Detecting platelets in a blood sample

Abstract

Provided are apparatuses and methods that include imaging a blood sample that is a suspension of cells deposited in a sample chamber. The cells are allowed to settle in the sample chamber to form a monolayer of cells. At least one microscopic image of the monolayer of cells is acquired using a microscope (24) while the microscope is focused at a monolayer depth level, and a first platelet count of platelets that have settled within the monolayer is determined. Additional microscopic images of the sample are acquired while the microscope is focused at depth levels different from the monolayer depth level, and a second platelet count of platelets that are not settled within the monolayer is determined. An output is generated based on the first platelet count and the second platelet count. Other applications are also described.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 946,998, filed December 12, 2019, entitled “Detecting Platelets in a Blood Sample”, which is incorporated herein by reference.

[0003] Field of the embodiments of the present invention

[0004] Some applications of the subject matter disclosed in this invention generally relate to the analysis of bodily samples, and particularly to the optical density and microscopic measurements of blood samples.

[0005] background

[0006] In some optical-based methods (e.g., diagnostic and / or analytical methods), the characteristics of biological samples, such as blood samples, are determined by performing optical measurements. For example, the density of a component (e.g., component count per unit volume) can be determined by counting the components within a microscopic image. Similarly, the concentration and / or density of a component can be measured by performing light absorption, transmission, fluorescence, and / or luminescence measurements on the sample. Typically, the sample is placed in a sample carrier, and a portion of the sample contained within the sample chamber of the sample carrier is measured. The measurements performed on the portion of the sample contained within the sample chamber of the sample carrier are analyzed to determine the characteristics of the sample.

[0007] Implementation Plan Overview

[0008] According to some applications of the invention, a portion of a blood sample comprising a cell suspension is placed in a sample chamber that is a cavity comprising a substrate surface. Typically, cells in the cell suspension are allowed to settle onto the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber. After the cells have settled onto the substrate surface of the sample chamber (e.g., by a predetermined time interval of settling), at least one microscopic image of at least a portion of the cell monolayer is typically acquired. Typically, more than one image of the monolayer is acquired, each image corresponding to an imaging field of view of a respective different region within the imaging plane of that monolayer. Typically, the optimal depth level for imaging the monolayer is determined, for example, using techniques such as those described in Greenfield's U.S. Patent US10,176,565, which is incorporated herein by reference. For some applications, the respective imaging fields have different optimal depth levels than each other. For some applications, platelets already settled within the monolayer are identified within at least one microscopic image of at least a portion of the cell monolayer.

[0009] The inventors of this application have noted that, typically, even after sedimentation using the techniques described herein to form a monolayer, not all platelets in a blood sample settle within the monolayer, and some cells remain suspended in the cell solution. More specifically, the inventors have found that, typically, even after approximately two minutes of sedimentation, between 10% and 70% of the platelets in the sample have not settled within the monolayer's focused field of view. Generally, if the monolayer is allowed to form over a longer period, more platelets settle within the monolayer's focused field of view. However, the inventors have found that even when the monolayer is allowed to form over a relatively long period (e.g., between 15 and 30 minutes), some platelets remain suspended in the solution, preventing them from being placed within the monolayer's focused field of view. Generally, the number of platelets remaining suspended in the solution (preventing them from being placed within the monolayer's focused field of view), apart from the height of the cavity in which the cell solution is placed, depends on the time allowed to form the monolayer.

[0010] Therefore, for some applications, in order to accurately estimate the number of platelets in a sample, it is necessary to identify platelets not only within the monolayer focused field of view, but also those suspended in the cell solution. Typically, such platelets are identified by focusing the microscope at a depth level beyond the depth level used to image the cell monolayer (referred to herein as the "monolayer depth level"), and acquiring images at these additional depth levels. Typically, platelets within the images acquired at these additional depth levels are identified and counted, and the total number of platelets suspended in the cell solution is estimated based on the platelet counts within these images.

[0011] For some applications, the platelet count that has settled within the monolayer is compared with the platelet count that has not yet settled within the monolayer. Typically, an output is generated in response to the comparison. For some applications, a clinical condition is derived and output to the user based on the comparison of the platelet counts that have settled within the monolayer and the platelet counts that have not yet settled within the monolayer. For example, the degree of platelet activation within the sample can be derived at least in part based on the comparison. Optionally or additionally, the clinical condition can be derived based on platelet size, shape, settling time, and / or settling kinetics. For some such applications, platelet settling kinetics are determined by performing more than one imaging of the same imaging field (with time intervals between each image acquisition), and / or by determining the height of platelets present in the sample chamber up to a specific time.

[0012] For some applications, the computer processor generates an output based on a comparison that indicates that the sample should be prepared in a way different from how it was prepared (e.g., by changing the diluent in which the sample is diluted, by adding a platelet activator and / or by adding a coagulant).

[0013] For some applications, based on a comparison between platelet counts that have settled within the monolayer and those that have not, a portion of a blood sample may be invalid for at least some measurements of the sample. For example, if the ratio of non-settled platelets to settled platelets is greater than a first specific threshold and / or less than a second specific threshold, this could be interpreted as indicating a problem with the sample and / or its preparation.

[0014] For some applications, sample parameters (such as platelet volume, mean platelet volume, and / or median platelet volume) are determined based on measurements taken from a portion of the sample. For other applications, measurements are calibrated based on a comparison between the characteristics of platelets that have already settled in the monolayer and those that have not yet settled in the monolayer.

[0015] Therefore, according to some applications of the present invention, a method is provided, the method comprising:

[0016] At least a portion of the blood sample, which is a cell suspension, will be placed in a sample chamber that includes a cavity with a base surface;

[0017] Cells in the cell suspension are allowed to settle onto the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber.

[0018] When the microscope is focused at the monolayer depth level, at least one first microscopic image of at least a portion of the cell monolayer is acquired using the microscope, wherein the monolayer is within the focal length of the microscope at the monolayer depth level.

[0019] Identify platelets that have settled in a monolayer within at least one microscopic image;

[0020] Based on platelets identified in at least one microscopic image, determine the first platelet count of platelets that have settled in the monolayer;

[0021] When the microscope is focused at a depth level different from the monolayer depth level, at least one additional microscopic image of a portion of the sample is obtained using the microscope.

[0022] Identify platelets that have not yet settled in the monolayer in at least one additional microscopic image;

[0023] Based on platelets identified in at least one additional microscopic image, a second platelet count is determined for platelets that have not yet settled within the monolayer; and

[0024] The output is generated based on the first platelet count and the second platelet count.

[0025] In some applications, the method also includes deriving an error about the blood sample based at least in part on comparing a first platelet count and a second platelet count with each other, wherein the generated output includes an indication of the generated error.

[0026] In some applications, the method also includes deriving an error in blood sample preparation based at least in part on comparing a first platelet count and a second platelet count with each other, wherein the generated output includes an indication of the generated error.

[0027] In some applications, the method also includes deriving the subject’s clinical condition based at least in part on comparing a first platelet count and a second platelet count with each other, wherein the output generated includes an indication of the clinical condition.

[0028] In some applications, the method also includes deriving a measure of platelet activation based at least in part on comparing a first platelet count and a second platelet count with each other, wherein the generated output includes an indication of platelet activation.

[0029] In some applications, identifying platelets that have not yet settled in a monolayer within at least one additional microscopic image includes taking into account the interference of leukocytes placed in the monolayer on platelet visibility.

[0030] In some applications, identifying platelets that have not yet settled in a monolayer within at least one additional microscopic image includes taking into account the interference of red blood cells placed in the monolayer on platelet visibility.

