101 that forms the cartridge 2100. In some embodiments, the detection angle θ is between about 1 degree and about 8 degrees. In other embodiments, the detection angle θ is between about 1.5 degrees and about 5 degrees. In other embodiments, the detection angle θ is about 2 degrees. In some embodiments, the detection surface 2161 and the first surface 2141 are integrally constructed, and the detection angle is the "bend" between the surfaces.

[0106] In some embodiments, the detection surface 2161 (and any detection surface described herein, including the detection surface 4161 described below) is configured to capture target particles TP. For example, in some embodiments, the detection surface 2161 can be modified to enhance the adhesion of the target particles TP. Such modifications can include, for example, chemical modifications, surface coatings, charging the detection surface 2161, or a combination of these modifications. For example, in some embodiments, the detection surface 2161 (and any detection surface described herein, including the detection surface 4161 described below) can include markings or textures that can help identify imaging boundaries. In other embodiments, the detection surface 2161 can be modified by corona treatment (also known as air plasma treatment). This process destroys the polymer chains at the surface, which can increase the surface energy of the detection surface 2161, thereby improving the surface adhesion properties of the detection surface 2161. In other embodiments, surface modification can include applying a charged coating, such as poly-L-lysine (PLL) or polyethyleneimine, which will bind negatively charged particles (e.g., bacteria). In some embodiments, the charged polymer may include (-NH 3 +). The charged coating may be applied in any suitable manner. In some embodiments, the charged polymer coating may be applied by attachment using a carbodiimide compound. Carbodiimide compounds are cross-linking compounds that facilitate the application of a charged polymer coating (e.g., PLL). In other embodiments, the charged polymer coating may be applied by photochemically induced polymerization and grafting of the desired coating. In addition, the detection surface 2161 may be modified using a combination of any suitable procedures to enhance the adhesion properties of the surface. For example, as shown in FIG. 18 (with reference to detection surface 4161), any detection surface described herein (including detection surface 2161) may be modified using a combination of corona treatment and PLL coating. Referring again to FIG. 6 , the positive charge of the detection surface 2161 is represented by (+), which improves the adhesion properties to capture target particles TP (e.g., bacteria) therein.

[0107] In some embodiments, the detection surface 2161 may be modified to have a surface finish (or roughness) that is favorable for capturing target particles TP. The surface finish (or roughness) may also be selected to provide identification and / or marking characteristics that do not interfere with the imaging process. In this way, the detection surface and other portions of the cartridge may include markings to aid in the collection and evaluation of imaging data.

[0108] The detection chamber 2163 has a depth D2 (also referred to herein as thickness) perpendicular to the first (detection) surface 2161 and the second surface 2162. In some embodiments, the depth D2 may be the same as the depth D1 of the separation chamber. In other embodiments, the depth D2 may be different from the depth D1 of the separation chamber.

[0109] The waste portion 2180 defines a waste chamber (or channel) 2183, which is bounded by a first surface 2181 (of the first member 2101) and a second surface 2182 (of the second member 2102). The first surface 2181 of the waste portion 2180 and the second surface 2142 of the separation portion 2140 are coplanar in a plane P. As described above, this arrangement provides improved capture efficiency in the detection chamber 2163. In other embodiments, the first surface 2181 of the waste portion 2180 is lower than the second surface 2142 of the separation portion 2140. The waste chamber 2183 has a depth D3 (also referred to herein as thickness) perpendicular to the first surface 2181 and the second surface 2182. In some embodiments, the depth D3 may be the same as the depth D1 of the separation chamber and / or the depth D2 of the detection chamber 2163. In other embodiments, the depth D3 may be different from one or both of the depth D1 of the separation chamber and / or the depth D2 of the detection chamber 2163. The depth D1, the depth D2, and / or the depth D3 may be any suitable value described herein. For example, in some embodiments, the depth D1, the depth D2, and / or the depth D3 may be less than about 0.6 mm. In other embodiments, the depth D1, the depth D2, and / or the depth D3 may be less than about 0.3 mm. In other embodiments, the depth D1, the depth D2, and / or the depth D3 may be about 0.250 mm. In other embodiments, the depth D1, the depth D2, and / or the depth D3 may be about 0.125 mm.

[0110] As shown, the sample may include a first portion (identified as S1 in FIG. 6 ), a target particle TP, and a second portion (identified as S2 in FIG. 6 ). The first portion S1 may include low-density contaminants, such as unbound dyes, cell fragments, proteins, or other matrix components. The second portion S2 may initially include target particles TP as well as other high-density contaminants, such as red blood cells, white blood cells, crystals, or large protein aggregates. In use, the effective centrifugal force causes the target particles TP and the high-density contaminants S2 to move in a radially outward direction, as shown by arrow R. The target particles TP will migrate until they contact the detection surface 2161. However, the low-density contaminants S1 are retained in the separation portion 2140 (e.g., blocked by a density medium or other mechanism), and are therefore prevented from reaching the detection surface 2161.

[0111] Although the cartridge 2100 is shown to have a depth D1 that is substantially equal to the depth D2, in other embodiments, the cartridge may include a detection chamber having a depth (or thickness) greater than the depth (or thickness) of the separation chamber. Similarly, in some embodiments, the cartridge may include a continuous channel (also referred to as a path or chamber) that diverges (i.e., the cross-sectional area increases) from the separation portion to the detection portion. For example, FIG. 7 shows a cross-sectional schematic diagram of a cartridge 3100 according to an embodiment. The cartridge 3100 includes a separation portion 3140, a detection (or imaging) portion 3160, and a waste (or collection) portion 3180. The separation portion 3140 defines a separation chamber (or channel) defined by a first surface 3141 (of the second member 2102) and a second surface 3142. The separation chamber has a depth D1 (also referred to herein as a thickness) that is perpendicular to the first surface 3141 and the second surface 3142. The depth D1 is also perpendicular to the radial direction R.

[0112] The detection portion 3160 defines a detection chamber (or channel) bounded by a first surface 3161 and a second surface 3162. The first surface 3161 (also referred to as a detection surface or imaging surface) is not parallel to at least one of the first surface 3141 or the second surface 3142, thereby allowing target particles (or cells) to impact on the detection surface 3161, as described in detail herein. Similarly, the detection surface 3161 and the first surface 3141 of the separation portion 3160 form a non-zero detection angle (not identified in FIG. 7). The detection chamber has a depth D2 (also referred to herein as thickness) that is at least perpendicular to the first (detection) surface 3161. As shown, the depth D2 is greater than the depth D1 of the separation chamber. Therefore, the entire chamber area from the separation chamber to the detection chamber diverges. Although the first surface 3161 is shown as not parallel to the second surface 3162, in other embodiments, the first surface 3161 may be parallel to the second surface 3162. Similarly, although the detection chamber is shown as having a diverging cross-sectional area, in other embodiments, the detection chamber may have a substantially constant cross-sectional area.

[0113] The waste portion 3180 defines a waste chamber (or channel) bounded by a first surface 3181 and a second surface 3182. The waste chamber 3183 has a depth D3 (also referred to herein as a thickness) perpendicular to the first surface 3181 and the second surface 3182. In some embodiments, the depth D3 may be the same as the depth D2 of the detection chamber 3163. In other embodiments, the depth D3 may be different from one or both of the depth D1 of the separation chamber and / or the depth D2 of the detection chamber 2163. The depth D1, the depth D2, and / or the depth D3 may be any suitable value as described herein.

