Explaining sources of error in optical measurements

Abstract

Determining the characteristics of a biological sample, such as a blood sample. An apparatus and method are described that include placing at least a portion of a blood sample in a sample chamber (52) and acquiring a microscopic image of the portion of the blood sample. Identifying candidates for a given entity within the blood sample within the microscopic image. Authenticating at least some of the candidates as the given entity by performing further analysis on the candidates. Comparing the number of candidates for the given entity with the number of authenticated candidates for the given entity, and invalidating use of at least a portion of the sample for performing at least some measurements on the sample based at least in part on the relationship between the number of candidates and the number of authenticated candidates. Other applications are also described.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. US62 / 924,229 to Pecker, entitled “Accounting for errors in optical measurements,” filed October 22, 2019.

[0002] This application claims priority to U.S. Provisional Patent Application No. US62 / 924,229, filed October 22, 2019, to Pecker, which is related to a PCT application entitled "Accounting for errors in optical measurements" filed on the same date.

[0003] The above-cited applications are incorporated herein by reference.

[0004] Some applications of the subject matter disclosed herein relate generally to the analysis of body samples, and specifically to optical density and microscopic measurements performed on blood samples. [Background technology]

[0005] In some optically based methods (e.g., diagnostic and / or analytical methods), properties of a biological sample, such as a blood sample, are determined by performing optical measurements. For example, component density (e.g., number of components per unit volume) can be determined by counting components in a microscopic image. Similarly, component concentration and / or density can be measured by performing optical absorption, transmittance, fluorescence, and / or luminescence measurements on the sample. Typically, the sample is loaded onto a sample carrier and measurements are performed on a portion of the sample contained within a chamber of the sample carrier. The measurements performed on the portion of the sample contained within the chamber of the sample carrier are analyzed to determine properties of the sample. Summary of the Invention

[0006] According to some applications of the invention, a biological sample (e.g., a blood sample) is placed in a sample carrier. While the sample is in the sample carrier, optical measurements are performed on the sample using one or more optical measurement devices. For example, the optical measurement devices may include a microscope (e.g., a digital microscope), a spectrophotometer, a photometer, a spectrometer, a camera, a spectral camera, a hyperspectral camera, a fluorometer, a fluorescence spectrometer, and / or a light detector (such as a photodiode, a photoresistor, and / or a phototransistor). In some applications, the optical measurement devices include a dedicated light source (such as a light emitting diode, an incandescent light source, and / or an optical element (such as a lens, a diffuser, a filter, and / or the like) for collecting and / or manipulating light emission. In some applications, a microscope system generally similar to that described in U.S. Patent Application Publication No. US2014 / 0347459 to Greenfield, which is incorporated herein by reference, is used.

[0007] Typically, a computer processor receives and processes the optical measurements performed by the optical measurement devices. More typically, the computer processor controls the acquisition of the optical measurements performed by one or more optical measurement devices. In some applications, the optical measurement devices are housed in an optical measurement unit. A sample carrier is placed in the optical measurement unit to perform optical measurements on the sample. Typically, the optical measurement unit includes a microscope system configured to perform microscopic imaging of a portion of the sample. In some applications, the microscope system includes a set of bright field light sources (e.g., light emitting diodes) configured to be used for bright field imaging of the sample, a set of fluorescent light sources (e.g., light emitting diodes) configured to be used for fluorescent imaging of the sample, and a camera (such as a CCD camera and / or a CMOS camera) configured to image the sample. Typically, the optical measurement unit also includes an optical density measurement unit configured to perform optical density measurements (e.g., optical absorption measurements) on a second portion of the sample. In some applications, the optical density measurement unit includes a set of optical density measurement light sources (e.g., light emitting diodes) and photodetectors configured to perform optical density measurements on the sample. In some applications, each of the aforementioned light source sets (i.e., the bright field light source set, the fluorescent light source set, and the optical density measurement light source set) includes multiple light sources (e.g., multiple light emitting diodes), each of which is configured to emit light at a respective wavelength or in a respective wavelength range.

[0008] According to some applications of the invention, various techniques are implemented (typically by a computer processor) to determine whether various errors have occurred and, optionally, to identify the source of the error if an error has occurred. Such errors may arise from the preparation of the entire sample. For example, the sample may have been left on the sample carrier for too long before a measurement was performed (which may result in the sample deteriorating and / or a dye mixed with one or both portions of the sample may be excessively absorbed by an entity within the sample). Alternatively, errors may arise from the preparation of a particular portion of the sample. Alternatively or additionally, errors may arise from errors in the sample carrier (such as the material of the sample carrier itself being dirty and / or there being dirt or spilled blood on the sample carrier), and / or errors in the microscope system itself, such as the illumination (e.g. light emitting diodes used for bright field imaging and / or light emitting diodes used during fluorescent imaging of the sample), and / or errors related to components or controllers of the optical path and / or motors and stages, and / or errors arising from the environment in which the device is placed (such as relative humidity, temperature, pressure, particle concentration, or some other environmental factor). Furthermore, alternatively or additionally, there may be inherent problems with the sample (such as a very low or high number of certain entities, or too long a time has passed since the sample collection was performed). This means that the computer processor cannot perform certain measurements with sufficient accuracy and / or the computer processor must flag this to the user.

[0009] In some applications, in response to identifying the error, the computer processor outputs a message indicating the error and / or indicating the source of the error. In some applications, in response to identifying the error, the computer processor does not perform a particular measurement on the blood sample. In some applications, in response to identifying the error, the computer processor does not perform a measurement on the sample, and / or informs the user that the sample is invalid, and / or instructs the user to repeat the sample preparation with a new test kit, and / or recollects the blood sample. Alternatively or additionally, to account for the cause of the error, the computer processor determines a particular parameter of the blood sample by calibrating the measurement performed on the blood sample.

[0010] Some applications account for one or more of the following causes of errors, for example in one or more of the ways described above:

[0011] Errors in microscope devices such as: - Step loss of the motor that moves the microscope stage -Changes in backlash of the microscope stage movement - Timing changes between the microscope camera and the microscope stage - Alignment between the microscope camera and the microscope stage (e.g. due to relative rotation between these elements) Problems with the optical system (e.g. changes in focus quality over time caused by the sample or by microscope stage debris (e.g. scratches)) -Leveling changes in microscope systems - the change in expected focus position along the z-axis (i.e. the optical axis) -Loss of communication between elements -Changes in camera linear response

[0012] Errors caused by environmental factors such as: -Devices outside the permitted temperature, humidity, altitude, etc. ranges -Specific components that are out of target range

[0013] Common errors caused by factors such as: - Scan time -Device boot time -Available working / storage memory

[0014] Some example techniques for identifying errors and explaining the causes of such errors are described below.

[0015] Thus, according to some applications of the present invention: Preparing a blood sample for analysis, comprising: placing a blood sample in a sample chamber; and placing a sample chamber containing a blood sample within a microscope unit; preparing the blood sample for analysis by acquiring, using a microscope of the microscope unit, one or more microscopic images of a sample chamber in which the blood sample is placed; determining, based on the one or more images, an amount of one or more cell types in the sample chamber that had already settled in the sample chamber prior to acquisition of the one or more microscopic images; determining a characteristic of the sample at least in part accordingly; and A method is provided that includes:

[0016] In some applications, preparing the blood sample for analysis further includes staining the blood sample with one or more dyes.

[0017] In some applications, Determining the amount of one or more cell types in the sample chamber that have already settled in the sample chamber prior to acquisition of the one or more microscopic images includes determining whether a threshold amount of red blood cells in the sample chamber have already settled in the sample chamber prior to acquisition of the one or more microscopic images; Determining the characteristics of the sample includes invalidating use of at least a portion of the sample to perform at least some measurements on the sample in response to a determination that a threshold amount of red blood cells in the sample chamber had already settled in the sample chamber prior to acquisition of the one or more microscopic images.

[0018] In some applications, the method further comprises: placing the sample chamber containing the blood sample in a microscope unit and then allowing one or more cell types to form a monolayer of cells in the sample chamber; acquiring one or more additional sets of microscopic images of the monolayer of cells; performing one or more measurements on the sample by analyzing the set of one or more additional microscopic images; Includes.

[0019] In some applications, the method further includes determining an indication of how long the blood sample was in the sample chamber prior to acquisition of the one or more microscopic images based on the amount of one or more cell types that had already settled in the sample chamber prior to acquisition of the one or more microscopic images.

[0020] In some applications, The method further includes performing one or more measurements on the sample; Determining the amount of one or more cell types in the sample chamber that have already settled in the sample chamber prior to acquisition of the one or more microscopic images includes determining whether a threshold amount of red blood cells in the sample chamber have already settled in the sample chamber prior to acquisition of the one or more microscopic images; Performing one or more measurements on the sample includes calibrating the measurements in response to determining that a threshold amount of red blood cells in the sample chamber have already settled in the sample chamber prior to acquisition of the one or more microscopic images.

[0021] In some applications, preparing the blood sample for analysis further includes staining the blood sample with one or more dyes, and calibrating the measurement includes calibrating the measurement to account for an amount of stained entities in the blood sample, as indicated by a threshold amount of red blood cells in the sample chamber having already settled in the sample chamber prior to acquisition of the one or more microscopic images.

[0022] Furthermore, in accordance with some applications of the present invention, placing a portion of the blood sample in a sample chamber; acquiring a microscopic image of red blood cells in the blood sample while the red blood cells in the blood sample are settling in a sample chamber; determining a sedimentation dynamics characteristic of the blood sample by analyzing the image; generating an output accordingly; and A method is provided that includes:

[0023] In some applications, disposing a portion of the blood sample in the sample chamber includes disposing an undiluted portion of the blood sample in the sample chamber, and obtaining a microscopic image of red blood cells in the blood sample includes obtaining a microscopic image of red blood cells in the undiluted blood sample.

[0024] In some applications, determining the sedimentation dynamics characteristics of the blood sample by analyzing the images includes determining the sedimentation dynamics characteristics of the blood sample in real time with respect to sedimentation of red blood cells within the blood sample.

[0025] In some applications, determining the sedimentation dynamics characteristics of the blood sample by analyzing the image includes determining the sedimentation dynamics characteristics of the blood sample while red blood cells within the blood sample are still sedimenting.

[0026] In some applications, determining a sedimentation dynamics characteristic of the blood sample by analyzing the image includes determining a sedimentation rate of red blood cells in the blood sample by analyzing the image.

[0027] Furthermore, according to some applications of the present invention: placing a first portion of the blood sample in a first sample chamber; placing a second portion of the blood sample in a second sample chamber; Obtaining a microscopic image of a first portion of the blood sample; performing an optical density measurement on a second portion of the blood sample; Detecting that a concentration of a given entity in a sample exceeds a threshold; determining the cause of the concentration of a given entity exceeding a threshold by comparing a parameter determined from the microscopic image of the first portion of the blood sample with a parameter determined from an optical density measurement performed on a second portion of the blood sample; A method is provided that includes:

[0028] In some applications, determining the cause of the concentration of a given entity exceeding a threshold value includes determining that the blood sample itself is the cause of the concentration of a given entity in the sample exceeding a threshold value by determining that the concentration of the entity shown by the microscopic image is similar to the concentration of the entity determined from the optical density measurement.

[0029] In some applications, determining the cause of the concentration of the given entity exceeding the threshold includes determining that the preparation of one of the portions of the blood sample is the cause of the concentration of the given entity in the sample exceeding the threshold by determining that the concentration of the entity indicated by the microscopic image is different from the concentration of the entity determined from the optical density measurement.

