Defocused microscopic images of samples

Abstract

Apparatus and methods for using body samples containing cells are described. A microscope (24) is focused such that the focal plane of the microscope (24) is at least approximately aligned with a horizontal plane in which at least some cells belonging to the sample are at least partially positioned. At least one focused microscopic image of the sample is acquired while the focal plane of the microscope (24) is approximately aligned with this horizontal plane. The microscope (24) is then focused such that the focal plane of the microscope is shifted relative to this horizontal plane, and at least one defocused microscopic image of the sample is acquired while the focal plane of the microscope (24) is shifted relative to this horizontal plane. Properties of at least a portion of the sample are determined at least partially based on the focused and defocused images. Other applications are also described.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 946,985, filed December 12, 2019, by Gluck et al., and U.S. Provisional Patent Application No. 63 / 048,692, filed July 7, 2020, both of which are entitled “Distinguishing between NRBCs and leukocytes”, and are incorporated herein by reference.

[0003] Field of the embodiments of the present invention

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

[0005] background

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

[0007] Red blood cells (erythrocytes) in mammals are the body's oxygen carriers. During maturation, red blood cells undergo enucleation, meaning the nucleus is completely removed from the cell. Normally, nucleated red blood cells (NRBCs) are absent in the peripheral blood of adult patients. However, in some cases (such as newborns, cancer patients, and patients with anemia), NRBCs are present in the peripheral blood.

[0008] Implementation Plan Overview

[0009] As described in the background section above, mammalian red blood cells are the body's oxygen carriers. Red blood cells undergo enucleation during maturation, meaning the nucleus is completely removed from the cell. Normally, nucleated red blood cells (NRBCs) are not present in the peripheral blood of adult patients. However, NRBCs are present in peripheral blood in some cases (such as neonates, cancer patients, and patients with anemia). Because NRBCs are similar in size and nucleus content to some white blood cells (and particularly lymphocytes), it is often difficult to distinguish NRBCs from white blood cells. In some applications of this invention, a complete blood count is performed on the blood sample. In the case of a complete blood count, it is often important to distinguish NRBCs from white blood cells to avoid overcounting white blood cells (especially in patients with low white blood cell counts) and to detect the presence of NRBCs (which may indicate an underlying condition).

[0010] According to some applications of the present invention, in order to distinguish leukocytes (e.g., lymphocytes) from NRBCs (e.g., in the case of detecting NRBC / leukocyte candidates (i.e., candidates for either NRBCs or leukocytes)), a microscopic image is acquired by illuminating the sample with light of a specific wavelength (at which hemoglobin has a high absorption level). Typically, violet light is used, for example, light with wavelengths in the range greater than 400 nm and / or less than 450 nm (e.g., 400 nm–450 nm). (Note that there are some variations in the literature regarding the wavelength range referred to as being in the violet light range. For the purposes of this application, violet light should be interpreted as light included in the 400 nm–450 nm range.) Within this wavelength range, hemoglobin absorption is relatively high compared to other wavelengths in the visible light spectrum. Typically, NRBCs have a high hemoglobin content (on the order of 30 picograms per cell), while leukocytes do not contain hemoglobin. Therefore, in images acquired under violet light illumination, NRBCs typically absorb light, while leukocytes do not.

[0011] Typically, NRBC / leukocyte candidates are classified as NRBCs or leukocytes, at least in part, based on the intensity of the candidates within an image acquired under violet illumination. For some applications, an intensity threshold is applied to the image (and / or a specific region or pixel thereof) acquired under violet light conditions, and NRBC / leukocyte candidates are classified as NRBCs or leukocytes based on whether their intensity exceeds the threshold.

[0012] For some applications, techniques similar to those described above are typically employed, but using light with wavelengths greater than 500 nm and / or less than 600 nm (e.g., 500 nm–600 nm). Within this wavelength range, the absorption of carbaminohemoglobin is relatively high compared to other wavelengths in the visible light spectrum.

[0013] According to some applications of the invention, a portion of a blood sample comprising a cell suspension is placed in a sample chamber of a carrier, the sample chamber being a cavity including a substrate surface. Typically, cells in the cell suspension are allowed to settle onto the substrate surface of the sample chamber to form a cell monolayer on the substrate surface of the sample chamber. After the cells have settled onto the substrate surface of the sample chamber (e.g., by a predetermined time interval of settling), at least one microscopic image of at least a portion of the cell monolayer is typically acquired.

[0014] For some applications, in addition to acquiring images of the microscope's focal plane set approximately to coincide with the monolayer focus level (such images are referred to herein as "on-focus" images), the microscope also acquires images of the microscope's focal plane set offset along the optical axis relative to the monolayer focus level (such images are referred to herein as "off-focus" images). Typically, such off-focus microscopic images are acquired when the microscope's focal plane is set closer to the microscope's objective lens than the monolayer focus level, although the scope of the invention includes acquiring off-focus images when the microscope's focal plane is set farther from the microscope's objective lens than the monolayer focus level. For some applications, off-focus microscopic images are acquired when the microscope's focal plane is offset by more than 20 micrometers and / or less than 100 micrometers (e.g., 20-100 micrometers) relative to the monolayer focus level. Optionally or additionally, off-focus microscopic images are acquired when the microscope's focal plane is offset by more than one microscope depth of focus and / or less than five microscope depths of focus (e.g., between one and five microscope depths of focus).

[0015] The inventors of this application have discovered that such defocused microscopic images can generate important data about a sample. Specifically, defocused images are commonly used to identify cell outlines and / or certain entities within a sample, such as leukocytes, leukocyte types (e.g., lymphocytes, granulocytes, monocytes, neutrophils, band neutrophils, eosinophils, basophils, macrophages, and / or blastocytes), erythrocytes, erythrocyte types (e.g., mature erythrocytes, NRBCs, echinocytes, sickle cells, teardrop cells), and / or platelets. For some applications, defocused images are used to enhance the visibility of such entities relative to other entities, allowing them to be identified with greater certainty. Optionally or additionally, defocused images are used to make such entities easier to distinguish from other entities that might otherwise be confused with them. For some applications, defocused images are used to characterize cellular features, such as the hemoglobin content of erythrocytes, other components of erythrocytes, and / or the maturity of cells (such as neutrophils).

[0016] Therefore, according to some applications of the present invention, a method for use on a body sample containing cells is provided, the method comprising:

[0017] The microscope is focused such that the focal plane of the microscope at least approximately coincides with the horizontal plane in which at least some of the cells belonging to the sample are at least partially positioned.

[0018] At least one focused microscopic image of the sample is acquired when the focal plane of the microscope approximately coincides with the horizontal plane.

[0019] The microscope is focused such that the focal plane of the microscope is shifted relative to the horizontal plane.

[0020] At least one defocused microscopic image of the sample is acquired when the focal plane of the microscope is shifted relative to the horizontal plane; and

[0021] The characteristics of at least a portion of the sample are determined based at least in part on the focused image and the defocused image.

[0022] In some applications, acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is shifted relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is shifted by a predetermined offset relative to the horizontal plane.

[0023] In some applications, determining at least a portion of the characteristics of the sample based at least in part on the focused image and the defocused image includes inputting the focused image and the defocused image into a machine learning classifier configured to determine at least a portion of the characteristics of the sample based at least in part on the focused image and the defocused image.

[0024] In some applications, determining at least a portion of the characteristics of the sample based at least in part on the focused image and the defocused image includes deriving one or more parameters from the focused image and the defocused image, and inputting the derived one or more parameters into a machine learning classifier configured to determine at least a portion of the characteristics of the sample based at least in part on the derived parameters.

[0025] In some applications, the sample includes a blood sample, and acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 505 nm and 535 nm.

[0026] In some applications, the sample includes a blood sample, and acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 400 nm and 450 nm.

[0027] In some applications, the sample includes a blood sample, and acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 620 nm and 640 nm.

[0028] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is set closer to the objective lens of the microscope than the horizontal position at least some of the cells belonging to the sample are at least partially positioned.

[0029] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is set further away from the objective lens of the microscope than the horizontal position at least some of the cells belonging to the sample are at least partially positioned.

[0030] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is at least partially positioned between 20 micrometers and 100 micrometers horizontally relative to at least some of the cells belonging to the sample.

[0031] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is at least partially positioned relative to at least some of the cells belonging to the sample between one and five focal depths of the microscope at a horizontal offset.

[0032] In some applications, the method further includes allowing some cells within the sample to settle in order to form a monolayer at a monolayer focal level, and focusing the microscope such that the focal plane of the microscope at least approximately coincides with the level at which at least some cells belonging to the sample are at least partially positioned, including focusing the microscope such that the focal plane of the microscope at least approximately coincides with the monolayer focal level.

[0033] In some applications, the method further includes allowing some cells within the sample to settle to form a monolayer at a single focal level, while other cells within the sample are suspended at at least one additional level within the sample. Focusing the microscope such that the focal plane of the microscope at least approximately coincides with the level at which at least some cells belonging to the sample are at least partially placed includes focusing the microscope such that the focal plane of the microscope at least approximately coincides with the additional level at which other cells within the sample are suspended.

[0034] In some applications, determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image includes normalizing the focused image and the defocused image relative to each other, and determining the characteristics of a portion of the sample based at least in part on the normalization.