[0114] In some embodiments, the first surface of the separation portion and the adjacent detection surface of any cartridge described herein may be integrally constructed. For example, in some embodiments, the cartridge 3100 may be formed of two separate pieces, similar to the first (or top) member 4101 and the second (or bottom) member 4102 shown with reference to the cartridge 4100. Moreover, in such embodiments, the area between the first surface and the detection surface may have a desired geometric size to produce a desired flow rate during the rotation cycle. For example, referring to Figures 8A and 8B, in some embodiments, the cartridge 3100 (and any cartridge described herein) may include a transition zone between any surfaces that form a boundary between the separation portion 3140, the detection portion 3160, and the waste portion 3180. The transition zone may be controlled to ensure the desired flow performance between the cartridge parts during the rotation cycle. For example, in some embodiments, the transition zone may be defined to produce a "snap-up" or otherwise limit a smooth transition that may produce undesirable particle and / or fluid movement within the cartridge 3100. As an example, FIG. 8A shows a transition zone between the second surface 3142 of the separation portion and the second surface 3162 of the detection portion, defining a radius of curvature R1. As shown, the radius of curvature R1 is relatively small, which produces a relatively sharp edge transition (compared to the cartridge 3100' in FIG. 8B). In some embodiments, the radius of curvature R1 is less than about 0.100 mm. In other embodiments, the radius of curvature R1 is less than about 0.050 mm. In other embodiments, the radius of curvature R1 is less than about 0.025 mm. Conversely, FIG. 8B shows a transition zone between the second surface 3142 of the separation portion and the second surface 3162 of the detection portion, defining a radius of curvature R2. As shown, the radius of curvature R2 is relatively large, which produces a relatively smooth transition (compared to the cartridge 3100 in FIG. 8A). In some embodiments, the radius of curvature R2 is greater than about 0.100 mm.

[0115] 9 to 17 show various views of a cartridge 4100 according to an embodiment. The cartridge 4100 may be included in any system and may be used in conjunction with any of the methods described herein. For example, as described herein, the cartridge 4100 may be rotated to produce centrifugal separation of a sample (e.g., blood, urine, or any other suitable biological sample) so that target particles (e.g., bacteria) are captured by a detection surface. The cartridge 4100 may be used with an optical system (e.g., optical system 410 described herein) to determine the concentration of target particles within a sample. Moreover, in some embodiments, the cartridge 4100 may be used in conjunction with other methods such as antibiotic sensitivity testing.

[0116] The cartridge 4100 is constructed of a first (or upper) member 4101 and a second (or lower) member 4102 that are coupled together to define the chambers and features described herein. The first member 4101 defines a pair of alignment openings 4106, and the second member 4102 includes a corresponding pair of alignment pins 4105 to help join the two members together. The first member 4101 and the second member 4102 may be joined together using any suitable technique described herein, including by ultrasonic welding (e.g., see welding protrusions 4108 in FIG. 14 ), by adhesive bonding, or other suitable methods. In other embodiments, the cartridge 4100 (or any other cartridge described herein) may be constructed as a whole. The cartridge 4100 includes (or defines) a central hub (or connection opening) 4110. The cartridge 4100 may be coupled to a rotating element (not shown) of an instrument via the central hub 4110. The cartridge 4100 may be rotated by the rotating element about a rotation axis Arot to perform any of the methods described herein.

[0117] As shown, the cartridge 4100 is symmetrical about the rotation axis Arot, such that the cartridge includes two inlet portions 4120, two separation portions 4140, two detection (or imaging) portions 4160, and two waste (or collection) portions 4180. Because the cartridge 4100 is symmetrical about the rotation axis Arot, only one of each of these portions is identified and described below. In other embodiments, the cartridge may be similar to the cartridge 4100, but need not be symmetrical, and may include only one inlet portion, separation portion, detection portion, and waste portion. As shown in FIG. 9 , the cartridge 4100 defines a radial axis Arad that is orthogonal to the rotation axis Arot.

[0118] The inlet portion 4120 defines an inlet reservoir 4122 (defined by the second member 4102) and an opening 4121 (defined by the first member 4101) through which a sample can be delivered to the inlet reservoir 4122. As shown in FIG. 15, the inlet reservoir includes a protrusion 4123. The protrusion 4123 can provide a structure that can enhance exhaust when the sample is delivered to the inlet reservoir. In some embodiments, the protrusion 4123 (or other structures within the reservoir, not shown) can enhance mixing (e.g., mixing of the sample and any other components added to the inlet reservoir) or other flow properties during the rotation cycle. The second member 4102 includes a welding protrusion 4124 for joining the inner surfaces together. The second member also defines a pair of valve openings 4125 that fluidly connect the inlet reservoir 4122 to the separation chamber 4143. The valve opening 4125 is configured to restrict the flow of the sample from the inlet reservoir 4122 into the separation chamber 4143 before the cartridge 4100 is rotated. Similarly, the size of the valve opening 4125 is set so that the sample is retained in the reservoir 4122 until the centrifugal force (or pressure in the sample) that pushes the sample outward along the radial axis Arad toward the separation channel 4143 (see Figure 11) exceeds a predetermined value. In this way, the valve opening 4125 acts as a capillary valve to restrict the flow of the sample until the fluid adhesion force exceeds during the rotation cycle. This arrangement allows the sample to be mixed with reagents in the inlet reservoir 4122, such as diluents, stains (or dyes) and any other suitable reagents. Although the valve opening 4125 is a "passive" valve that does not change its structure during use, in other embodiments, the cartridge 4100 may include any suitable type of valve or flow restrictor to limit the flow between the inlet reservoir 4122 and the separation chamber 4143. Such a valve may include, for example, an active valve, such as a check valve arrangement activated by centrifugal force (e.g., when the cartridge exceeds a predetermined rotation speed), a frangible valve that breaks or punctures during use, etc.

[0119] The separation portion 4140 defines a separation chamber (or channel) 4143 defined by a first surface 4141 (of the first member 4101) and a second surface 4142 (of the second member 4102). The second member 4102 defines two reservoirs 4144, and the density medium in the reservoirs can be maintained in the cartridge. This arrangement ensures that a sufficient amount of density medium is present in the cartridge 4100 to ensure accurate collection and / or capture of target cells on the detection surface 4161. For example, the reservoir 4144 can provide an additional volume between about 25 μL and about 75 μL to ensure that a sufficient amount of density medium is available after long-term storage (e.g., up to about 1 year or up to about 2 years). The additional volume can account for potential evaporation of the density medium. Although the separation portion 4140 is shown as including two separate (i.e., non-continuous) reservoirs 4144, in other embodiments, any arrangement of reservoirs for maintaining density medium can be used. For example, in some embodiments, the separation portion 4140 may include a single reservoir.

[0120] The density media can be any density media of the type shown and described herein. For example, FIG. 21 shows the effectiveness of the density media in filtering low density contaminants as tested with E. coli. In some embodiments, the density media can have a density of about 1.01 g / cm 3 and about 1.13 g / cm 3 In other embodiments, the density of the medium may be about 1.06 g / cm 3 and about 1.09 g / cm 3 In other embodiments, the density of the medium may be about 1.07 g / cm 3 and about 1.08 g / cm 3 The separation chamber 4143 may have any suitable depth as disclosed herein. For example, in some embodiments, the separation chamber 4143 may have a depth (also referred to herein as thickness) of 0.250 mm or 0.125 mm, or any value therebetween.