[0030] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample in a sample chamber; obtaining a microscopic image of said portion of the blood sample; identifying at least one type of entity selected from the group consisting of echinocytes, spherocytes, and crenulated red blood cells within the microscopic image; Measuring the number of entities of a selected type; and generating an output accordingly; and A method is provided that includes:

[0031] In some applications, identifying the at least one type of entity includes identifying sea urchin red blood cells. In some applications, identifying the at least one type of entity includes identifying spherocytes. In some applications, identifying the at least one type of entity includes identifying serrated red blood cells.

[0032] In some applications, generating the output includes disabling use of at least the portion of the blood sample to perform at least some measurements on the blood sample based at least in part on the number of entities of the selected type exceeding a threshold. In some applications, generating the output includes generating an indication of the number to a user. In some applications, generating the output includes generating an indication of the age of the portion of the sample to a user.

[0033] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample in a sample chamber; obtaining a microscopic image of said portion of the blood sample; identifying at least one type of entity selected from the group consisting of echinocytes, spherocytes, and crenulated red blood cells within the microscopic image; Measuring the number of entities of a selected type; and determining an indication of the age of the portion of the sample based at least in part on the number; and A method is provided that includes:

[0034] In some applications, the method further includes measuring a parameter of the sample by analyzing the microscopic image, where measuring the parameter of the sample includes performing a measurement on the microscopic image and calibrating the measurement based on the determined indication of age of the portion of the sample.

[0035] In some applications, the method further includes measuring a parameter of the sample by performing an optical density measurement on a second portion of the blood sample, where measuring the parameter of the sample includes calibrating the optical density measurement based on the determined indication of age of the second portion of the sample.

[0036] In some applications, identifying the at least one type of entity includes identifying sea urchin red blood cells. In some applications, identifying the at least one type of entity includes identifying spherocytes. In some applications, identifying the at least one type of entity includes identifying serrated red blood cells.

[0037] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample within a sample chamber, the sample chamber being a cavity including a base surface; allowing cells in the cell suspension to settle on the base surface of the carrier to form a monolayer of cells on the base surface of the carrier; obtaining at least one microscopic image of at least a portion of the monolayer of cells; Identifying hemolyzed red blood cells in the microscopic image; Measuring the number of identified hemolyzed red blood cells; generating an output based on the number of identified hemolyzed red blood cells; A method is provided that includes:

[0038] In some applications, the method further includes estimating a total number of hemolyzed red blood cells in the blood sample that is greater than the number of identified hemolyzed red blood cells based on the number of identified hemolyzed red blood cells, and generating the output includes generating an indication of the estimated total number of hemolyzed red blood cells to a user.

[0039] In some applications, generating the output includes disabling use of the portion of the sample to perform at least some measurements on the sample based at least in part on the number of identified hemolyzed red blood cells exceeding a threshold value.

[0040] In some applications, disabling the use of the sample for performing at least some measurements on the sample includes estimating, based on the number of identified hemolyzed red blood cells, a total number of hemolyzed red blood cells in the sample that is greater than the number of identified hemolyzed red blood cells.

[0041] In some applications, the method further includes staining the blood sample, and identifying the at least partially hemolyzed red blood cells includes distinguishing between hemolyzed and non-hemolyzed red blood cells by identifying red blood cells stained with the dye as hemolyzed.

[0042] For some applications, staining the blood sample includes staining the blood sample with Hoechst reagent.

[0043] In some applications, acquiring at least one microscopic image of at least a portion of the monolayer of cells includes acquiring at least one bright field microscopic image of at least a portion of the monolayer of cells, and identifying red blood cells stained with the dye includes identifying red blood cells having a visible outline in the bright field microscopic image and an interior similar to the background of the bright field microscopic image.

[0044] In some applications, acquiring at least one microscopic image of at least a portion of the monolayer of cells includes acquiring at least one fluorescent microscopic image of at least a portion of the monolayer of cells, and identifying red blood cells stained with the dye includes identifying red blood cells that appear as bright circles in the image.

[0045] Furthermore, in accordance with some applications of the present invention, disposing a biological sample in a sample chamber having a plurality of regions each defining a different height; measuring a parameter indicative of light transmission through the sample chamber at each region; Using a computer processor, normalizing the parameters measured in each region relative to each other; detecting a presence of a bubble in the sample chamber at least in part in response thereto; performing an action in response to detecting the presence of a bubble in the sample chamber; A method is provided that includes:

[0046] Furthermore, in accordance with some applications of the present invention, disposing a blood sample in a sample chamber having a plurality of regions each defining a different height; measuring a parameter indicative of light transmission through the sample chamber at each region; Using a computer processor, calculating a hemoglobin concentration in the sample based on the absolute values ​​of the parameters in the selected regions; normalizing the parameters measured in each region relative to each other; validating the calculated hemoglobin concentration based on the normalized parameters; A method is provided that includes:

[0047] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample in a sample chamber; obtaining a microscopic image of said portion of the blood sample; Identifying within the microscopic image candidates for a given entity within the blood sample; authenticating at least some of the candidates as the given entity by performing further analysis on the candidates; Comparing the number of candidates for a given entity with the number of authenticated candidates for a given entity; Disabling use of at least a portion of the sample to perform at least some measurements on the sample based at least in part on a relationship between the number of candidates and the number of authenticated candidates; A method is provided that includes:

[0048] In some applications, Identifying candidates for a given entity in the blood sample within the microscopic image includes identifying candidates for a white blood cell in the blood sample within the microscopic image; Authenticating at least some of the candidates as the given entity by performing further analysis on the candidates includes authenticating at least some of the platelet candidates as platelets by performing further analysis on the candidates; Disabling use of at least a portion of the sample for performing at least some measurements on the sample includes disabling use of at least a portion of the sample for performing at least some measurements on the sample based at least in part on a relationship between the number of platelet candidates and the number of authenticated platelet candidates.

[0049] In some applications, Identifying candidates for a given entity in the blood sample in the microscopic image includes identifying candidates for a white blood cell in the blood sample in the microscopic image; authenticating at least some of the candidates as the given entity by performing further analysis on the candidates includes authenticating at least some of the white blood cell candidates as white blood cells by performing further analysis on the candidates; Disabling use of at least a portion of the sample to perform at least some measurements on the sample includes disabling use of at least a portion of the sample to perform at least some measurements on the sample based at least in part on a relationship between the number of white blood cell candidates and the number of authenticated white blood cell candidates.

[0050] In some applications, disabling use of at least a portion of the sample to perform at least some measurements on the sample includes disabling use of at least a portion of the sample to perform at least some measurements on the sample based on a ratio of the number of authenticated candidates to the number of candidates above a maximum threshold.

[0051] In some applications, disabling use of at least a portion of the sample to perform at least some measurements on the sample includes disabling use of at least a portion of the sample to perform at least some measurements on the sample based on a ratio of the number of authenticated candidates to the number of candidates below a minimum threshold.

[0052] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample in a sample chamber; obtaining a microscopic image of said portion of the blood sample; identifying, within the microscopic image, potential white blood cells within the blood sample; performing further analysis on the leukocyte candidates to identify at least some of the leukocyte candidates as leukocytes of a given type; comparing the number of candidate white blood cells with the number of candidate white blood cells authenticated as a given type of white blood cell; Disabling use of at least a portion of the sample to perform at least some measurements on the sample based at least in part on a relationship between the number of leukocyte candidates and the number of leukocyte candidates authenticated as a given type of leukocyte; A method is provided that includes:

[0053] Furthermore, in accordance with some applications of the present invention, staining the blood sample with one or more fluorescent dyes; obtaining one or more microscopic images of the blood sample; identifying contaminating bodies in the sample by identifying stained bodies having irregular shapes; performing a count of one or more entities located within the sample by performing a microscopic analysis on the sample; Disabling the inclusion in the count of areas of the sample located within a given distance of an identified contaminant object; A method is provided that includes:

[0054] In some applications, staining the blood sample with one or more dyes includes staining the blood sample with acridine orange and Hoechst reagent, and identifying the debris includes identifying stained objects having an irregular shape and stained with both acridine orange and Hoechst reagent.

[0055] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample in a sample chamber; performing an optical measurement on the sample; determining, based on the optical measurement, that one or more gas bubbles are present in the sample chamber; generating an output based at least in part on determining that one or more gas bubbles are present in the sample chamber; A method is provided that includes:

[0056] In some applications, the method further includes determining one or more parameters of the sample based on the optical measurements and disabling use of at least a portion of the optical measurements to determine the one or more parameters of the sample based on a determination that one or more gas bubbles are present in the sample chamber.

[0057] In some applications, generating the output includes disabling use of the portion of the blood sample to perform at least some measurements on the sample based on a determination that one or more air bubbles are present in the sample chamber.

[0058] In some applications, determining the presence of one or more gas bubbles in the sample chamber includes analyzing an optical absorption profile of the sample along a given direction along the sample chamber.

[0059] In some applications, performing optical measurements on the sample includes performing one or more optical absorption measurements at wavelengths at which hemoglobin does not absorb light, and determining that one or more gas bubbles are present in the sample chamber includes analyzing the one or more optical absorption measurements at wavelengths at which hemoglobin does not absorb light.

[0060] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample within a sample chamber, the sample chamber being a cavity including a base surface; allowing cells in the cell suspension to settle on the base surface of the carrier to form a monolayer of cells on the base surface of the carrier; obtaining at least one microscopic image of at least a portion of the monolayer of cells; Identifying areas within the microscopic image where no sample is present; Disabling at least the identified area from use in microscopic analysis of this portion of the sample; A method is provided that includes:

[0061] In some applications, identifying the sample-free region includes identifying an interface between a wet region and a dry region on a base surface of the sample chamber.

[0062] In some applications, identifying regions where no sample is present includes distinguishing between regions where no sample is present and one or more regions where sample is present and where the sample has a low cell density.

[0063] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample within a sample chamber, the sample chamber being a cavity including a base surface and a top cover; allowing cells in the cell suspension to settle on the base surface of the carrier to form a monolayer of cells on the base surface of the carrier; acquiring at least one microscopic image of at least a portion of the monolayer of cells while the at least a portion of the monolayer of cells is located within the monolayer focal plane and the microscope is focused on the monolayer focal plane; Identifying the presence of debris under the top cover or base surface by identifying areas in which entities are visible at a focal plane different from the single layer focal plane; Disabling at least the identified area from use in microscopic analysis of this portion of the sample; A method is provided that includes:

[0064] Furthermore, in accordance with some applications of the present invention, placing at least a portion of the blood sample within a sample chamber, the sample chamber being a cavity including a base surface and a top cover; allowing cells in the cell suspension to settle on the base surface of the carrier to form a monolayer of cells on the base surface of the carrier; acquiring at least one microscopic image of at least a portion of the monolayer of cells while the at least a portion of the monolayer of cells is located within the monolayer focal plane and the microscope is focused on the monolayer focal plane; By identifying areas where the background intensity of the microscope image indicates the presence of dirt under the top cover or base surface; Disabling at least the identified area from use in microscopic analysis of this portion of the sample; A method is provided that includes:

[0065] Furthermore, in accordance with some applications of the present invention, staining at least a portion of the blood sample with a fluorescent dye; identifying stained cells within the blood sample by acquiring a plurality of fluorescent microscopic images of the portion of the blood sample with a microscope unit by illuminating the portion of the sample with a light source emitting light in a given spectral band; identifying one or more fluorescent regions visible in a microscopic image acquired under illumination by the light source other than the stained cells; determining a characteristic of the light source based on the identified fluorescent region; A method is provided that includes:

[0066] In some applications, determining the characteristics of the light source based on the identified fluorescent regions includes determining whether the spatial distribution of illumination by the fluorescent light source has changed since a previous measurement.