[0031] In some applications:

[0032] Acquiring at least one first microscopic image of a portion of a cell monolayer includes: acquiring a first number of images of the monolayer at respective imaging fields of view while the microscope is focused at one or more monolayer depth levels, wherein the monolayer is within the focal length of the microscope at the monolayer depth levels;

[0033] Acquiring at least one additional microscopic image of a portion of the sample includes acquiring a second number of images at their respective imaging fields of view while the microscope is focused at a depth level different from the monolayer depth level; and

[0034] The ratio between the first quantity and the second quantity is greater than 2:1.

[0035] In some applications:

[0036] Acquiring at least one additional microscopic image of a portion of the sample includes acquiring more than one additional microscopic image while the microscope is focused at a respective depth level different from the monolayer depth level; and

[0037] Identifying platelets that have not yet settled in a monolayer within at least one additional microscopic image includes avoiding duplicate counting of platelets in images acquired at adjacent depth levels.

[0038] In some applications, avoiding duplicate platelet counts within images acquired at adjacent depth levels involves taking into account lateral platelet movement between acquisitions of images at adjacent depth levels.

[0039] Therefore, according to some applications of the present invention, a method is also provided, the method comprising:

[0040] At least a portion of the blood sample, which is a cell suspension, will be placed in a sample chamber that includes a cavity with a base surface;

[0041] Cells in the cell suspension are allowed to settle onto the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber.

[0042] When the microscope is focused at the monolayer depth level, at least one microscopic image of at least a portion of a cell monolayer is acquired using the microscope, wherein the monolayer is within the focal length of the microscope at the monolayer depth level.

[0043] Identify platelets that have settled in a monolayer within at least one microscopic image;

[0044] When the microscope is focused at a depth level different from the monolayer depth level, at least one additional microscopic image of a portion of the sample is obtained using the microscope.

[0045] Identify platelets that have not yet settled in the monolayer in at least one additional microscopic image; and

[0046] The output is generated at least in part based on platelets identified as having settled within the monolayer and platelets identified as not yet settled within the monolayer.

[0047] In some applications, the method also includes estimating the platelet count of a blood sample based on platelets identified as having settled within the monolayer and platelets identified as not having settled within the monolayer, wherein the generated output includes an indication of the generated platelet count.

[0048] In some applications, the method also includes deriving platelet sedimentation characteristics of a sample based on platelets identified as having settled within a monolayer and platelets identified as not having settled within a monolayer, wherein the generated output includes an indication of the generated platelet sedimentation characteristics.

[0049] In some applications, the method also includes deriving platelet characteristics within a sample based on a combination of platelets identified as having settled within the monolayer and platelets identified as not having settled within the monolayer, wherein the generated output includes an indication of the generated characteristics.

[0050] Therefore, according to some applications of the present invention, a method is also provided, the method comprising:

[0051] At least a portion of the suspension sample will be placed in a sample chamber that includes a cavity with a substrate surface;

[0052] Allow solids in the cell suspension to settle onto the substrate surface of the sample chamber to form a solid monolayer on the substrate surface of the sample chamber;

[0053] When the microscope is focused at the monolayer depth level, at least one microscopic image of at least a portion of a monolayer is acquired using the microscope, wherein the monolayer is within the focal length of the microscope at the monolayer depth level.

[0054] Identify a specific type of entity that has settled within a single layer within at least one microscopic image;

[0055] When the microscope is focused at a depth level different from the monolayer depth level, at least one additional microscopic image of a portion of the sample is obtained using the microscope.

[0056] Identify specific types of entities that have not yet settled within a monolayer in at least one additional microscopic image; and

[0057] Output is generated at least in part based on specific types of entities identified as having settled within a single layer and specific types of entities identified as not having settled within a single layer.

[0058] Therefore, according to some applications of the present invention, an apparatus is also provided, the apparatus comprising:

[0059] The sample chamber is a cavity including a substrate surface and is configured to receive at least a portion of a blood sample that is a cell suspension, and to allow cells in the cell suspension to settle on the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber.

[0060] The microscope is configured as follows:

[0061] When the microscope is focused at the monolayer depth level, at least one first microscopic image of at least a portion of the cell monolayer is acquired, wherein the monolayer is within the focal length of the microscope at the monolayer depth level.

[0062] Acquire at least one additional microscopic image of a portion of the sample while the microscope is focused at a depth level different from the monolayer depth level; and

[0063] The computer processor is configured as follows:

[0064] Identify platelets that have settled within the monolayer in at least one of the first microscopic images.

[0065] The first platelet count is determined based on platelets identified in at least one microscopic image, indicating that the platelets have settled within the monolayer.

[0066] Identify platelets that have not yet settled in the monolayer in at least one additional microscopic image.

[0067] Based on platelets identified in at least one additional microscopic image, a second platelet count is determined for platelets that have not yet settled within the monolayer; and

[0068] The output is generated based on the first platelet count and the second platelet count.

[0069] Therefore, according to some applications of the present invention, an apparatus is also provided, the apparatus comprising:

[0070] The sample chamber is a cavity including a substrate surface and is configured to receive at least a portion of a blood sample that is a cell suspension, and to allow cells in the cell suspension to settle on the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber.

[0071] The microscope is configured as follows:

[0072] When the microscope is focused at the monolayer depth level, at least one first microscopic image of at least a portion of the cell monolayer is acquired, wherein the monolayer is within the focal length of the microscope at the monolayer depth level.

[0073] Acquire at least one additional microscopic image of a portion of the sample while the microscope is focused at a depth level different from the monolayer depth level; and

[0074] The computer processor is configured as follows:

[0075] Identify platelets that have settled within the monolayer in at least one of the first microscopic images.

[0076] And platelets that have not yet settled in the monolayer were identified in at least one additional microscopic image, and

[0077] The output is generated at least in part based on platelets identified as having settled within the monolayer and platelets identified as not yet settled within the monolayer.

[0078] Therefore, according to some applications of the present invention, an apparatus is also provided, the apparatus comprising:

[0079] A sample chamber, the sample chamber being a cavity including a substrate surface and configured to receive at least a portion of a sample being a cell suspension, and to allow cells in the cell suspension to settle onto the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber.

[0080] The microscope is configured as follows:

[0081] When the microscope is focused at the monolayer depth level, at least one first microscopic image of at least a portion of the cell monolayer is acquired, wherein the monolayer is within the focal length of the microscope at the monolayer depth level.

[0082] Acquire at least one additional microscopic image of a portion of the sample while the microscope is focused at a depth level different from the monolayer depth level; and

[0083] The computer processor is configured as follows:

[0084] Identify specific types of entities that have settled within a single layer within at least one first microscopic image.

[0085] Identify specific types of entities that have not yet settled within a monolayer in at least one additional microscopic image, and

[0086] Output is generated at least in part based on specific types of entities identified as having settled within a single layer and specific types of entities identified as not having settled within a single layer.

[0087] The invention will be more fully understood from the following detailed description of embodiments thereof, taken in conjunction with the accompanying drawings, in which: Brief description of the attached diagram

[0089] Figure 1 This is a block diagram illustrating the components of a biological sample analysis system according to some applications of the present invention;

[0090] Figure 2A , Figure 2B and Figure 2C This is a schematic diagram of an optical measurement unit according to some applications of the present invention;

[0091] Figure 3A , Figure 3B and Figure 3C This is a schematic diagram of respective views of a sample carrier for both microscopic and optical density measurements according to some applications of the present invention.