[0121] As shown in FIG. 11 , the detection portion 4160 defines a detection chamber (or channel) 4163 bounded by a first surface 4161 (of the first member 4101) and a second surface 4162 (of the second member 4102). In some embodiments, the second surface 4162 may include ribs 4107 that may limit or prevent bulk flow of a portion of the sample during a rotation cycle. In other embodiments, the second surface 4162 does not include these ribs. The first surface 4161 (also referred to as the detection surface or imaging surface) is non-parallel to at least one of the first surface 4141 or the second surface 4142, thereby allowing target particles (or cells) to impinge on the detection surface 4161, as described in detail herein. Similarly, with reference to FIG. 11 , the detection surface 4161 and the first surface 4141 of the separation portion 4160 form a non-zero detection angle θ. The detection angle θ is along the radial axis of the cartridge 4100 (the radial direction is indicated by the arrow R), i.e., the axis that intersects the rotation axis Arot of the cartridge in the radial direction. Specifically, the detection angle θ is within a cross-sectional plane defined by the rotation axis Arot and the radial axis Arad. In some embodiments, the detection angle θ is between about 1 degree and about 8 degrees. In other embodiments, the detection angle θ is between about 1.5 degrees and about 5 degrees. In other embodiments, the detection angle θ is about 2 degrees. In some embodiments, the detection surface 4161 and the first surface 4141 are integrally constructed, and the detection angle is the "bend" between the surfaces.

[0122] In some embodiments, the detection surface 4161 (and any detection surface described herein, including the detection surface 4161 described below) is configured to capture target particles TP. For example, in some embodiments, the detection surface 4161 can be modified to enhance the adhesion of the target particles TP. Such modifications can include, for example, chemical modifications, surface coatings, charging the detection surface 4161, or a combination of these modifications. For example, in some embodiments, the detection surface 4161 (and any detection surface described herein, including the detection surface 4161 described below) can include markings or textures that can help identify imaging boundaries. In other embodiments, the detection surface 4161 can be modified by corona treatment (also known as air plasma treatment). This process destroys the polymer chains at the surface, which can increase the surface energy of the detection surface 4161, thereby improving the surface adhesion properties of the detection surface 4161. In other embodiments, surface modification can include applying a charged coating, such as poly-L-lysine (PLL) or polyethyleneimine, which will bind negatively charged particles (e.g., bacteria). In some embodiments, the charged polymer may include (-NH 3+ ). The charged coating may be applied in any suitable manner. In some embodiments, the charged polymer coating may be applied by attachment using a carbodiimide compound. Carbodiimide compounds are cross-linking compounds that facilitate the application of a charged polymer coating (e.g., PLL). In other embodiments, the charged polymer coating may be applied by photochemically induced polymerization and grafting of the desired coating. In addition, the detection surface 4161 may be modified using a combination of any suitable procedures to enhance the adhesion properties of the surface. For example, as shown in FIG. 18 (with reference to the detection surface 4161), any detection surface described herein (including the detection surface 4161) may be modified using a combination of corona treatment and PLL coating. The positive charge of the detection surface 4161 is shown by (+), which improves the adhesion properties to capture target particles (e.g., bacteria) therein.

[0123] In some embodiments, the detection surface 4161 may be modified to have a surface finish (or roughness) that is favorable for capturing target particles TP. The surface finish (or roughness) may also be selected to provide identification and / or marking characteristics that do not interfere with the imaging process. In this way, the detection surface and other portions of the cartridge may include markings to aid in the collection and evaluation of imaging data.

[0124] As shown, the detection angle θ is in a downward direction. In this manner, the detection surface 4161 is on the first member 4101 forming the cartridge 4100. Referring again to FIG. 11 , the outer surface 4171 of the first member 4101 further includes an angled surface 4172 that defines an imaging (or optical) path through the first member 4101 together with the detection surface 4161. The angle of the surface 4172 is the same as the detection angle θ. Similarly, the outer surface 4172 is parallel to the detection surface 4161.

[0125] Detection chamber 4163 (see FIG. 11 ) can have any suitable depth as disclosed herein. For example, in some embodiments, the depth (also referred to herein as thickness) of detection chamber 4163 can be 0.250 mm or 0.125 mm, or any value therebetween. In some embodiments, the depth of detection chamber 4163 can be the same as the depth of separation chamber 4143. In other embodiments, the depth of detection chamber 4163 can be different from the depth of separation chamber 4143.

[0126] 11 , the waste portion 4180 defines a waste chamber (or channel) 4183, which is bounded by a first surface 4181 (of the first member 4101) and a second surface 4182 (of the second member 4102). In some embodiments, the first surface 4181 of the waste portion 4180 and the second surface 4142 of the separation portion 4140 are coplanar. In other embodiments, the first surface 4181 of the waste portion 4180 is lower than the second surface 4142 of the separation portion 4140. The depth (also referred to herein as thickness) of the waste chamber 4183 is 0.250 mm or 0.125 mm, or any value therebetween.

[0127] 17, the size (i.e., surface area or volume) of the detection chamber 4163 and the size (i.e., surface area or volume) of the separation chamber 4143 can be selected so that the concentration of target particles captured on the detection surface 4161 is greater than the concentration of target particles in the initial sample. Similarly, the size (i.e., surface area or volume) of the detection chamber 4163 and the size (i.e., surface area or volume) of the separation chamber 4143 can be selected to produce "upward concentration" of target particles in the detection chamber 4163. For example, in some embodiments, the ratio of the volume of the separation chamber 4143 to the volume of the detection chamber 4163 is at least about 2.0. In other embodiments, the ratio of the volume of the separation chamber 4143 to the volume of the detection chamber 4163 is at least about 2.5. In addition, the volume of the chamber can promote a desired volume ratio of the initial sample to the density medium in the cartridge 4100. For example, in some embodiments, the ratio of the sample volume to the volume of the density medium is about 100:45. Specifically, for a sample volume of 100 microliters, the cartridge 4100 can contain approximately 45 microliters of density medium to ensure that the detection chamber 4163 is completely filled with density medium. The ratio of density medium to inlet sample can also produce "upward concentration" of target particles within the detection chamber 4163.

[0128] The cartridge 4100 including the first member 4101 and the second member 4102 may be constructed of any suitable material. For example, in some embodiments, the cartridge 4100 may be constructed of polymethyl methacrylate (PMMA). In other embodiments, the cartridge 4100 may be composed of cyclic olefin copolymers (COC), including such polymers manufactured by Topas® of Advanced Polymers. Moreover, any material forming the cartridge 4100 may include the surface modification of the detection surface 4161 described above. Figures 19 and 20 are graphs showing experimental data associated with surface treatment on the detection surface 4161 of the cartridge formed by PMMA and COC. As shown, there are many different possible methods for applying the charged coating to the detection surface 4161, and some of these methods are effective for PMMA and / or COC. However, surface treatment using nonionic surfactant F68 does not enhance the adhesion of bacteria to the detection surface.

