Methods and devices for biological sample preparation and microscopic analysis

Abstract

A method of analyzing a sample fluid is disclosed. The method comprises providing a disposable cartridge having a receiving inlet and an analyzing chamber, receiving a volume of a fluid having one or more components of a sample fluid and a buffer in the receiving inlet of the disposable cartridge; tagging the fluid moving through the disposable cartridge with dried tagging reagents disposed along the sample pathway; and forming a monolayer of the tagged fluid in the analyzing chamber for microscopic analysis. The disposable cartridge moves the sample fluid through a sample pathway between the receiving inlet and the analyzing chamber by a rotating motor.

Description

TITLE

[0001] Methods and Devices for Biological Sample Preparation and Microscopic AnalysisCROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U. S. Provisional Patent Application No. 63 / 514,978 filed July 21, 2023 entitled “Methods and Systems for Biological Sample Preparation and Microscopic Analysis”, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] The present disclosure generally relates to devices and methods for handling biological samples and, in some embodiments, to devices and methods for handling biological samples using microfluidics and processing and quantifying the sample characteristics using microscopic analysis.SUMMARY

[0004] A method for biological sample preparation and microscopic analysis may be disclosed herein. In certain embodiments, the method comprises providing a disposable cartridge comprising a receiving inlet and an analyzing chamber, the disposable cartridge configured to move a sample fluid through a sample pathway between the receiving inlet and the analyzing chamber, receiving a volume of a fluid having one or more components of the sample fluid and a buffer in the receiving inlet of the disposable cartridge, tagging the fluid moving through the disposable cartridge with dried tagging reagents disposed along the sample pathway; and forming a monolayer of the tagged fluid in the analyzing chamber for microscopic analysis. In certain embodiments, the disposable cartridge moves the fluid from the receiving inlet to the analyzing chamber by a rotating motor.

[0005] In certain embodiments, the fluid is moved between the receiving inlet and the analyzing chamber by centrifugal and inertial forces generated by the rotating motor. In certain embodiments, the disposable cartridge is a disk comprising at least one testing chamber, the testing chamber testing one or more of white blood cells, red blood cells, hemoglobin, platelets and / or hematocrit in the sample fluid. In certain embodiments, the test determines one or more of a number of cells, a density or concentration of small molecules, and morphological features of a cell such as its granularity, size,circularity, nuclei size, shape, and nucleic concentrations. In certain embodiments, the fluid comprises the sample fluid and the buffer, and in certain embodiments a volume of the buffer and a volume of the sample fluid are received in a buffer chamber and sample chamber, respectively, of each testing chamber.

[0006] In certain embodiments, the testing chamber configured to test white blood cells and platelets receives about 120 pL of the buffer into the buffer chamber through a buffer inlet and about 60 pL of the sample fluid into the sample chamber through a sample inlet. In certain embodiments, the testing chamber configured to test red blood cells receives about 300 pL of the buffer into the buffer chamber through a buffer inlet and about 15 pL of the sample fluid into the sample chamber through a sample inlet. In certain embodiments, the testing chamber configured to test hemoglobin receives about 190 pL of the buffer into the buffer chamber through a buffer inlet and about 35 pL of the sample fluid into the sample chamber through a sample inlet. In certain embodiments, the testing chamber configured to test hematocrit receives about 25 uL of the buffer into the buffer chamber through a buffer inlet and about 25 uL of the sample fluid into the sample chamber through a sample inlet.

[0007] In certain embodiments, the disk is rotated in a first direction at a rotational speed of about 400 rpm to meter the volume of the sample fluid and the buffer. In certain embodiments, the testing chamber configured to test white blood cells contains about 120 pL of the buffer and about 24 pL of the sample fluid after metering. In certain embodiments, the testing chamber configured to test red blood cells contains about 200 pL of the buffer and about 2 pL of the sample fluid after metering. In certain embodiments, the testing chamber configured to test hemoglobin contains about 130 pL of the buffer and about 8.5 pL of the sample fluid after metering.

[0008] In certain embodiments, an excess amount of the sample fluid and the buffer flows into an overflow chamber. In certain embodiments, rotation of the disk in a first direction causes the sample fluid and the buffer to flow into a mixing chamber. In certain embodiments, the disk is rotated in the first direction at a rotational speed of about 600 rpm to cause the sample fluid to flow into the mixing chamber. In certain embodiments, the sample fluid flows into the mixing chamber at approximately the same time in each testing chamber. In certain embodiments, the disk is rotated in the first direction at a rotational speed of about 700 rpm to cause the buffer to flow into the mixing chamber. In certain embodiments, the mixing chamber contains at least one reagent.

[0009] In certain embodiments, the at least one reagent comprises one or more of lyo bead, ACO bead, and / or CholOxidase bead. In certain embodiments, the reagent is present in liquid state. Incertain embodiments, the reagent is present in the form of a coating on an inner surface of the mixing chamber. In certain embodiments, the disk is rotated in the first direction and then suddenly stopped to mix a first reagent of the at least one reagent into the sample to deplete a first target content of the sample. In certain embodiments, the disk is rotated in the first direction and then suddenly stopped to mix a second reagent of the at least one reagent into the sample to deplete a second target content of the sample. In certain embodiments, the disk is rotated in the first direction at a rotational speed of about 600 rpm and then suddenly stopped to mix the sample fluid and the buffer within the mixing chamber.

[0010] In certain embodiments, mixing of the sample fluid and the buffer hydrates and reconstitutes the reagent in the mixing chamber. In certain embodiments, reconstitution of the reagent causes the sample fluid to become translucent. In certain embodiments, reconstitution of the reagent depletes small molecules, analytes or interferents present in the sample fluid. In certain embodiments, reconstitution of the reagent causes red blood cells in the sample to lyse. In certain embodiments, reconstitution of the reagent causes white blood cells in the sample to be tagged. In certain embodiments, reconstitution of the reagent causes platelets in the sample to be tagged. In certain embodiments, reconstitution and mixing causes the red blood cells to become spherical in shape.

[0011] In certain embodiments, rotation of the disk in a second direction causes the sample fluid and buffer to flow from the mixing chamber into the analyzing chamber. In certain embodiments, the disk is rotated to position, in succession, the analyzing chamber proximate an imager for imaging. In certain embodiments, the imaging content is analyzed to identify at least one a number of cells, a density or concentration of small molecules, morphological features, fluorescence signatures, scatter signatures and absorbance signatures. In certain embodiments, the cells are grouped based on the identified properties. In certain embodiments, rotation of the disk in a second direction at a rotational speed of about 7,000 rpm separates a plasma from the sample fluid. In certain embodiments, the disk is rotated to position the plasma in the analyzing chamber proximate the imager for imaging.

[0012] A device for biological sample preparation and microscopic analysis may be disclosed herein. In certain embodiments, the device comprises a base; a spindle rotatably coupled to the base; a motor configured to rotate a disk disposed on the spindle, the disk configured to receive a volume of a fluid comprising one or more components of the sample fluid; an imager coupled to the base on a first side relative to the disk and configured to collect image data of the sample fluid in the disk; a lens moveably coupled to the base on the first side relative to the disk, the lens configured to focus an optical lens disposed therein relative to the disk as the imager collects image data; an illumination system coupledto the base, the illumination system emitting light on the disk at different wavelength and angles; at least one disk guide coupled to the base, the disk guide configured to engage an edge of the disk to minimize movement of the disk relative to the lens; and a controller configured to control each of the spindle, imager, lens, illumination ring and disk guide.