[0035] In some applications, the sample includes a blood sample, and determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image includes identifying one or more entities within the blood sample based at least in part on the focused image and the defocused image, the one or more entities being selected from the group consisting of: platelets, leukocytes, lymphocytes, granulocytes, monocytes, neutrophils, band neutrophils, eosinophils, basophils, and macrophages.

[0036] In some applications, the sample includes a blood sample, and determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image includes identifying blast cells within the blood sample based at least in part on the focused image and the defocused image.

[0037] In some applications, determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image includes identifying the outlines of one or more entities within the sample based at least in part on the focused image and the defocused image.

[0038] In some applications, determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image also includes estimating parameters of the one or more entities based at least in part on the identified contours, the parameters being selected from the group consisting of cell area and cell volume.

[0039] In some applications, determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image also includes estimating parameters of the sample based at least in part on the identified contour, the at least one parameter being selected from the group consisting of: mean cell area and mean cell volume.

[0040] According to some applications of the present invention, an apparatus for use with body samples containing cells is also provided, the apparatus comprising:

[0041] microscope; and

[0042] Computer processor, the computer processor being configured to:

[0043] The microscope is focused such that the focal plane of the microscope at least approximately coincides with the horizontal plane in which at least some of the cells belonging to the sample are at least partially positioned.

[0044] When the focal plane of the microscope approximately coincides with the horizontal plane, the microscope is driven to acquire at least one focused microscopic image of the sample.

[0045] The microscope is focused such that the focal plane of the microscope is shifted relative to the horizontal plane.

[0046] When the focal plane of the microscope is shifted relative to the horizontal plane, the microscope is driven to acquire at least one defocused microscopic image of the sample, and

[0047] The characteristics of at least a portion of the sample are determined based at least in part on the focused image and the defocused image.

[0048] According to some applications of the present invention, a method for use on a body sample containing cells is also provided, the method comprising:

[0049] The microscope is focused such that the focal plane of the microscope is at least partially offset horizontally relative to at least some of the cells belonging to the sample.

[0050] At least one defocused microscopic image of the sample is acquired when the focal plane of the microscope is shifted relative to the horizontal plane; and

[0051] Based at least in part on the defocused image, one or more operations selected from the group consisting of: identifying entities placed within the level, determining the outline of entities placed within the level, determining parameters of entities placed within the level, determining parameters of the sample, and any combination thereof.

[0052] In some applications, acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is shifted relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is shifted by a predetermined offset relative to the horizontal plane.

[0053] In some applications, performing one or more operations at least in part based on the defocused image includes feeding the defocused image into a machine learning classifier configured to perform one or more operations at least in part based on the defocused image.

[0054] In some applications, performing the one or more operations at least in part based on the defocused image includes deriving one or more parameters from the defocused image and inputting the derived one or more parameters into a machine learning classifier configured to perform the one or more operations at least in part based on the derived parameters.

[0055] In some applications, the sample includes a blood sample, and acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 505 nm and 535 nm.

[0056] In some applications, the sample includes a blood sample, and acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 400 nm and 450 nm.

[0057] In some applications, the sample includes a blood sample, and acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the horizontal plane includes acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 620 nm and 640 nm.

[0058] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is set closer to the objective lens of the microscope than the horizontal position at least some of the cells belonging to the sample are at least partially positioned.

[0059] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is set further away from the objective lens of the microscope than the horizontal position at least some of the cells belonging to the sample are at least partially positioned.

[0060] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is at least partially positioned between 20 micrometers and 100 micrometers horizontally relative to at least some of the cells belonging to the sample.

[0061] In some applications, focusing the microscope such that the focal plane of the microscope is offset relative to the horizontal includes focusing the microscope such that the focal plane of the microscope is at least partially positioned relative to at least some of the cells belonging to the sample between one and five focal depths of the microscope at a horizontal offset.

[0062] In some applications, the method further includes allowing some cells within the sample to settle in order to form a monolayer at a monolayer focal level, and focusing the microscope such that the focal plane of the microscope is shifted relative to the level includes focusing the microscope such that the focal plane of the microscope is shifted relative to the monolayer focal level.

[0063] In some applications, the method further includes allowing some cells within the sample to settle to form a monolayer at a single focal level, while other cells within the sample are suspended at at least one additional level within the sample, and focusing the microscope such that the focal plane of the microscope is shifted relative to the level includes focusing the microscope such that the focal plane of the microscope is shifted relative to the additional level to which other cells within the sample are suspended.

[0064] In some applications, determining the characteristics of a portion of the sample based at least in part on the defocused image includes normalizing the defocused image relative to another image and determining the characteristics of a portion of the sample based at least in part on the normalization.

[0065] In some applications, the sample includes a blood sample, and determining the characteristics of a portion of the sample based at least in part on the defocused image includes identifying one or more entities within the blood sample based at least in part on the defocused image, the one or more entities being selected from the group consisting of: platelets, leukocytes, lymphocytes, granulocytes, monocytes, neutrophils, band neutrophils, eosinophils, basophils, and macrophages.

[0066] In some applications, the sample includes a blood sample, and determining the characteristics of a portion of the sample based at least in part on the defocused image includes identifying blast cells within the blood sample based at least in part on the defocused image.

[0067] In some applications, determining the characteristics of a portion of the sample based at least in part on the defocused image includes identifying the outlines of one or more entities within the sample based at least in part on the defocused image.

[0068] In some applications, determining the characteristics of a portion of the sample based at least in part on the defocused image also includes estimating parameters of the one or more entities based at least in part on the identified contours, the parameters being selected from the group consisting of cell area and cell volume.

[0069] In some applications, determining the characteristics of a portion of the sample based at least in part on the defocused image also includes estimating parameters of the sample based at least in part on the identified contour, the at least one parameter being selected from the group consisting of: mean cell area and mean cell volume.

[0070] According to some applications of the present invention, an apparatus for use with body samples containing cells is also provided, the apparatus comprising:

[0071] microscope; and

[0072] Computer processor, the computer processor being configured to:

[0073] The microscope is focused such that the focal plane of the microscope is at least partially shifted horizontally relative to at least some of the cells belonging to the sample.

[0074] When the focal plane of the microscope is shifted relative to the horizontal plane, the microscope is driven to acquire at least one defocused microscopic image of the sample, and

[0075] Based at least in part on the defocused image, one or more operations selected from the group consisting of: identifying entities placed within the level, determining the outline of entities placed within the level, determining parameters of entities placed within the level, determining parameters of the sample, and any combination thereof.

[0076] According to some applications of the present invention, a method for use on a body sample containing cells is also provided, the method comprising:

[0077] The microscope is focused such that the focal plane of the microscope at least approximately coincides with the horizontal plane in which at least some of the cells belonging to the sample are at least partially positioned;

[0078] At least one focused microscopic image of the sample is acquired when the focal plane of the microscope approximately coincides with the horizontal plane.

[0079] The microscope is focused such that the focal plane of the microscope is shifted relative to the horizontal plane.

[0080] When the focal plane of the microscope is shifted by a predetermined offset relative to the horizontal plane, at least one defocused microscopic image of the sample is acquired; and

[0081] By analyzing the focused image and the defocused image, it is verified that the focal plane of the microscope coincides with the horizontal plane in the focused image.

[0082] According to some applications of the present invention, an apparatus for use with body samples containing cells is also provided, the apparatus comprising:

[0083] microscope; and

[0084] Computer processor, the computer processor being configured to:

[0085] The microscope is focused such that the focal plane of the microscope at least approximately coincides with the horizontal plane in which at least some of the cells belonging to the sample are at least partially positioned.

[0086] When the focal plane of the microscope approximately coincides with the horizontal plane, the microscope is driven to acquire at least one focused microscopic image of the sample;

[0087] The microscope is focused such that the focal plane of the microscope is shifted relative to the horizontal plane.

[0088] When the focal plane of the microscope is shifted by a predetermined offset relative to the horizontal plane, the microscope is driven to acquire at least one defocused microscopic image of the sample, and

[0089] By analyzing the focused image and the defocused image, it is verified that the focal plane of the microscope coincides with the horizontal plane in the focused image.

[0090] According to some applications of the present invention, a method for using blood samples is also provided, the method comprising:

[0091] The NRBC / leukocyte candidate is identified in one or more microscopic images of the blood sample based on NRBC / leukocyte candidates having characteristics that indicate that the NRBC / leukocyte candidate may be an NRBC or a leukocyte.

[0092] The NRBC / leukocyte candidates were identified in microscopic images acquired under illumination in the wavelength range between 400 nm and 450 nm and / or between 500 nm and 600 nm.

[0093] The NRBC / leukocyte candidates are classified as NRBCs or leukocytes based at least in part on the light absorption levels of the candidates in microscopic images acquired under illumination in the wavelength range between 400 nm and 450 nm and / or between 500 nm and 600 nm; and

[0094] At least in part, this is based on classifying the NRBC / leukocyte candidates as NRBCs or leukocyte production outputs.