[0129] In some embodiments, the cartridge 4100 may include a stain within the inlet reservoir 4122 or other portion of the cartridge. The stain is formulated to bind and enhance detection of target cells. In this manner, the stain can improve the specificity of detecting (e.g., imaging) target particles in a sample containing contaminants. Specifically, in the case where certain contaminants of target particles of similar size, density, and / or shape are also captured on the detection surface 4161, the use of a stain can allow these contaminants to be distinguished from the target particles. In some embodiments, the stain may include a DNA dye that is effective for both Gram-positive and Gram-negative organisms. Such a stain may include ethylenediaminetetraacetic acid (EDTA) to promote disruption of the cell wall of Gram-negative organisms, thereby promoting equalization of results. In some embodiments, the stain may include any suitable Syto™ nucleic acid stain, including those shown in FIG. 22, which is a graph of experimental data associated with various stains. In other embodiments, the stain may include a DAPI fluorescent stain or Hoechst 33258. Such stains can be used to label DNA within target cells (eg, bacterial particles) while leaving platelets (which do not contain DNA) unstained.

[0130] In some embodiments, the stain can be prepackaged in the cartridge 4100, and the stain can be mixed with the sample after the sample is introduced into the inlet reservoir 4122. For example, in some embodiments, the stain can be stored in a dry state (e.g., powdered or lyophilized) in the inlet reservoir, and can be reconstituted by the sample when the sample is added to the inlet reservoir. In other embodiments, the stain can be stored in the inlet reservoir in a liquid state, and the opening 4121 can be covered to prevent spillage during storage and transportation before use. In other embodiments, the cartridge 4100 may include one or more separate reagent reservoirs (not shown), and the stain can be contained in these reagent reservoirs. In use, the reagent reservoir can be placed in fluid communication with the inlet reservoir 4122 (e.g., via a valve, a pierced frangible seal, etc.) to allow the stain to mix with the biological sample.

[0131] In other embodiments, the stain may be included as part of a kit that includes cartridge 4100 (or any cartridge described herein) and all necessary items for completing any of the methods described herein. Such kits are described in more detail below.

[0132] In some embodiments, the cartridge 4100 may include a diluent in the inlet reservoir 4122 or in other parts of the cartridge. The diluent is formulated to improve the properties of the biological sample to facilitate detection according to the methods described herein. For example, in some embodiments, the diluent may include any of a defoamer, a wetting agent, a dispersant, or an emulsifier. In some embodiments, the diluent may include a poloxamer, such as F-68 Pluronic. In some embodiments, the poloxamer may include poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). In some embodiments, the diluent may include sodium azide. In some embodiments, the diluent may be pre-packaged in the cartridge 4100 and may be mixed with the sample after the sample is introduced into the inlet reservoir 4122. For example, in some embodiments, the diluent may be stored in the inlet reservoir in a liquid state, and the opening 4121 may be covered to prevent spillage during storage and transportation before use. In other embodiments, the cartridge 4100 may include one or more separate reagent reservoirs (not shown) in which the diluent may be contained. In use, the reagent reservoir can be placed in fluid communication with the inlet reservoir 4122 (eg, via a valve, a pierced frangible seal, etc.) to allow the diluent to mix with the biological sample.

[0133] In other embodiments, the diluent may be included as part of a kit that includes cartridge 4100 (or any cartridge described herein) and all necessary items for completing any method described herein. Such kits are described in more detail below.

[0134] Although described as including separate reagents and / or components, such as stains, diluents, and density media, in some embodiments, certain components and / or functions of one of these components may be performed by any other component. For example, in some embodiments, the density media may include a poloxamer, such as the types described above.

[0135] As described above with respect to cartridge 2100, the sample may include a first low-density portion and a second high-density portion. The first portion may include low-density contaminants, such as unbound dyes (stains), cell fragments, proteins, or other matrix components. The second portion may initially include target particles, as well as other high-density contaminants, such as red blood cells, white blood cells, crystals, or large protein aggregates. In use, the effective centrifugal force causes the target particles and high-density contaminants to migrate in a radially outward direction. The target particles will migrate until they contact the detection surface 4161. However, the low-density contaminants are retained in the separation portion 4140 (e.g., blocked by the density medium), and are therefore prevented from reaching the detection surface 4161.

[0136] 23 is a flow chart of method 10 for detecting the concentration of target cells in a sample according to an embodiment. In some embodiments, method 10 (and the cartridge described herein) can determine the concentration of target cells within a lower limit of 10^3 colony forming units (CFU) per milliliter (mL) and an upper limit of 10^9 CFU per milliliter (mL). In other embodiments, the lower limit is 10^3 colony forming units (CFU) per milliliter (mL). Although described in conjunction with cartridge 4100, method 10 can be performed using any of the cartridges, systems and / or components described herein. In addition, method 10 can be included as part of other methods for determining treatment regimens, etc. In some embodiments, the method can optionally include processing the original biological sample to produce a sample suitable for use in cartridge 4100. Specifically, method 10 can optionally include processing the original cultured blood sample at operation 12 to produce a sample suitable for input into the inlet reservoir 4122 of cartridge 4100. Examples of suitable sample preparation are depicted in FIGS. 24A and 24B , which show sample preparation methods 20 and 30 that include initial centrifugation.

[0137] The method may also optionally include mixing a stain with the sample at operation 13. The stain may be any of the stains described herein and is formulated to bind and enhance detection of target cells. As described above, in some embodiments, the stain may be stored in a cartridge and mixing may occur in the inlet reservoir after the sample is transported to the reservoir. In other embodiments, the stain may be stored separately from the cartridge and may be added to the sample outside the cartridge or after the sample is in the inlet reservoir.

[0138] The method includes delivering a sample to an inlet reservoir of a cartridge at operation 14. The cartridge may be similar to cartridge 4100 and include a separation portion and a detection portion. The separation portion includes a first surface and a second surface defining a separation chamber, which may be fluidly coupled to the inlet reservoir (e.g., via a passive valve or an active valve). The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidly coupled to the separation chamber, and the detection surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture target cells.

[0139] The cartridge is coupled to a rotating element of the instrument at 16. The instrument may be any of the instruments described herein. The instrument is then activated at operation 18 to cause the instrument to perform separation and detection functions. Specifically, after activation, at operation 18A, the instrument rotates the cartridge at a rotational speed sufficient to cause at least a portion of the sample to flow, be transported or otherwise move from the inlet reservoir through the separation chamber and into the detection chamber, and the target cells are captured on the detection surface. In some embodiments, the cartridge may include a density medium of the type described herein to facilitate separation of the sample to isolate the target cells.

[0140] The rotation speed and time can be any suitable time for the target cells to be captured by the detection surface. For example, in some embodiments, the rotation speed can be maintained for at least one minute, at least two minutes, at least three minutes, and at least five minutes. In some embodiments, the rotation speed can be at least 3000 RPM. In other embodiments, the rotation speed can be between about 3000 RPM and about 15,000 RPM. In other embodiments, the rotation speed can be between about 5000 RPM and about 9000 RPM. In other embodiments, the rotation speed can be between about 6000 RPM and about 7000 RPM.

[0141] At operation 18B, the instrument then receives a signal indicating the number of target cells captured on the detection surface via a detector of the instrument. The detector can be any suitable detector. For example, in some embodiments, the detector is an optical detector and the signal is an optical (optical) signal. At operation 18C, the instrument then determines the concentration of target cells in the sample based on the signal.