[0013] In certain embodiments, the disk has one or more wells at a first common radius relative to a rotation axis of the disk, and wherein each of the one or more wells is configured to receive a volume of a fluid including one or more components of the sample fluid. In certain embodiments, the device further comprising: a light source coupled to the base on a second side relative to the disk and configured to illuminate the sample fluid in the disk. In certain embodiments, the light source is movable between an engaged position and a disengaged position.

[0014] In certain embodiments, the light source illuminates the sample fluid in perpendicular and oblique angles, thereby enabling measurement of direct absorption, fluorescence measurement as well as scattering analysis of the sample fluid in the disk. In certain embodiments, the optical lens may flex relative to the imager to focus the disk on the imager when the light source is in the engaged position. In certain embodiments, the device further comprising: an imaging filter having a plurality of optical components disposed thereon, the imaging filter being moveable relative to the imager to align an optical component between the imager and the sample fluid in the disc.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following detailed description of embodiments of the device will be better understood when read in conjunction with the appended drawings of an exemplary method and device for biological sample preparation and microscopic analysis. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0016] In the drawings:

[0017] Fig. 1 is a top perspective view of a device for biological sample preparation and microscopic analysis in accordance with an exemplary embodiment of the present invention;

[0018] Fig. 2 is an exploded view of the device of Fig. 1;

[0019] Fig. 3 is a top perspective view of the device of Fig. 1, showing the light source in a disengaged position;

[0020] Fig. 4 is a top view of a disk of the device of Fig. 1;

[0021] Fig. 5 is a top view of a disk of the device of Fig. 1, showing a sample and a buffer being received in the disk;

[0022] Fig. 6 is a top view of a disk of the device of Fig. 1, showing a sample and a buffer being metered in the disk;

[0023] Fig. 7 is a top view of a disk of the device of Fig. 1, showing a sample and a buffer being received in a mixing chamber of the disk;

[0024] Fig. 8 is a top view of a disk of the device of Fig. 1, showing a sample and a buffer being mixed in the mixing chamber of the disk;

[0025] Fig. 9 is a top view of a disk of the device of Fig. 1, showing a mixed fluid being received in one or more wells of the disk;

[0026] Fig. 10 is a view of the analyzed sample measuring each component fluorescence level against its scattering levels;

[0027] Fig. 11 is a cross-section view of the lens of the device of Fig. 1, showing scatter light originating from a cell passing through the lens;

[0028] Fig. 12 is a cross-section view of the lens of the device of Fig. 1;

[0029] Fig. 13 is a top perspective view of a spindle of the device of Fig. 1;

[0030] Fig. 14 is a side cross-section view of the spindle of the device of Fig. 1; and

[0031] Fig. 15 is a perspective view of a filter wheel of the device of Fig. 1.DETAILED DESCRIPTION

[0032] Currently, about 70% of all medical decisions rely on lab based diagnostics. However, as it stands today, the diagnostic process is disjointed from how care is delivered. The primary care system requires patients to travel to external phlebotomy sites to draw blood, which is sent to labs via courier, and subsequently processed. This means that lab results reach health care professionals long after the patient has left. This friction in care delivery and disease management leads to tremendous waste in the healthcare system, including: (i) patients delay getting lab tests or fail to adhere to lab testing, or subsequent care recommendations; (ii) the gap in the diagnostic process leads to missed tests, missed diagnosis, a lack of intervention, and ultimately poor outcomes; and (iii) healthcare professionalswaste time tracing lab orders to patient encounter notes. When intervention is needed, more time is wasted in reaching out to patients and driving subsequent steps in the patient’s care pathway.

[0033] These problems are even more acute when caring for rural populations or patients belonging to groups facing adverse social-determinants of health, where there are many challenges in ensuring successful follow ups from an initial patient encounter.

[0034] Several point-of-care instruments have been developed to bridge this divide. However, these instruments are limited to single types of tests and fail to completely meet the workflow needs of primary care providers. As such, there is a need for a single system that produces simple, comprehensive, and fast test results. This is achieved through a highly automated workflow and through the use of centrifugal microfluidics disks.

[0035] Centrifugal microfluidics are used in clinical chemistry, immunoassays, hematology, medicine, biomedical research and other fields. These applications often require metering, transferring, mixing fluids and / or other processes. Many of these applications also require detection of concentrations and reactions. However, achieving effective control on the metering, transferring and mixing of the fluids and precise measurement of concentrations and reactions can be a challenge in microfluidics, because the behavior of liquids can be significantly different from their bulk counterparts due to the small scales involved. Accordingly, there remains a need for improved devices and methods in centrifugal microfluidics to address these and other needs in the art.

[0036] Referring to the drawings in detail, wherein like reference numerals indicate like elements throughout, there is shown in Figs. 1-15 a device, generally designated 10, in accordance with an exemplary embodiment of the present invention. As discussed in more detail below, the device 10 may effectively control the metering, transferring and mixing of a fluid to generate efficient and accurate test results.

[0037] As shown in Figs. 1-3, the device 10 may be used for analyzing a sample fluid. The device 10 may include a base 12 to which one or more features may be coupled. The base 12 may be a generally rectangular shape. The base 12 may include a plurality of holes extending therethrough to receive additional components, as described in more detail below. The base 12 may be formed from a metal or a plastic with sufficient resiliency as to withstand movement of components relative to the base 12 during use. Movement of the components will be described in more detail below.

[0038] The base 12 may be coupled to a plate 88. The plate 88 may be configured to couple the device 10 to a separate device or structure. The plate 88 may include a number of apertures extending therethrough to receive hardware therethrough to couple the device 10 to another device or structure.

[0039] As shown in Figs. 1-3, the device 10 may include a spindle 14 rotatably coupled to the base 12. The spindle 14 may extend from the base 12. The disk 18, described below in more detail, may include an aperture 36 extending therethrough. The spindle 14 may extend through the aperture 36 thereby coupling the disk 18 to the base 12.

[0040] As shown in Figs. 1-3, the device 10 may include a motor 16 configured to rotate a disk 18 disposed on the spindle 14. The motor 16 may be a brushless direct current (DC) motor. In one embodiment, the motor 16 is driven by a sub-motor 30. The sub-motor 30 may be a precision stepper motor. The motor 16 may rotate the disk 18 at rotational speeds between 100-7000 rpm.

[0041] The motor 16 may rotate the disk 18 at a speed of at least about 100 revolutions per minute (rpm). The motor 16 may rotate the disk 18 at a speed of at least about 150 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 200 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 250 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 300 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 350 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 400 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 450 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 500 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 600 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 700 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 800 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 900 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 1000 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 1200 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 1400 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 1600 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 1800 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 2000 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 2200 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 2400 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 2600 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 2800 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 2900 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 3000 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 3500 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 4000 rpm. The motor 16 may rotate the disk 18 at a speed of at least about4500 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 5000 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 5500 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 6000 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 6500 rpm. The motor 16 may rotate the disk 18 at a speed of at least about 7000 rpm.

[0042] As shown in Figs. 4-9, a disposable cartridge may be configured to receive a volume of a fluid comprising one or more components of the sample fluid. The disposable cartridge may be a disk 18. The disk 18 may comprise a plurality of wells 32. Each of the plurality of wells 32 may be configured to hold at least one of white blood cells (WBC), red blood cells (RBC), hemoglobin, platelets and / or hematocrit.