[0092] Figure 4A and Figure 4B These are microscopic images acquired from a single layer depth level using their respective imaging parameters in bright-field imaging according to some applications of the present invention;

[0093] Figure 5 These are fluorescence micrographs obtained from a single layer depth level according to some applications of the present invention;

[0094] Figure 6A and Figure 6BThese are microscopic images obtained from a different depth level (i.e., not a single-layer depth level) using their respective imaging parameters in bright-field imaging according to some applications of the present invention.

[0095] Figure 7 These are fluorescence micrographs obtained from a different depth level according to some applications of the present invention;

[0096] Figure 8 This is a flowchart illustrating the steps of a method performed according to some applications of the present invention;

[0097] Figure 9 This is a flowchart illustrating the steps of another method performed according to some applications of the present invention; and

[0098] Figure 10 This is a flowchart illustrating the steps of another method performed according to some applications of the present invention.

[0099] Detailed implementation plan

[0100] Now for reference Figure 1 This is a block diagram illustrating the components of a biological sample analysis system 20 according to some applications of the present invention. Typically, a biological sample (e.g., a blood sample) is placed in a sample carrier 22. While the sample is placed in the sample carrier, optical measurements are performed on the sample using one or more optical measuring devices 24. For example, the optical measuring devices may include microscopes (e.g., digital microscopes), spectrophotometers, photometers, spectrometers, cameras, spectral cameras, hyperspectral cameras, fluorometers, fluorescence spectrophotometers, and / or photodetectors (such as photodiodes, photoresistors, and / or phototransistors). For some applications, the optical measuring devices include dedicated light sources (such as light-emitting diodes, incandescent light sources, etc.) and / or optical elements (such as lenses, diffusers, filters, etc.) for operating light collection and / or light emission.

[0101] Computer processor 28 typically receives and processes optical measurements performed by optical measuring devices. Furthermore, computer processor typically controls the acquisition of optical measurements performed by one or more optical measuring devices. Computer processor communicates with memory 30. A user (e.g., a laboratory technician or an individual from whom a sample is taken) sends instructions to computer processor via user interface 32. For some applications, the user interface includes a keyboard, mouse, joystick, touchscreen device (such as a smartphone or tablet), touchpad, trackball, voice command interface, and / or other types of user interfaces known in the art. Typically, computer processor generates output via output device 34. Furthermore, output device typically includes a display, such as a monitor, and output includes output displayed on the display. For some applications, the processor generates output on different types of visual, text, graphic, tactile, audio, and / or video output devices (e.g., speakers, headphones, smartphones, or tablets). For some applications, user interface 32 serves as both an input interface and an output interface; that is, it serves as an input / output interface. For some applications, the processor generates output on computer-readable media (e.g., non-transitory computer-readable media) such as a disk or portable USB drive, and / or on a printer.

[0102] Now for reference Figure 2A , Figure 2B and Figure 2C , Figure 2A , Figure 2B and Figure 2C This is a schematic diagram of an optical measurement unit 31 according to some applications of the present invention; Figure 2A An oblique view of the exterior of the fully assembled device is shown, while Figure 2B and Figure 2C The respective oblique views of the apparatus are shown, with the covers made transparent to allow the components within the apparatus to be seen. For some applications, one or more optical measuring devices 24 (and / or computer processor 28 and memory 30) are housed within an optical measuring unit 31. For optical measurements of a sample, a sample carrier 22 is placed within the optical measuring unit. For example, the optical measuring unit may define a slot 36 through which the sample carrier is inserted. Typically, the optical measuring unit includes a stage 64 configured to support the sample carrier 22 within the optical measuring unit. For some applications, a screen 63 on the cover of the optical measuring unit (e.g., a screen on the front cover of the optical measuring unit, as shown) functions as a user interface 32 and / or an output device 34.

[0103] Typically, the optical measurement unit includes a microscope system 37 (in Figures 2B-2CAs shown in the diagram, it is configured to perform microscopic imaging of a portion of a sample. For some applications, the microscope system includes a set of light sources 65 (which typically includes a set of bright-field light sources (e.g., light-emitting diodes) configured for bright-field imaging of the sample, a set of fluorescent light sources (e.g., light-emitting diodes) configured for fluorescence imaging of the sample, and a camera (e.g., a CCD camera or a CMOS camera) configured to image the sample. Typically, the optical measurement unit also includes an optical density measurement unit 39 (in... Figure 2C As shown in the diagram, it is configured to perform optical density measurement (e.g., light absorption measurement) on a second portion of the sample. For some applications, the optical density measurement unit includes a set of optical density measurement light sources (e.g., light-emitting diodes) and a photodetector, which are configured to perform optical density measurement on the sample. For some applications, each of the above-described light source groups (i.e., bright-field light source group, fluorescence light source group, and optical density measurement light source group) includes more than one light source (e.g., more than one light-emitting diode), wherein each light source is configured to emit light at its respective wavelength or in its respective wavelength band.

[0104] Now for reference Figure 3A and Figure 3B , Figure 3A and Figure 3B These are schematic diagrams of various views of a sample carrier 22 according to some applications of the present invention. Figure 3A A top view of the sample carrier is shown (for illustrative purposes, the top cover of the sample carrier is shown). Figure 3A (shown as opaque in the image), and Figure 3B The bottom view is shown (where relative to) Figure 3A The view shown indicates the sample carrier has been rotated about its short edge. Typically, a sample carrier comprises one or more sample chambers in a first set 52 for microscopic analysis of the sample and sample chambers in a second set 54 for optical density measurements of the sample. Typically, the sample chambers of the sample carrier are filled with a bodily sample, such as blood, via a sample inlet orifice 38. For some applications, the sample chambers define one or more outlet orifices 40. The outlet orifices are configured to assist in the filling of the sample chambers with the bodily sample by allowing air present in the sample chambers to escape from the sample chambers. Typically, as shown, the outlet orifices are positioned longitudinally opposite the inlet orifices (relative to the sample chambers of the sample carrier). For some applications, the outlet orifices thus provide a more efficient air escape mechanism than if the outlet orifices were placed closer to the inlet orifices.

[0105] refer to Figure 3CThis illustration shows an exploded view of a sample carrier 22 according to some applications of the invention. For some applications, the sample carrier comprises at least three components: a molding assembly 42, a glass layer 44 (e.g., a glass plate), and an adhesive layer 46 configured to bond the glass layer to the underside of the molding assembly. The molding assembly is typically made of a polymer (e.g., plastic) and is molded (e.g., via injection molding) to provide a sample chamber having a desired geometry. For example, as shown, the molding assembly is typically molded to define an inlet orifice 38, an outlet orifice 40, and a groove 48 surrounding a central portion of each sample chamber. The groove typically assists in filling the sample chamber by allowing air to flow to the outlet orifice and / or by allowing body sample to flow around the central portion of the sample chamber.

[0106] For some applications, when performing a complete blood count on a blood sample, using methods such as... Figures 3A-3C The sample carrier is shown in the diagram. For some such applications, the sample carrier is used in conjunction with an optical measurement unit 31, which typically references... Figures 2A-2C The configuration is shown and described. For some applications, a first portion of the blood sample is placed in the sample chamber of the first set 52 (which is used, for example, with the microscope system 37). Figures 2B-2C (As shown in the diagram) the sample is subjected to microscopic analysis, and a second portion of the blood sample is placed in the sample chamber of the second set 54 (which is used, for example, with the optical density measurement unit 39). Figure 2C (As shown in the diagram) Optical density measurement of the sample. For some applications, the sample chambers of the first group 52 include more than one sample chamber, while the sample chambers of the second group 54 include only a single sample chamber, as shown. However, the scope of this application includes the use of any number of sample chambers (e.g., a single sample chamber or more than one sample chamber) within the first group of sample chambers, the second group of sample chambers, or any combination thereof. The first portion of the blood sample is typically diluted relative to the second portion of the blood sample. For example, the diluent may contain pH buffers, staining agents, fluorescent staining agents, antibodies, sphering agents, lysis agents, etc. Typically, the second portion of the blood sample placed in the sample chamber of the second group 54 is a natural, undiluted blood sample. Optionally or additionally, the second portion of the blood sample may be a sample that has undergone some modification, including one or more of the following: for example, dilution (e.g., controlled dilution), addition of components or reagents, or fractionation.