[0095] In some applications:

[0096] Identification of the NRBC / leukocyte candidates within microscopic images acquired under illumination in the wavelength range between 400 nm and 450 nm and / or between 500 nm and 600 nm includes identification of the NRBC / leukocyte candidates within violet light microscopic images acquired under violet light illumination in the wavelength range between 400 nm and 450 nm; and

[0097] Classifying the NRBC / leukocyte candidates as NRBC or leukocytes includes classifying the NRBC / leukocyte candidates as NRBC or leukocytes based at least in part on the light absorption level of the NRBC / leukocyte candidates in the violet light micrographs obtained under illumination in the wavelength range between 400 nm and 450 nm.

[0098] In some applications, classifying the NRBC / leukocyte candidates as NRBCs or leukocytes involves applying an intensity threshold to the violet light microscopy image and classifying the NRBC / leukocyte candidates as leukocytes based on the intensity of candidates exceeding the threshold.

[0099] In some applications, the method further includes, in response to the detection of the NRBC / leukocyte candidate, selecting to acquire a violet light microscopy image of the imaging field in which the NRBC / leukocyte candidate is present.

[0100] In some applications, classifying the NRBC / leukocyte candidates as NRBC or leukocytes also includes analyzing one or more additional characteristics of candidates selected from the group consisting of: candidate size, candidate nucleus size, candidate intensity in a fluorescence image, candidate cytoplasm intensity, candidate cytoplasm area, candidate ellipticity, candidate nucleus ellipticity, candidate nucleus roundness, and combinations thereof.

[0101] In some applications, the method further includes adjusting the detection threshold for detecting NRBCs in the blood sample in response to the concentration of NRBCs detected in the blood sample exceeding a threshold, so as to increase the sensitivity of NRBC detection.

[0102] In some applications, the method further includes adjusting a detection threshold for detecting one or more entities other than NRBCs in the blood sample in response to the concentration of NRBCs detected in the blood sample exceeding a threshold.

[0103] In some applications, the method further includes reanalyzing at least some of the NRBC / leukocyte candidates in response to a concentration of NRBCs detected in the blood sample exceeding a first threshold and a white blood cell count in the sample being less than a second threshold.

[0104] In some applications, the method further includes generating an output indicating that the NRBC count may be erroneous in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a white blood cell count in the sample being less than a second threshold.

[0105] In some applications, the method further includes generating an output indicating that the white blood cell count may be erroneous in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a white blood cell count in the sample being less than a second threshold.

[0106] In some applications, the method further includes generating an output indicating that the specific type of white blood cell count may be erroneous in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a count of a specific type of white blood cell in the sample being less than a second threshold.

[0107] According to some applications of the present invention, an apparatus for using blood samples is also provided, the apparatus comprising:

[0108] A microscope configured to acquire microscopic images of the blood sample; and

[0109] Computer processor, the computer processor being configured to:

[0110] The NRBC / leukocyte candidate is identified in one or more microscopic images of the blood sample based on NRBC / leukocyte candidates having characteristics that indicate that the NRBC / leukocyte candidate may be an NRBC or a leukocyte.

[0111] The NRBC / leukocyte candidates were identified in microscopic images acquired under illumination in the wavelength range between 400 nm and 450 nm and / or between 500 nm and 600 nm.

[0112] The NRBC / leukocyte candidates are classified as NRBCs or leukocytes based at least in part on the light absorption levels of the candidates in microscopic images acquired under illumination in the wavelength range between 400 nm and 450 nm and / or between 500 nm and 600 nm; and

[0113] At least in part, this is based on classifying the NRBC / leukocyte candidates as NRBCs or leukocyte production outputs.

[0114] Therefore, according to some applications of the present invention, a method for using blood samples is provided, the method comprising:

[0115] NRBC / leukocyte candidates are identified within one or more microscopic images of the blood sample based on NRBC / leukocyte candidates that have characteristics indicating that the NRBC / leukocyte candidate may be an NRBC or a leukocyte.

[0116] NRBC / leukocyte candidates were identified in violet light microscopy images acquired under violet light illumination in the wavelength range between 400 nm and 450 nm.

[0117] Based at least in part on the light absorption levels of NRBC / leukocyte candidates within ultraviolet light microscopy images, NRBC / leukocyte candidates are classified as either NRBCs or leukocytes; and

[0118] At least in part, this is based on classifying the NRBC / leukocyte candidates as NRBCs or leukocyte production outputs.

[0119] For some applications, classifying the NRBC / leukocyte candidates as NRBCs or leukocytes involves applying an intensity threshold to the violet light microscopy image and classifying the NRBC / leukocyte candidates as leukocytes based on the intensity of the candidates exceeding the threshold.

[0120] For some applications, the method further includes, in response to the detection of the NRBC / leukocyte candidate, selecting to acquire a violet light microscopy image of the imaging field in which the NRBC / leukocyte candidate is present.

[0121] For some applications, classifying the NRBC / leukocyte candidates as NRBC or leukocytes also includes analyzing one or more additional characteristics of candidates selected from the group consisting of: candidate size, candidate nucleus size, candidate intensity in a fluorescence image, candidate cytoplasm intensity, candidate cytoplasm area, candidate ellipticity, candidate nucleus ellipticity, candidate nucleus roundness, and combinations thereof.

[0122] For some applications, the method further includes adjusting the detection threshold for detecting NRBCs in the blood sample in response to the concentration of NRBCs detected in the blood sample exceeding a threshold, so as to increase the sensitivity of NRBC detection.

[0123] For some applications, the method further includes adjusting a detection threshold for detecting one or more entities other than NRBCs in the blood sample in response to the concentration of NRBCs detected in the blood sample exceeding a threshold.

[0124] For some applications, the method further includes reanalyzing at least some of the NRBC / leukocyte candidates in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a white blood cell count in the sample being less than a second threshold.

[0125] For some applications, the method further includes generating an output indicating that the NRBC count may be erroneous in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a white blood cell count in the sample being less than a second threshold.

[0126] For some applications, the method further includes generating an output indicating that the white blood cell count may be erroneous in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a white blood cell count in the sample being less than a second threshold.

[0127] For some applications, the method further includes generating an output indicating that the specific type of white blood cell count may be erroneous in response to a concentration of NRBC detected in the blood sample exceeding a first threshold and a specific type of white blood cell count in the sample being less than a second threshold.

[0128] According to some applications of the present invention, a method for using blood samples is also provided, the method comprising:

[0129] At least one ultraviolet light micrograph of the sample was acquired under ultraviolet light illumination in the wavelength range between 400 nm and 450 nm.

[0130] Identify red blood cells in at least one ultraviolet light micrograph of the sample;

[0131] Based on the red blood cells identified in at least one ultraviolet light micrograph of the sample, the statistical hemoglobin-related characteristics of the sample are determined; and

[0132] The output is generated at least in part based on the statistical hemoglobin-related characteristics of the determined samples.

[0133] According to some applications of the present invention, a method for using blood samples is further provided, the method comprising:

[0134] The NRBC / leukocyte candidate is identified in one or more microscopic images of the blood sample based on NRBC / leukocyte candidates having characteristics that indicate that the NRBC / leukocyte candidate may be an NRBC or a leukocyte.

[0135] The NRBC / leukocyte candidates were identified in microscopic images acquired under illumination in the wavelength range between 500 nm and 600 nm.

[0136] The NRBC / leukocyte candidates are classified as NRBCs or leukocytes, at least in part, based on the light absorption levels of the NRBC / leukocyte candidates within the microscopic images; and

[0137] At least in part, this is based on classifying the NRBC / leukocyte candidates as NRBCs or leukocyte production outputs.

[0138] According to some applications of the present invention, a method for using blood samples is also provided, the method comprising:

[0139] At least one microscopic image of the sample is acquired under illumination by light in the wavelength range between 500 nm and 600 nm.

[0140] Identify red blood cells in at least one microscopic image of the sample;

[0141] Based on the red blood cells identified in at least one microscopic image of the sample, the statistical hemoglobin-related characteristics of the sample are determined; and

[0142] The output is generated at least in part based on the statistical hemoglobin-related characteristics of the determined samples.

[0143] The invention will be more fully understood from the following detailed description of embodiments thereof, taken in conjunction with the accompanying drawings, in which: Brief description of the attached diagram

[0145] Figure 1 This is a block diagram illustrating the components of a biological sample analysis system according to some applications of the present invention;

[0146] Figure 2A , Figure 2B and Figure 2C This is a schematic diagram of an optical measurement unit according to some applications of the present invention;

[0147] Figure 3A , Figure 3B and Figure 3CThis is a schematic diagram of respective views of a sample carrier for both microscopic and optical density measurements according to some applications of the present invention.

[0148] Figure 4A These are microscopic images of entities used in some applications of the present invention as NRBC / leukocyte candidates;

[0149] Figure 4B These are microscopic images of NRBCs obtained under ultraviolet illumination conditions according to some applications of the present invention;

[0150] Figure 4C These are microscopic images of leukocytes obtained under ultraviolet light illumination, based on some applications of the present invention.

[0151] Figure 5 This is a flowchart illustrating the steps of a method for identifying NRBC / leukocyte candidates within one or more microscopic images of a blood sample, according to some applications of the present invention.

[0152] Figure 6A , Figure 6B and Figure 6C These are microscopic images of red blood cells obtained under red, green, and violet light illumination conditions, respectively, according to some applications of the present invention.

[0153] Figure 7 This is a flowchart illustrating the steps of a method for identifying red blood cells within one or more microscopic images of a blood sample, according to some applications of the present invention.