[0142] In some embodiments, the instrument may include a processor, memory, and other electronic components to perform any portion of method 10 (and any other method described herein). The processor of the instrument may be configured to run and / or execute application modules, processes, and / or functions associated with the instrument. For example, the processor may be configured to run and / or execute an image capture module that facilitates capturing and processing images generated during method 10. The processor may be, for example, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), a digital signal processor (DSP), etc. The processor may be configured to retrieve data from a storage device (not shown) and / or write data to a storage device.

[0143] The memory (not shown) can be, for example, a random access memory (RAM), a memory buffer, a hard drive, a database, an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), a read-only memory (ROM), flash memory, a hard disk, a floppy disk, cloud storage, etc. In some embodiments, the memory stores instructions to cause the processor to execute modules, processes, and / or functions associated with the instrument. For example, the memory can store instructions to cause the processor to execute an image capture module.

[0144] In some embodiments, any of the methods, cartridges, and kits for detecting target cell concentrations within a sample may be used as part of an assay to determine the sensitivity of target cells to a treatment regimen. For example, in some embodiments, any of the methods, cartridges, and kits for detecting target cell concentrations may be used as part of an antibiotic sensitivity test (AST). For example, FIG. 25 is a flow chart of a method 40 for determining the sensitivity of target cells to a treatment regimen according to an embodiment. The method includes delivering a first portion of the sample to a cartridge at operation 41. The cartridge may be similar to cartridge 4100 and include a separation portion and a detection portion. The separation portion includes a first surface and a second surface defining a separation chamber. The detection portion includes a detection surface that forms a boundary of the detection chamber. The detection chamber is fluidically connected to the separation chamber, and the detection surface and the first surface of the separation portion form a non-zero detection angle. The detection surface is configured to capture a plurality of target cells.

[0145] The cartridge is then coupled to the first instrument. At operation 42, the first instrument is activated to allow the instrument to perform a variety of functions. At operation 42A, the first instrument rotates the cartridge at a rotational speed sufficient to cause the first portion of the sample to flow, be transported or otherwise move through the separation chamber and into the detection chamber, and the target cells are captured on the detection surface. At operation 42B, the instrument then receives a signal indicating the number of target cells captured on the detection surface via a detector of the instrument. Finally, at operation 42C, the first instrument then determines the concentration of target cells in the sample based on the signal. The method also includes processing the second portion of the sample based on the concentration of target cells in the first portion of the sample at operation 43. The processing may include, for example, diluting the second portion of the sample to ensure that the concentration of the target cells is within the desired range. The second portion of the sample is then transported to the reaction chamber at 44. The method then includes activating the second instrument at operation 45 to perform a sensitivity test on the second portion of the sample to determine the sensitivity of the target cells to the treatment regimen.

[0146] In some embodiments, the kit includes a cartridge of the type shown and described herein, and other materials for performing any of the methods described herein. For example, in some embodiments, the kit may include a cartridge (e.g., cartridge 4100) and any of the reagents and / or density media described herein. In some embodiments, a predetermined amount of density media (e.g., an amount sufficient to fill the cartridge to a desired level without overfilling) may be packaged in a container separate from the cartridge. In some embodiments, the volume of the density media may be less than fifty percent of the volume of the cartridge. In some embodiments, a predetermined amount of diluent (e.g., an amount sufficient to fill the cartridge to a desired level without overfilling) and / or stain (e.g., an amount sufficient to produce the desired staining of the target particle) may be packaged in a container separate from the cartridge. In some embodiments, stains and diluents may be stored together in a single reagent container. In other embodiments, each composition (e.g., density media, diluent, stain) may be stored in a separate container. By packaging density media, reagents, and cartridges separately, the kit may extend shelf life. Additionally, if some tests require one density medium or reagent and other tests require a second, different density medium or a second, different reagent, packaging the density medium, reagents, and cartridges separately can allow the same cartridge to be used in different kits (each kit having different reagents, density medium, etc.).

[0147] In some embodiments, the test kit may include a sample transfer device (e.g., a pipette) and / or other items for sample processing. For example, in some embodiments, the test kit may include a pipette and a mixing or sample processing container. In use, the sample may be mixed with one or both of a diluent and a stain in a mixing container. The pipette may be used to transport a predetermined amount of a desired reagent from a storage container to a mixing container. The pipette may also be used to transfer the sample to a mixing container, and may also pump or mix the components. Finally, the pipette may transfer the mixture of the sample and the reagent from the mixing container to a cartridge.

[0148] In some embodiments, additional elements include a vapor resistant material, such as an adhesive foil, over the inlet hole(s). The cartridges may have an extended shelf life by storing them in a secondary sealed bag. For biosafety, the inlet hole(s) may be covered during rotation.

[0149] However, in other embodiments, the kit may include a cartridge pre-filled with a density medium and / or any of the reagents described herein.

[0150] FIG. 26A and FIG. 26B are graphs showing experimental data for cell concentration associated with the cartridges and methods described herein. These data show the correlation between reference samples and test results performed on a two-dimensional prototype cartridge (2D cartridge). The 2D cartridge includes a single parallel channel through which the sample is transported during a rotation cycle. The effect of an angled detection surface can be simulated in a 2D cartridge by orienting the cartridge at a non-zero angle relative to the radial axis. In these tests, the reference sample included bacteria (Staphylococcus aureus or Escherichia coli) spiked into a blood culture supernatant (containing blood and culture medium). The reference results were obtained by using a blood cell count. These results demonstrate the feasibility of the cartridges and methods described herein to determine cell concentrations with a lower limit of detection of about 10^5 CFU / mL and an upper limit of about 10^9 CFU / mL.

[0151] FIG. 27 is a graph showing experimental data for particle concentration associated with the cartridges and methods described herein. Specifically, FIG. 27 shows experimental data for particle concentration of different particles according to tests performed on cartridges of the type shown and described herein. Specifically, the test cartridges included a separation surface and a detection surface that formed a non-zero detection angle. Reference samples for the results shown in FIG. 27 included simulated beads (1 µm, fluorescent beads) for a lower concentration range and bacteria for a higher concentration range. These results demonstrate the feasibility of the cartridges and methods described herein to determine cell concentrations, with a lower limit of detection of approximately 10^3 CFU / mL and an upper limit of approximately 10^9 CFU / mL.

[0152] 28 is a graph showing experimental data for Staphylococcus aureus and E. coli concentrations associated with the cartridges and methods described herein.

[0153] Figures 29A and 29B are graphs showing experimental data of particle concentration associated with the cartridges and methods described herein. Specifically, Figure 29A shows the relevant curves for Gram-negative bacteria, including Acinetobacter baumannii (expressed as Abi), Escherichia coli (expressed as Eco), Klebsiella pneumoniae (denoted as Kpn) and Pseudomonas aeruginosa (expressed as Pae). Figure 29B shows the relevant curves for Gram-positive bacterial genera, including Enterococcus faecalis (denoted as Efs) and Staphylococcus epidermidis (denoted as Sep). These results were generated using an overnight culture procedure that involved inoculating the bacteria in a 1:4 mixture of whole blood and blood culture medium. The samples were incubated overnight at 37°C with shaking. The overnight cultures were then centrifuged at 3000 rpm for 10 minutes in a serum separator. The serum separator tube was then vortexed to resuspend the bacteria before transferring the sample to a cartridge (similar to Cartridge 4100).