[0043] During a detection period, as described below, the motor 16 may rotate the disk 18 to move a well 32 of the one or more wells 32 proximate the imager 20 such that the lens 22 is between the well 32 and the imager 20. In some embodiment, the sub-motor 30 drives the motor 16 to rotate the disk 18 to move a well 32 of the one or more wells 32 proximate the imager 20 such that the lens 22 is between the well 32 and the imager 20. During a subsequent detection period, the motor 16 may rotate the disk 18 to move another well 32 of the one or more wells 32 proximate the imager 20 such that the lens 22 is between the well 32 and the imager 20. The well 32 holding the red blood cells, white blood cells, platelets, hemoglobin, or hematocrit may be imaged by the imager 20 when the lens 22 is between such well 32 and the imager 20. The well 32 holding the hemoglobin may be imaged by the imager 20 first. The well 32 holding the red blood cells may be imaged by the imager 20 second. The well 32 holding the white blood cells and platelets may be imaged by the imager 20 third. The order of imaging of the red blood cells, white blood cells, platelets, hemoglobin, or hematocrit may vary depending on the specific tests and measurements being performed. In some embodiments, all of the wells 32 are imaged. In some embodiments, only a subset of the wells 32 are imaged.

[0044] The sample fluid may comprise one or more of blood, sera, plasma, bone marrow, lymph, saliva, sputum, mucus, respiratory or nasal secretion, oropharyngeal swab, nasopharyngeal swab, oral swab, ductal lavage, bronchoalveolar lavage, cerebrospinal fluid, skin swab, vaginal swab, gastric juice, ascites, peritoneal fluid, pleural fluid, gynecological fluids, pus, perspiration, tears, urine, and stool, or a combination thereof. In one embodiment, the sample fluid comprises blood. In one embodiment, one or more components of the sample fluid comprise K-EDTA anticoagulated whole blood.

[0045] As shown in Figs. 1-3, the device 10 may include an imager 20 coupled to the base 12. The imager 20 may be fixed relative to the base 12. The imager may be spaced apart from the axis AD. The imager 20 may be located on a first side relative to the disk 18. The imager 20 may be configured to collect image data of the sample fluid in the disk 18. The imager 20 may only collect image data when the light source 26 is in an engaged position, as described below in more detail. The imager 20 may perform high-resolution fluorescent microscopy, brightfield imaging, darkfield scattering, red fluorescence, and / or green fluorescence on the sample fluid. The imager 20 may be based on a complementary metal oxide semiconductor (CMOS) or charge-coupled device (CCD) sensor. The imager 20 may be a monochromatic or color sensor.

[0046] The imager 20 may capture up to 90 fields of view at each well of the one or more wells 32. The imager 20 may capture at least one of brightfield images, darkfield scattering, red fluorescence, and green fluorescence in each field of view. The imager 20 may collect approximately 30 fields of view during collection of image data for the well 32 holding red blood cells. The imager 20 may collect approximately 60 fields of view during collection of image data for the well 32 holding white blood cells. The imager 20 may collect approximately 8 fields of view during collection of image data for the well 32 holding platelets. The imager 20 may collect approximately 10 fields of view during collection of image data for the well 32 holding hemoglobin.

[0047] As shown in Figs. 1-3, the device 10 may include a lens 22 moveably coupled to the base 12. The lens 22 may be located on the first side relative to the disk 18. The lens 22 may be configured to focus relative to the disk 18 as the imager 20 collects image data. The lens 22 may be in line with the imager 20 along the axis Ai. The lens 22 may be considered an objective lens because it may react to movement of the disk 18.

[0048] As shown in Figs. 11-12, the lens 22 may flex relative to the imager 20 to focus the imager 20 on the disk 18 when the light source 26 is in the engaged position. The lens 22 may refract the light traveling therethrough to relay the light from the disk 18 to the imager 20. The lens 22 may include a plurality of optical lenses 23. Each optical lens 23 may have a generally concave or convex shape. The optical lenses 23 may be disposed within the lens 22 in a layered configuration such that a portion of each optical lens 23 fits at least partially within the adjacent optical lens 23. The lens 22 and / or optical lenses 23 may be moved or flexed relative to the imager 20 by an actuating mechanism 25. The actuating mechanism 25 may include, for example, a piezoelectric, mechanical gear-coupled, or voice-coil motor (Fig. 12). The actuating mechanism 25 may be a torque-based system configured toactively flex and align the optical lenses 23 within the lens 22. The actuating mechanism 25 may be driven by the sub-motor 30 or a separate sub-motor (not shown).

[0049] The actuating mechanism 25 may utilize a lever system for precise adjustment of the lens 22. The actuating mechanism 25 may include a lever with one end fixed to a base plate via a hinge, allowing pivotal movement. The opposite end of the lever may include a threaded hole configured to receive a lead screw, which is driven by the sub-motor 30. As the sub-motor 30 rotates the lead screw, it causes the end of the lever to move relative to the base plate.

[0050] The lens 22 may be mounted on a lens holder that sits within a flexture 29 positioned between the hinge and the lead screw above the lever. The flexture 29 may be configured to stabilize the lens holder and reduce tilt in the lens 22. The lens holder may be directly coupled to the lever. As the lever is actuated by the sub-motor 30 and lead screw, the lens holder and lens 22 may move correspondingly to achieve focus. The range of the lens 22 may be determined by the length of the lever, the pitch of the lead screw, and the step size of the sub-motor 30. A limit switch may be incorporated to home the focusing mechanism, providing a reference position for the system. Both the motor and the limit switch are governed by the controller 27, which orchestrates the movement and ensures accurate focusing.

[0051] In some embodiments, a liquid lens is included in the lens 22 and its focal length may be tuned with electrical stimulus. In some embodiments, the lens 22 includes one or a combination of at least one of dichroic mirrors, bandpass filters, longpass filters, shortpass filters, neutral density filters, polarizing filters, interference filters, hot mirrors, cold mirrors, or similar in place of or in combination with the optical lenses 23.

[0052] As shown in Fig. 15, the device 10 may include a filter wheel 100. The filter wheel 100 may be disposed between the lens 22 and the imager 20. The filter wheel 100 may be a disc-shaped member with multiple ports 101 spaced radially around its center. The ports 101 may be sized and shaped for mounting optical components 102 thereto. The optical components 102 installed in the ports 101 may be positioned in the optical path between the lens 22 and the imager 20. The filter wheel 100 may rotate relative to the imager 20 by a filter wheel motor 103. The filter wheel motor 103 may be controlled by the controller 27. The filter wheel 100 may have a notched outer circumference 104 that is engageable by an engagement element 105 of the filter wheel motor 103. In some embodiment, the filter wheel 100 and filter wheel motor 103 have a frictional engagement. A portion of the filter wheel 100 may be received in the filter wheel motor 103.

[0053] The controller 27 may cause the filter wheel motor 102 to position each optical component 102 in the optical path as required for different types of imaging. The optical components 102 installed in the filter wheel 100 may be selected to enable various imaging techniques, including at least brightfield imaging, darkfield scattering, and fluorescence imaging. The contrast of each captured image is calculated and associated with an index at the time of image capture. A target position of lens 22 may then be determined by identifying the index corresponding to the image with the greatest contrast. The focusing process can be performed multiple times, adjusting the step size and the number of steps to achieve both coarse and fine focus. This multi-stage focusing process can facilitate precise image focusing.