[0067] In some applications, determining the characteristics of the light source based on the identified fluorescent regions includes determining whether the spatial uniformity of the illumination by the fluorescent light source has changed since a previous measurement.

[0068] In some applications, determining the characteristics of the light source based on the identified fluorescent regions includes determining whether the spatial location of illumination by the fluorescent light source has changed since a previous measurement.

[0069] In some applications, determining the characteristics of the light source based on the identified fluorescent regions includes determining whether the vignetting effect of the illumination by the fluorescent light source has changed since a previous measurement.

[0070] In some applications, determining the characteristics of the light source based on the identified fluorescent regions includes determining whether the spectral distribution of the illumination by the fluorescent light source has changed since a previous measurement.

[0071] In some applications, determining the characteristic of the light source based on the identified fluorescent regions includes determining whether the intensity of illumination by the fluorescent light source has changed since a previous measurement.

[0072] In some applications, the method further includes determining a parameter of the blood sample by performing a measurement on the stained cells and normalizing the measurement based on the determined characteristic of the light source.

[0073] For some applications, the method further includes disabling performing at least some measurements on the blood sample based on the determined characteristic of the light source.

[0074] In some applications, identifying one or more fluorescent regions visible in a microscopic image acquired under illumination by a light source other than stained cells includes identifying intercellular regions within a blood sample.

[0075] In some applications, identifying one or more fluorescent regions visible in a microscopic image acquired under illumination by a light source other than stained cells includes identifying one or more fluorescent regions of a microscope unit.

[0076] In some applications, acquiring multiple fluorescent microscopic images of a portion of the blood sample includes acquiring multiple fluorescent microscopic images of a portion of the blood sample while the blood sample is contained within a sample carrier, and identifying one or more fluorescent regions visible in the microscopic image acquired under illumination by the light source other than stained cells includes identifying one or more fluorescent regions of the sample carrier.

[0077] In some applications, the sample carrier includes a glass layer and a plastic layer bonded together via a fluorescent pressure-sensitive adhesive, and identifying one or more fluorescent regions visible in a microscopic image obtained under illumination by the light source other than the stained cells includes identifying the pressure-sensitive adhesive.

[0078] Furthermore, in accordance with some applications of the present invention, staining at least a portion of the blood sample with a fluorescent dye; identifying stained cells within the blood sample by acquiring a plurality of fluorescent microscopic images of a portion of the blood sample by illuminating the portion of the sample with a light source emitting light in a given spectral band; identifying one or more fluorescent regions visible in a microscopic image acquired under illumination by the light source other than the stained cells; normalizing the fluorescence of the stained cells based on the identified fluorescent areas; A method is provided that includes:

[0079] In some applications, identifying one or more fluorescent regions visible in a microscopic image acquired under illumination by a light source other than stained cells includes identifying intercellular regions within a blood sample.

[0080] In some applications, identifying one or more fluorescent regions visible in a microscopic image acquired under illumination by a light source other than stained cells includes identifying one or more fluorescent regions of a microscope unit.

[0081] In some applications, acquiring multiple fluorescent microscopic images of a portion of the blood sample includes acquiring multiple fluorescent microscopic images of a portion of the blood sample while the blood sample is contained within a sample carrier, and identifying one or more fluorescent regions visible in the microscopic image acquired under illumination by the light source other than stained cells includes identifying one or more fluorescent regions of the sample carrier.

[0082] In some applications, the sample carrier includes a glass layer and a plastic layer bonded together via a fluorescent pressure-sensitive adhesive, and identifying one or more fluorescent regions visible in a microscopic image obtained under illumination by the light source other than the stained cells includes identifying the pressure-sensitive adhesive.

[0083] Furthermore, in accordance with some applications of the present invention, identifying entities within the blood sample by obtaining a plurality of bright field microscopic images of at least a portion of the blood sample by illuminating the portion of the blood sample with light from a bright field light source; analyzing a bright field region of light emitted by the bright field light source in the absence of a blood sample; determining a characteristic of the bright field light source based on analyzing the bright field region of the light; A method is provided that includes:

[0084] In some applications, determining the characteristics of the light source based on the identified bright field regions includes determining whether the spatial distribution of illumination by the bright field light source has changed since a previous measurement.

[0085] In some applications, determining the characteristics of the light source includes determining whether the spatial uniformity of the illumination by the bright field light source has changed since a previous measurement.

[0086] In some applications, determining the characteristics of the light source includes determining whether the spatial location of illumination by the bright field light source has changed since a previous measurement.

[0087] In some applications, determining the characteristics of the light source includes determining whether the vignetting effect of the illumination by the bright field light source has changed since a previous measurement.

[0088] In some applications, determining the characteristics of the light source includes determining whether the spectral distribution of the illumination by the bright field light source has changed since a previous measurement.

[0089] In some applications, determining the characteristic of the light source includes determining whether the intensity of illumination by the bright field light source has changed since a previous measurement.

[0090] In some applications, analyzing the bright field region of light emitted by the bright field light source in the absence of the blood sample includes periodically analyzing the bright field region of light emitted by the bright field light source in the absence of the blood sample at fixed time intervals.

[0091] In some applications, analyzing the bright field region of light emitted by the bright field light source in the absence of a blood sample includes analyzing the bright field region of light emitted by the bright field light source after imaging a given number of blood samples with the bright field light source.

[0092] In some applications, the method further includes determining a parameter of the blood sample by performing a measurement on an entity within the blood sample and normalizing the measurement based on the determined characteristic of the bright field light source.

[0093] For some applications, the method further includes disabling performing at least some measurements on the blood sample based on the determined characteristics of the bright field light source.

[0094] According to some applications of the present invention, staining a portion of each blood sample with at least one type of fluorescent dye; obtaining, using a microscope unit, a fluorescent microscope image of a portion of each blood sample by illuminating the portion of the sample with a plurality of light sources emitting light in each spectral band; Detecting the presence of errors in at least some of the microscopic images; Classifying sources of error, In response to detecting that an error occurred from a given time point, identifying the fluorescent dye as a source of the error; In response to detecting a gradual increase in the error over time, identifying debris within the microscope unit as a source of the error; in response to detecting that the error is present only in images captured under illumination by a given one of the light sources, identifying the given one of the light sources as a source of the error; Classifying sources of error by A method is provided that includes:

[0095] The present invention will be more fully understood from the following detailed description of the embodiments considered in conjunction with the drawings. [Brief description of the drawings]

[0096] [Figure 1] 1 is a block diagram illustrating components of a biological sample analysis system according to some applications of the present invention. [Figure 2A] 1A-1C are schematic diagrams of various views of a sample carrier used to perform both microscopic and optical density measurements in accordance with some applications of the present invention. [Figure 2B] 1A-1C are schematic diagrams of various views of a sample carrier used to perform both microscopic and optical density measurements in accordance with some applications of the present invention. [Figure 2C] 1A-1C are schematic diagrams of various views of a sample carrier used to perform both microscopic and optical density measurements in accordance with some applications of the present invention. [Figure 3A] 1 is a microscopic image of a blood sample containing hemolyzed red blood cells obtained according to some applications of the present invention. [Figure 3B] 1 is a microscopic image of a blood sample containing hemolyzed red blood cells obtained according to some applications of the present invention. [Figure 3C] 1 is a microscopic image of a blood sample containing hemolyzed red blood cells obtained according to some applications of the present invention. [Figure 4] 1 is a graph showing normalized optical transmission intensity profiles recorded along the length of a sample chamber in accordance with certain applications of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] Reference is now made to FIG. 1, a block diagram illustrating 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 in the sample carrier, optical measurements are performed on the sample using one or more optical measurement devices 24. For example, the optical measurement devices may include a microscope (e.g., a digital microscope), a spectrophotometer, a photometer, a spectrometer, a camera, a spectral camera, a hyperspectral camera, a fluorometer, a fluorescence spectrometer, and / or a light detector (such as a photodiode, a photoresistor, and / or a phototransistor). In some applications, the optical measurement devices include a dedicated light source (such as a light emitting diode, an incandescent light source, and / or optical elements (such as lenses, diffusers, filters, and the like) for collecting and / or manipulating the light emission. In some applications, a microscope system generally similar to that described in U.S. Patent Application Publication No. US2014 / 0347459 to Greenfield, which is incorporated herein by reference, is used.

[0098] Typically, a computer processor 28 receives and processes the optical measurements performed by the optical measurement devices. More typically, the computer processor controls the acquisition of the optical measurements performed by one or more optical measurement devices. The computer processor communicates with a memory 30. A user (e.g., a laboratory technician or an individual from whom a sample is taken) sends instructions to the computer processor via a user interface 32. In some applications, the user interface includes a keyboard, a mouse, a joystick, a touch screen device (such as a smartphone or tablet computer), a touch pad, a trackball, a voice command interface, and / or other types of user interfaces known in the art. Typically, the computer processor generates output via an output device 34. More typically, the output device includes a display, such as a monitor, and the output includes output displayed on the display. In some applications, the processor generates output on different types of visual, textual, graphical, tactile, auditory, and / or video output devices, such as, for example, speakers, headphones, a smartphone, or a tablet computer. In some applications, the user interface 32 functions as both an input interface and an output interface, i.e., an input / output interface. In some applications, the processor generates output on a computer-readable medium (e.g., a non-transitory computer-readable medium), such as a disk or a portable USB drive, and / or generates output on a printer.

[0099] In some applications, the optical measurement device 24 (and / or the computer processor 28 and memory 30) are housed in an optical measurement unit 31. The sample carrier 22 is placed in the optical measurement unit to perform optical measurements on the sample. Typically, the optical measurement unit includes a microscope system configured to perform microscopic imaging of a portion of the sample. In some applications, the microscope system includes a set of bright field light sources (e.g. light emitting diodes) configured to be used for bright field imaging of the sample, a set of fluorescent light sources (e.g. light emitting diodes) configured to be used for fluorescent imaging of the sample, and a camera (such as a CCD camera or a CMOS camera) configured to image the sample. Typically, the optical measurement unit also includes an optical density measurement unit configured to perform optical density measurements (e.g. light absorption measurements) on a second portion of the sample. In some applications, the optical density measurement unit includes a set of optical density measurement light sources (e.g. light emitting diodes) and photodetectors configured to perform optical density measurements on the sample. In some applications, each of the aforementioned light source sets (i.e., the bright field light source set, the fluorescent light source set, and the optical density measurement light source set) includes multiple light sources (e.g., multiple light emitting diodes), each of which is configured to emit light at a respective wavelength or in a respective wavelength range.

[0100] Reference is now made to Figures 2A and 2B, which are schematic illustrations of views of a sample carrier 22 according to some applications of the present invention. Figure 2A is a top view of the sample carrier (for illustration purposes, the top cover of the sample carrier is illustrated as opaque in Figure 2A) and Figure 2B is a bottom view (the sample carrier is rotated about a short edge relative to the view illustrated in Figure 2A). Typically, the sample carrier includes a first set 52 of one or more chambers used to perform microscopic analysis on the samples and a second set 54 of one or more chambers used to perform optical density measurements on the samples. Typically, the chambers of the sample carrier are filled with a body sample, such as blood, via a sample inlet hole 38. In some applications, the chambers define one or more outlet holes 40. The outlet holes are configured to facilitate filling of the chambers with a body sample by releasing air present in the chamber from the chamber. Typically, as illustrated, the outlet holes are located longitudinally opposite the inlet holes (with respect to the sample chambers of the sample carrier). In some applications, the exit holes may thereby provide a more efficient air relief mechanism than if they were located closer to the inlet holes.