[0154] 30A-30C are graphs showing experimental data for bilirubin (FIG. 30A), lipid (FIG. 30B), and hemoglobin (FIG. 30C) interference associated with the cartridges and methods described herein.

[0155] 31 is a perspective view of a cartridge according to an embodiment.

[0156] 32A and 32B are schematic diagrams of cartridges according to various embodiments.

[0157] Although various embodiments have been described above, it should be understood that these embodiments are exemplary only and not limiting. Where the above methods and / or schematics indicate certain events and / or flow patterns occurring in certain sequences, the order of certain events and / or operations may be modified. Although embodiments have been specifically shown and described, it should be understood that various changes in form and detail may be made.

[0158] Any of the cartridges described herein may be manufactured according to any suitable process. For example, in some embodiments, the top and bottom plates of the cartridge may be assembled using ultrasonic welding, adhesives, press fits, or by other means.

[0159] In some embodiments, any of the cartridges described herein may include removable or permanent seals, markings, disposal accessories, and the like.

[0160] Some embodiments include symmetrical cartridges, where both ends of the cartridge are functional, although not necessarily identical. For example, different ends of the cartridge may contain different density media, different dyes, different charged surfaces, etc. Embodiments also include cartridges with only a single functional end. For this, the other end may be viewed as primarily a counterweight. The balance of weights within the cartridge may be selected so that the cartridge is balanced before, during, or after rotation. In one embodiment, the cartridge is balanced after rotation.

[0161] Some embodiments include an instrument that uses the same motor to rotate the cartridge to also position the cartridge within the imaging system, so that the imaging system can image multiple different locations at the end of the cartridge. That is, during the imaging phase of the system, only a single motor is used to both rotate and position the cartridge.

[0162] Any of the methods described herein can be performed on any suitable biological sample. The biological sample can include body fluids, such as blood, urine, nasal swabs, vaginal swabs, etc. In some embodiments, the sample can be a blood culture medium.

[0163] Any of the methods described herein may be performed to detect any suitable target particle. In some embodiments, the target particle may include a cell. Furthermore, in some embodiments, the target cell may include a bacterial cell. Thus, the methods described herein may be used in conjunction with an assay to determine the sensitivity of a bacterial cell to treatment with a range of antibiotics. The target bacteria may include, for example, Enterobacter, Pseudomonas, Acinetobacter, Staphylococcus, Streptococcus or Enterococcus bacteria.

[0164] Some embodiments described herein relate to a computer storage product having a non-transitory computer-readable medium (also referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that the computer-readable medium (or processor-readable medium) itself does not include transient propagating signals (e.g., propagating electromagnetic waves that carry information on a transmission medium such as space or cables). The medium and computer code (also referred to as code) may be designed or constructed for one or more specific purposes. Examples of non-transitory computer-readable media include, but are not limited to: magnetic storage media such as hard disks, floppy disks, and magnetic tapes; optical storage media such as compact disks / digital video disks (CD / DVD), compact disk-read only memories (CD-ROMs), and holographic devices; magneto-optical storage media such as optical disks; carrier signal processing modules; and hardware devices specially configured to store and execute program code, such as application specific integrated circuits (ASICs), programmable logic devices (PLDs), read-only memories (ROMs), and random access memory (RAM) devices.

[0165] Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions (such as those generated by a compiler), code for generating Web services, and files containing higher-level instructions executed by a computer using an interpreter. For example, these embodiments may be implemented using an imperative programming language (e.g., C, Fortran, etc.), a functional programming language (Haskell, Erlang, etc.), a logical programming language (e.g., Prolog), an object-oriented programming language (e.g., Java, C++, etc.), or other suitable programming languages ​​and / or development tools. Other examples of computer code include, but are not limited to, control signals, encryption codes, and compression codes.

[0166] Although various embodiments have been described as having specific features and / or combinations of components, other embodiments may also have any features and / or combinations of components from any of the embodiments described above. These aspects have been described in the general context of determining the concentration of cells, particularly bacterial cells, but these aspects of the invention are not necessarily limited to use in molecular diagnostics, health services, and / or medical devices.

Claims

1. A kit for performing a method for determining the concentration of bacterial target cells in a sample, comprising: A cartridge configured to be removably coupled to an instrument configured to rotate the cartridge about an axis of rotation and detect a plurality of bacterial target cells within a sample, the cartridge comprising: an inlet portion defining an inlet reservoir configured to receive the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidly coupled to the inlet reservoir such that when the cartridge is rotated, at least a portion of the sample can reach the separation chamber from the inlet reservoir; and a detection portion, the detection portion defining a detection chamber, the detection portion including a detection surface forming a boundary of the detection chamber, the detection chamber being fluidly coupled to the separation chamber so that at least the plurality of bacterial target cells can pass through the separation chamber and enter the detection chamber, the detection surface being directly connected to the first surface of the separation portion to form a non-zero detection angle, the detection surface being configured to capture the plurality of bacterial target cells; A density medium formulated to separate bacterial target cells from contaminants, the density medium having a density between 1.01 g / cm 3 and 1.13 g / cm 3 between; and a stain formulated to bind to and enhance detection of the plurality of bacterial target cells, The first surface of the separation portion and the detection surface are integrally constructed, and the non-zero detection angle between the detection surface and the first surface of the separation portion is between 1 degree and 8 degrees.

2. The kit according to claim 1, characterized in that It further comprises a diluent.

3. The kit according to any one of claims 1 to 2, characterized in that The detection surface includes surface modifications to enhance adhesion of the plurality of bacterial target cells.

4. The kit according to any one of claims 1 to 2, characterized in that The detection angle is 2 degrees.

5. The kit according to claim 3, characterized in that The detection surface includes chemical modifications to enhance adhesion of the plurality of bacterial target cells.

6. The kit according to claim 3, characterized in that The surface modification includes a coating comprising a charged polymer.

7. The kit according to claim 1 or 2, characterized in that The density medium is stored in a pre-filled sealed container that is separate from the cartridge and has a volume that is less than fifty percent of the volume of the cartridge.

8. The kit according to claim 1 or 2, characterized in that The density medium comprises any one of a defoamer, a wetting agent, a dispersant or an emulsifier.

9. The kit according to claim 2, characterized in that The diluent is stored in a pre-filled container that is separate from the cartridge, and the diluent contains any one of a defoaming agent, a wetting agent, a dispersing agent, or an emulsifying agent.

10. A method for detecting the concentration of a plurality of bacterial target cells in a sample for non-diagnostic purposes, the method comprising: delivering the sample into an inlet reservoir of a cartridge, the cartridge further comprising a separation portion and a detection portion, the separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidly coupled to the inlet reservoir, the detection portion defining a detection chamber and comprising a detection surface forming a boundary of the detection chamber, the detection chamber being fluidly coupled to the separation chamber, the detection surface being directly connected to the first surface of the separation portion to form a non-zero detection angle, the detection surface being configured to capture the plurality of bacterial target cells; coupling the cartridge to a rotating element of an instrument; as well as Start the instrument so that it performs the following operations: The cartridge is rotated at a speed sufficient to cause a) at least a portion of the sample to be transported from the inlet reservoir through the separation chamber and into the detection chamber; and b) the plurality of bacterial target cells to be captured on the detection surface, wherein the cartridge contains a density medium in the separation chamber, the density medium being formulated to separate bacterial target cells from contaminants, the density medium having a density between 1.01 g / cm 3 and 1.13 g / cm 3 between; receiving, via a detector of the instrument, a signal indicative of a number of the plurality of bacterial target cells captured on the detection surface; and Based on the signal, determining that the concentration of the plurality of bacterial target cells in the sample is within a lower limit of 10^3 colony forming units (CFU) per milliliter (mL) and an upper limit of 10^9 CFU per mL, The first surface of the separation portion and the detection surface are integrally constructed, and the non-zero detection angle between the detection surface and the first surface of the separation portion is between 1 degree and 8 degrees.