[0107] For some applications, a first portion of the blood sample (placed in the sample chamber of group 52) is stained with one or more staining substances before microscopic imaging of the sample. For example, the staining substance may be configured to preferentially stain DNA relative to other cellular components. Optionally, the staining substance may be configured to preferentially stain all cellular nucleic acids relative to other cellular components. For example, the sample may be stained with acridine orange reagent, Hoechst reagent, and / or any other staining substance configured to preferentially stain DNA and / or RNA in the blood sample. Optionally, the staining substance is configured to stain all cellular nucleic acids, but staining of DNA and RNA is more clearly visible under certain lighting and filtering conditions, as is known for, for example, acridine orange. Images of the sample can be acquired using imaging conditions that allow detection of cells (e.g., bright field) and / or imaging conditions that allow visualization of stained bodies (e.g., appropriate fluorescent illumination). Typically, the first portion of the sample is stained with acridine orange reagent and Hoechst reagent. For example, a first (diluted) portion of a blood sample can be prepared using techniques such as those described in Pollak's US 9,329,129, which is incorporated herein by reference and describes a method for preparing a blood sample for analysis, including a dilution step that facilitates the identification and / or counting of components within a microscopic image of the sample. For some applications, the first portion of the sample is stained with one or more staining agents that make platelets within the sample visible under bright-field imaging conditions and / or fluorescence imaging conditions, for example, as described above. For example, the first portion of the sample can be stained with methylene blue and / or Romanowsky stains.

[0108] Same reference Figures 2B-2C Typically, the sample carrier 22 is supported within the optical measurement unit by a stage 64. Furthermore, the stage typically has a forked design, such that the sample carrier is supported by the stage around its edge, but without interfering with the visibility of the sample chamber of the sample carrier to the optical measurement apparatus. For some applications, the sample carrier is held within the stage such that the molding assembly 42 of the sample carrier is positioned above the glass layer 44, and the objective lens 66 of the microscope unit of the optical measurement unit is positioned below the glass layer of the sample carrier. Typically, at least some light sources 65 used during microscopic measurements of the sample (e.g., light sources used during bright-field imaging) illuminate the sample carrier from above the molding assembly. Additionally, typically, at least some additional light sources (not shown) illuminate the sample carrier from below (e.g., via the objective lens). For example, a light source used to excite the sample during fluorescence microscopy can illuminate the sample carrier from below (e.g., via the objective lens).

[0109] Typically, prior to microscopic imaging, a first portion of the blood (placed in the sample chamber of the first set 52) ​​is allowed to settle, such as to form a cell monolayer, for example using techniques described in, for example, Pollak’s US 9,329,129, which is incorporated herein by reference. Note that in the context of this application, the term monolayer is used to refer to a layer of cells that has settled, such as those placed in a single focused field of view of a microscope. Within a monolayer, some cell overlap may exist, such that two or more overlapping cell layers exist in certain areas. For example, red blood cells may overlap each other within a monolayer, and / or platelets may overlap with or be positioned above red blood cells within a monolayer.

[0110] For some applications, the microscopic analysis of the first portion of a blood sample is performed on a cellular monolayer. Typically, the first portion of the blood sample is imaged under bright-field imaging, i.e., under illumination from one or more light sources (e.g., one or more light-emitting diodes, which typically emit light in their respective spectral bands). Additionally, the first portion of the blood sample is often additionally imaged under fluorescence imaging. Fluorescence imaging is typically performed by directing light of a known excitation wavelength (i.e., wavelengths at which the stained object emits fluorescence if excited with light of those wavelengths) onto the sample to excite the stained object within the sample (i.e., the object that has absorbed the staining agent) and detecting the fluorescence. Typically, for fluorescence imaging, a set of separate light sources (e.g., one or more light-emitting diodes) is used to illuminate the sample at known excitation wavelengths.

[0111] As described with reference to Pollak's US 2019 / 0302099 (which is incorporated herein by reference), for some applications, sample chambers belonging to group 52 (for microscopic measurements) have different heights to facilitate the measurement of different measurands using microscopic images from each sample chamber, and / or different sample chambers for microscopic analysis of different sample types. For example, if a blood sample and / or a monolayer formed from the sample has a relatively low red blood cell density, measurements can be taken in a sample chamber of the sample carrier with a greater height (i.e., the sample chamber of the sample carrier has a greater height relative to different sample chambers with relatively low heights), such that sufficient cell density is present, and / or sufficient cell density is present in the monolayer formed from the sample to provide statistically reliable data. Such measurements may include, for example, red blood cell density measurements, measurements of other cellular properties (such as the count of abnormal red blood cells, the count of red blood cells including endosomes (e.g., pathogens, Howo-Jones bodies, etc.), and / or hemoglobin concentration. Conversely, if the blood sample and / or the monolayer formed from the sample has a relatively high density of red blood cells, such measurements can be performed on sample chambers of the sample carrier with a relatively low height, for example, such that there are sufficiently sparse cells, and / or such that there are sufficiently sparse cells within the cell monolayer formed from the sample, allowing for cell identification within a microscopic image. For some applications, this method can be performed even if the height variations between sample chambers belonging to group 52 are not precisely known.

[0112] For some applications, the sample chamber within the sample carrier on which optical measurements are performed is selected based on the object being measured. For example, a sample chamber with a greater height in the sample carrier can be used for white blood cell counting (e.g., to reduce statistical errors that may be caused by low counts in shallower regions), white blood cell differentiation, and / or detection of rarer forms of white blood cells. Conversely, to determine mean corpuscular hemoglobin content (MCH), mean corpuscular volume (MCV), erythrocyte distribution width (RDW), erythrocyte morphology characteristics, and / or erythrocyte abnormalities, microscopic images can be obtained from a sample chamber with a relatively lower height in the sample carrier, because in such a sample chamber, cells are relatively sparsely distributed throughout the area and / or form a monolayer in which cells are relatively sparsely distributed. Similarly, for platelet counting, platelet classification, and / or extraction of any other platelet properties (such as volume), microscopic images can be obtained from a sample chamber with a relatively low height in the sample carrier, because in such a sample chamber, there are fewer red blood cells (completely or partially) overlapping with platelets in the microscopic image and / or in a monolayer.

[0113] According to the embodiments described above, it is preferable to use a sample chamber with a lower height of the sample carrier for optical measurements of some metric objects within a sample (such as a blood sample), while it is preferable to use a sample chamber with a higher height of the sample carrier for optical measurements of other metric objects within such a sample. Therefore, for some applications, a first metric object within the sample is measured by performing a first optical measurement (e.g., by acquiring a microscopic image of the sample) on a portion of the sample placed in a first sample chamber belonging to group 52 of the sample carrier, and a second metric object of the same sample is measured by performing a second optical measurement (e.g., by acquiring a microscopic image of the sample) on a portion of the sample placed in a second sample chamber belonging to group 52 of the sample carrier. For some applications, the first and second metric objects are normalized relative to each other, for example, using techniques such as those described in Zait's US 2019 / 0145963, which is incorporated herein by reference.