[0154] Figure 8 This is a flowchart illustrating the steps of a method for using a body sample containing cells according to some applications of the present invention;

[0155] Figure 9A and Figure 9B Examples of bright-field microscopic images of cell monolayers of blood samples obtained using ultraviolet illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cell monolayer. Figure 9A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 9B );

[0156] Figure 9C According to some applications of the present invention, after staining samples with acridine orange and Hoechst's reagent and exciting the samples with UV light to induce platelet fluorescence, such as... Figure 9A and Figure 9B Fluorescence micrographs of the same portion of the sample shown in the image;

[0157] Figure 10A and Figure 10BExamples of bright-field microscopic images of cell monolayers of blood samples obtained using ultraviolet illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cell monolayer. Figure 10A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 10B );

[0158] Figure 11A and Figure 11B Examples of bright-field microscopic images of a cell monolayer of a blood sample obtained using green light illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cell monolayer. Figure 11A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 11B );and

[0159] Figure 12A , Figure 12B and Figure 12C Examples of bright-field microscopic images of a cell monolayer of a blood sample obtained using green light illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cell monolayer. Figure 12A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 12B and Figure 12C ).

[0160] Detailed implementation plan

[0161] Now for reference Figure 1 This is a block diagram illustrating the components of a biological sample analysis system 20 according to some applications of the present invention. Typically, a biological sample (e.g., a blood sample) is placed in a sample carrier 22. While the sample is placed in the sample carrier, optical measurements are performed on the sample using one or more optical measuring devices 24. For example, the optical measuring devices may include microscopes (e.g., digital microscopes), spectrophotometers, photometers, spectrometers, cameras, spectral cameras, hyperspectral cameras, fluorometers, fluorescence spectrophotometers, and / or photodetectors (such as photodiodes, photoresistors, and / or phototransistors). For some applications, the optical measuring devices include dedicated light sources (such as light-emitting diodes, incandescent light sources, etc.) and / or optical elements (such as lenses, diffusers, filters, etc.) for operating light collection and / or light emission.

[0162] Computer processor 28 typically receives and processes optical measurements performed by optical measuring devices. Furthermore, computer processor typically controls the acquisition of optical measurements performed by one or more optical measuring devices. Computer processor communicates with memory 30. A user (e.g., a laboratory technician or an individual from whom a sample is taken) sends instructions to computer processor via user interface 32. For some applications, the user interface includes a keyboard, mouse, joystick, touchscreen device (such as a smartphone or tablet), touchpad, trackball, voice command interface, and / or other types of user interfaces known in the art. Typically, computer processor generates output via output device 34. Furthermore, output device typically includes a display, such as a monitor, and output includes output displayed on the display. For some applications, the processor generates output on different types of visual, text, graphic, tactile, audio, and / or video output devices (e.g., speakers, headphones, smartphones, or tablets). For some applications, user interface 32 serves as both an input interface and an output interface; that is, it serves as an input / output interface. For some applications, the processor generates output on computer-readable media (e.g., non-transitory computer-readable media) such as a disk or portable USB drive, and / or on a printer.

[0163] Now for reference Figure 2A , Figure 2B and Figure 2C , Figure 2A , Figure 2B and Figure 2C This is a schematic diagram of an optical measurement unit 31 according to some applications of the present invention; Figure 2A An oblique view of the exterior of the fully assembled device is shown, while Figure 2B and Figure 2C The respective oblique views of the apparatus are shown, with the covers made transparent to allow the components within the apparatus to be seen. For some applications, one or more optical measuring devices 24 (and / or computer processor 28 and memory 30) are housed within an optical measuring unit 31. For optical measurements of a sample, a sample carrier 22 is placed within the optical measuring unit. For example, the optical measuring unit may define a slot 36 through which the sample carrier is inserted. Typically, the optical measuring unit includes a stage 64 configured to support the sample carrier 22 within the optical measuring unit. For some applications, a screen 63 on the cover of the optical measuring unit (e.g., a screen on the front cover of the optical measuring unit, as shown) functions as a user interface 32 and / or an output device 34.

[0164] Typically, the optical measurement unit includes a microscope system 37 (in Figures 2B-2CAs shown in the diagram, it is configured to perform microscopic imaging of a portion of a sample. For some applications, the microscope system includes a set of light sources 65 (which typically includes a set of bright-field light sources (e.g., light-emitting diodes) configured for bright-field imaging of the sample, a set of fluorescent light sources (e.g., light-emitting diodes) configured for fluorescence imaging of the sample, and a camera (e.g., a CCD camera or a CMOS camera) configured to image the sample. Typically, the optical measurement unit also includes an optical density measurement unit 39 (in... Figure 2C As shown in the diagram, it is configured to perform optical density measurement (e.g., light absorption measurement) on a second portion of the sample. For some applications, the optical density measurement unit includes a set of optical density measurement light sources (e.g., light-emitting diodes) and a photodetector, which are configured to perform optical density measurement on the sample. For some applications, each of the above-described light source groups (i.e., bright-field light source group, fluorescence light source group, and optical density measurement light source group) includes more than one light source (e.g., more than one light-emitting diode), wherein each light source is configured to emit light at its respective wavelength or in its respective wavelength band.

[0165] Now for reference Figure 3A and Figure 3B , Figure 3A and Figure 3B These are schematic diagrams of various views of a sample carrier 22 according to some applications of the present invention. Figure 3A A top view of the sample carrier is shown (for illustrative purposes, the top cover of the sample carrier is shown). Figure 3A (shown as opaque in the image), and Figure 3B The bottom view is shown (where relative to) Figure 3A The view shown indicates the sample carrier has been rotated about its short edge. Typically, a sample carrier comprises one or more sample chambers in a first set 52 for microscopic analysis of the sample and sample chambers in a second set 54 for optical density measurements of the sample. Typically, the sample chambers of the sample carrier are filled with a bodily sample, such as blood, via a sample inlet orifice 38. For some applications, the sample chambers define one or more outlet orifices 40. The outlet orifices are configured to assist in the filling of the sample chambers with the bodily sample by allowing air present in the sample chambers to escape from the sample chambers. Typically, as shown, the outlet orifices are positioned longitudinally opposite the inlet orifices (relative to the sample chambers of the sample carrier). For some applications, the outlet orifices thus provide a more efficient air escape mechanism than if the outlet orifices were placed closer to the inlet orifices.

[0166] refer to Figure 3CThis illustration shows an exploded view of a sample carrier 22 according to some applications of the invention. For some applications, the sample carrier comprises at least three components: a molding assembly 42, a glass layer 44 (e.g., a glass plate), and an adhesive layer 46 configured to bond the glass layer to the underside of the molding assembly. The molding assembly is typically made of a polymer (e.g., plastic) and is molded (e.g., via injection molding) to provide a sample chamber having a desired geometry. For example, as shown, the molding assembly is typically molded to define an inlet orifice 38, an outlet orifice 40, and a groove 48 surrounding a central portion of each sample chamber. The groove typically assists in filling the sample chamber by allowing air to flow to the outlet orifice and / or by allowing body sample to flow around the central portion of the sample chamber.

[0167] For some applications, when performing a complete blood count on a blood sample, using methods such as... Figures 3A-3C The sample carrier is shown in the diagram. For some such applications, the sample carrier is used in conjunction with an optical measurement unit 31, which typically references... Figures 2A-2C The configuration is shown and described. For some applications, a first portion of the blood sample is placed in the sample chamber of the first set 52 (which is used, for example, with the microscope system 37). Figures 2B-2C (As shown in the diagram) the sample is subjected to microscopic analysis, and a second portion of the blood sample is placed in the sample chamber of the second set 54 (which is used, for example, with the optical density measurement unit 39). Figure 2C (As shown in the diagram) Optical density measurement of the sample. For some applications, the sample chambers of the first group 52 include more than one sample chamber, while the sample chambers of the second group 54 include only a single sample chamber, as shown. However, the scope of this application includes the use of any number of sample chambers (e.g., a single sample chamber or more than one sample chamber) within the first group of sample chambers, the second group of sample chambers, or any combination thereof. The first portion of the blood sample is typically diluted relative to the second portion of the blood sample. For example, the diluent may contain pH buffers, staining agents, fluorescent staining agents, antibodies, sphering agents, lysis agents, etc. Typically, the second portion of the blood sample placed in the sample chamber of the second group 54 is a natural, undiluted blood sample. Optionally or additionally, the second portion of the blood sample may be a sample that has undergone some modification, including one or more of the following: for example, dilution (e.g., controlled dilution), addition of components or reagents, or fractionation.

[0168] For some applications, a first portion of the blood sample (placed in the sample chamber of group 52) is stained with one or more staining substances before microscopic imaging of the sample. For example, the staining substance may be configured to preferentially stain DNA relative to other cellular components. Optionally, the staining substance may be configured to preferentially stain all cellular nucleic acids relative to other cellular components. For example, the sample may be stained with acridine orange reagent, Hoechst reagent, and / or any other staining substance configured to preferentially stain DNA and / or RNA in the blood sample. Optionally, the staining substance is configured to stain all cellular nucleic acids, but staining of DNA and RNA is more clearly visible under certain lighting and filtering conditions, as is known for, for example, acridine orange. Images of the sample can be acquired using imaging conditions that allow detection of cells (e.g., bright field) and / or imaging conditions that allow visualization of stained bodies (e.g., appropriate fluorescent illumination). Typically, the first portion of the sample is stained with acridine orange reagent and Hoechst reagent. For example, a first (diluted) portion of a blood sample can be prepared using techniques such as those described in Pollak's US 9,329,129, which is incorporated herein by reference and describes a method for preparing a blood sample for analysis, including a dilution step that facilitates the identification and / or counting of components within a microscopic image of the sample. For some applications, the first portion of the sample is stained with one or more staining agents that make platelets within the sample visible under bright-field imaging conditions and / or fluorescence imaging conditions, for example, as described above. For example, the first portion of the sample can be stained with methylene blue and / or Romanowsky stains.