11. The method according to claim 10, characterized in that The lower limit is 10^5 CFU per mL.

12. The method according to any one of claims 10 to 11, characterized in that The portion of the sample that is transported from the inlet reservoir through the separation chamber and into the detection chamber is the second portion; and the density of the density medium is greater than the density of the first portion of the sample and less than the density of the second portion of the sample, and after rotating the cartridge, the first portion of the sample is retained in the separation chamber.

13. The method according to any one of claims 10 to 11, characterized in that The portion of the sample that is transported from the inlet reservoir through the separation chamber and into the detection chamber is a second portion, the method further comprising: A density medium having a density greater than that of a first portion of the sample and less than that of a second portion of the sample is delivered to the cartridge before the sample is delivered to the inlet reservoir, and after rotating the cartridge, the first portion of the sample is retained within the separation chamber.

14. The method according to claim 13, characterized in that It further includes: A diluent is delivered into the cartridge, at least one of the diluent or the density medium comprising any of a defoaming agent, a wetting agent, a dispersing agent, or an emulsifier.

15. The method according to claim 14, characterized in that Delivering the diluent includes mixing the diluent with the sample and delivering the diluent and the sample to the inlet reservoir of the cartridge in the same operation.

16. The method according to claim 14, characterized in that The diluent is delivered into the cartridge together with the density medium, at least one of the diluent or the density medium comprising any one of a defoaming agent, a wetting agent, a dispersing agent or an emulsifier.

17. The method according to claim 12, characterized in that The density of the density medium is between 1.05 g / cm 3 and 1.09 g / cm 3 between.

18. The method according to any one of claims 14 to 16, characterized in that At least one of the diluent or the density medium comprises a poloxamer.

19. The method according to any one of claims 10 to 11, characterized in that The sample is a body fluid.

20. The method according to claim 19, characterized in that The sample is any one of a blood sample or a urine sample.

21. The method according to claim 20, characterized in that The blood sample is a cultured blood sample; and the plurality of bacterial target cells are a plurality of bacterial cells.

22. The method according to claim 21, characterized in that The plurality of bacterial cells include any one of the genera Enterobacteriaceae, Pseudomonas, Acinetobacter, Staphylococcus, Streptococcus, or Enterococcus.

23. The method according to any one of claims 21 to 22, characterized in that It further includes: Prior to delivering the cultured blood sample into the inlet reservoir of the cartridge, an original cultured blood sample is processed to produce the cultured blood sample.

24. The method according to any one of claims 10 to 11, characterized in that It further includes: A stain is mixed with the sample, the stain formulated to bind to and enhance detection of the plurality of bacterial target cells.

25. The method according to claim 24, characterized in that The stain is a fluorescent stain formulated to stain the plurality of bacterial target cells.

26. The method according to any one of claims 10 to 11, characterized in that The instrument rotates the cartridge at a rotation speed of between 3000 rpm and 15,000 rpm for a period of at least 1 minute.

27. The method according to claim 26, characterized in that The rotation speed is between 6000 rpm and 7000 rpm.

28. A method for detecting the concentration of a plurality of bacterial target cells in a sample, the method comprising: transferring a density medium from a container into a cartridge, the density medium being formulated to separate bacterial target cells from contaminants, the cartridge comprising an inlet portion, a separation portion, and a detection portion, the inlet portion defining an inlet reservoir, the separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being fluidly coupled to the inlet reservoir, the detection portion comprising a detection surface forming a boundary of a detection chamber, the detection chamber being fluidly coupled to the separation chamber, the detection surface being directly coupled to the first surface of the separation portion to form a non-zero detection angle, the detection surface being configured to capture the plurality of bacterial target cells; The transferring is performed such that the density medium is contained within the separation chamber, the density of the density medium is greater than the density of the first portion of the sample and less than the density of the second portion of the sample and the density of the density medium is between 1.01 g / cm 3 and 1.13 g / cm 3 The second portion of the sample comprises the plurality of bacterial target cells; delivering the sample into the inlet reservoir; Rotating the cartridge within the instrument at a speed and for a duration sufficient to cause a) a portion of the sample to pass from the inlet reservoir through the density medium in the separation chamber and into the detection chamber, the portion being the second portion of the sample; and a remainder of the sample to be retained within the separation chamber, the remainder being the first portion of the sample, and b) the plurality of bacterial target cells to be captured on the detection surface; receiving, via a detector of the instrument, a signal indicative of a number of the plurality of bacterial target cells captured on the detection surface; as well as determining the concentration of the plurality of bacterial target cells in the sample based on the signal, The first surface of the separation portion and the detection surface are integrally constructed, and the non-zero detection angle between the detection surface and the first surface of the separation portion is between 1 degree and 8 degrees.

29. The method according to claim 28, characterized in that The instrument determines the concentration of the plurality of bacterial target cells to be within a lower limit of 10^3 colony forming units (CFU) per milliliter (mL) and an upper limit of 10^9 CFU per mL.

30. The method according to any one of claims 28 to 29, characterized in that The container and the cartridge are packaged together in a kit.

31. The method according to any one of claims 28 to 29, characterized in that It further includes: A diluent is delivered into the cartridge, at least one of the diluent or the density medium comprising any of a defoaming agent, a wetting agent, a dispersing agent, or an emulsifier.

32. The method according to claim 31, characterized in that At least one of the diluent or the density medium comprises a poloxamer.

33. The method according to claim 31, characterized in that The delivering the diluent includes mixing the diluent with the sample and delivering the diluent and the sample to the inlet reservoir of the cartridge in the same operation.

34. An apparatus comprising: A cartridge configured to be removably coupled to a rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of bacterial target cells within a sample, the cartridge comprising: an inlet portion defining an inlet reservoir configured to receive the sample; A separation section comprising a first surface and a second surface defining a separation chamber, the separation section being configured to contain a density medium, the density medium being configured to separate bacterial target cells from contaminants and having a density greater than a density of the first portion of the sample and less than a density of the second portion of the sample and having a density between 1.01 g / cm 3 and 1.13 g / cm 3 wherein the second portion includes the plurality of bacterial target cells, the separation chamber being configured to be fluidly coupled to the inlet reservoir such that when the cartridge is rotated, at least the second portion of the sample can reach the separation chamber from the inlet reservoir; and a detection portion, the detection portion comprising a detection surface forming a boundary of a detection chamber, the detection chamber being fluidly coupled to the separation chamber so that after rotating the cartridge, the second portion of the sample can pass through the density medium and enter the detection chamber and the first portion of the sample is retained in the separation chamber, the detection surface being directly connected to and non-parallel to at least one of the first surface or the second surface so that the plurality of bacterial target cells impinge on the detection surface when entering the detection chamber, the detection surface being configured to capture the plurality of bacterial target cells, The detection surface and the directly connected first surface or the second surface are integrally constructed, and a detection angle between the detection surface and the directly connected first surface or the second surface is between 1 degree and 8 degrees.