[0114] Typically, in order to perform optical density measurements on a sample, it is desirable to know as accurately as possible the optical path length, volume, and / or thickness of the portion of the sample to be optically measured. Optical density measurements are usually performed on a second portion of the sample (which is typically placed in the sample chamber of the second set 54 in undiluted form). For example, the concentration and / or density of a component can be measured by measuring the sample's light absorption, transmission, fluorescence, and / or luminescence.

[0115] Same reference Figure 3BFor some applications, the sample chamber belonging to group 54 (for optical density measurement) defines at least a first region 56 (which is typically deeper) and a second region 58 (which is typically shallower). The height of the sample chamber varies between the first and second regions in a predefined manner, for example, as described in Pollak's US 2019 / 0302099, which is incorporated herein by reference. The heights of the first region 56 and the second region 58 of the sample chamber are defined by a lower surface defined by a glass plate and an upper surface defined by a molding assembly. The upper surface at the second region is stepped relative to the upper surface at the first region. The step between the upper surfaces at the first and second regions provides a predetermined height difference Δh between the regions, such that even if the absolute height of the known regions is not sufficiently accurate (e.g., due to tolerances in the manufacturing process), the height difference Δh is known to be sufficiently accurate to determine the parameters of the sample using the techniques described herein and as described in Pollak's US 2019 / 0302099, which is incorporated herein by reference. For some applications, the height of the sample chamber varies from a first region 56 to a second region 58, and then similarly from a second region to a third region 59, such that along the sample chamber, the first region 56 defines the maximum height region, the second region 58 defines the medium height region, and the third region 59 defines the minimum height region. For some applications, further variations in height occur along the length of the sample chamber, and / or the height varies gradually along the length of the sample chamber.

[0116] As described above, optical measurements of the sample are performed using one or more optical measuring devices 24 while the sample is placed in a sample carrier. Typically, the sample is observed through a glass layer via the optical measuring devices, the glass being transparent at least to the wavelengths typically used by the optical measuring devices. Typically, during optical measurements, the sample carrier is inserted into an optical measuring unit 31 that houses the optical measuring devices. Typically, the optical measuring unit houses the sample carrier such that the molding layer is positioned above the glass layer and the optical measuring unit is positioned below the glass layer of the sample carrier, enabling optical measurements of the sample through the glass layer. The sample carrier is formed by adhering the glass layer to the molding assembly. For example, the glass layer and the molding assembly may be bonded together during manufacturing or assembly (e.g., using thermal bonding, solvent-assisted bonding, ultrasonic welding, laser welding, thermal riveting, adhesives, mechanical clamping, and / or other substances). For some applications, the glass layer and the molding assembly are bonded together during manufacturing or assembly using an adhesive layer 46.

[0117] According to some applications of the invention, a portion of a blood sample containing a cell suspension is placed in a sample chamber comprising a cavity 55 including a base surface 57. Figure 3C(As shown in the diagram). Depending on the application, the cavity may be a closed (i.e., covered) cavity or an open (i.e., uncovered) cavity. For example, as described above, a first portion of a blood sample is typically placed in the sample chamber of the first group 52. For some applications, the first portion of the blood sample is placed in the sample chamber of the first group 52 after it has been diluted. Typically, cells in the cell suspension are allowed to settle on the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber. As described above, in the context of this application, the term monolayer is used to refer to a layer of cells that has settled, such as in a single focusing field of view (referred to herein as a “monolayer focusing field of view”) of a microscope. There may be some cell overlap within a monolayer, such that two or more overlapping cell layers exist in certain areas. For example, red blood cells may overlap each other within a monolayer, and / or platelets may overlap with or be positioned above red blood cells within a monolayer.

[0118] After cells have settled on the substrate surface of the sample chamber (e.g., by a predetermined time interval of settling), at least one microscopic image of at least a portion of the cell monolayer is typically acquired. More than one image of the monolayer is typically acquired, each corresponding to an imaging field of view of a different region within the imaging plane of that monolayer. Typically, the optimal depth level for imaging the monolayer is determined using techniques such as those described in Greenfield's US 10,176,565, which is incorporated herein by reference. For some applications, the respective imaging fields have different optimal depth levels than each other. For some applications, platelets settled within the monolayer are identified within at least one microscopic image of at least a portion of the cell monolayer.

[0119] The inventors of this application have noted that, typically, even after sedimentation using the techniques described herein to form a monolayer, not all platelets in a blood sample settle within the monolayer, and some cells remain suspended in the cell solution. More specifically, the inventors have found that, typically, even after approximately two minutes of sedimentation, between 10% and 70% of the platelets in the sample have not settled within the monolayer's focused field of view. Generally, if the monolayer is allowed to form over a longer period, more platelets settle within the monolayer's focused field of view. However, the inventors have found that even when the monolayer is allowed to form over a relatively long period (e.g., between 15 and 30 minutes), some platelets remain suspended in the solution, preventing them from being placed within the monolayer's focused field of view. Generally, the number of platelets remaining suspended in the solution (preventing them from being placed within the monolayer's focused field of view), apart from the height of the cavity in which the cell solution is placed, depends on the time allowed to form the monolayer.

[0120] Therefore, for some applications, in order to accurately estimate the number of platelets in a sample, it is necessary to identify platelets not only within the monolayer focused field of view, but also those suspended in the cell solution. Typically, such platelets are identified by focusing the microscope at a depth level beyond the depth level used to image the cell monolayer (referred to herein as the "monolayer depth level"), and acquiring images at these additional depth levels. Typically, platelets within the images acquired at these additional depth levels are identified and counted, and the total number of platelets suspended in the cell solution is estimated based on the platelet counts within these images.

[0121] As described above, platelets suspended in a cell solution are typically identified by acquiring microscopic images at additional depth levels. The height intervals between successive additional depth levels at which the cell solution is imaged typically depend on the focusing depth of the microscope optics. For example, depending on the focusing depth of the microscope optics, the microscope may focus on additional depth levels separated from each other at a specific height difference (e.g., between 1 micrometer and 10 micrometers) at the height of the sample portion within the sample chamber. Typically, within a single depth level, microscopic images of the sample chamber are acquired from a relatively large number of imaging fields, as described above. For example, microscopic images of a single layer may be acquired from between 100 and 500 imaging fields. Typically, between 5 and 100 (e.g., between 10 and 50) of these imaging fields are optimized for platelet identification by acquiring these images with an increased exposure time relative to other images. For some applications, for each additional depth level, microscopic images are acquired only from a subset of the imaging fields to reduce the amount of time required to image the sample portion relative to if microscopic images were acquired from all imaging fields. For example, microscopic images of between 3 and 10 imaging fields can be acquired at each additional depth level. For some applications, the ratio of (a) the number of imaging fields optimized for single-layer imaging and platelet identification (e.g., by increasing exposure time) to (b) the number of imaging fields for imaging at each additional depth level is between 2:1 and 10:1. For some applications, the ratio is 1:1.

[0122] For some applications, portions of the sample are imaged at a different depth level under bright-field imaging, for example, as described above. Optionally or additionally, portions of the sample are imaged at a different depth level under fluorescence imaging, for example, as described above. For some applications, a first portion of the sample is stained with one or more staining agents that make platelets within the sample visible under bright-field imaging and / or fluorescence imaging conditions, for example, as described above. For example, the first portion of the sample may be stained with methylene blue and / or Roche stain. For some applications, for example, images with imaging parameters that are not necessarily optimized for platelet detection are acquired at a different depth level in order to detect other entities that might otherwise be confused with platelets.