[0054] The imager 20 may capture a z-stack of images for detailed analysis, specifically for platelet analysis. For each field of view (FOV), a stack of, for example, five brightfield images of platelets may be captured. These images can be analyzed to determine platelet morphology and other relevant characteristics.

[0055] As shown in Figs. 1-3, the device 10 may include at least one disk guide 24 coupled to the base 12. In some embodiments, more than one disk guide 24 are coupled to the base 12 spaced apart from each other. The disk guide 24 may be configured to engage an edge of the disk 18. Engagement of the disk guide 24 and the disk 18 may minimize movement of the disk 18 relative to the lens 22. The disk guide 24 may be a generally bobbin shape, including a top, a bottom and a middle. The top may have a diameter similar to that of the bottom. The top and bottom may have a diameter that is larger than the middle. The disk guide 24 may include a taper toward the middle from the top and bottom. When the disk 18 is received in the disk guide 24, the taper may minimize movement of the disk relative to the lens 22. The disk guide 24 may receive the edge of the disk 18 in the middle. The top and bottom of the disk guide 24 may prevent the disk 18 from moving relative to the disk guide 24.

[0056] In some embodiment, the device 10 includes two disk guides 24. As shown in Figs. 1-3, the two disk guides 24 may be disposed on both sides of the lens 22. Providing two disk guides 24 may minimize movement of the disk 18 relative to the lens 22. In some embodiments, the device 10 includes three disk guides 24. In some embodiments, the device 10 includes four disk guides 24. In some embodiments, the device 10 includes five disk guides 24. In some embodiments, the device 10 includes six disk guides 24.

[0057] The disk guide 24 may include an axis AG extending through the top and bottom thereof. Axis AG may be parallel to a rotation axis AD. The disk guide 24 may be configured to rotate about the axis AG. In one embodiment, the disk guide 24 is fixed relative to the axis AG.

[0058] For the imager 20 to collect accurate data, a light may be provided on an opposite side of the disk 18 to illuminate the target area. As shown in Figs. 1-3, the device 10 may include a light source 26 coupled to the base 12. The light source 26 may be coupled on a second side relative to the disk 18. The light source 26 may be configured to illuminate the sample fluid in the disk 18. The light source 26 may comprise a light ring. The light source 26 may emit light on the sample fluid at different wavelengths and angles. The light source 26 may illuminate the sample fluid in the disk 18 in perpendicular angles and oblique angles, thereby enabling measurement of direct absorption, fluorescence measurement and scattering analysis of the sample fluid within the well 32. In some embodiments, the light source 26 emits a polarized light to, for example, differentiate between various types of cells. For example, polarized light may be used to identify various types of white blood cells, as lymphocytes cause depolarization of plane-polarized incident light, which can be detected and used to identify them among other white blood cells.

[0059] The light source 26 may be movable between an engaged position and a disengaged position. The light source 26 may be radially aligned with an axis Ai extending between the lens 22 and the imager 20 when the light source 26 is in the engaged position. As shown in Fig. 3, the light source 26 may be radially spaced apart from the axis Ai when the light source 26 is in the disengaged position. The light source 26 may be radially spaced apart from the axis Ai in the disengaged position such that the light source 26 does not extend over any part of the disk 18. The light source 26 may be prevented from illuminating in the disengaged position.

[0060] The light source 26 is slidably coupled to a guide rail 28 extending on a plane parallel to a plane of the base 12. In some embodiments, more than one guide rail 28 extends on a plane parallel to a plane of the base 12. The guide rail 28 may be a generally cylindrical rod. The guide rail 28 may include ribbing on one side. In one embodiment, the guide rail 28 may be threaded along a portion thereof. The light source 26 may include a sub-motor 30 configured to move the light source 26 between the engaged position and the disengaged position. The sub-motor 30 may include a gear or a threaded shaft (not shown) that is powered by the sub-motor 30 to move the light source 26 between the engaged position and the disengaged position. In some embodiments, the sub-motor 30 is an actuator coupled to the light source 26 and the base 12 that extends and retracts to move the light source 26 between the engaged position and the disengaged position, respectively. In someembodiments, the sub-motor 30 is a lever that moves the light source 26 between the engaged position and the disengaged position.

[0061] The sub-motor 30 may be spaced apart from the light source 26 by an extension 44. The extension may be coupled to the sub-motor 30 and the light source 26. The extension 44 may have a length sufficient to allow the sub-motor 30 to interact with the guide rail 28 and the disk guides 24 to interact with the disk 18. The extension 44 may include a first receiver 46 configured to receive a guide rail 28 of the base 12. The first receiver 46 may increase the stability of the light source 26 as it moves between the engaged position and the disengaged position. The extension 44 may include a second receiver 48 configured to receive a rail 50 of the base 12. The second receiver 48 may increase the stability of the light source 26 as it moves between the engaged position and the disengaged position.

[0062] The disk guide 24 may be coupled to the light source 26. In some embodiments, the disk guide 24 is coupled to the base 12. The disk guide 24 may extend from a guide arm 66 extending from the light source 26. The disk guide 24 may engage the edge of the disk 18 when the light source 26 is in the engaged position. In one embodiment, the engaged position may be considered the point at which contact is maintained between the disk 18 and the disk guide 24. The light source 26 may include a hood 64 covering an emitter (not shown). The hood 64 may prevent the emitter(s) from shining a light outside of the intended target area.

[0063] To facilitate a uniform temperature of the sample fluid in the disk 18 heat may be provided to the disk. As shown in Figs. 1-3, the device 10 may include a heater 52. The heater 52 may provide radiant heat to the disk 18 when the disk 18 is disposed on the spindle 14. The heater 52 may bring the fluid sample to a desired temperature through constant heat. In some embodiments, the heater 52 may bring the fluid sample to a desired temperature through pulsing heat. In some embodiments, rotation of the disk 18 relative to the heater may generate air circulation between the heater 52 and the disk 18 to lower the temperature of the fluid sample in the disk 18.

[0064] The heater 52 may be coupled to the base 12. The heater 52 may be disposed between the base 12 and the disk 18. In some embodiments, the disk 18 is disposed between the base 12 and the heater 52. In some embodiments, the heater 52 is disposed within the base 12. The heater 52 may be a generally rectangular shape. The heater 52 may be integrated on a heater printed circuit board assembly (PCBA) 58. The heater 52 may define an opening 56 in a center thereof. The opening 56 may be shaped and sized as to fit around the spindle 14. The base 12 may include a recess 60 definedtherein. The recess 60 may have a generally circular shape. The recess may be shaped and sized to receive a portion of the heater 52 therein. The recess 60 may extend around a portion of the base 12. The recess 60 may be defined around the spindle 14 extending from the base 12.

[0065] The heater 52 may include at least one heating element 54. As shown in Figs. 1-3, the at least one heating element 54 may extend beyond an edge of the heater PCBA 58. The at least one heating element 54 may extend circumferentially around the opening 56. The heater 52 may include at least four heating elements 54. The heater 52 may include at least one heating element 54. The heater 52 may include at least two heating elements 54. The heater 52 may include at least three heating elements 54. The heater 52 may include at least five heating elements 54. The heater 52 may include at least six heating elements 54. The heater 52 may include at least ten heating elements 54. The heater 52 may include at least fifteen heating elements 54. The heater 52 may include at least twenty heating elements 54.