[0101] Please refer to FIG. 2C, which shows an exploded view of sample carrier 22 according to some applications of the present invention. In some applications, the sample carrier includes at least three components: a molded component 42, a glass sheet 44, and an adhesive layer 46 configured to attach the glass sheet to the underside of the molded component. The molded component is typically made of a polymer (e.g., plastic) that is molded (e.g., by injection molding) to provide chambers having the desired geometric shapes. For example, as shown, the molded component is typically molded to define an inlet hole 38, an outlet hole 40, and a groove 48 that surrounds the center of each chamber. The groove typically facilitates filling of the chambers with the body sample by directing air to the outlet hole and / or by directing the body sample to flow around the center of the chamber.

[0102] In some applications, a sample carrier such as that shown in Figures 2A-2C is used in performing a complete blood count on a blood sample. In some applications, a first portion of the blood sample is placed in a first set of chambers 52 (used to perform a microscopic analysis on the sample) and a second portion of the blood sample is placed in a second set of chambers 54 (used to perform an optical density measurement on the sample). In some applications, as shown, the first set of chambers 52 includes multiple chambers and the second set of chambers 54 includes only one chamber. However, the scope of the invention includes any number of chambers (e.g., a single chamber or multiple chambers) in either the first set of chambers or the second set of chambers, or any combination thereof. For example, the diluent may include a pH buffer, a dye, a fluorescent dye, an antibody, a sphering agent, a lysing agent, and the like. Typically, the second portion of the blood sample placed in the second set of chambers 54 is a natural undiluted blood sample. Alternatively or additionally, the second portion of the blood sample may be a sample that has been altered in some way, including, for example, one or more of dilution (e.g., dilution in a controlled manner), addition of components or reagents, or fractionation.

[0103] In some applications, a first portion of the blood sample (disposed in the first set of chambers 52) is stained with one or more staining substances before the sample is imaged under a microscope. For example, the staining substances may be configured to preferentially stain DNA over other cellular components. Alternatively, the staining substances may be configured to preferentially stain all cellular nucleic acids over other cellular components. For example, the sample may be stained with acridine orange reagent, Hoechst reagent, and / or any other staining substances configured to preferentially stain DNA and / or RNA in a blood sample. Optionally, the staining substances are configured to stain all cellular nucleic acids, but under certain lighting and filter conditions, the respective staining of DNA and RNA is more prominently visible, for example as known for acridine orange. Images of the sample may be acquired using imaging conditions that allow detection of cells (e.g. bright field) and / or visualization of stained portions (e.g. suitable fluorescent illumination). Typically, the first portion of the sample is stained with acridine orange and Hoechst reagent. For example, a first (diluted) portion of the blood sample may be prepared using techniques such as those described in U.S. Patent Application Publication No. US2015 / 0316477 to Pollak, which is incorporated herein by reference. U.S. Patent Application Publication No. US2015 / 0316477 describes a method of preparing a blood sample for analysis, which includes a dilution step that facilitates identification and / or counting of components in a microscopic image of the sample.

[0104] Typically, prior to microscopic imaging, the first portion of blood (placed in the first set of chambers 52) is allowed to settle, e.g., to form a monolayer of cells, using techniques such as those described in U.S. Pat. No. 9,329,129 to Pollak (hereby incorporated by reference). In some applications, microscopic analysis of the first portion of the blood sample is performed on the monolayer of cells. 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, typically emitting in respective spectral bands). More typically, the first portion of the blood sample is further imaged under fluorescence imaging. Typically, fluorescence imaging is performed by exciting dyed objects in the sample by directing light of known excitation wavelengths (i.e., wavelengths at which dyed objects (i.e., objects that have absorbed one or more dyes) are known to emit fluorescent light when excited with light of these wavelengths) toward the sample, and detecting the fluorescent light. Typically, in fluorescence imaging, a set of separate light sources (eg, one or more light emitting diodes) is used to illuminate the sample at known excitation wavelengths.

[0105] It is noted that in the context of this application, the term monolayer is used to describe a layer of cells that have settled and are located within a single focal field of, for example, a microscope. There may be some overlap of cells within the monolayer, e.g., there may be two or more overlapping layers of cells in a particular area. For example, red blood cells may overlap each other within the monolayer, and / or platelets may overlap or rest on red blood cells within the monolayer.

[0106] As described with reference to US Patent Application Publication No. US2019 / 0302099 to Pollack, which is incorporated herein by reference, in some applications, the chambers belonging to set 52 (used for measurements under a microscope) have different heights from each other. The purpose of this is to facilitate the measurement of different measurement quantities using a microscopic image of each chamber and / or to use a different chamber for the microscopic analysis of each sample type. For example, if a blood sample and / or a monolayer formed by the sample has a relatively low density of red blood cells, performing the measurement in a chamber of the sample carrier having a larger height (i.e., a chamber of the sample carrier having a larger height compared to a different chamber having a smaller height) can ensure that a sufficient density of cells is present and / or that a sufficient density of cells is present in the monolayer formed by the sample to provide statistically reliable data. Such measurements can include, for example, density measurements of red blood cells, measurements of other cell attributes (number of abnormal red blood cells, number of red blood cells containing intracellular bodies (e.g., pathogens, Howell-Jolly bodies), etc.), and / or measurements of hemoglobin concentration. Conversely, if the blood sample and / or the monolayer formed by the sample has a relatively high density of red blood cells, such measurements may be performed in chambers of the sample carrier of a relatively small height, for example, such that there is sufficient sparsity of cells and / or there is sufficient sparseness of cells within the monolayer of cells formed by the sample to identify the cells in the microscope image. In some applications, such methods are performed even if the height difference between the chambers of set 52 is not precisely known.

[0107] In some applications, a chamber in the sample carrier in which an optical measurement is performed is selected based on the measurand to be measured. For example, a chamber in a sample carrier with a large height may be used to perform white blood cell counting (e.g., to reduce statistical errors that may arise from low numbers in shallow areas), white blood cell differentiation, and / or detection of rare morphologies of white blood cells. Conversely, microscopic images may be acquired from a chamber in a sample chamber with a relatively small height to determine mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red blood cell distribution width (RDW), red blood cell morphological characteristics, and / or red blood cell anomalies, because in such chambers the cells are relatively sparsely distributed in the area of ​​the region and / or form a monolayer in which the cells are relatively sparsely distributed. Similarly, microscopic images may be acquired from a chamber in a sample chamber with a relatively small height to perform platelet counting, platelet classification, and / or extraction of any other attributes of platelets (such as volume). This is because in such chambers fewer red blood cells overlap (fully or partially) with the platelets in the microscopic image and / or in the monolayer.

[0108] According to the above example, it is preferable to use a chamber of a sample carrier with a small height to perform optical measurements to measure one measurand in a sample (such as a blood sample), but a chamber of a sample carrier with a large height to perform optical measurements to measure another measurand in such a sample. Thus, in some applications, a first measurand in a sample is measured by performing a first optical measurement on a portion of the sample placed in a first chamber belonging to the set of sample carriers 52 (e.g., by acquiring a microscopic image of the portion), and a second measurand of the same sample is measured by performing a second optical measurement on a portion of the sample placed in a second chamber of the set of sample carriers 52 (e.g., by acquiring a microscopic image of the portion). In some applications, the first and second measurands are normalized relative to each other, for example, using techniques such as those described in U.S. Patent Application Publication No. US2019 / 0145963 to Zait, which is incorporated herein by reference.

[0109] Typically, to perform an optical density measurement on a sample, it is desirable to know as precisely as possible the optical path length, volume, and / or thickness of the portion of the sample on which the optical measurement was performed. Typically, the optical density measurement is performed on a second portion of the sample (typically disposed in the second set of chambers 54 in undiluted form). For example, optical absorption, transmittance, fluorescence, and / or luminescence measurements can be performed on the sample to measure the concentration and / or density of components.

[0110] Referring again to FIG. 2A, in some applications, the chambers of set 54 (used for optical density measurements) typically define at least a first region 56 (typically deep) and a second region 58 (typically shallow). The heights of the chambers in the first and second regions are different in a predetermined manner, as described, for example, in International Publication No. WO 17 / 195205 to Pollack, which is incorporated herein by reference. The heights of the first and second regions 56 and 58 of the sample chamber are defined by a lower surface defined by the glass sheet and an upper surface defined by the molding component. The upper surface of the second region is stepped relative to the upper surface of the first region. The step between the top surfaces of the first and second regions provides a predetermined height difference Δh between the regions, so that even if the absolute heights of the regions are not known with sufficient precision (e.g., due to manufacturing process tolerances), the height difference Δh is known with sufficient precision to determine parameters of the sample using techniques such as those described herein and in U.S. Patent Application Publication No. US2019 / 0302099 to Pollack, which is incorporated herein by reference. In some applications, the height of the chamber varies from the first region 56 to the second region 58, and again from the second region 58 to the third region 59, such that along the sample chamber, the first region 56 defines a maximum height region, the second region 58 defines an intermediate height region, and the third region 59 defines a minimum height region. In some applications, there is further height variation along the length of the chamber and / or there is a gradual variation in height along the length of the chamber.

[0111] As described above, while the sample is disposed on the sample carrier, optical measurements are performed on the sample using one or more optical measurement devices 24. Typically, the sample is interrogated by the optical measurement device through the glass layer. The glass is transparent at least to the wavelengths typically used by the optical measurement device. Typically, while performing the optical measurements, the sample carrier is inserted into an optical measurement unit 31 housing the optical measurement device. Typically, the optical measurement unit houses the sample carrier such that the molded layer is disposed above the glass layer and such that the optical measurement unit is disposed below the glass layer of the sample carrier so that the optical measurement unit can perform optical measurements on the sample through the glass layer. The sample carrier is formed by attaching a glass sheet to a molded component. For example, the glass sheet and the molded component can be bonded to each other during manufacturing or assembly (e.g., using thermal bonding, bonding with a solvent, ultrasonic welding, laser welding, heat staking, adhesives, mechanical clamps, and / or additional substrates). In some applications, the glass layer and the molded component are bonded to each other during manufacturing or assembly using an adhesive layer 46. In some applications, the absolute height of the sample chamber is not known due to manufacturing process tolerances. As described above, the step between the top surfaces of the first and second regions provides a predetermined height difference Δh between these regions, so that even if the absolute heights of these regions are not known with sufficient precision (e.g., due to manufacturing process tolerances), the height difference Δh is known with sufficient precision to determine the parameters of the sample. Instead of or in addition to the step between the first and second regions, height variations (e.g., step height variations or gradual height variations) along the length of the chamber may alternatively or additionally be used, for example, using techniques such as those described herein and in U.S. Patent Application Publication No. US2019 / 0302099 to Pollack, which is incorporated herein by reference.

[0112] In some applications, a portion of the sample carrier 22 is configured to fluoresce at least under certain conditions. For example, a portion of the sample carrier can be configured to fluoresce when illuminated with light emitted by the optical measurement device 24 (e.g., bright field light or fluorescent light emitted by a microscope system). Alternatively, a portion of the sample carrier can be configured to fluoresce when placed in an optical measurement unit 31 in which the optical measurement device 24 is housed. As described above, in some applications, the sample carrier 22 includes an adhesive layer 46. In some applications, the adhesive layer or a portion thereof is configured to fluoresce as described above (e.g., an adhesive material in the adhesive layer is configured to fluoresce, the adhesive layer contains an additional material configured to fluoresce, and / or the adhesive layer is coated with such a material). In some applications, the adhesive layer is a pressure-sensitive adhesive, at least a portion of which is configured to fluoresce. For example, the pressure-sensitive adhesive is an acrylic pressure-sensitive adhesive, at least a portion of which is configured to fluoresce.