[0123] Now for reference Figure 4A and Figure 4B , Figure 4A and Figure 4B These are microscopic images acquired at a monolayer depth level using respective imaging parameters in bright-field imaging, according to some applications of the invention. As can be observed, platelets 60 can be identified within the image, and the platelets are surrounded by red blood cells 61. The red blood cells are in focus because the image is acquired at a monolayer depth level where the red blood cells have settled. See also... Figure 5 , Figure 5 These are fluorescence micrographs obtained at a single-layer depth level according to some applications of the present invention. Similarly, platelets 60 can be identified within the image, and the platelets are surrounded by faintly visible red blood cells.

[0124] Additionally, see reference Figure 6A and Figure 6B , Figure 6A and Figure 6B These are microscopic images acquired from a different depth level (i.e., not a single-layer depth level) using respective imaging parameters in bright-field imaging according to some applications of the invention. As can be observed, platelets 60 are identifiable within the image, and red blood cells are visible in the background. The red blood cells are out of focus because the image was not acquired at a single-layer depth level where the red blood cells have already settled. See also... Figure 7 , Figure 7 These are fluorescence micrographs obtained from a different depth level according to some applications of the present invention. Similarly, platelets 60 can be identified within the images.

[0125] Typically, platelets that have not yet settled within the monolayer are identified in images acquired at a different depth level. (Note that the identified platelets may include out-of-focus platelets in the other depth level.) Furthermore, the count of platelets not yet settled within the monolayer is typically estimated based on the platelets identified in images acquired at a different depth level. For some applications, to perform the estimation described above, the computer processor is configured to avoid double-counting platelets in images acquired at adjacent depth levels by identifying platelets in images placed at similar locations within the imaging plane and determining whether the platelets identified at each depth level likely correspond to a single (i.e., the same) platelet. For some applications, in determining whether the platelets identified at each adjacent depth level likely correspond to a single (i.e., the same) platelet, the computer processor considers lateral movement of platelets between images acquired at each adjacent depth level. For some applications, to perform the estimation described above, the computer processor is configured to consider the interference of leukocytes placed within the monolayer and / or suspended in the cell solution on platelet visibility. For example, in fluorescence images acquired at other depth levels, fluorescing leukocytes within a monolayer are typically visible and interfere with the visibility of platelets positioned above them. Therefore, platelets positioned above leukocytes within a monolayer may not be included in the platelet count. Since platelets within the volume above leukocytes are not included in the count, the number of platelets within these volumes can be estimated. Optionally or additionally, for the estimation described above, the computer processor is configured to account for the interference of red blood cells positioned within a monolayer that may interfere with platelet identification in bright-field images on platelet visibility.

[0126] For some applications, platelet counts within a single depth level and / or at additional depth levels are normalized, and / or other statistical analysis techniques are applied to one or both of these counts. For example, platelet counts within a specific imaging field of view can be normalized relative to the red blood cell count within that field of view, the time dynamics of platelet sedimentation can be incorporated into estimates of platelets that did not settle within the monolayer, and / or outliers can be removed from the estimates. For some applications, additional imaging fields are imaged if low platelet counts are present, if outlier fields of view exist (e.g., with very high or very low platelet counts), and / or if imaging fields at additional depth levels are rejected due to errors.

[0127] For some applications, the platelet count that has settled within the monolayer is compared with the platelet count that has not yet settled within the monolayer. Typically, an output is generated in response to the comparison. For some applications, a clinical condition is derived and output to the user based on the comparison of the platelet counts that have settled within the monolayer and the platelet counts that have not yet settled within the monolayer. For example, the degree of platelet activation within the sample can be derived at least in part based on the comparison. Optionally or additionally, the clinical condition can be derived based on platelet size, shape, settling time, and / or settling kinetics. For some such applications, platelet settling kinetics are determined by performing more than one imaging of the same imaging field (with time intervals between each image acquisition), and / or by determining the height of platelets present in the sample chamber up to a specific time.

[0128] For some applications, the computer processor generates an output based on a comparison that indicates that the sample should be prepared in a way different from how it was prepared (e.g., by changing the diluent in which the sample is diluted, by adding a platelet activator and / or by adding a coagulant).

[0129] For some applications, a portion of a blood sample is invalid for performing at least some measurements on the sample, based on a comparison between platelet counts that have settled within the monolayer and those that have not. For example, if the ratio of non-settled platelets to settled platelets is greater than a first specific threshold and / or less than a second specific threshold, this can be interpreted as indicating a problem with the sample and / or its preparation.

[0130] For some applications, sample parameters (such as platelet volume, mean platelet volume, and / or median platelet volume) are determined based on measurements taken on a first portion of the sample. For other applications, measurements are calibrated based on a comparison between the characteristics of platelets that have already settled in the monolayer and those that have not yet settled in the monolayer.

[0131] Now for reference Figure 8 and Figure 9 , Figure 8 and Figure 9This is a flowchart illustrating the steps of a method for processing platelets in a blood sample according to some applications of the present invention. For some applications, a cell suspension is placed in a sample chamber (step 70) and allowed to settle to form a monolayer (step 71). Subsequently, with the microscope focused at the monolayer depth level, at least one image is acquired (step 72), and platelets already settled in the monolayer are identified in the image (step 74). Additionally, with the microscope focused at a depth level different from the monolayer depth level, at least one microscopic image of the sample is acquired (step 73), and platelets not yet settled in the monolayer are identified in at least one microscopic image at a depth level different from the monolayer depth level (step 75). As shown, an output is generated at least in part based on platelets identified as having settled in the monolayer and platelets identified as not yet settled in the monolayer (step 79). For some applications, such as… Figure 9 As shown, after identifying platelets in the image (steps 74 and 75), a first platelet count of platelets that have settled within the monolayer is determined (step 76), and a second count of platelets that have not yet settled within the monolayer is determined (step 77). An output is then generated based on the first and second platelet counts (step 78). For example, according to the method described above, the output can be generated based on a comparison between the first and second platelet counts.

[0132] Generally, it should be noted that although some applications of the invention have been described with respect to platelets in blood samples, the scope of the invention includes applications of the apparatus and methods described herein, with necessary modifications, to various entities in various samples. For example, the apparatus and methods described for identifying platelets not yet settled in a monolayer can be modified to identify other entities in blood samples. Such entities may include leukocytes, abnormal leukocytes, circulating tumor cells, erythrocytes, reticulocytes, Howell-Jolly bodies, foreign bodies (such as bacteria, fungi, yeast, or parasites), entities added to the diluent (such as beads), etc.

[0133] Now for reference Figure 10 , Figure 10 This is a flowchart illustrating the steps of a method for processing entities within a blood sample according to some applications of the present invention. (Refer to the above text.) Figure 8 and Figure 9The process involves placing a cell suspension in a sample chamber (step 70) and allowing it to settle to form a monolayer (step 71). Subsequently, with the microscope focused at the monolayer depth level, at least one image is acquired (step 72), and specific types of entities are identified in the image (step 92). Additionally, with the microscope focused at a depth level different from the monolayer depth level, at least one microscopic image of the sample is acquired (step 73), and specific types of entities not yet settled within the monolayer are identified in at least one microscopic image at a depth level different from the monolayer depth level (step 94). As shown, an output is generated based at least in part on the specific types of entities identified as having settled within the monolayer and the specific types of entities identified as not yet settled within the monolayer (step 96).