35. An apparatus comprising: A cartridge configured to be removably coupled to a rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of bacterial target cells within a sample, the cartridge comprising: an inlet portion defining an inlet reservoir configured to receive the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidly coupled to the inlet reservoir such that when the cartridge is rotated, at least a portion of the sample can be transferred from the inlet reservoir to the separation chamber; and a detection portion defining a detection chamber, the detection portion including a detection surface forming a boundary of the detection chamber, the detection chamber being fluidly coupled to the separation chamber so that at least the plurality of bacterial target cells can be transported through the separation chamber and into the detection chamber, the detection surface being directly coupled to the first surface of the separation portion to form a non-zero detection angle, the detection surface being configured to capture the plurality of bacterial target cells, the ratio of the volume of the separation chamber to the volume of the detection chamber being at least 2.0, wherein the first surface of the separation portion and the detection surface are integrally constructed, wherein the non-zero detection angle between the detection surface and the first surface of the separation portion is between 1 degree and 8 degrees, The cartridge contains a density medium in the separation chamber, wherein the density medium is formulated to separate bacterial target cells from contaminants, and the density of the density medium is between 1.01 g / cm 3 and 1.13 g / cm 3 between.

36. An apparatus comprising: A cartridge configured to be removably coupled to a rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of bacterial target cells within a sample, the cartridge comprising: an inlet portion defining an inlet reservoir configured to receive the sample; A separation section comprising a first surface and a second surface defining a separation chamber, the separation section being configured to contain a density medium, the density medium being configured to separate bacterial target cells from contaminants and having a density greater than a density of the first portion of the sample and less than a density of the second portion of the sample and having a density between 1.01 g / cm 3 and 1.13 g / cm 3 wherein the second portion includes the plurality of bacterial target cells, the separation chamber being configured to be fluidly coupled to the inlet reservoir such that when the cartridge is rotated, at least the second portion of the sample can reach the separation chamber from the inlet reservoir; and a detection portion, the detection portion comprising a detection surface forming a boundary of a detection chamber, the detection chamber being fluidly coupled to the separation chamber such that upon rotating the cartridge, the second portion of the sample can pass through the density medium and enter the detection chamber and the first portion of the sample is retained within the separation chamber, wherein the detection surface of the detection portion is directly connected to the first surface of the separation portion to form a non-zero detection angle such that the plurality of bacterial target cells impinge on the detection surface upon entering the detection chamber, the detection surface being configured to capture the plurality of bacterial target cells; and a diluent, wherein at least one of the diluent or the density medium comprises any one of a defoamer, a wetting agent, a dispersant or an emulsifier, The first surface of the separation portion and the detection surface are integrally constructed, and the non-zero detection angle between the detection surface and the first surface of the separation portion is between 1 degree and 8 degrees.

37. The device according to claim 36, characterized in that The detection angle is 2 degrees.

38. The device according to any one of claims 36 to 37, characterized in that The cartridge defines a radial axis that intersects the rotational axis; and the detection angle is along the radial axis and within a cross-sectional plane defined by the radial axis and the rotational axis.

39. The device according to any one of claims 36 to 37, characterized in that The inlet portion defines an opening through which the sample can be delivered into the inlet reservoir; and the detection surface is angled in a downward direction relative to the opening.

40. The device according to any one of claims 36 to 37, characterized in that The ratio of the volume of the separation chamber to the volume of the detection chamber is at least 2.

0.

41. The device according to claim 40, characterized in that The ratio of the volume of the separation chamber to the volume of the detection chamber is at least 2.

5.

42. The device according to any one of claims 36 to 37, characterized in that A distance between the first surface of the separation portion and the second surface of the separation portion defines a thickness of the separation chamber, which is less than 0.6 mm.

43. The device according to claim 42, characterized in that The thickness of the separation chamber is less than 0.3 mm.

44. The device according to claim 36, characterized in that The density of the density medium is between 1.05 g / cm 3 and 1.09 g / cm 3 between.

45. The device according to claim 36, characterized in that The separation portion defines a density medium reservoir, and the density medium is contained within the density medium reservoir.

46. ​​The device according to any one of claims 36 to 37, characterized in that At least one of the diluent or the density medium comprises a poloxamer.

47. The device according to claim 46, characterized in that The poloxamers include poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO).

48. The device according to any one of claims 36 to 37, characterized in that It further comprises a stain formulated to bind to and enhance detection of the plurality of bacterial target cells.

49. The device according to any one of claims 36 to 37, characterized in that The detection surface includes surface modifications to enhance adhesion of the plurality of bacterial target cells.

50. The device according to claim 49, characterized in that The surface modification includes a coating comprising a charged polymer.

51. The device according to claim 50, characterized in that The coating is a + ) charged poly-L-lysine.

52. An apparatus comprising: A cartridge configured to be removably coupled to a rotating element configured to rotate the cartridge about a rotation axis to capture a plurality of bacterial target cells within a sample, the cartridge comprising: an inlet portion defining an inlet reservoir configured to receive the sample; a separation portion comprising a first surface and a second surface defining a separation chamber, the separation chamber being configured to be fluidly coupled to the inlet reservoir such that when the cartridge is rotated, at least a portion of the sample can be transferred from the inlet reservoir to the separation chamber; and a detection portion defining a detection chamber, the detection portion including a detection surface forming a boundary of the detection chamber, the detection chamber being fluidly coupled to the separation chamber so that at least the plurality of bacterial target cells can be transported through the separation chamber and into the detection chamber, the detection surface being directly coupled to the first surface of the separation portion to form a non-zero detection angle, the detection surface including a surface modification to enhance adhesion of the plurality of bacterial target cells, the ratio of the volume of the separation chamber to the volume of the detection chamber being at least 2.0, wherein the first surface of the separation portion and the detection surface are integrally constructed, wherein the non-zero detection angle between the detection surface and the first surface of the separation portion is between 1 degree and 8 degrees, The cartridge contains a density medium in the separation chamber, wherein the density medium is formulated to separate bacterial target cells from contaminants, and the density of the density medium is between 1.01 g / cm 3 and 1.13 g / cm 3 between.

53. The device according to claim 52, characterized in that The cartridge is configured to focus the plurality of bacterial target cells captured on the detection surface.

54. The device according to any one of claims 52 to 53, characterized in that The ratio of the volume of the separation chamber to the volume of the detection chamber is between 2.0 and 5.

0.

55. The device according to claim 54, characterized in that The ratio of the volume of the separation chamber to the volume of the detection chamber is 2.

5.

56. The device according to any one of claims 52 to 53, characterized in that: The detection surface includes surface modifications to enhance adhesion of the plurality of bacterial target cells.

57. The device according to claim 56, characterized in that: The detection angle is 2 degrees.

58. The device according to claim 57, characterized in that: A transition zone between the first surface of the separating portion and the detection surface defines a maximum radius of curvature of less than 0.1 mm.

59. The device according to any one of claims 52 to 53, characterized in that A distance between the first surface of the separation portion and the second surface of the separation portion defines a thickness of the separation chamber, which is less than 0.6 mm.

60. The device according to claim 56, characterized in that The surface modification includes a coating comprising a charged polymer.

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