[0113] According to some applications of the invention, various techniques are implemented (typically by a computer processor) to determine whether various errors have occurred and, optionally, to identify the source of the errors if they have occurred. Such errors may result from the preparation of the entire sample. For example, the sample may have been left on the sample carrier for too long before a measurement was performed (which may result in the sample deteriorating and / or dyes mixed with one or both parts of the sample being excessively absorbed by entities in the sample). Alternatively, errors may result from the preparation of a particular part of the sample. For example, an error may have occurred in the preparation of a first part (which is typically diluted and placed in the first set of chambers 52 to be analyzed by the microscope). This may be, for example, an error in diluting the first part of the sample, introducing an air bubble into the first set of chambers 52, or contamination of this part of the sample. Alternatively or additionally, an error may have occurred in the preparation of a second part (which is typically not diluted and placed in the second set of chambers 54 to be analyzed by optical density measurement). This may be, for example, contamination of this part or introducing an air bubble into the second set of chambers 54.

[0114] Alternatively or additionally, errors may arise from errors in the sample carrier (such as the material of the sample carrier itself (e.g. the substrate) being dirty and / or there being dirt or spilled blood on the sample carrier), and / or errors in the microscope system itself, such as the illumination (e.g. light emitting diodes used for bright field imaging and / or light emitting diodes used during fluorescent imaging of the sample), and / or errors related to the optical path and / or motor and stage components or controllers, and / or errors arising from the environment in which the device is placed (such as relative humidity, temperature, pressure, particle concentration, or some other environmental factor). Further, alternatively or additionally, there may be inherent problems with the sample (such as too few or too many specific entities, or too long a time has passed since the sample collection was performed). This means that the computer processor cannot perform a specific measurement with sufficient accuracy and / or the computer processor must inform the user of this.

[0115] In some applications, in response to identifying the error, the computer processor outputs a message indicating the error and / or indicating the source of the error. In some applications, in response to identifying the error, the computer processor does not perform a particular measurement on the blood sample. In some applications, in response to identifying the error, the computer processor does not perform a measurement on the sample, and / or informs a user that the sample is invalid, and / or instructs a user to repeat the sample preparation with a new test kit, and / or recollects the blood sample. Alternatively or additionally, to account for the cause of the error, the computer processor determines a particular parameter of the blood sample by calibrating the measurement performed on the blood sample.

[0116] Some applications account for one or more of the following causes of errors, for example in one or more of the ways described above:

[0117] Errors in microscope devices such as: -Loss of synchronism in the motor that moves the microscope stage -Variation of the translational play of the microscope stage (e.g. as detailed below) - Timing changes between the microscope camera and the microscope stage (e.g., as described in more detail below) - Alignment between the microscope camera and the microscope stage (e.g. due to relative rotation between these elements) - Problems with the optical system (e.g. changes in focus quality over time caused by the sample or by microscope stage debris (e.g. scratches), as detailed below) -Leveling changes in microscope systems - the change in expected focus position along the z-axis (i.e. the optical axis) -Loss of communication between elements -Changes in camera linear response

[0118] Errors caused by environmental factors such as: -Devices outside the permitted temperature, humidity, altitude, etc. ranges -Specific components that are out of target range

[0119] Common errors caused by factors such as: - Scan time -Device boot time -Available working / storage memory

[0120] Some example techniques for identifying errors and explaining the causes of such errors are described below.

[0121] As mentioned above, typically a first portion of the blood sample is analyzed under a microscope while disposed within the first set of sample chambers 52. Typically, prior to imaging under a microscope, the first portion of blood (disposed in the first set of chambers 52) is allowed to settle, e.g., to form a monolayer of cells, using techniques such as those described in U.S. Pat. No. US9,329,129 to Pollak, which is incorporated herein by reference.

[0122] In some applications, the computer processor is configured to obtain one or more microscopic images of the sample after the sample carrier is placed in the microscope unit and to determine whether one or more cell types (e.g., red blood cells) in the sample chamber had already settled in the sample chamber before the sample carrier was placed in the microscope unit by analyzing the one or more images.

[0123] Typically, if it is determined that all of the red blood cells have already settled before the microscopic image is acquired, this indicates that the blood sample was left in the sample chamber for too long before placing the sample carrier in the microscope unit. It should be noted that although analysis of the microscopic image is typically performed on a monolayer of settled cells, it is nevertheless desirable to place the sample carrier in the microscope unit when some red blood cells are still settling, as this indicates that the sample was not left in the sample carrier for too long before being placed in the microscope unit. Conversely, if all of the red blood cells (or a sufficiently large percentage of the red blood cells) have already settled, the sample may be degraded and / or the dye may have been excessively absorbed by entities in the sample. Thus, the degree to which the cells are still settling may be used as a measure of how recently the sample was taken from the subject and / or how recently the sample was placed in the sample carrier or sample chamber (i.e., the freshness of the sample). Thus, in some applications, in response to a determination that a threshold amount of red blood cells in the sample chamber have already settled in the sample chamber prior to acquisition of one or more microscopic images, the sample (or a portion thereof) is disabled from use for performing at least some measurements on the sample.

[0124] In some applications, the indication of age of the blood sample is determined based at least in part on determining whether one or more cell types in the sample chamber have already settled in the sample chamber prior to acquisition of the one or more microscopic images. In some applications, the indication of age of the blood sample is determined based at least in part on the amount (or percentage) of one or more cell types in the sample chamber that have not yet settled in the sample chamber prior to acquisition of the one or more microscopic images.

[0125] In some applications, a second portion of the sample (typically placed in the second set of chambers 54 in undiluted form) is subjected to an analysis generally similar to that described in the paragraph above (to determine how recently the sample was taken from the subject and / or how recently the sample was placed in the sample carrier or sample chamber).

[0126] It should be noted that typically, after the computer processor has determined (e.g., using the analytical techniques described above) an indication of how recently the sample was taken from the subject and / or how recently the sample was placed in the sample carrier or sample chamber, the same carrier is left in place within the microscope unit for a few minutes (e.g., 2-10 minutes, e.g., about 5 minutes) before further imaging of a first portion of the blood sample (which imaging is performed for the purposes of performing microscopic analysis of the blood sample) to allow time for the first portion of the blood sample to settle and form a monolayer.

[0127] In some applications, measurements performed on the sample are calibrated in response to determining that a threshold amount of red blood cells in the sample chamber have already settled in the sample chamber prior to acquisition of the one or more microscopic images. For example, such measurements may be calibrated to account for the amount of stained entities in the blood sample that are indicated by a threshold amount of red blood cells in the sample chamber have already settled in the sample chamber prior to acquisition of the one or more microscopic images. In some applications, measurements performed on the sample are calibrated based on the amount (or percentage) of one or more cell types in the sample chamber that have not yet settled in the sample chamber prior to acquisition of the one or more microscopic images.

[0128] In some applications, a computer processor determines a sedimentation dynamics characteristic (e.g., erythrocyte sedimentation rate) of a blood sample by acquiring microscopic images of red blood cells in the blood sample while the red blood cells are settling in a sample chamber and analyzing these images. Typically, the sedimentation dynamics characteristic (e.g., erythrocyte sedimentation rate) of a blood sample is determined by a computer processor in real time with respect to the sedimentation of the red blood cells (i.e., while the red blood cells are settling). This is in contrast to other techniques for determining a sedimentation dynamics characteristic (e.g., erythrocyte sedimentation rate) of a blood sample, which measure the total time it takes for the red blood cells to sediment. It should be noted that if such measurements are performed on a diluted portion of the blood sample, the effect of proteins on the sedimentation time of the red blood cells is weakened. Thus, in some applications, such measurements are performed on an undiluted portion of the blood sample.

[0129] In some applications, the above-described analyses for determining an indication of how recently the sample was taken from the subject and / or how recently the sample was placed on the sample carrier and / or for determining sedimentation dynamics characteristics are corrected using sample-by-sample information, such as red blood cell or platelet mean cell volume, mean red blood cell hemoglobin concentration, or other such sample indicator measures. Alternatively or additionally, the above-described analyses for determining an indication of how recently the sample was taken from the subject and / or how recently the sample was placed on the sample carrier and / or for determining sedimentation dynamics characteristics are corrected using single-cell level information (i.e., by extracting data for individual cells and correcting the determined sedimentation dynamics characteristics based on these data).

[0130] In some applications, in response to determining that the concentration of a given entity in the blood sample exceeds a threshold, the computer processor identifies the cause of the exceeding of the threshold by comparing parameters determined from the microscopic image of a first portion of the blood sample with parameters determined from an optical density measurement performed on a second portion of the blood sample. For example, in response to detecting that a first portion of the sample (typically diluted and placed in the first set of chambers 52) has too many red blood cells or too few red blood cells, the computer processor may perform this comparison to determine whether this is due to an error in the preparation of this portion of the sample (e.g., dilution of the first portion). The computer processor typically determines that the exceeding of the threshold concentration of a given entity in the sample is due to the blood sample itself by determining that the concentration of the entity shown by the microscopic image is similar to the concentration of the entity determined from the optical density measurement. More typically, the computer processor determines that the preparation of the portion of the blood sample is the cause of the concentration of a given entity in the sample exceeding a threshold by determining that the concentration of the entity indicated by the microscopic image is different from the concentration of the entity determined from the optical density measurement.

[0131] In some applications, a similar analysis to that described in the paragraph above is performed using parameters of the sample other than concentration (e.g., mean cell volume, mean red blood cell hemoglobin, etc.) In some applications, in response to identifying a difference between the parameters measured in the portions of the sample, it is determined that the portions of the sample are likely to be portions of two different samples (e.g., from two different patients).

[0132] In some applications, the parameters determined from each portion of the sample are used to compensate for one another, for example, if the values ​​of a parameter measured at each of the sample portions are different from one another, but there is a third value (or range of values) of that parameter that is within the error range for both of the values ​​measured at each of the sample portions, then it may be determined that this third value (or range of values) is likely to be correct.

[0133] In some applications, in response to one or more parameters of the sample being outside of the normal range, the computer processor compares those parameters to other parameters of the sample, and in response to all of the parameters being outside of the normal range (or being erroneous in correlation with the error in one or more parameters), the computer processor can determine that the cause is a calculation error affecting all of those parameters.

[0134] In some applications, the computer processor is configured to identify sea urchins, spherocytes, and / or crenulated red blood cells in the microscopic image of the first portion of the sample. Typically, the presence of such entities is an indication that this portion of the sample has deteriorated due to age and / or sample storage conditions. In some such applications, the computer processor determines the number of such entities and disables using at least this portion of the blood sample to perform at least some measurements on the blood sample based at least in part on the number of entities of the selected type exceeding a threshold. In some applications, the computer processor generates an indication of the number and / or an indication of an associated clinical condition to the user. Alternatively or additionally, the computer processor determines an indication of the age of this portion of the sample based at least in part on the number. In some applications, measurements are performed on the microscopic image to determine a parameter of the sample and the measurements are calibrated based on the determined indication of the age of this portion of the sample and / or based on the number of said entities. In some applications, a parameter of the sample is determined by performing an optical density measurement on a second portion of the blood sample (typically disposed in the second set of sample chambers 54) and calibrating the optical density measurement based on the determined indication of the age of this portion of the sample and / or based on the number of the aforementioned entities. (In some applications, the optical density measurement is calibrated based on one or more other factors, such as red blood cell morphology, red blood cell volume, platelet count, level of debris in the sample, and / or the presence or amount of objects or attributes that may affect scattering.)