[0066] The device 10 may include a controller 27 configured to control each of at least the spindle 14, imager 20, lens 22, disk guide 24, light source 26, actuating mechanism 25 and filter wheel 100. The controller 27 may be integrated on a main printed circuit board 37. The controller 27 may be configured to transmit the collected image data to a processor 34. The processor 34 may identify at least one of lymphocytes, granulocytes, monocytes, platelets, hemoglobin, and / or hematocrit in the sample fluid. The processor 34 may transmit the collected image data to a graphic user interface (not shown).

[0067] A method for analyzing a sample fluid may be disclosed herein. The method may include inserting the disk 18 in the device 10. As shown in Figs. 4-9, the disk 18 may have one or more wells 32 at a first common radius relative to a rotation axis AD of the disk. Each of the one or more wells 32 may be configured to receive a volume of a fluid including one or more components of the sample fluid. Each of the one or more wells 32 may be a transparent imaging channel. Each of the one or more wells 32 may have a depth to allow only a single layer of cells to spread thereon.

[0068] The depth of each of the one or more wells 32 may be between 50 pm and 600 pm. The depth of each of the one or more wells 32 may be between 100 pm and 550 pm. The depth of each of the one or more wells 32 may be between 150 pm and 500 pm. The depth of each of the one or more wells 32 may be between 200 pm and 450 pm. The depth of each of the one or more wells 32 may be between 250 pm and 400 pm. The depth of each of the one or more wells 32 may be between 300pm and 350 pm. Each of the one or more wells 32 may have a range of depths, such as a 50 um region as well as a 600 um region.

[0069] The disk 18 may include a plurality of testing chambers 68. Each of the plurality of testing chambers 68 may include a sample inlet 70 for receiving the sample fluid, a buffer inlet 72 for receiving a buffer, a mixing chamber 74 for combining the sample and the buffer, and one of the one or more wells 32. The sample inlet 70, the buffer inlet 72 and the mixing chamber 74 may be in fluid communication. In one embodiment, the disk 18 includes three testing chambers 68. Each of the three testing chambers 68 may be configured to test one of white blood cells, red blood cells, platelets, and / or hemoglobin. The test may determine one or more of a number of cells, a density or concentration of small molecules, and morphological features of a cell such as its granularity, size, circularity, nuclei size, shape, and nucleic concentrations as well as fluorescence, scatter and absorbance signatures. The testing chamber 68 configured to test red blood cells may also be configured to test hematocrit. In some embodiments, the disk 18 includes a hematocrit chamber 85 configured to test hematocrit. While reference throughout may be made to a single testing chamber 68, it should be appreciated that each of the plurality of testing chambers may include such features.

[0070] As shown in Fig. 5, the method may include receiving, in each of the testing chambers 68, a volume of the sample into a sample chamber 78 through the sample inlet 70 and a volume of the buffer into a buffer chamber 80 through the buffer inlet 72. The sample chamber 78 and the buffer chamber 80 of each testing chamber 68 may be a different size and shape to accommodate a volume necessary for a specific application. In some embodiments, the sample chamber 78 and the buffer chamber 80 of each testing chamber 68 are the same size and shape.

[0071] The testing chamber 68 configured to test white blood cells may receive about 120 pL of the buffer and about 53 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 80 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 90 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 100 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 110 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 120 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 130 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 140 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 150 pL of the buffer. The testing chamber 68 configured to test white blood cells may receive at least 160 pL of the buffer. The testing chamber 68 configured to test white blood cellsmay receive at least 35 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 40 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 45 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 50 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 55 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 60 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 65 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 70 pL of the sample. The testing chamber 68 configured to test white blood cells may receive at least 75 pL of the sample.

[0072] The testing chamber 68 configured to test red blood cells may receive about 600 pL of the buffer and about 18 pL of the sample . The testing chamber 68 configured to test red blood cells may receive at least 400 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 450 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 500 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 550 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 600 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 650 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 700 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 750 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 800 pL of the buffer. The testing chamber 68 configured to test red blood cells may receive at least 5 pL of the sample. The testing chamber 68 configured to test red blood cells may receive at least 10 pL of the sample. The testing chamber 68 configured to test red blood cells may receive at least 15 pL of the sample. The testing chamber 68 configured to test red blood cells may receive at least 20 pL of the sample. The testing chamber 68 configured to test red blood cells may receive at least 25 pL of the sample. The testing chamber 68 configured to test red blood cells may receive at least 30 pL of the sample. The testing chamber 68 configured to test red blood cells may receive at least 35 pL of the sample.

[0073] The testing chamber 68 configured to test hemoglobin may receive about 120 pL of the buffer and about 20 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 80 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 90 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 100 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 110pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 120 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 130 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 140 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 150 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 160 pL of the buffer. The testing chamber 68 configured to test hemoglobin may receive at least 5 pL of the sample.The testing chamber 68 configured to test hemoglobin may receive at least 10 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 15 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 20 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 25 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 30 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 35 pL of the sample. The testing chamber 68 configured to test hemoglobin may receive at least 40 pL of the sample.

[0074] The fluid may be prevented from flowing from each of the sample chamber 78 and the buffer chamber 80 into the mixing chamber by a flow valve 76a, 76b, respectively. Flow valves 76a, 76b may be sized and shaped such that the surface tension of the fluid prevents flow therethrough.

[0075] As shown in Fig. 6, the method may include operating the motor 16 of the device 10 to rotate the disk 18 in a first direction around the spindle 14 to meter a volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of about 100-650 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 100 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 150 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 200 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 250 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 300 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 350 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 400 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 450 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 500 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 550 rpm to meter, or measure,the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 600 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 650 rpm to meter, or measure, the volume of the sample and the buffer. The disk 18 may be rotated at a rotational speed of at least 700 rpm to meter, or measure, the volume of the sample and the buffer.

[0076] The testing chamber 68 configured to test white blood cells may contain about 110 pL of the buffer and about 30 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 80 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 90 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 100 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 110 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 120 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 130 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 140 pL of the buffer after metering. The testing chamber 68 configured to test white blood cells may contain at least 15 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 20 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 25 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 30 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 35 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 40 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 45 pL of the sample after metering. The testing chamber 68 configured to test white blood cells may contain at least 50 pL of the sample after metering.

[0077] The testing chamber 68 configured to test red blood cells may contain about 500 pL of the buffer and about 3.86 pL of the sample after metering. The testing chamber 68 configured to test red blood cells may contain at least 300 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 350 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 400 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 450 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 500 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain atleast 550 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 600 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 650 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 700 pL of the buffer after metering. The testing chamber 68 configured to test red blood cells may contain at least 2.5 pL of the sample after metering. The testing chamber 68 configured to test red blood cells may contain at least 3 pL of the sample after metering. The testing chamber 68 configured to test red blood cells may contain at least 3.5 pL of the sample after metering. The testing chamber 68 configured to test red blood cells may contain at least 4 pL of the sample after metering. The testing chamber 68 configured to test red blood cells may contain at least 4.5 pL of the sample after metering. The testing chamber 68 configured to test red blood cells may contain at least 5 pL of the sample after metering.

[0078] The testing chamber 68 configured to test hemoglobin may contain about 100 pL of the buffer and about 5.8 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 80 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 85 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 90 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 95 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 100 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 105 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 110 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 115 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 120 pL of the buffer after metering. The testing chamber 68 configured to test hemoglobin may contain at least 3.5 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 4 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 4.5 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 5 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 5.5 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 6 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 6.5 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 7 pL of the sample after metering. The testing chamber 68 configured to testhemoglobin may contain at least 7.5 pL of the sample after metering. The testing chamber 68 configured to test hemoglobin may contain at least 8 pL of the sample after metering.