[0134] For some applications, the devices and methods described herein are modified as necessary for use with biological samples, such as blood, saliva, semen, sweat, sputum, vaginal fluid, feces, breast milk, bronchoalveolar lavage fluid, gastric lavage fluid, tears, and / or nasal discharge. Biological samples can be derived from any organism and are typically from warm-blooded animals. For some applications, biological samples are from mammals, such as human samples. For some applications, samples are taken from any domesticated animal, zoo animal, and farm animal, including but not limited to dogs, cats, horses, cattle, and sheep. Optionally or additionally, biological samples are taken from animals used as disease vectors, including deer or rats.

[0135] For some applications, the devices and methods described herein are applied to non-human samples. With necessary modifications, for some applications, the sample is an environmental sample, such as a water (e.g., groundwater) sample, a surface swab, a soil sample, an air sample, or any combination thereof. In some embodiments, the sample is a food sample, such as a meat sample, a dairy product sample, a water sample, a detergent sample, a beverage sample, and / or any combination thereof.

[0136] For some applications, such as those described herein, the sample is a sample that includes blood or its components (e.g., diluted or undiluted whole blood samples, samples that mainly consist of red blood cells, or diluted samples that mainly consist of red blood cells), and parameters related to components in the blood (such as platelets, white blood cells, abnormal white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howell-Jolly bodies, etc.) are determined.

[0137] The applications of the invention described herein can take the form of a computer program product accessible from a computer-usable or computer-readable medium (e.g., a non-transitory computer-readable medium) that provides program code used by or in conjunction with a computer or any instruction execution system (such as computer processor 28). For the purposes of this description, a computer-usable or computer-readable medium can be any device that may include, store, communicate, propagate, or transmit a program used by or in conjunction with an instruction execution system, device, or apparatus. The medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or device or apparatus) or a propagation medium. Typically, a computer-usable or computer-readable medium is a non-transitory computer-usable or non-transitory computer-readable medium.

[0138] Examples of computer-readable media include semiconductor or solid-state memory, magnetic tape, removable computer disks, random access memory (RAM), read-only memory (ROM), hard disks, and optical discs. Current examples of optical discs include optical disc read-only memory (CD-ROM), optical disc read / write (CD-R / W), and DVDs.

[0139] A data processing system suitable for storing and / or executing program code will include at least one processor (e.g., computer processor 28) directly or indirectly coupled to a memory element (e.g., memory 30) via a system bus. The memory element may include local memory, mass storage, and cache memory used during actual execution of the program code, the cache memory providing transient storage of at least some of the program code to reduce the number of times code must be retrieved from mass storage during execution. The system can read the instructions of the present invention on a program storage device and follow those instructions to perform the methods of embodiments of the present invention.

[0140] A network adapter can be coupled to a processor, enabling the processor to become coupled to other processors or remote printers or storage devices via an intervening private or public network. Modems, cable modems, and Ethernet cards are just a few of the types of network adapters currently available.

[0141] The computer program code used to perform the operations of this invention can be written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Java, Smalltalk, C++, etc., and conventional programming languages ​​such as C or similar programming languages.

[0142] It should be understood that the algorithms described herein can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via a computer processor (e.g., computer processor 28) or the processor of the other programmable data processing apparatus, create means for implementing the functions / operations specified in the algorithms described herein. These computer program instructions can also be stored in a computer-readable medium (e.g., a non-transitory computer-readable medium) that can instruct a computer or other programmable data processing apparatus to operate in a particular manner, such that the instructions stored in the computer-readable medium produce an instruction means including means for implementing the flowchart blocks and the functions / operations specified in the algorithm. The computer program instructions can also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process, such that the instructions, which execute on the computer or other programmable apparatus, provide processing for implementing the functions / operations specified in the algorithms described herein.

[0143] Computer processor 28 is typically a hardware device programmed with computer program instructions to produce a dedicated computer. For example, when programmed to perform the algorithms described herein, computer processor 28 is typically used as a dedicated sample analysis computer processor. Generally, the operations performed by computer processor 28 described herein convert the physical state of memory 30 (which is a real physical object) into different magnetic polarities, charges, etc., depending on the memory technology used.

[0144] The apparatus and methods described herein may be used in conjunction with the apparatus and methods described in any of the following patents or patent applications, all of which are incorporated herein by reference:

[0145] Bachelet, US 9,522,396;

[0146] Greenfield’s US 10,176,565;

[0147] Pollak's US 10,640,807;

[0148] Pollak's US 9,329,129;

[0149] Pollak's US 10,093,957;

[0150] Yorav Raphael’s US 10,831,013;

[0151] Bachelet, US 10,843,190;

[0152] Yorav Raphael's US 10,482,595;

[0153] Eshel's US 10,488,644;

[0154] Eshel's WO 17 / 168411;

[0155] Pollak's US 2019 / 0302099;

[0156] Zait's US 2019 / 0145963; and

[0157] Yorav-Raphael's WO 19 / 097387.

[0158] Those skilled in the art will understand that the present invention is not limited to what has been specifically shown and described above. Rather, the scope of the invention includes combinations and sub-combinations of the various features described above, as well as variations and modifications thereof that do not exist in the prior art and will come to the mind of those skilled in the art upon reading the foregoing description.

Claims

1. A method of detecting platelets in a blood sample, the method comprising: placing at least a portion of a blood sample that is a cell suspension in a sample chamber that is a cavity comprising a base surface; allowing cells in the cell suspension to settle on the base surface of the sample chamber to form a cell monolayer on the base surface of the sample chamber; acquiring, using a microscope, at least one first microscopic image of at least a portion of the cell monolayer while the microscope is focused at a monolayer depth level at which the monolayer is within a focal distance of the microscope; identifying, within the at least one first microscopic image, platelets that have settled within the monolayer; determining, based on the platelets identified within the at least one first microscopic image, a first platelet count of platelets that have settled within the monolayer; acquiring, using the microscope, at least one additional microscopic image of the portion of the sample while the microscope is focused at a depth level that is different from the monolayer depth level; identifying, within the at least one additional microscopic image, platelets that have not settled within the monolayer; determining, based on the platelets identified within the at least one additional microscopic image, a second platelet count of platelets that have not settled within the monolayer; comparing the first platelet count and the second platelet count to one another; and generating an output based on the comparison of the first platelet count and the second platelet count to one another.

2. The method of claim 1, further comprising deriving an error regarding the blood sample based at least in part on comparing the first platelet count and the second platelet count to one another, wherein generating the output comprises generating an indication of the error.

3. The method of claim 1, further comprising deriving an error regarding the blood sample preparation based at least in part on comparing the first platelet count and the second platelet count to one another, wherein generating the output comprises generating an indication of the error.

4. The method of claim 1, further comprising deriving a clinical condition of a subject based at least in part on comparing the first platelet count and the second platelet count to one another, wherein generating the output comprises generating an indication of the clinical condition.

5. The method of claim 1, further comprising deriving a measure of platelet activation based at least in part on comparing the first platelet count and the second platelet count to one another, wherein generating the output comprises generating an indication of the platelet activation.

6. The method of claim 1, wherein identifying, within the at least one additional microscopic image, platelets that have not settled within the monolayer comprises accounting for interference with platelet visibility by white blood cells disposed within the monolayer.

7. The method of claim 1, wherein identifying, within the at least one additional microscopic image, platelets that have not settled within the monolayer comprises accounting for interference with platelet visibility by red blood cells disposed within the monolayer.