[0135] In some applications, the computer processor estimates the volume of echinocytes, spherocytes, and / or crenocytes, and in some such applications, incorporates the volume of such red blood cells into an overall measure of mean corpuscular volume in the sample.

[0136] Reference is now made to Figures 3A and 3B, which are bright-field microscopic images acquired under violet and green LED illumination, respectively, according to some applications of the present invention. As described above, typically, a microscopic image of a first portion of a blood sample is acquired to analyze a monolayer of cells within the first portion. Typically, no spherification technique is used on the red blood cells within the sample prior to imaging this portion of the sample. The inventors of the present application have discovered that when this portion of the sample is imaged under a microscope using the techniques described herein, some hemolyzed red blood cells are visible within the microscopic image. Typically, after staining the sample with Hoechst reagent (or any fluorescent or non-fluorescent dye with affinity for cell membranes), the outlines of the cells are visible under bright-field imaging, while the remainder of the cells look similar to the image background. This effect can be observed in Figures 3A and 3B. Red blood cells 60 are visible and the outlines of hemolyzed red blood cells 62 are visible, but the interiors of these cells look similar to the background and the outlines look like "empty" cells, which typically have roughly the same shape and size as red blood cells.

[0137] See also FIG. 3C, which is a fluorescence microscopy image obtained according to some applications of the present invention. This image was recorded after staining the blood sample with Hoechst reagent and exciting with UV illumination centered at about 360 nm. It can be observed that unlike red blood cells 60, which appear as hazy dark circles, hemolyzed red blood cells 62 appear as bright circles having roughly the same shape and size as red blood cells. It is hypothesized that hemolyzed red blood cells are stained by Hoechst reagent binding with remnants remaining on the membrane of the hemolyzed red blood cells. This makes them appear as "empty" cells in bright field images and / or as bright circles in fluorescence images. It is further hypothesized that the reason red blood cells appear as dark circles in fluorescence images is because there is a background emission of free Hoechst reagent throughout the sample, but this emission is attenuated by hemoglobin within the red blood cells, making the red blood cells appear darker than the background.

[0138] Thus, according to some applications of the present invention, hemolyzed red blood cells are identified in a non-sphered blood sample. Typically, the sample is stained with a dye such as Hoechst reagent (or any fluorescent or non-fluorescent dye with affinity for cell membranes). Hemolyzed red blood cells are identified in a bright field image of the stained sample by identifying cells that have a visible outline but whose interior generally looks similar to the background (appearing as "empty" outline cells). Alternatively or additionally, hemolyzed red blood cells are identified in a fluorescent image of the stained sample. Typically, hemolyzed red blood cells have a generally similar shape and size to red blood cells in the image.

[0139] In some applications, the visible hemolyzed red blood cells make up only a small proportion of the total number of hemolyzed red blood cells present in this portion of the sample. This is because some of the hemolyzed red blood cells are typically not visible. In some applications, the computer processor identifies the visible hemolyzed red blood cells and measures the number of identified hemolyzed red blood cells in this portion of the sample. Typically, based on the number of identified hemolyzed red blood cells, the computer processor estimates the total number of hemolyzed red blood cells in this portion of the sample, which is greater than the number of identified hemolyzed red blood cells. Alternatively or additionally, based on the number of identified hemolyzed red blood cells, the computer processor estimates the ratio of hemolyzed red blood cells to non-hemolyzed red blood cells in the sample. Typically, this ratio is calculated by estimating the total number of hemolyzed red blood cells in this portion of the sample, which is greater than the number of identified hemolyzed red blood cells. In some applications, the computer processor outputs to the user an indication of the estimated total number of hemolyzed red blood cells or the ratio of hemolyzed red blood cells to non-hemolyzed red blood cells in the blood sample. Alternatively or additionally, the computer processor disables the use of the sample to perform at least some measurements on the sample based at least in part on the number of identified hemolyzed red blood cells exceeding a threshold value or on said ratio being above a threshold value. In some applications, the disablement of the sample is based on an estimate of the total number of hemolyzed red blood cells in this portion of the sample being greater than the number of identified hemolyzed red blood cells.

[0140] In some applications, to identify a given entity (such as a platelet, red blood cell, white blood cell, etc.) in a blood sample, a computer processor first identifies candidates for the given entity in the blood sample by analyzing a microscopic image of a first portion of the blood sample, and then the computer processor authenticates at least some of the candidates as the given entity by performing further analysis on these candidates.

[0141] In some applications, the computer processor compares the number of candidates for the given entity to the number of authenticated candidates for the given entity and disables using at least this portion of the sample to perform at least some measurements on the sample based at least in part on the relationship between the number of candidates and the number of authenticated candidates. For example, if the ratio of the number of authenticated candidates to the number of candidates is above a maximum threshold, this indicates that there are too many authenticated candidates, indicating an error, and the computer processor can disable using at least this portion of the sample to perform at least some measurements on the sample. Alternatively or additionally, if the ratio of the number of authenticated candidates to the number of candidates is below a minimum threshold, this indicates that there are too few authenticated candidates, indicating an error, and the computer processor can disable using at least this portion of the sample to perform at least some measurements on the sample. In some applications, if the ratio of the number of authenticated platelets to the number of platelet candidates is below a minimum threshold, this indicates that there are too few platelet candidates authenticated as platelets, indicating an error, and the computer processor can disable using at least this portion of the sample to perform a platelet count. For example, this error may occur due to debris (or other contaminants) being erroneously identified as platelets or as potential platelets. Note that the source of error may be in the preparation of this portion of the sample, in the sample carrier, in parts of the microscope unit, and / or in the blood itself. In some applications, similar techniques are performed for red blood cells, white blood cells, and / or other entities within the sample (e.g., abnormal white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howell-Jolly bodies, etc.).

[0142] In some applications, the computer processor is configured to identify leukocyte candidates in the blood sample and then authenticate at least some of the leukocyte candidates as a given type of leukocyte (e.g., neutrophils, lymphocytes, eosinophils, monocytes, blasts, immature cells, atypical lymphocytes, and / or basophils) by performing further analysis on the leukocyte candidates. In some applications, the computer processor compares the number of leukocyte candidates to the number of leukocyte candidates authenticated as the given type of leukocyte and disables using at least this portion of the sample to perform at least some measurements on the sample based at least in part on the relationship between the number of leukocyte candidates and the number of leukocyte candidates authenticated as the given type of leukocyte. For example, if the ratio between the number of leukocyte candidates authenticated as the given type of leukocyte and the number of leukocyte candidates is above a maximum threshold, which indicates too many authenticated candidates and indicates an error, the computer processor can disable using at least this portion of the sample to perform at least some measurements on the sample. Alternatively or additionally, if the ratio between the number of leukocyte candidates authenticated as a given type of leukocyte and the number of leukocyte candidates is below a minimum threshold, this indicates that too few candidates are authenticated, indicating an error, and the computer processor may disable the use of at least this portion of the sample to perform at least some measurements on the sample, whereby the source of error may be in the preparation of this portion of the sample, in the sample carrier, in parts of the microscope unit, and / or in the blood itself.

[0143] In some applications, the computer processor is configured to identify debris (or other contaminants) in the sample by identifying stained objects having an irregular shape (e.g., fibrous, non-circular, and / or elongated shape). As described above, typically the computer processor performs a count of one or more entities located in the sample by performing a microscopic analysis on the sample. In some applications, the computer processor disables including in the count an area of ​​the sample located within a given distance from the identified debris (or other contaminants). Typically, the debris is stained with a dye, and more typically, the debris is stained with both acridine orange and Hoechst reagent. In some applications, the computer processor identifies the debris (or other contaminants) by identifying objects having an irregular shape and / or stained with a dye (e.g., both acridine orange and Hoechst reagent).

[0144] Reference is now made to FIG. 4, which is a graph illustrating a normalized logarithmic profile of optical transmission intensity measured along the length of a sample chamber belonging to the second set of sample chambers 54, according to some applications of the present invention. As described above, typically the computer processor is configured to perform an optical measurement (e.g., an optical density measurement) on a second portion of the sample disposed in the second set of sample chambers 54. In some applications, light in a spectral band in which hemoglobin absorbs light (e.g., light from an LED) is transmitted through the chambers, and the intensity of the transmitted light is detected by a photodetector. According to the Beer-Lambert law, the amount of light absorbed is interpreted as indicative of the hemoglobin concentration in the second portion of the sample.

[0145] In some applications, the computer processor determines that one or more bubbles are present in one of the sample chambers of the second set based on the optical measurements. For example, due to regions in the sample chamber having different heights, the normalized logarithm profile of the transmitted intensity along the length of the chamber (related to the hemoglobin absorption profile) is expected to have a given shape. That is, there are regions where the logarithm of the optical transmitted intensity is substantially constant, with differences between these regions (due to the step in the chamber). This is shown in FIG. 4 by the solid curve 70. The curve 70 is a combination of the normalized logarithm of the transmitted intensity recorded along the length of the sample chamber for several different samples. As shown, along the minimum height region 56 of the sample chamber, the transmitted intensity logarithm is substantially constant. (In practice, there is a slight slope along the length of this region due to manufacturing tolerances of the sample carrier as discussed above.) The logarithm of the transmission intensity then falls as the distance along the sample chamber transitions to the mid-height region 58 (where hemoglobin absorption increases and therefore light transmission decreases), and then falls further as the distance along the sample chamber transitions to the maximum height region 59 (where hemoglobin absorption increases further and therefore light transmission decreases further). Note that the manner in which the transmission was normalized was to take the average of the logarithm of the light transmission intensity within a given portion of the maximum height region and assign this a value of 1, take the average of the logarithm of the light transmission intensity within a given portion of the minimum height region and assign this a second value, and then normalize the other values ​​relative to these two values. (In some cases, the logarithm of the light transmission intensity within a given portion of the minimum height region is assigned the value of Euler's number (i.e., 2.718), but this is not the case in the example shown in FIG. 4.) It is expected that the normalized profile of any sample filling the sample chamber will have a similar profile regardless of the absolute hemoglobin absorption of the sample. This is because the shape of the profile depends on the relative absorption along different regions of the sample chamber.

[0146] The dotted thin curve 72 in FIG. 4 shows the normalized logarithm of the transmission intensity recorded along the length of the sample chamber for a given sample. Within the minimum height region, it can be observed that there is a relatively flat portion of the curve (below curve 70) followed by a peak (above curve 70). Furthermore, along the mid-height region, curve 72 is below curve 70. Typically, such a profile indicates the presence of bubbles (e.g., air bubbles and / or the presence of different substances) within the minimum height region. The peak of curve 72 occurs at the location of the bubble, since the light transmission increases at this location. At other locations (e.g., within other parts of the minimum height region) and throughout the mid-height region, the presence of bubbles within the minimum height region will cause the normalized logarithm of the transmission intensity to decrease compared to a sample without bubbles. Similarly, if bubbles are present in other regions of the sample chamber (or along the entire region), this will result in a different normalized logarithm of the transmission intensity profile. For example, if bubbles are present along the entire minimum height region, this will cause the normalized logarithm of the transmission intensity within the mid-height region to decrease compared to a sample without bubbles. In some applications, the chamber height will vary, but will generally follow similar techniques mutatis mutandis.