[0079] An excess amount of the sample and the buffer may flow into an overflow chamber 82, 84, respectively, in fluid communication with the testing chamber 68.

[0080] As shown in Figs. 7-8, the method may include operating the motor 16 of the device 10 to rotate the disk 18 in a first direction around the spindle 14 to cause the sample and the buffer to flow into the mixing chamber. The disk 18 may be rotated at a rotational speed of about 600 rpm to allow the metered sample to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of about 700-1500 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 700 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 800 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 900 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1000 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1100 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1200 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1300 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1400 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1500 rpm to allow the buffer to flow into the mixing chamber 74. The disk 18 may be rotated at a rotational speed of at least 1600 rpm to allow the buffer to flow into the mixing chamber 74. The metered sample and buffer may flow into the mixing chamber 74 at approximately the same time in each of the plurality of testing chambers 68.

[0081] The mixing chamber 74 may include a reagent 86 disposed therein. The reagent 86 may comprise one or more lyophilized (lyo) beads bead, Acetyl-CoA Carboxylase (ACO) beads and / or Cholesterol Oxidase (CholOxidase) bead. In some embodiments, the reagent 86 is present in a liquid state. In some embodiments, the reagent 86 is present in the form of a coating on an inner surface of the mixing chamber 74. The disk 18 may be rotated in the first direction and then suddenly stopped to mix a first reagent 86 into the sample to deplete a first target content (e.g., one or more types of lipids, small molecules, analytes or interferents) from the sample. The disk 18 may then be rotated in the first direction and then suddenly stopped to mix a second reagent 86 into the sample to deplete a second target content (e.g., cell types such as RBCs, small molecules, analytes or interferents) fromthe sample. The disk 18 may be rotated in the first direction at a rotational speed of about 600 rpm and then suddenly stopped to mix the sample and the buffer within the mixing chamber 74. The disk 18 may be rotated in the first direction at a rotational speed of at least 300 rpm and then suddenly stopped to mix the sample and the buffer within the mixing chamber 74. The disk 18 may be rotated in the first direction at a rotational speed of at least 400 rpm and then suddenly stopped to mix the sample and the buffer within the mixing chamber 74. The disk 18 may be rotated in the first direction at a rotational speed of at least 500 rpm and then suddenly stopped to mix the sample and the buffer within the mixing chamber 74. The disk 18 may be rotated in the first direction at a rotational speed of at least 600 rpm and then suddenly stopped to mix the sample and the buffer within the mixing chamber 74. The disk 18 may be rotated in the first direction at a rotational speed of at least 700 rpm and then suddenly stopped to mix the sample and the buffer within the mixing chamber 74.

[0082] The mixing chamber 74 may have a rounded wall to facilitate mixing of the sample and the buffer within the mixing chamber 74. As shown in Fig. 8, mixing of the sample and the buffer hydrates and reconstitutes the reagent 86 in the mixing chamber 74. Reconstitution of the lyo bead may cause the sample to become translucent. Reconstitution of the lyo bead may cause the red blood cells in the sample to lyse by virtue of agents capable of selectively lysing red blood cells only. Reconstitution of the lyo bead may cause the red blood cells to become spherical in shape. Some examples of such agents are saponin, ammonium chloride, non-ionic and zwitterionic surfactants. Reconstitution of the lyo bead may cause the white blood cells in the sample to be fixed. Reconstitution of the lyo bead may cause the white blood cells in the sample to be tagged. Tagging agents may include organic fluorescent dyes with specific acidophilic, basophilic or neutrophilic properties, dyes with affinity to single-stranded and / or double stranded nucleic acids, dyes with affinity to lipids. Tagging agents may include binders with high specificity (monoclonal antibodies, antibody single-chain fragments, aptamers, peptides) conjugated with fluorescent dyes. In other embodiments, other fluorescent or chromatic markers such as quantum dots, gold nanoparticles or polymer nanoparticles may be used. Reconstitution of the lyo bead may cause platelets in the sample to be tagged with agents similar to the ones described above.

[0083] As shown in Fig. 9, the method may include operating the motor 16 of the device 10 to rotate the disk 18 in a second direction around the spindle 14 to cause the sample and buffer to flow from the mixing chamber 74 into one of the one or more wells 32. Each testing chamber 68 may have a different number of wells 32 depending on the sample provided to such testing chamber 68 and a specific application. Each testing chamber may each have wells 32 of differing shapes and sizes.Each testing chamber 68 may include at least one well 32. In some embodiments, each of the testing chambers 68 includes the same number of wells 32. In some embodiments, each of the wells 32 are the same shape and size. The disk 18 may be rotated at a rotational speed of about 1 ,000 rpm to allow the metered sample to flow into one of the one or more wells 32.

[0084] The method may include operating the motor 16 of the device 10 to rotate the disk 18 in a second direction around the spindle 14 to cause the one or more wells 32 to stop between the imager 20 and the light source 26. The imager 20 may collect image data of the sample fluid. The imager 20 may capture image data of the sample in the well 32 before the motor 16 rotates the disk 18 to position a next of the one or more wells 32 between the imager 20 and the light source 26. As shown in Fig. 9, the disk 18 may include a homing recess 90. The homing recess may be a generally circular recess on the edge of the disk 18. The homing recess 90 may be detected by a positioning sensor of the controller 27 such that the relative location of each of the one or more wells 32 can be tracked during rotation of the disk 18. The disk 18 may include a plurality of homing recesses 90. The homing recess 90 may protrude from the disk 18 in some embodiments.

[0085] The disk 18 may also include light waveguiding features 91 (Fig. 9) extending through the disk 18. The light waveguiding features 91 may be transparent and configured to guide the light from the light source 26 through the imager 20. The light waveguiding features 91 may allow the device 10 to perform alignment and homing of the disk 18. The imager 20 may detect an amount of light input provided by light waveguiding features 91 that is larger than the light input provided by the rest of the disk 18 (e.g., the wells 32). This detection may serve as an angular reference of the disk 18 for the device 10. By developing an angular reference of the disk 18, the one or more wells 32 along the imaging radius of the disk 18 can be identified using their known angular position information.

[0086] Upon completion of imaging the one or more wells 32, the method may include operating the motor 16 of the device 10 to rotate the disk 18 in one of the first direction or the second direction around the spindle 14 to cause plasma separation in the hematocrit chamber 85. The hematocrit chamber 85 may receive about 25 uL of the buffer and about 25 uL of the sample. The hematocrit chamber 85 may receive at least 15 uL of the buffer. The hematocrit chamber 85 may receive at least 20 uL of the buffer. The hematocrit chamber 85 may receive at least 25 uL of the buffer. The hematocrit chamber 85 may receive at least 30 uL of the buffer. The hematocrit chamber 85 may receive at least 35 uL of the buffer. The hematocrit chamber 85 may receive at least 15 uL of the sample. The hematocrit chamber 85 may receive at least 20 uL of the sample. The hematocritchamber 85 may receive at least 25 uL of the sample. The hematocrit chamber 85 may receive at least 30 uL of the sample. The hematocrit chamber 85 may receive at least 35 uL of the sample.