[0169] Same reference Figures 2B-2C Typically, the sample carrier 22 is supported within the optical measurement unit by a stage 64. Furthermore, the stage typically has a forked design, such that the sample carrier is supported by the stage around its edge, but without interfering with the visibility of the sample chamber of the sample carrier to the optical measurement apparatus. For some applications, the sample carrier is held within the stage such that the molding assembly 42 of the sample carrier is positioned above the glass layer 44, and the objective lens 66 of the microscope unit of the optical measurement unit is positioned below the glass layer of the sample carrier. Typically, at least some light sources 65 used during microscopic measurements of the sample (e.g., light sources used during bright-field imaging) illuminate the sample carrier from above the molding assembly. Additionally, typically, at least some additional light sources (not shown) illuminate the sample carrier from below (e.g., via the objective lens). For example, a light source used to excite the sample during fluorescence microscopy can illuminate the sample carrier from below (e.g., via the objective lens).

[0170] Typically, prior to microscopic imaging, a first portion of the blood (placed in the sample chamber of the first group 52) is allowed to settle, such as to form a cell monolayer, for example using techniques described in, for example, Pollak’s US 9,329,129, patent application which is incorporated herein by reference. For some applications, the first portion of the blood is a cell suspension, and each chamber belonging to the first group 52 is customarily defined to include a cavity 55 (in a base surface 57) Figure 3C (As shown in the figure). Typically, cells in a cell suspension are allowed to settle onto the substrate surface of the sample chamber of the carrier to form a cell monolayer on the substrate surface of the sample chamber. After the cells have settled on the substrate surface of the sample chamber (e.g., by a predetermined time interval of settling), at least one microscopic image of at least a portion of the cell monolayer is typically acquired. Typically, more than one image of the monolayer is acquired, each image corresponding to an imaging field of view of a different region within the imaging plane of that monolayer. Typically, the optimal depth level for focusing microscopy to image the monolayer is determined, for example, using techniques such as those described in Greenfield’s U.S. Patent US 10,176,565, which is incorporated herein by reference. For some applications, the optimal depth levels for each imaging field of view are different from each other.

[0171] Note that, in the context of this application, the term monolayer is used to refer to a layer of cells that has settled, such as at a single focus level (referred to herein as the "monolayer focus level") in a microscope. Within a monolayer, some cells may overlap, resulting in two or more overlapping cell layers in certain areas. For example, red blood cells may overlap each other within a monolayer, and / or platelets may overlap with or be positioned above red blood cells within a monolayer.

[0172] For some applications, the microscopic analysis of the first portion of a blood sample is performed on a cellular monolayer. Typically, the first portion of the blood sample is imaged under bright-field imaging, i.e., under illumination from one or more light sources (e.g., one or more light-emitting diodes, which typically emit light in their respective spectral bands). Additionally, the first portion of the blood sample is often additionally imaged under fluorescence imaging. Fluorescence imaging is typically performed by directing light of a known excitation wavelength (i.e., wavelengths at which the stained object emits fluorescence if excited with light of those wavelengths) onto the sample to excite the stained object within the sample (i.e., the object that has absorbed the staining agent) and detecting the fluorescence. Typically, for fluorescence imaging, a set of separate light sources (e.g., one or more light-emitting diodes) is used to illuminate the sample at known excitation wavelengths.

[0173] As described with reference to Pollak's US 2019 / 0302099 (which is incorporated herein by reference), for some applications, sample chambers belonging to group 52 (for microscopic measurements) have different heights to facilitate the measurement of different measurands using microscopic images from each sample chamber, and / or different sample chambers for microscopic analysis of different sample types. For example, if a blood sample and / or a monolayer formed from the sample has a relatively low red blood cell density, measurements can be taken in a sample chamber of the sample carrier with a greater height (i.e., the sample chamber of the sample carrier has a greater height relative to different sample chambers with relatively low heights), such that sufficient cell density is present, and / or sufficient cell density is present in the monolayer formed from the sample to provide statistically reliable data. Such measurements may include, for example, red blood cell density measurements, measurements of other cellular properties (such as the count of abnormal red blood cells, the count of red blood cells including endosomes (e.g., pathogens, Howo-Jones bodies, etc.), and / or hemoglobin concentration. Conversely, if the blood sample and / or the monolayer formed from the sample has a relatively high density of red blood cells, such measurements can be performed on sample chambers of the sample carrier with a relatively low height, for example, such that there are sufficiently sparse cells, and / or such that there are sufficiently sparse cells within the cell monolayer formed from the sample, allowing for cell identification within a microscopic image. For some applications, this method can be performed even if the height variations between sample chambers belonging to group 52 are not precisely known.

[0174] For some applications, the sample chamber within the sample carrier on which optical measurements are performed is selected based on the object being measured. For example, a sample chamber with a greater height in the sample carrier can be used for white blood cell counting (e.g., to reduce statistical errors that may be caused by low counts in shallower regions), white blood cell differentiation, and / or detection of rarer forms of white blood cells. Conversely, to determine mean corpuscular hemoglobin content (MCH), mean corpuscular volume (MCV), erythrocyte distribution width (RDW), erythrocyte morphology characteristics, and / or erythrocyte abnormalities, microscopic images can be obtained from a sample chamber with a relatively lower height in the sample carrier, because in such a sample chamber, cells are relatively sparsely distributed throughout the area and / or form a monolayer in which cells are relatively sparsely distributed. Similarly, for platelet counting, platelet classification, and / or extraction of any other platelet properties (such as volume), microscopic images can be obtained from a sample chamber with a relatively low height in the sample carrier, because in such a sample chamber, there are fewer red blood cells (completely or partially) overlapping with platelets in the microscopic image and / or in a monolayer.

[0175] According to the embodiments described above, it is preferable to use a sample chamber with a lower height of the sample carrier for optical measurements of some metric objects within a sample (such as a blood sample), while it is preferable to use a sample chamber with a higher height of the sample carrier for optical measurements of other metric objects within such a sample. Therefore, for some applications, a first metric object within the sample is measured by performing a first optical measurement (e.g., by acquiring a microscopic image of the sample) on a portion of the sample placed in a first sample chamber belonging to group 52 of the sample carrier, and a second metric object of the same sample is measured by performing a second optical measurement (e.g., by acquiring a microscopic image of the sample) on a portion of the sample placed in a second sample chamber belonging to group 52 of the sample carrier. For some applications, the first and second metric objects are normalized relative to each other, for example, using techniques such as those described in Zait's US 2019 / 0145963, which is incorporated herein by reference.

[0176] Typically, in order to perform optical density measurements on a sample, it is desirable to know as accurately as possible the optical path length, volume, and / or thickness of the portion of the sample to be optically measured. Optical density measurements are usually performed on a second portion of the sample (which is typically placed in the sample chamber of the second set 54 in undiluted form). For example, the concentration and / or density of a component can be measured by measuring the sample's light absorption, transmission, fluorescence, and / or luminescence.

[0177] Same reference Figure 3BFor some applications, the sample chamber belonging to group 54 (for optical density measurement) defines at least a first region 56 (which is typically deeper) and a second region 58 (which is typically shallower). The height of the sample chamber varies between the first and second regions in a predefined manner, for example, as described in Pollak's US 2019 / 0302099, which is incorporated herein by reference. The heights of the first region 56 and the second region 58 of the sample chamber are defined by a lower surface defined by a glass layer and an upper surface defined by a molding assembly. The upper surface at the second region is stepped relative to the upper surface at the first region. The step between the upper surfaces at the first and second regions provides a predetermined height difference Δh between the regions, such that even if the absolute height of the known regions is not sufficiently accurate (e.g., due to tolerances in the manufacturing process), the height difference Δh is known to be sufficiently accurate to determine the parameters of the sample using the techniques described herein and as described in Pollak's US 2019 / 0302099, which is incorporated herein by reference. For some applications, the height of the sample chamber varies from a first region 56 to a second region 58, and then similarly from a second region to a third region 59, such that along the sample chamber, the first region 56 defines the maximum height region, the second region 58 defines the medium height region, and the third region 59 defines the minimum height region. For some applications, further variations in height occur along the length of the sample chamber, and / or the height varies gradually along the length of the sample chamber.