8. The method of claim 1, wherein: Acquiring the at least one first microscopic image of the portion of the cell monolayer includes acquiring, at respective imaging fields of view, a first number of images of the monolayer while the microscope is focused at one or more monolayer depth levels at which the monolayer is within a focal distance of the microscope; Acquiring the at least one additional microscopic image of the portion of the sample includes acquiring, at respective imaging fields of view, a second number of images while the microscope is focused at depth levels different from the monolayer depth levels; and A ratio between the first number and the second number is greater than 2:

1.

9. The method of any one of claims 1-8, wherein: Acquiring the at least one additional microscopic image of the portion of the sample includes acquiring more than one additional microscopic image while the microscope is focused at respective depth levels different from the monolayer depth levels; and Identifying, within the at least one additional microscopic image, platelets that have not settled within the monolayer includes refraining from double counting platelets within images acquired at adjacent depth levels.

10. The method of claim 9, wherein refraining from double counting platelets within images acquired at adjacent depth levels includes accounting for lateral movement of platelets between acquisition of images acquired at adjacent depth levels.

11. A method of detecting platelets in a blood sample, the method comprising: placing at least a portion of a blood sample that is a cell suspension in a sample chamber that is a cavity comprising a base surface; allowing cells in the cell suspension to settle on the base surface of the sample chamber to form a cell monolayer on the base surface of the sample chamber; acquiring, using a microscope, at least one microscopic image of at least a portion of the cell monolayer while the microscope is focused at a monolayer depth level at which the monolayer is within a focal distance of the microscope; identifying, within the at least one microscopic image, platelets that have settled within the monolayer; acquiring, using the microscope, at least one additional microscopic image of the portion of the sample while the microscope is focused at a depth level different from the monolayer depth level; identifying, within the at least one additional microscopic image, platelets that have not settled within the monolayer; comparing platelets identified as having settled within the monolayer to platelets identified as not having settled within the monolayer to one another; and generating an output based at least in part on the comparison of platelets identified as having settled within the monolayer to platelets identified as not having settled within the monolayer to one another.

12. The method of claim 11, further comprising estimating a platelet count of the blood sample based on platelets identified as having settled within the monolayer and platelets identified as not having settled within the monolayer, wherein generating an output includes generating an indication of the platelet count.

13. The method of claim 11 or claim 12, further comprising deriving a platelet sedimentation signature of the sample based on the platelets identified as having settled within the monolayer and the platelets identified as not having settled within the monolayer, wherein generating an output comprises generating an indication of the platelet sedimentation signature.

14. The method of claim 11 or claim 12, further comprising deriving a platelet signature within the sample based on a combination of the platelets identified as having settled within the monolayer and the platelets identified as not having settled within the monolayer, wherein generating an output comprises generating an indication of the derived signature.

15. The method of claim 13, further comprising deriving a platelet signature within the sample based on a combination of the platelets identified as having settled within the monolayer and the platelets identified as not having settled within the monolayer, wherein generating an output comprises generating an indication of the derived signature.

16. A method of detecting entities in a blood sample, the method comprising: placing at least a portion of a blood sample that is a suspension in a sample chamber that is a cavity comprising a base surface; allowing entities in the cell suspension to settle on the base surface of the sample chamber to form an entity monolayer on the base surface of the sample chamber; acquiring at least one microscopic image of at least a portion of the entity monolayer using a microscope while the microscope is focused at a monolayer depth level at which the monolayer is within a focal distance of the microscope; identifying entities within the monolayer that have settled within the monolayer within the at least one microscopic image; acquiring at least one additional microscopic image of the portion of the sample using the microscope while the microscope is focused at a depth level different from the monolayer depth level; identifying the entities that have not settled within the monolayer within the at least one additional microscopic image; comparing the entities identified as having settled within the monolayer to the entities identified as not having settled within the monolayer to one another; and generating an output based at least in part on the comparison of the entities identified as having settled within the monolayer to the entities identified as not having settled within the monolayer to one another, wherein the entities are selected from the group consisting of white blood cells, circulating tumor cells, red blood cells, and Howell-Jolly bodies.

17. The method of claim 16, wherein the entities are abnormal white blood cells or reticulocytes.

18. The method of claim 16 or 17, wherein identifying entities within the monolayer that have settled within the monolayer within the at least one microscopic image comprises identifying entities within the monolayer that have settled within the monolayer within the at least one microscopic image.

19. An apparatus for detecting platelets in a blood sample, the apparatus comprising: a sample chamber that is a cavity comprising a base surface and configured to receive at least a portion of a blood sample that is a cell suspension and allow cells in the cell suspension to settle on the base surface of the sample chamber to form a cell monolayer on the base surface of the sample chamber; a microscope configured to: acquiring at least one first microscopic image of at least a portion of the cell monolayer, the monolayer being within a focal distance of the microscope at a monolayer depth level, and acquiring at least one further microscopic image of the portion of the sample while the microscope is focused at a depth level different from the monolayer depth level; and a computer processor configured to: identify, within the at least one first microscopic image, platelets that have settled within the monolayer, determine, based on the platelets identified within the at least one microscopic image, a first platelet count of platelets that have settled within the monolayer, identify, within the at least one further microscopic image, platelets that have not settled within the monolayer, determine, based on the platelets identified within the at least one further microscopic image, a second platelet count of platelets that have not settled within the monolayer; compare the first platelet count and the second platelet count to each other; and generate an output based on the comparison of the first platelet count and the second platelet count to each other.

20. An apparatus for detecting platelets in a blood sample, the apparatus comprising: a sample chamber, the sample chamber being a cavity comprising a base surface and configured to receive at least a portion of a blood sample that is a cell suspension and to allow cells in the cell suspension to settle on the base surface of the sample chamber to form a cell monolayer on the base surface of the sample chamber; a microscope, the microscope being configured to: acquire at least one first microscopic image of at least a portion of the cell monolayer, the monolayer being within a focal distance of the microscope at a monolayer depth level, and acquire at least one further microscopic image of the portion of the sample while the microscope is focused at a depth level different from the monolayer depth level; and a computer processor configured to: identify, within the at least one first microscopic image, platelets that have settled within the monolayer, identify, within the at least one further microscopic image, platelets that have not settled within the monolayer, compare the platelets identified as having settled within the monolayer to the platelets identified as not having settled within the monolayer to each other; and generate an output based at least in part on the comparison of the platelets identified as having settled within the monolayer and the platelets identified as not having settled within the monolayer to each other.

21. An apparatus for detecting entities in a blood sample, the apparatus comprising: a sample chamber, the sample chamber being a cavity comprising a base surface and configured to receive at least a portion of a blood sample that is a cell suspension and to allow cells in the cell suspension to settle on the base surface of the sample chamber to form a cell monolayer on the base surface of the sample chamber; a microscope, the microscope being configured to: acquiring at least one first microscopic image of at least a portion of the cell monolayer, the monolayer being within a focal distance of the microscope at a monolayer depth level when the microscope is focused at the monolayer depth level, and acquiring at least one further microscopic image of the portion of the sample when the microscope is focused at a depth level different from the monolayer depth level; and a computer processor configured to: identify, within the at least one first microscopic image, entities that have settled within the monolayer, identify, within the at least one further microscopic image, the entities that have not settled within the monolayer, compare the entities identified as having settled within the monolayer with the entities identified as not having settled within the monolayer to each other; and generate an output based at least in part on the comparison of the entities identified as having settled within the monolayer and the entities identified as not having settled within the monolayer to each other, wherein the entities are selected from the group consisting of white blood cells, circulating tumor cells, red blood cells, and Howell-Jolly bodies.

22. The apparatus of claim 21, wherein the entities are abnormal white blood cells or reticulocytes.

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