[0087] The disk 18 may be rotated at a rotational speed of about 7,000 rpm to cause a plasma to separate from the sample. The disk 18 may be rotated at a rotational speed of at least 5,000 rpm to cause a plasma to separate from the sample. The disk 18 may be rotated at a rotational speed of about 6,000 rpm to cause a plasma to separate from the sample. The disk 18 may be rotated at a rotational speed of about 7,000 rpm to cause a plasma to separate from the sample. The disk 18 may be rotated at a rotational speed of about 8,000 rpm to cause a plasma to separate from the sample. The disk 18 may be rotated at a rotational speed of about 9,000 rpm to cause a plasma to separate from the sample.

[0088] The hematocrit chamber 85 may have different cross-sectional areas to accommodate the target sample volume within a small area of the disk 18. Varying the cross sectional area of the hematocrit chamber 85 may improve resolution of the image data collected by the imager 20. As shown in Fig. 9, the hematocrit chamber 85 may include a column 87. The column 87 may be imaged using an external imaging device (e.g., imager 20) and then fed to an image analysis algorithm where the length of the plasma in the column 87 is compared to the length of the column of solid matter sedimented at the bottom of the column 87 due to centrifugal force. The sample may be diluted with appropriate buffers, as discussed above in more detail, to speed up the sedimentation process.

[0089] As shown in Figs. 13-14, the spindle 14 may comprise a locking element 38 configured to prevent the disk 18 from rotating relative to the spindle 14. The locking element 38 may include at least one protrusion 53 extending radially therefrom. The locking element 38 may include at least two protrusions 53 extending radially therefrom. The locking element 38 may include at least four protrusions 53 extending radially therefrom. The locking element 38 may include at least six protrusions 53 extending radially therefrom. The locking element 38 may include at least ten protrusions 53 extending radially therefrom. The spindle 14 may include a collar 40 extending radially therefrom. The collar 40 may be a generally circular shape. The collar 40 may have a diameter greater than that of the aperture 36 to prevent the disk 18 from passing over the collar 40. The collar 40 may include a deformable ring 61 disposed thereon. The deformable ring 61 may be comprised of a rubber or other deformable material. The deformable ring 61 may extend around at least a portion of the collar 40. In some embodiments, the deformable ring extends around only a portion of the collar 40. The deformable ring 61 may provide friction between the spindle 14 and the disk 18 to ensure that the torque generated by rotation of the spindle 14 is efficiently transferred to thedisk 18 during operation of the device 10. The deformable ring 61 may extend from the collar 40 to engage the disk 18 when the disk 18 is disposed on the spindle 14.

[0090] The locking element 38 may include at least one locking pin 57 extending therefrom. The locking element 38 may include at least two locking pins 57 extending therefrom. The locking element 38 may include at least five locking pins 57 extending therefrom. The locking element 38 may include at least ten locking pins 57 extending therefrom. The locking pin 57 may extend from the locking element 38 proximate the collar 40. The locking pin 57 may be received in a detent 59 of the disk 18. The bottom of the disk 18 may include at least as many detents 59 as the spindle 14 has locking pins 57. The detent 59 may extend into the disk 18 from the aperture 36. The detent 59 may be shaped and sized to receive at least a portion of the locking pin 57. The locking pin 57 may prevent the disk 18 from moving relative to the spindle 14 during operation of the device 10. In a case where more than one locking pin 57 is included on the spindle 14, the locking pins 57 may be unevenly spaced apart to ensure only one possible orientation of the disk 18 on the spindle 14. This orientation may ensure the disk 18 accurately receives the fluid into the testing chamber 68.

[0091] To ensure the locking pin 57 is aligned with the detent 59 when the disk 18 is received in the device 10, a spindle setting mechanism may be used. The spindle 14 may be coupled to the motor 16 with a fastener 63. The spindle 14 may be fixed to the motor 16. The motor 16 may have a starting position, which corresponds to the position of the locking pin 57. The starting position may be electrically programmed into the controller 27 which moves the motor 16 into the starting position before the disk 18 is received in the device 10.

[0092] As shown in Figs. 13-14, the spindle 14 may include a disk lock 65 spaced apart from the collar 40. The disk lock 65 may be spaced apart from the collar 40 such that the disk 18 can fit therebetween. The disk lock 65 may be a ball pin disposed at an end of the locking element 38. The spindle 14 may include as many disk lock 65 as it includes locking pins 57. In some embodiments, there are more disk locks 65 than locking pins 57. In some embodiments, there are less disk locks 65 than locking pins 57. The disk lock 65 may include a ball 67 and a spring 69. The ball 67 and spring 69 may be received in a cavity 71 of the locking element 38. The cavity 71 may be a generally cylindrical space defined by the locking element 38 and a lock top 73 coupled to the end of the locking element 38. The lock top 73 may be fixed to the locking element 38 by, for example, a screw. The spring 69 may urge the ball 67 out of the cavity 71. The cavity 71 may include an opening 75 that has a smaller diameter than the ball 67 to prevent the ball 67 from moving out of the cavity 71. The ball 67 may retract in response to a force by, for example, the disk 18 being disposed on the spindle 14.The ball 67 may extend when the force is released, for example, the disk 18 has been received on the spindle 14. The ball 67 may enact a force on the disk 18 when the disk is received on the spindle 14. The ball 67 may urge the disk 18 toward the collar 40. The disk lock 65 may prevent the disk 18 from moving relative to the collar 40 along the axis As during operation of the device 10.

[0093] In one embodiment, the device 10 includes one or more computers having one or more processors and memory (e.g., one or more nonvolatile storage devices). In some embodiments, memory or computer readable storage medium of memory stores programs, modules and data structures, or a subset thereof for a processor to control and run the various systems and methods disclosed herein. In one embodiment, a non-transitory computer readable storage medium having stored thereon computer-executable instructions which, when executed by a processor, perform one or more of the methods disclosed herein.

[0094] Once the images of various testing chambers 68 are taken with appropriate metadata, they may be fed to a computer algorithm for downstream analysis. The automated algorithm can measure the number of cells within the well 32, their physical properties such as a number of cells, a density or concentration of small molecules, and morphological features of a cell such as its granularity, size, circularity, nuclei size, shape, and nucleic concentrations as well as fluorescence, scatter and absorbance signatures. All these data may be used to differentiate between various types of cells within the sample. Fig 10 illustrates an example of various types of Lukocytes observed in a well 32 (e.g., a RBC imaging chamber) showing fluorescent and scatter light originating from each cell passing through the lens 22 and their difference in fluorescence response and scattering properties. Each cluster illustrates one cell type.

[0095] The term “about” or “approximately” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number, which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. It should be appreciated that all numerical values and ranges disclosed herein are approximate values and ranges, whether “about” is used in conjunction therewith. It should also be appreciated that the term “about,” as used herein, in conjunction with a numeral refers to a value that may be ±0.01% (inclusive), ±0.1% (inclusive), ±0.5% (inclusive), ±1% (inclusive) of that numeral, ±2% (inclusive) of that numeral, ±3% (inclusive) of that numeral, ±5% (inclusive) of that numeral, ±10% (inclusive) of that numeral, or±15% (inclusive) of that numeral. It should further be appreciated that when a numerical range is disclosed herein, any numerical value falling within the range is also specifically disclosed.

[0096] It will be appreciated by those skilled in the art that changes could be made to the exemplary embodiments shown and described above without departing from the broad inventive concepts thereof. It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways.

[0097] Specific features of the exemplary embodiments may or may not be part of the claimed invention and various features of the disclosed embodiments may be combined. Unless specifically set forth herein, the terms “a”, “an” and “the” are not limited to one element but instead should be read as meaning “at least one”. Finally, unless specifically set forth herein, a disclosed or claimed method should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the steps may be performed in any practical order.