[0178] As described above, optical measurements of the sample are performed using one or more optical measuring devices 24 while the sample is placed in a sample carrier. Typically, the sample is observed through a glass layer via the optical measuring devices, the glass being transparent at least to the wavelengths typically used by the optical measuring devices. Typically, during optical measurements, the sample carrier is inserted into an optical measuring unit 31 that houses the optical measuring devices. Typically, the optical measuring unit houses the sample carrier such that the molding layer is positioned above the glass layer and the optical measuring unit is positioned below the glass layer of the sample carrier, enabling optical measurements of the sample through the glass layer. The sample carrier is formed by adhering the glass layer to the molding assembly. For example, the glass layer and the molding assembly may be bonded together during manufacturing or assembly (e.g., using thermal bonding, solvent-assisted bonding, ultrasonic welding, laser welding, thermal riveting, adhesives, mechanical clamping, and / or other substances). For some applications, the glass layer and the molding assembly are bonded together during manufacturing or assembly using an adhesive layer 46.

[0179] Now for reference Figures 4A-4C , Figures 4A-4CThese are microscopic images obtained according to some applications of the present invention. As described in the background section above, mammalian red blood cells are the body's oxygen carriers. Red blood cells undergo enucleation during maturation, meaning the nucleus is completely removed from the cell. Normally, nucleated red blood cells (NRBCs) are not present in the peripheral blood of adult patients. However, NRBCs are present in peripheral blood in some cases (such as newborns, cancer patients, anemic patients, etc.). Because NRBCs are similar in size and nucleus content to some white blood cells (and particularly lymphocytes), it is often difficult to distinguish NRBCs from white blood cells. For example, Figure 4A A combined image is shown, comprising a fluorescence microscopy image superimposed on a bright-field microscopy image. The bright-field image was acquired under ultraviolet illumination, and the microscope was defocused relative to the cellular monolayer within the sample during the acquisition of the bright-field image. The sample was stained with Hoechst's reagent, which has an affinity for DNA, and the Hoechst reagent was excited before the fluorescence image was acquired. (As described above, additional bright-field and / or fluorescence images of the sample are typically acquired. For example, it is common practice to stain the sample with acridine orange and acquire a fluorescence image in which acridine orange is excited.) Entity 60 is visible in the image, and the entity has a size indicating that it may be an NRBC or a leukocyte. Furthermore, the central portion of the entity fluoresces, indicating the presence of a cell nucleus, which would be expected if the entity is an NRBC or if the entity is a leukocyte.

[0180] As described above, in some applications of the present invention, a complete blood count is performed on a blood sample. In the case of a complete blood count, it is often important to distinguish NRBCs from white blood cells in order to avoid overcounting white blood cells (especially in patients with low white blood cell counts) and to detect the presence of NRBCs (which may indicate an underlying condition).

[0181] According to some applications of the invention, in order to distinguish between leukocytes (e.g., lymphocytes) and NRBCs (e.g., in the case of detecting NRBC / leukocyte candidates (i.e., candidates for either NRBCs or leukocytes)), a microscopic image is acquired by illuminating the sample with light of a specific wavelength (at which hemoglobin has a high absorption level). Typically, violet light is used, for example, light with wavelengths in the range greater than 400 nm and / or less than 450 nm (e.g., 400 nm–450 nm). Within this wavelength range, hemoglobin absorption is relatively high compared to other wavelengths in the visible light spectrum. Typically, NRBCs have a high hemoglobin content (on the order of 30 picograms per cell), while leukocytes do not contain hemoglobin. Therefore, in images acquired under violet illumination, NRBCs typically absorb light, while leukocytes do not. For some applications, light with wavelengths greater than 500 nm and / or less than 600 nm (e.g., 500 nm–600 nm) is used. Within this wavelength range, the absorption of carbamoylhemoglobin is relatively high compared to other wavelengths in the visible light spectrum. Therefore, in images acquired under illumination within the aforementioned wavelength range, NRBCs typically absorb light, while leukocytes do not.

[0182] Now for reference Figure 4B and Figure 4C , Figure 4B The image shows NRBC 62 within an image acquired under violet illumination, while Figure 4C The image shows a white blood cell 67 within an image acquired under violet illumination. As shown, the entire NRBC appears dark because hemoglobin within it absorbs light, while the center of the white blood cell does not appear dark because hemoglobin is absent within the white blood cell. Therefore, typically, an entity is classified as either an NRBC or a white blood cell, at least in part, based on the intensity of the entity within an image acquired under violet illumination. For some applications, an intensity threshold is applied to the image (and / or a specific region or pixel thereof) acquired under violet light conditions, and the entity is classified as either an NRBC or a white blood cell based on whether its intensity exceeds the threshold.

[0183] Typically, entities that are NRBC / leukocyte candidates (i.e., candidates can be either NRBCs or leukocytes) are first identified based on bright-field images acquired at wavelengths other than violet light and / or based on fluorescence images. In response to the identification of such candidates, images acquired under violet illumination are analyzed (e.g., as described above) to distinguish NRBCs from leukocytes. For some applications, if one or more (e.g., a specific minimum number) NRBC / leukocyte candidates are identified within a particular imaging field, an image of that imaging field is acquired only under violet illumination. That is, the computer processor drives the microscope to acquire images under violet illumination only in response to the detection of a need to do so (by identifying NRBC / leukocyte candidates (and / or more than a specific number or concentration of NRBC / leukocyte candidates) within that imaging field).

[0184] For some applications, additional features are used to distinguish NRBCs from leukocytes. For example, such additional features may include cell size, nuclear size, fluorescence intensity, cytoplasmic intensity, cytoplasmic area, cell ellipticity, nuclear ellipticity, nuclear roundness, and / or any combination of the above features. For some applications, one or more images of NRBC / leukocyte candidates are acquired under different bright-field illumination conditions, such as red and / or green light, and the classification of the candidates is verified by analyzing one or more additional images. For some applications, images acquired under violet illumination and / or one or more additional images are defocused bright-field images. For some applications, machine learning classifiers (e.g., convolutional neural network classifiers, decision tree classifiers, regression analysis classifiers, Bayesian network classifiers, and / or support network vector classifiers) are applied to one or more of the described features of the NRBC / leukocyte candidates to classify the NRBC / leukocyte candidates as NRBCs or leukocytes. Optionally or additionally, a neural network classifier can be applied to the original image (such images typically include those acquired under violet illumination conditions) to classify NRBC / leukocyte candidates as NRBC or leukocytes.

[0185] As mentioned above, NRBCs are typically not present in the blood of healthy adults, and their presence indicates an underlying clinical condition. The presence of one or more NRBCs in a subject's blood may indicate an increased likelihood that another entity in the blood sample is an NRBC rather than a white blood cell (white blood cells are always present in blood). For some applications, in response to the detection of even a single NRBC (and / or in response to the detection of a specific number or concentration of NRBCs), one or more thresholds used to identify an entity as an NRBC are adjusted, thereby increasing the sensitivity of the computer processor to NRBCs. Typically, in response to the identification of one or more NRBCs (e.g., in response to the detection of more than a specific number and / or more than a specific concentration of NRBCs), output is generated to the user, marking the identified NRBCs and / or indicating the concentration or relative concentration of the identified NRBCs.

[0186] For some applications, in response to detecting more than a certain number and / or concentration of NRBCs, the computer processor adjusts the threshold used to detect other entities and / or generates output indicating that the counts of other entities may be incorrect. For some applications, in response to detecting a combination of relatively high NRBC counts and relatively low white blood cell counts, the computer processor interprets this as indicating that some NRBC / white blood cell candidates have been misclassified. In response, the computer processor typically reanalyzes at least some of the candidates and / or generates output indicating that there may be errors in the NRBC and / or white blood cell counts. For some applications, the computer processor detects that only one specific type (or more specific types) of white blood cells has a low concentration, indicating that only this type (or these types) of white blood cells are incorrectly distinguished from NRBCs. In response, the computer processor typically generates output indicating that there may be errors in the count of that specific type (or more types) of white blood cells.

[0187] Now for reference Figure 5 , Figure 5 This is a flowchart illustrating the steps of a method for identifying NRBC / leukocyte candidates within one or more microscopic images of a blood sample, according to some applications of the present invention. (Refer to the above text.) Figures 4A-4C Describe and in Figure 5As shown in the flowchart, NRBC / leukocyte candidates are identified in one or more microscopic images of a blood sample (step 100), and then identified in microscopic images acquired under illumination in the wavelength range of 400 nm to 450 nm or under illumination in the wavelength range of 500 nm to 600 nm (step 102). Then, based on the light absorption level of the NRBC / leukocyte candidates in the microscopic images, the NRBC / leukocyte candidates are classified as NRBCs or leukocytes (step 104), and an output is generated based on the classification of the NRBC / leukocyte candidates as NRBCs or leukocytes (step 106).

[0188] Now for reference Figure 6A , Figure 6B and Figure 6C , Figure 6A , Figure 6B and Figure 6C These are microscopic images of red blood cells acquired under red, green, and violet illumination conditions, respectively, according to some applications of the present invention. Typically, hemoglobin variants absorb in the violet range (e.g., greater than 400 nm and / or less than 450 nm (e.g., between 400 nm and 450 nm)) at a rate between one and three orders of magnitude greater than their absorption at any other wavelength in the visible spectrum. For example, in Figures 6A-6C It can be observed in Figure 6C Contrast ratio between red blood cells and background (obtained under ultraviolet illumination) Figure 6A (obtained under red light illumination) and Figure 6B (Acquired under green light illumination) The contrast between red blood cells and the background is much greater.