[0147] Thus, according to some applications of the invention, in addition to measuring the absolute value of the parameter indicative of the light transmission along the length of the sample chamber, a normalized value of the parameter indicative of the light transmission along the sample chamber (e.g., a normalized logarithm of the light transmission intensity) is determined. Based on the normalized value of the parameter, the computer processor determines that there is a high probability of bubbles (e.g., the presence of an air bubble or a different substance) in the sample chamber. In some applications, in response to determining that there is a high probability of bubbles being present in the sample chamber, the computer processor generates an output. For example, the computer processor can generate an error message (e.g., a message indicating that the sample chamber should be refilled), invalidate the sample, and / or invalidate some of the measurements performed on the sample.

[0148] In some applications, the computer processor performs light absorption measurements, but uses only regions of the chamber that are free of bubbles to do so. Thus, in some applications, the computer processor calculates the hemoglobin concentration in the sample based on the absolute values ​​of the parameters indicative of the light transmission of at least some regions in the sample chamber. Furthermore, the computer processor normalizes the parameters measured at each region in the sample chamber relative to each other. Depending at least in part on the normalization parameters, the computer processor determines which regions to use to calculate the hemoglobin concentration in the sample. Alternatively, the computer processor can disable the use of the sample to calculate the hemoglobin concentration in the sample and / or generate an error message (e.g., a message indicating that the sample chamber must be refilled).

[0149] Along the width of the chamber, the sample is expected to have a substantially constant absorption profile. In some applications, the computer processor is configured to interpret an unexpectedly low absorption level (not consistent with the above-mentioned profile) as an indication of the presence of air bubbles. In some applications, to determine whether air bubbles are present, one or more light absorption measurements are performed at a wavelength at which hemoglobin does not absorb light (e.g., using green light). Alternatively or additionally, a camera (e.g., a CCD camera or CMOS camera of a microscope) is used to image a second portion of the blood sample, and the computer processor determines whether air bubbles are present based on the one or more images.

[0150] Typically, one or more parameters of the sample are determined by a computer processor based on the optical measurements. In some applications, based on a determination that one or more gas bubbles are present in the chamber, the computer processor disables use of at least a portion of the optical measurements to determine one or more parameters of the sample. In some applications, based on a determination that one or more gas bubbles are present in the sample chamber, the computer processor generates an output indicating the disablement of use of the portion of the blood sample to perform at least some measurements of the sample.

[0151] In some applications, a generally similar technique is performed on a first portion of the blood sample placed in the first set of sample chambers 52. For example, the computer processor may be configured to identify regions in a microscopic image of the sample chamber where no sample is present and to disable at least the identified regions from use in microscopic analysis of the portion of the sample. In some applications, the computer processor is configured to identify such regions by identifying an interface between wet and dry regions on a base surface of the sample chamber. Typically, in identifying such regions, the computer processor is configured to distinguish between regions where no sample is present and regions where the sample has a low cell density.

[0152] In some applications, the computer processor is configured to identify the presence of debris (e.g., spilled blood) on the exterior surface of the sample carrier. As described above, typically, at least one microscopic image of a monolayer of cells settled on the base surface of the sample carrier is acquired. Typically, the monolayer of cells is positioned in the monolayer focal plane and the image is acquired while the microscope is focused on the monolayer focal plane. In some applications, the computer processor identifies that debris is under the top cover or base surface of the sample carrier by identifying an area where an entity is visible in a focal plane different from the monolayer focal plane. Alternatively or additionally, the computer processor identifies an area where the background intensity of the microscopic image (acquired in the monolayer focal plane) indicates the presence of debris under the top cover or base surface. In some applications, the computer processor responsively disables at least this identified area from use in the microscopic analysis of this portion of the sample.

[0153] As described above, typically, a first portion of the blood sample is imaged under bright field imaging, i.e., under illumination from one or more bright field light sources (e.g., one or more bright field light emitting diodes typically emitting in each spectral band). More typically, the first portion of the blood sample is further imaged under fluorescent imaging. Typically, fluorescent imaging is performed by exciting stained objects in the sample by directing light of known excitation wavelengths (i.e., wavelengths at which the stained objects are known to fluoresce when excited with light of these wavelengths) toward the sample. Typically, in fluorescent imaging, a separate set of one or more light emitting diodes is used to illuminate the sample with known excitation wavelengths.

[0154] In some applications, the computer processor analyzes the light emitted by the bright field light source and determines characteristics of the bright field light source. For example, the computer processor may periodically determine whether the spatial distribution of illumination by the bright field light source has changed since a previous measurement (e.g., whether the spatial uniformity or spatial location of the illumination has changed since a previous measurement), whether the vignetting effect of illumination by the bright field light source has changed since a previous measurement, whether the spectral distribution of illumination by the bright field light source has changed since a previous measurement, and / or whether the intensity of illumination by the bright field light source has changed since a previous measurement. As described above, the computer processor typically determines parameters of the blood sample by performing measurements on entities identified in the microscopic image of the blood sample. In some applications, such measurements are normalized based on the determined characteristics of the bright field light source. In some applications, the computer processor disables performing at least some measurements on the blood sample based on the determined characteristics of the bright field light source.

[0155] In some applications, to determine the characteristics of the bright field light source, the computer processor analyzes light emitted by the bright field light source in the absence of the sample carrier. Alternatively or additionally, the computer processor analyzes light emitted by the bright field light source that passes through intercellular regions in the blood sample.

[0156] In some applications, the computer processor analyzes the light emitted by the fluorescent light source and determines characteristics of the fluorescent light source. For example, the computer processor can periodically determine whether the spatial distribution of the illumination by the fluorescent light source has changed since a previous measurement (e.g., whether the spatial uniformity or spatial location of the illumination has changed since a previous measurement), whether the vignetting effect of the illumination by the fluorescent light source has changed since a previous measurement, whether the spectral distribution of the illumination by the fluorescent light source has changed since a previous measurement, and / or whether the intensity of the illumination by the fluorescent light source has changed since a previous measurement. As described above, the computer processor typically determines parameters of the blood sample by performing measurements on entities identified in the microscopic image of the blood sample. In some applications, such measurements are normalized based on the determined characteristics of the fluorescent light source. In some applications, the computer processor disables performing at least some measurements on the blood sample based on the determined characteristics of the fluorescent light source.

[0157] In some applications, to determine the characteristic of the fluorescent light source, the computer processor identifies one or more fluorescent regions visible in the microscopic image acquired under illumination by the fluorescent light source other than the stained cells, and determines the characteristic of the fluorescent light source based on the identified fluorescent regions. For example, such fluorescent regions may include intercellular regions in the blood sample, one or more fluorescent regions of the microscope unit, and / or one or more fluorescent regions of the sample carrier (e.g., the pressure-sensitive adhesive of the sample carrier described above). In some applications, the sample carrier is placed on a stage in the microscope unit, the stage including a fluorescent region for performing the measurements described above. Alternatively or additionally, the sample carrier includes a fluorescent region (e.g., a fluorescent patch) for performing the measurements described above.

[0158] In some applications, the computer processor is configured to acquire microscopic images in the absence of the sample carrier and detect whether there is dirt or scratches on parts of the microscope unit (such as the CCD camera, the CMOS camera, the lens, and / or the stage for holding the sample carrier) either directly or by identifying scratches in such images. In some applications, such images are acquired periodically (e.g. at fixed time intervals) to determine whether the microscope unit, parts of the microscope unit, have dirt or scratches. In some applications, in response to detecting such dirt and / or scratches, a user is notified. Alternatively or additionally, areas in the images corresponding to the location of the dirt and / or scratches are not included in at least a part of the image analysis of the sample. Further, alternatively or additionally, measurements performed on areas in the images corresponding to the location of the dirt and / or scratches are calibrated to account for the dirt and / or scratches.

[0159] For some applications, the computer processor is configured to detect whether a portion of the sample carrier has dirt or scratches using techniques generally similar to those described above. For example, the sample carrier can be imaged in the absence of a sample. For some applications, the computer processor is configured to detect whether a portion of the sample carrier fluoresces irregularly, for example by imaging the sample carrier in the absence of a sample.

[0160] In some applications, in response to detecting that an error is present in at least a portion of the fluorescent microscope images, the computer processor classifies the source of the error as follows: In response to detecting that the error occurred from a given time, the computer processor identifies the dye as the source of the error, since this indicates that the error occurred as a result of using a new dye; In response to detecting that the error gradually increased over time, the computer processor identifies debris in the microscope unit as the source of the error, since such debris typically causes gradual degradation over time; In response to detecting that the error is present only in images acquired under illumination by a given one of the light sources (e.g., a light emitting diode), the computer processor identifies the given one of the light sources as the source of the error.

[0161] In some applications, the optical measurement unit includes a microscope, which typically includes a microscope stage on which the sample carrier is placed. Typically, a computer processor drives the microscope stage to move, with one or more motors used to drive the movement of the mechanical element. In some cases, there is some degree of play in the movement of the mechanical element. For example, if the direction of the movement is initiated or reversed, there may be a delay between when the computer-implemented command is sent to the motor and when the movement of the mechanical element is performed. In some applications, the computer processor accounts for this effect when performing the movement (for example, by assuming that there is a given fixed delay, or by periodically measuring the delay and accounting for the most recently measured delay). In some applications, the amount of play is quantified using the microscope system and is interpreted as indicating the state of the mechanical element and / or one or more motors. For example, the amount of play can be quantified by observing how many motor steps are required to generate a distinguishable movement in the microscope system and / or by repeating the same measurement in two different movement directions and correlating the images or metrics extracted from these images related to the movements in these two different directions. In some applications, a state of the mechanical element and / or one or more motors is determined based on the measurements. In some applications, an output is generated in response to the determined state of the mechanical element and / or one or more motors. For example, analysis of at least some images of the sample (and / or data related to the sample) may be refused, other parts of the microscope and / or optical measurement unit may be locked from use, and / or an alarm may be generated (e.g., an alarm indicating that servicing is required and / or an alarm indicating that preemptive servicing is desirable).

[0162] In some applications, microscope images (and / or other signals) are acquired while the stage is moving. In some such applications, there may be a timing discrepancy between the reported or interpolated stage position at a given time and the actual timing of acquisition by the camera or sensor at that position. This can introduce errors in the position where a given image (or data) is assumed to have been acquired, which may introduce further errors if this position is then used for other purposes (e.g., if this image (or data) and its corresponding position are used as input for focusing a microscope). In some applications, such timing discrepancies are measured by measuring the same target twice, for example as follows: a first image of the target (or a data set associated with the target) is acquired using a static acquisition along a mechanical axis, and a second image of the target (or a data set associated with the target) is acquired by acquisition while the stage is moving. A difference between these images, or between distances extracted from the images (and / or a difference between the two data sets), is detected, and if such a difference is detected, the computer processor uses this as an input to determine that a timing discrepancy exists and / or to correct the timing discrepancy. In some applications, such measurements are performed periodically and used as input to determine the status of parts of the optical measurement system (such as parts of the microscope system including image acquisition, mechanical elements, and / or motors). In some applications, an output is generated in response to a detected timing discrepancy and / or difference from a previous measurement. For example, analysis of at least some images of the sample (and / or data relating to the sample) may be refused, the microscope and / or other parts of the optical measurement unit may be locked from use, and / or an alarm may be generated (e.g., an alarm indicating that after-sales service is required and / or an alarm indicating that pre-service service is advisable).