Claims

CLAIMSWhat is claimed is:

1. A method of analyzing a sample fluid, the method comprising: providing a disposable cartridge comprising a receiving inlet and an analyzing chamber, the disposable cartridge configured to move a sample fluid through a sample pathway between the receiving inlet and the analyzing chamber; receiving a volume of a fluid having one or more components of the sample fluid and a buffer in the receiving inlet of the disposable cartridge; tagging the fluid moving through the disposable cartridge with dried tagging reagents disposed along the sample pathway; and forming a monolayer of the tagged fluid in the analyzing chamber for microscopic analysis, wherein the disposable cartridge moves the fluid from the receiving inlet to the analyzing chamber by a rotating motor.

2. The method of claim 1 , wherein the disposable cartridge is a disk comprising at least one testing chamber, the testing chamber testing one or more of white blood cells, red blood cells, hemoglobin, platelets and / or hematocrit in the sample fluid.

3. The method of claim 2, wherein the test determines one or more of a number of cells, a density or concentration of small molecules, and morphological features of a cell such as its granularity, size, circularity, nuclei size, shape, and nucleic concentrations.

4. The method of claim 2, wherein the fluid comprises the sample fluid and the buffer, and wherein a volume of the buffer and a volume of the sample fluid are received in a buffer chamber and sample chamber, respectively, of each testing chamber.

5. The method of claim 4, wherein the testing chamber configured to test white blood cells and platelets receives about 120 pL of the buffer into the buffer chamber through a buffer inlet and aboutwherein the testing chamber configured to test red blood cells receives about 300 pL of the buffer into the buffer chamber through a buffer inlet and about 15 pL of the sample fluid into the sample chamber through a sample inlet, and wherein the testing chamber configured to test hemoglobin receives about 190 pL of the buffer into the buffer chamber through a buffer inlet and about 35 pL of the sample fluid into the sample chamber through a sample inlet. wherein the testing chamber configured to test hematocrit receives about 25 uL of the buffer into the buffer chamber through a buffer inlet and about 25 uL of the sample fluid into the sample chamber through a sample inlet.

6. The method of claim 3, wherein the disk is rotated in a first direction at a rotational speed of about 400 rpm to meter the volume of the sample fluid and the buffer.

7. The method of claim 5, wherein the testing chamber configured to test white blood cells contains about 120 pL of the buffer and about 24 pL of the sample fluid after metering, wherein the testing chamber configured to test red blood cells contains about 200 pL of the buffer and about 2 pL of the sample fluid after metering, and wherein the testing chamber configured to test hemoglobin contains about 130 pL of the buffer and about 8.5 pL of the sample fluid after metering.

8. The method of claim 6, wherein an excess amount of the sample fluid and the buffer flows into an overflow chamber.

9. The method of claim 8, wherein rotation of the disk in a first direction causes the sample fluid and the buffer to flow into a mixing chamber.

10. The method of claim 9, wherein the mixing chamber contains at least one reagent.

11. The method of claim 10, wherein the disk is rotated in the first direction at a rotational speed of about 600 rpm and then suddenly stopped to mix the sample fluid and the buffer within the mixing chamber.

12. The method of claim 11, wherein mixing of the sample fluid and the buffer hydrates and reconstitutes the reagent in the mixing chamber.

13. The method of claim 12, wherein reconstitution of the reagent causes the sample fluid to become translucent, wherein reconstitution of the reagent depletes small molecules, analytes or interferents present in the sample fluid, wherein reconstitution of the reagent causes red blood cells in the sample to lyse, wherein reconstitution of the reagent causes white blood cells in the sample to be tagged, wherein reconstitution of the reagent causes platelets in the sample to be tagged, and wherein reconstitution and mixing causes the red blood cells to become spherical in shape.

14. The method of claim 13, wherein rotation of the disk in a second direction causes the sample fluid and buffer to flow from the mixing chamber into the analyzing chamber.

15. The method of claim 14, wherein the disk is rotated to position, in succession, the analyzing chamber proximate an imager for imaging.

16. The method of claim 15, wherein the imaging content is analyzed to identify at least one a number of cells, a density or concentration of small molecules, morphological features, fluorescence signatures, scatter signatures and absorbance signatures.

17. The method of claim 16, wherein the cells are grouped based on the identified properties.

18. The method of claim 15, wherein rotation of the disk in a second direction at a rotational speed of about 7,000 rpm separates a plasma from the sample fluid.

19. The method of claim 18, wherein the disk is rotated to position the plasma in the analyzing chamber proximate the imager for imaging.

20. The method of claim 10, wherein the at least one reagent comprises one or more of lyo bead, ACO bead, and / or CholOxidase bead, wherein the reagent is present in liquid state, orwherein the reagent is present in the form of a coating on an inner surface of the mixing chamber.

21. The method of claim 11, wherein the disk is rotated in the first direction and then suddenly stopped to mix a first reagent of the at least one reagent into the sample to deplete a first target content of the sample.

22. The method of claim 21, wherein the disk is rotated in the first direction and then suddenly stopped to mix a second reagent of the at least one reagent into the sample to deplete a second target content of the sample.

23. The method of claim 9, wherein the disk is rotated in the first direction at a rotational speed of about 600 rpm to cause the sample fluid to flow into the mixing chamber.

24. The method of claim 9, wherein the sample fluid flows into the mixing chamber at approximately the same time in each testing chamber.

25. The method of claim 9, wherein the disk is rotated in the first direction at a rotational speed of about 700 rpm to cause the buffer to flow into the mixing chamber.

26. The method of claim 1 , wherein the fluid is moved between the receiving inlet and the analyzing chamber by centrifugal and inertial forces generated by the rotating motor.

27. A device for analyzing a sample fluid, the device comprising: a base; a spindle rotatably coupled to the base; a motor configured to rotate a disk disposed on the spindle, the disk configured to receive a volume of a fluid comprising one or more components of the sample fluid; an imager coupled to the base on a first side relative to the disk and configured to collect image data of the sample fluid in the disk; a lens moveably coupled to the base on the first side relative to the disk, the lens configured to focus an optical lens disposed therein relative to the disk as the imager collects image data;an illumination system coupled to the base, the illumination system emitting light on the disk at different wavelength and angles; at least one disk guide coupled to the base, the disk guide configured to engage an edge of the disk to minimize movement of the disk relative to the lens; and a controller configured to control each of the spindle, imager, lens, illumination ring and disk guide.

28. The device of claim 27, wherein the disk has one or more wells at a first common radius relative to a rotation axis of the disk, and wherein each of the one or more wells is configured to receive a volume of a fluid including one or more components of the sample fluid.

29. The device of claim 28 further comprising: a light source coupled to the base on a second side relative to the disk and configured to illuminate the sample fluid in the disk.

30. The device of claim 29, wherein the light source is movable between an engaged position and a disengaged position.

31. The device of claim 29, wherein the light source illuminates the sample fluid in perpendicular and oblique angles, thereby enabling measurement of direct absorption, fluorescence measurement as well as scattering analysis of the sample fluid in the disk.

32. The device of claim 29, wherein the optical lens may flex relative to the imager to focus the disk on the imager when the light source is in the engaged position.

33. The device of claim 27 further comprising: an imaging filter having a plurality of optical components disposed thereon, the imaging filter being moveable relative to the imager to align an optical component between the imager and the sample fluid in the disc.

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