[0189] Therefore, for some applications, to determine the hemoglobin-related properties of a blood sample, the blood sample is imaged under violet illumination. For example, the hemoglobin content of a single red blood cell can be determined by measuring the absorption of violet light. For some applications, absorption measurements under violet illumination are combined with additional absorption measurements under different illumination conditions (e.g., red or green light). Typically, this is applied to more than one cell and determines the statistical hemoglobin-related properties of the sample, such as mean corpuscular hemoglobin (MCH) and hemoglobin distribution width. For some applications, cell volume data are additionally determined. For example, the volume of individual cells within the sample and / or the mean cell volume of red blood cells can be determined. Based on the MCH and cell volume data, a computer processor determines the MCH concentration of the sample. For some applications, light with wavelengths greater than 500 nm and / or less than 600 nm (e.g., 500 nm–600 nm) is used. Within this wavelength range, the absorption of carbamoylhemoglobin is relatively high compared to other wavelengths in the visible light spectrum.

[0190] For some applications, erythrocyte populations are classified into subpopulations, such as reticulocytes, NRBCs, and / or cells with certain morphological characteristics (e.g., sickle cells, oval cells, target cells, acanthocytes, etc.). For some applications, statistical hemoglobin-related properties of the sample described above are determined for one or more erythrocyte subpopulations. For example, a computer processor can determine mean reticulocyte hemoglobin, or mean sickle cell hemoglobin, mean acanthocyte hemoglobin concentration, etc.

[0191] Now for reference Figure 7 , Figure 7 This is a flowchart illustrating the steps of a method for identifying red blood cells within one or more microscopic images of a blood sample, according to some applications of the present invention. (Refer to the above text) Figures 6A-6C Describe and in Figure 7 As shown in the flowchart, microscopic images of blood samples are acquired under illumination in the wavelength range of 400 nm to 450 nm or under illumination in the wavelength range of 500 nm to 600 nm (step 110), and red blood cells are identified within the microscopic images (step 112). The hemoglobin-related properties of the blood samples are determined based on the identified red blood cells (step 114), and an output is generated based on the determined hemoglobin-related properties (step 116).

[0192] As described above, for some applications, cells in a cell suspension are allowed to settle onto the substrate surface of the sample chamber of carrier 22 to form a cell monolayer on the substrate surface of the sample chamber. After the cells have settled onto the substrate surface of the sample chamber (e.g., by a predetermined time interval of settling), at least one microscopic image of at least a portion of the cell monolayer is typically acquired. As stated above, in the context of this application, the term monolayer is used to refer to a cell layer that has settled, such as within a single focal level of the microscope (referred to herein as a “monolayer focal level”). For some applications, in addition to acquiring images of a microscope focal plane set approximately to coincide with the monolayer focal level (such images are referred to herein as “focused” images), the microscope also acquires images of a microscope focal plane set offset along the optical axis relative to the monolayer focal level (such images are referred to herein as “defocused” images). Typically, such defocused microscopic images are acquired when the microscope focal plane is set closer to the microscope objective than the monolayer focal level, although the scope of the invention includes acquiring defocused images when the microscope focal plane is set farther from the microscope objective than the monolayer focal level. Typically, defocused microscopic images are acquired with the microscope's focal plane set at a predetermined offset relative to the monolayer focal level. For some applications, defocused microscopic images are acquired with the microscope's focal plane set at an offset of more than 20 micrometers and / or less than 100 micrometers (e.g., 20-100 micrometers) relative to the monolayer focal level. Optionally or additionally, defocused microscopic images are acquired with the microscope's focal plane set at an offset of more than one microscope depth of focus and / or less than five microscope depths of focus (e.g., between one and five microscope depths of focus).

[0193] The inventors of this application have discovered that such defocused microscopic images can generate important data about a sample. Specifically, defocused images are commonly used to identify cell outlines and / or certain entities within a sample, such as leukocytes, leukocyte types (e.g., lymphocytes, granulocytes, monocytes, neutrophils, band neutrophils, eosinophils, basophils, macrophages, and / or blastocytes), erythrocytes, erythrocyte types (e.g., mature erythrocytes, NRBCs, acanthocytes, sickle cells, teardrop cells), and / or platelets. For some applications, defocused images are used to enhance the visibility of such entities relative to other entities, allowing them to be identified with greater certainty. Optionally or additionally, defocused images are used to make such entities easier to distinguish from other entities that might otherwise be confused with them. For some applications, defocused images are used to characterize cellular features, such as the hemoglobin content of erythrocytes, other components of erythrocytes, and / or the maturity of cells (such as neutrophils).

[0194] First refer to Figure 8, Figure 8 This is a flowchart illustrating the steps of a method for using a body sample containing cells according to some applications of the present invention. As described above and... Figure 8 As shown in the flowchart, the microscope is focused to its "focusing" focal plane, where the focal plane at least approximately coincides with the horizontal plane at which at least some cells belonging to the sample are at least partially positioned (step 120). Then, a focused microscopic image is acquired when the focal plane is "focused" such that the focal plane of the microscope approximately coincides with the horizontal plane at which at least some cells belonging to the sample are at least partially positioned (step 122). Additionally, the microscope is focused to its "defocusing" focal plane, where the focal plane of the microscope is offset relative to the horizontal plane at which at least some cells belonging to the sample are at least partially positioned (i.e., offset relative to the "focusing" focal plane) (step 124). Then, a defocusing microscopic image is acquired when the focal plane of the microscope is offset relative to the horizontal plane at which at least some cells belonging to the sample are at least partially positioned (step 126). Then, characteristics of at least a portion of the sample are determined at least partially based on the focused and defocused images (step 128).

[0195] Note that, typically, defocused images are acquired not only at wavelengths where hemoglobin absorbs light poorly (e.g., red light, such as light in the approximately 620nm–640nm wavelength range), but also at wavelengths where hemoglobin absorbs light slightly (e.g., green light, such as light in the approximately 505nm–535nm or 520nm–530nm wavelength range), or even at wavelengths where hemoglobin absorbs light heavily (e.g., violet light, such as light in the wavelength range greater than 400nm and / or less than 450nm (e.g., 400nm–450nm)). See below for reference. Figures 9A-12C Describe some examples of using such images.

[0196] Now for reference Figure 9A and Figure 9B , Figure 9A and Figure 9B Examples of bright-field microscopic images of cell monolayers of blood samples obtained using ultraviolet illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cell monolayer. Figure 9A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 9B In the focused image ( Figure 9A In the image, erythrocytes 80 and acanthocytes 82 are visible. Additionally, a bright area 84 can be observed. This bright area represents platelets. However, based on the original focused image, it may be difficult to identify platelets and to distinguish them from other entities such as intraerythrocyte parasites or leukocytes. As can also be observed in the defocused image (…),… Figure 9BIn this context, the bright area is less visible. For some applications, the computer processor normalizes the images based on the focused and defocused images (e.g., by subtracting one image from the other, or by dividing one image by the other). Typically, based on normalization, the bright area is more clearly visible, and the computer processor can identify the bright area as a platelet with a greater degree of certainty than it could do based solely on the original focused image. (Note that for some applications, the two images are not actually normalized relative to each other, but rather the computer processor performs a processing step equivalent to normalizing the images or portions thereof relative to each other). Now refer to Figure 9C , Figure 9C According to some applications of the present invention, after staining samples with acridine orange and Hoechst's reagent and exciting the samples with UV light to induce platelet fluorescence, such as... Figure 9A and Figure 9B The image shown is a fluorescence micrograph of the same portion of the sample. In this image, platelets are clearly visible and distinguishable from the surrounding tissue. Figure 9A and Figure 9B As shown, normalization based on focused and defocused images is an alternative or other method to enhance platelet visibility.

[0197] Now for reference Figure 10A and Figure 10B , Figure 10A and Figure 10B Examples of bright-field microscopic images of cell monolayers of blood samples obtained using ultraviolet illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cell monolayer. Figure 10A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 10B In the focused image ( Figure 10A In the image, a bright, circular entity 90 is visible. This entity is a white blood cell. However, based on the original focused image, it may be difficult to identify the white blood cell and to distinguish it from other entities such as platelets or platelet clusters. As can also be observed in the defocused image ( Figure 10BIn this context, the bright entity is less visible. Furthermore, it may be difficult to classify white blood cells into specific types (e.g., lymphocytes, granulocytes, monocytes, neutrophils, zona neutrophils, eosinophils, basophils, macrophages, and / or blast cells). For some applications, the computer processor performs normalization based on the focused and defocused images (e.g., by subtracting one image from the other, or by dividing one image by the other). Generally, based on normalization, the bright entity is more clearly visible, and the computer processor can identify the bright entity as a white blood cell and / or classify the white blood cell into specific types with a greater degree of certainty than it can do based on the original focused image. (Note that for some applications, the two images are not actually normalized relative to each other, but rather the computer processor performs a processing step equivalent to normalizing the images or portions thereof relative to each other).

[0198] Based on the above Figures 9A-9B and Figures 10A-10B The description states that, for some applications, a computer processor identifies one or more entities within a sample based at least in part on a defocused image of a cellular monolayer within the sample (e.g., by distinguishing one or more entities from other entities). For some such applications, normalization is performed based on both focused and defocused images, and the computer processor identifies one or more entities within the sample based on the normalization (e.g., by distinguishing one or more entities from other entities). Although Figures 9A-9B and Figures 10A-10B The examples shown involve platelets and white blood cells, but for some applications, similar techniques are often used to identify other entities such as abnormal white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howejo bodies, sickle cells, teardrop cells, etc.