[0163] In some applications, the condition of the optical measurement unit (e.g., the microscope of the optical measurement system and / or the light absorption measurement portion of the optical measurement unit) is estimated by imaging a target that is installed on the optical measurement unit itself (or routinely input into the optical measurement unit). For example, a scratched glass surface, a printed glass surface, one or more fluorescent or non-fluorescent beads, a pinhole, or any other resolution target can be used. In some applications, by acquiring and analyzing images of such targets (and / or data related to such targets), the computer processor derives optical attributes of the optical measurement unit, such as aberration, resolution, contrast, scattering, and attenuation. In some applications, the computer processor periodically performs such an analysis and compares the determined attributes to predetermined values. In some applications, an output is generated in response to detecting a difference between one or more of the determined attributes and the predetermined value. For example, the analysis of at least some images of the sample (and / or data related to the sample) can be refused, the microscope and / or other parts of the optical measurement unit can be locked from use, and / or an alarm can be generated (e.g., an alarm indicating that after-sales service is required and / or an alarm indicating that pre-service is advisable). In some applications, based on the determined attributes, the computer processor corrects the extracted image features, the measurements (in the case of non-imaging measurements), and / or the measured quantities of the sample. For example, absorption measurements can be corrected for changes in the apparent contrast of the target.

[0164] In some applications, the sample described herein is a sample containing blood or a component thereof (e.g., a diluted or undiluted whole blood sample, a sample containing primarily red blood cells, or a diluted sample containing primarily red blood cells), and parameters related to components of blood, such as, for example, platelets, white blood cells, abnormal white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howell-Jolly bodies, etc., are determined.

[0165] Generally, although some applications of the present invention have been described in relation to blood samples, it should be noted that the scope of the present invention includes the application of the devices and methods described herein to a variety of samples. In some applications, the sample is a biological sample, such as blood, saliva, semen, sweat, sputum, vaginal fluid, feces, breast milk, bronchoalveolar lavage fluid, gastric lavage fluid, tears, and / or nasal secretions. The biological sample can be taken from any living creature, typically from a warm-blooded animal. In some applications, the biological sample is from a mammal, e.g., a human body sample. In some applications, the sample is taken from captive animals, zoo animals, and farm animals, including but not limited to dogs, cats, cows, and sheep. Alternatively or additionally, the biological sample is taken from animals that act as disease vectors, including deer or mice.

[0166] In some applications, techniques similar to those described above are applied to non-bodily samples. In 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 sample, a water sample, a washings sample, a beverage sample, and / or any combination thereof.

[0167] Applications of the invention described herein may 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 for use by or in association with any instruction execution system, such as a computer or computer processor 28. For purposes of this description, a computer usable or computer readable medium may be any apparatus that can contain, store, communicate, propagate, or transfer a program for use by or in association with an instruction execution system, apparatus, or device. The medium may be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device), or a propagation medium. Typically, the computer usable or computer readable medium is a non-transitory computer usable or computer readable medium.

[0168] Examples of a computer-readable medium include a semiconductor or solid state memory, magnetic tape, a removable computer diskette, a random access memory (RAM), a read-only memory (ROM), a rigid magnetic disk, and an optical disk. Current examples of optical disks include compact disk-read-only memory (CD-ROM), compact disk-read / write (CD-R / W), and DVD.

[0169] A data processing system suitable for storing and / or executing program code includes at least one processor (e.g., computer processor 28) coupled directly or indirectly to memory elements (e.g., memory 30) via a system bus. The memory elements may include local memory used during the actual execution of the program code, mass storage devices, and cache memories that provide temporary storage of at least some of the program code to reduce the number of times the code must be retrieved from mass storage devices during execution. The system is capable of reading instructions of the present invention on a program storage device and performing the methods of embodiments of the present invention in accordance with these instructions.

[0170] A network adapter may be coupled to the processor to enable the processor to be coupled to other processors or remote printers or storage devices over a private or public network. Modems, cable modems, and Ethernet cards are just a few of the currently available types of network adapters.

[0171] Computer program code for carrying out operations of the present invention can be written in any combination of one or more programming languages, including object-oriented programming languages, such as Java, Smalltalk, C++, and conventional procedural programming languages, such as the C programming language or a similar programming language.

[0172] It will be understood that the algorithms described herein may be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine whereby the instructions executed by the processor of the computer (e.g., computer processor 28) or other programmable data processing apparatus produce means for performing the functions / acts specified in the algorithms described herein. These computer program instructions may also be stored on a computer readable medium (e.g., a non-transitory computer readable medium) to instruct the computer or other programmable data processing apparatus to function in a particular manner such that the instructions stored on the computer readable medium produce an article of manufacture including the blocks of the flowcharts and instruction means for performing the functions / acts specified in the algorithms. The computer program instructions may also be loaded into a computer or other programmable data processing apparatus to perform a series of operational steps on the computer or other programmable apparatus to produce a computer implemented process whereby the instructions executing on the computer or other programmable apparatus provide a process for performing the functions / acts specified in the algorithms described herein.

[0173] Computer processor 28 is typically a hardware device that is programmed with computer program instructions to create a special purpose computer. For example, when programmed to execute the algorithms described herein, computer processor 28 typically functions as a special purpose sample analysis computer processor. Typically, the operations described herein performed by computer processor 28 change the physical state of memory 30, which is an actual physical item, to have a different magnetic polarity, charge, etc. depending on the memory technology used.

[0174] The apparatus and methods described herein may be used in conjunction with the apparatus and methods described in any one of the following patent applications, all of which are incorporated herein by reference: U.S. Patent Application Publication No. US2012 / 0169863 to Bachelet U.S. Patent Application Publication No. US2014 / 0347459 to Greenfield U.S. Patent Application Publication No. US2015 / 0037806 to Pollak U.S. Patent Application Publication No. US2015 / 0316477 to Pollak U.S. Patent Application Publication No. US2016 / 0208306 to Pollak U.S. Patent Application Publication No. US2016 / 0246046 to Yorav Raphael U.S. Patent Application Publication No. US2016 / 0279633 to Bachelet U.S. Patent Application Publication No. US2018 / 0246313 to Eshel International Publication No. WO16 / 030897 to Yorav Raphael International Publication No. WO17 / 046799 to Eshel International Publication No. WO17 / 168411 to Eshel International Publication No. WO17 / 195205 to Pollack U.S. Patent Application Publication No. US2019 / 0145963 to Zait International Publication No. US19 / 097387 to Yorav-Raphael

[0175] It will be appreciated by those skilled in the art that the present invention is not limited to what has been specifically shown and described above, and the scope of the present invention includes both combinations and subcombinations of the various features described above that would occur to one skilled in the art upon reading the foregoing description, as well as variations and modifications thereof that are not present in the prior art.

Claims

1. placing at least a portion of the blood sample in a sample chamber; obtaining at least one microscopic image of the portion of the blood sample; using a computer processor in the optical measurement unit, identifying within the microscopic image candidates for a given entity within the blood sample; authenticating at least some of the candidates as the given entity by performing further analysis on the candidates; determining a ratio between the number of candidates for the given entity and the number of authenticated candidates for the given entity; Disabling use of at least the portion of the sample for performing at least some measurements on the sample based on the ratio between the number of candidates and the number of validated candidates; using a computer processor, The method includes:

2. Identifying candidates for a given entity in the blood sample within the microscopic image includes identifying candidates for a given entity in the blood sample within the microscopic image; authenticating at least some of the candidates as the given entity by performing further analysis on the candidates includes authenticating at least some of the platelet candidates as platelets by performing further analysis on the platelet candidates; 2. The method of claim 1, wherein disabling use of at least the portion of the sample to perform at least some measurements on the sample comprises disabling use of at least the portion of the sample to perform at least some measurements on the sample based at least in part on a ratio between the number of the platelet candidates and the number of the authenticated platelet candidates.

3. Identifying candidates for a given entity in the blood sample in the microscopic image includes identifying candidates for a white blood cell in the blood sample in the microscopic image; authenticating at least some of the candidates as the given entity by performing further analysis on the candidates includes authenticating at least some of the white blood cell candidates as white blood cells by performing further analysis on the candidates; 2. The method of claim 1, wherein disabling use of at least the portion of the sample to perform at least some measurements on the sample comprises disabling use of at least the portion of the sample to perform at least some measurements on the sample based at least in part on a ratio between the number of white blood cell candidates and the number of authenticated white blood cell candidates.

4. 4. The method of claim 1, wherein disabling use of at least the portion of the sample to perform at least some measurements on the sample comprises disabling use of at least the portion of the sample to perform at least some measurements on the sample based on the ratio between the number of authenticated candidates and the number of candidates that are above a maximum threshold.

5. 4. The method of claim 1, wherein disabling use of at least the portion of the sample to perform at least some measurements on the sample comprises disabling use of at least the portion of the sample to perform at least some measurements on the sample based on the ratio between the number of authenticated candidates and the number of the candidates less than a minimum threshold.

6. placing at least a portion of the blood sample in a sample chamber; obtaining at least one microscopic image of the portion of the blood sample; using a computer processor in the optical measurement unit, identifying, within the microscopic image, potential white blood cells within the blood sample; performing further analysis on the white blood cell candidates to identify at least some of the white blood cell candidates as white blood cells of a given type; determining a ratio between the number of said white blood cell candidates and the number of said white blood cell candidates authenticated as said given type of white blood cell; invalidating the use of at least the portion of the sample for performing at least some measurements on the sample based on the ratio between the number of the white blood cell candidates and the number of the white blood cell candidates authenticated as the given type of white blood cell; using a computer processor, The method includes:

7. a sample chamber configured to receive at least a portion of the blood sample; An optical measurement unit, a microscope configured to acquire at least one microscopic image of the portion of the blood sample; and and an optical measurement unit including at least one computer processor, The at least one computer processor: identifying within the microscopic image candidates for a given entity within the blood sample; authenticating at least some of the candidates as the given entity by performing further analysis on the candidates; determining a ratio between the number of candidates for the given entity and the number of authenticated candidates for the given entity; and disabling use of at least the portion of the sample for performing at least some measurements on the sample based on the ratio between the number of candidates and the number of authenticated candidates.

8. The computer processor includes: identifying candidates for a given entity in the blood sample within the microscopic image by identifying candidates for white blood cells in the blood sample within the microscopic image; authenticating at least some of the platelet candidates as platelets by performing further analysis on the platelet candidates; 8. The device of claim 7, configured to disable using at least the portion of the sample for performing at least some measurements on the sample based at least in part on a ratio between the number of the white blood cell candidates and the number of the authenticated white blood cell candidates.

9. The computer processor includes: identifying, within the microscopic image, potential white blood cells within the blood sample; authenticating at least some of the leukocyte candidates as leukocytes by performing further analysis on the candidates; 8. The device of claim 7, configured to disable using at least the portion of the sample to perform at least some measurements on the sample based at least in part on a ratio between the number of white blood cell candidates and the number of authenticated white blood cell candidates.

10. 10. The apparatus of claim 7, wherein the computer processor is configured to disable using at least the portion of the sample to perform at least some measurements on the sample based on the ratio between the number of authenticated candidates and the number of the candidates that are above a maximum threshold.

11. 10. The apparatus of claim 7, wherein the computer processor is configured to disable using at least the portion of the sample to perform at least some measurements on the sample based on the ratio between the number of authenticated candidates and the number of the candidates below a minimum threshold.

12. a sample chamber configured to receive at least a portion of the blood sample; An optical measurement unit, a microscope configured to acquire at least one microscopic image of the portion of the blood sample; and and an optical measurement unit including at least one computer processor, The at least one computer processor: identifying, within the microscopic image, potential white blood cells within the blood sample; performing further analysis on the white blood cell candidates to identify at least some of the white blood cell candidates as white blood cells of a given type; determining a ratio between the number of the white blood cell candidates and the number of the white blood cell candidates that are identified as the given type of white blood cell; The device is configured to disable using at least the portion of the sample to perform at least some measurements on the sample based at least in part on the ratio between the number of the white blood cell candidates and the number of the white blood cell candidates authenticated as the given type of white blood cell.

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