[0199] Same reference Figures 9A-9B and Figures 10A-10B Furthermore, it can be observed that the outlines of cells (and particularly erythrocytes, acanthocytes, and other erythrocyte types) are more clearly visible in defocused images than in focused images. For some applications, the outlines are more clearly visible in defocused images due to reduced refraction and / or diffraction effects compared to focused images. Several other examples of this exist... Figures 11A-12C As shown in the image.

[0200] Now for reference Figures 11A-11B These figures are examples of bright-field microscopic images of cellular monolayers of blood samples obtained using green light illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cellular monolayer. Figure 11A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 11BAs can be observed, the same phenomenon occurs in defocused images acquired under green illumination, where the outlines of cells (and particularly erythrocytes, acanthocytes, and other erythrocyte types) are more clearly visible than in focused images.

[0201] Now for reference Figures 12A-12C These figures are examples of bright-field microscopic images of cellular monolayers of blood samples obtained using green light illumination according to some applications of the present invention, wherein the microscope focal plane is set to coincide with the cellular monolayer. Figure 12A ), and setting the microscope focal plane to be defocused relative to the cell monolayer ( Figure 12B and Figure 12C ). Figure 12B Acquired when the microscope focal plane is set closer to the microscope objective than the cell monolayer is, and Figure 12C This is achieved when the microscope focal plane is set further away from the microscope objective than the cell monolayer. As can be observed, in defocused images acquired by setting the microscope focal plane closer to or farther away from the microscope objective than the cell monolayer, the outlines of the cells (and especially erythrocytes, acanthocytes, and other erythrocyte types) are more clearly visible than in focused images.

[0202] Based on the above Figures 9A-9B , Figures 10A-10B and Figures 11A-12C As described above, for some applications of the invention, a computer processor determines the contours of one or more entities in a blood sample based on a defocused image. For some applications, the computer processor determines additional parameters of one or more entities based on the determined contours. For example, the computer processor may determine cell volume, cell area, mean cell volume, and / or mean cell area based on the determined contours.

[0203] For some applications, a computer processor uses a machine learning classifier, such as a neural network (e.g., a convolutional neural network), to determine one or more parameters of a sample (and / or an entity placed within the sample). For some applications, parameters derived from one or more defocused images are used as input to a machine learning classifier, and the parameters of the sample (and / or the entity placed within the sample) are determined accordingly. For example, parameters derived from defocused images can be used as input to a classifier that estimates cytohemoglobin, cell volume, mean cytohemoglobin, mean cell volume, and / or other parameters.

[0204] Typically, even after a blood sample has been allowed to settle using the techniques described herein, such as to form a monolayer, not all platelets in the blood sample settle within the monolayer, and some cells remain suspended in the cell solution. For some applications, to accurately estimate the number of platelets in a sample, it is necessary to identify platelets suspended in the cell solution in addition to identifying those within the monolayer focal level. Typically, such platelets are identified by focusing the microscope at a depth level beyond the depth level used to image the cell monolayer, and acquiring images at these additional depth levels. Typically, platelets in the images acquired at these additional depth levels are identified and counted, and the total number of platelets suspended in the cell solution is estimated based on the platelet counts in these images.

[0205] For some applications, in addition to acquiring focused images at each additional depth level, defocused images (i.e., images offset along the optical axis relative to the focal plane of that additional depth level) are also acquired at that additional depth level. Typically, defocused microscopic images are acquired with the microscope's focal plane set at a predetermined offset relative to that additional depth level. For some applications, defocused microscopic images are acquired with the microscope's focal plane set offset by more than 20 micrometers and / or less than 100 micrometers (e.g., 20-100 micrometers) relative to that additional depth level. Optionally or additionally, defocused microscopic images are acquired with the microscope's focal plane set offset by more than one microscope depth of focal length and / or less than five microscope depths of focal length (e.g., between one and five microscope depths of focal lengths). For some such applications, platelets are identified at that additional depth level at least in part based on the defocused images from that additional depth level, for example, according to the techniques described above.

[0206] Based on the above description, it is generally expected that certain effects exist when comparing a focused image of a specific region of a monolayer with a defocused image of the same region. For some applications, to verify that a focused image of a specific region of a monolayer is optimally focused relative to the monolayer, a defocused image of that region is acquired (typically by setting the microscope's focal plane at a predetermined offset relative to the focal plane of the focused image). The defocused and focused images are then analyzed, and based at least in part on the analysis, the computer processor determines whether the focused image is indeed optimally focused relative to the monolayer. For example, in response to detecting that the outline of a cell is not more clearly visible in the defocused image than in the focused image, the computer processor can determine that the focused image is not optimally focused relative to the monolayer. In response, the computer processor can refocus the microscope and then acquire a new focused image.

[0207] For some applications, such as those described herein, the sample is a sample that includes blood or its components (e.g., diluted or undiluted whole blood samples, samples that mainly consist of red blood cells, or diluted samples that mainly consist of red blood cells), and parameters related to components in the blood (such as platelets, white blood cells, abnormal white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howell-Joe bodies, sickle cells, teardrop cells, etc.) are determined.

[0208] For some applications, the devices and methods described herein, with necessary modifications, can be applied to biological samples other than blood, such as saliva, semen, sweat, sputum, vaginal fluid, feces, breast milk, bronchoalveolar lavage fluid, gastric lavage fluid, tears, and / or nasal discharge. Biological samples can be from any organism and are typically from warm-blooded animals. For some applications, biological samples are from mammals, such as human samples. For some applications, samples are taken from any domestic animal, zoo animal, and farm animal, including but not limited to dogs, cats, horses, cattle, and sheep. Optionally or additionally, biological samples are taken from animals used as disease vectors, including deer or rats.

[0209] For some applications, the devices and methods described herein are applied to non-human samples. With necessary modifications, for some applications, the sample is an environmental sample, such as a water (e.g., groundwater) sample, a surface swab, a soil sample, an air sample, or any combination thereof. In some embodiments, the sample is a food sample, such as a meat sample, a dairy product sample, a water sample, a detergent sample, a beverage sample, and / or any combination thereof.

[0210] The applications of the invention described herein can take the form of a computer program product accessible from a computer-usable or computer-readable medium (e.g., a non-transitory computer-readable medium) that provides program code used by or in conjunction with a computer or any instruction execution system (such as computer processor 28). For the purposes of this description, a computer-usable or computer-readable medium can be any device that may include, store, communicate, propagate, or transmit a program used by or in conjunction with an instruction execution system, device, or apparatus. The medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or device or apparatus) or a propagation medium. Typically, a computer-usable or computer-readable medium is a non-transitory computer-usable or non-transitory computer-readable medium.

[0211] Examples of computer-readable media include semiconductor or solid-state memory, magnetic tape, removable computer disks, random access memory (RAM), read-only memory (ROM), hard disks, and optical discs. Current examples of optical discs include optical disc read-only memory (CD-ROM), optical disc read / write (CD-R / W), and DVDs.

[0212] A data processing system suitable for storing and / or executing program code will include at least one processor (e.g., computer processor 28) directly or indirectly coupled to a memory element (e.g., memory 30) via a system bus. The memory element may include local memory, mass storage, and cache memory used during actual execution of the program code, the cache memory providing transient storage of at least some of the program code to reduce the number of times code must be retrieved from mass storage during execution. The system can read the instructions of the present invention on a program storage device and follow those instructions to perform the methods of embodiments of the present invention.

[0213] A network adapter can be coupled to a processor, enabling the processor to become coupled to other processors or remote printers or storage devices via an intervening private or public network. Modems, cable modems, and Ethernet cards are just a few of the types of network adapters currently available.

[0214] The computer program code used to perform the operations of this invention can be written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Java, Smalltalk, C++, etc., and conventional programming languages ​​such as C or similar programming languages.

[0215] It should be understood that the algorithms described herein can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via a computer processor (e.g., computer processor 28) or the processor of the other programmable data processing apparatus, create means for implementing the functions / operations specified in the algorithms described herein. These computer program instructions can also be stored in a computer-readable medium (e.g., a non-transitory computer-readable medium) that can instruct a computer or other programmable data processing apparatus to operate in a particular manner, such that the instructions stored in the computer-readable medium produce an instruction means including means for implementing the flowchart blocks and the functions / operations specified in the algorithm. The computer program instructions can also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process, such that the instructions, which execute on the computer or other programmable apparatus, provide processing for implementing the functions / operations specified in the algorithms described herein.

[0216] Computer processor 28 is typically a hardware device programmed with computer program instructions to produce a dedicated computer. For example, when programmed to perform the algorithms described herein, computer processor 28 is typically used as a dedicated sample analysis computer processor. Generally, the operations performed by computer processor 28 described herein convert the physical state of memory 30 (which is a real physical object) into different magnetic polarities, charges, etc., depending on the memory technology used.

[0217] The apparatus and methods described herein may be used in conjunction with the apparatus and methods described in any of the following patents or patent applications, all of which are incorporated herein by reference:

[0218] Bachelet, US 9,522,396;

[0219] Greenfield’s US 10,176,565;

[0220] Pollak's US 10,640,807;

[0221] Pollak's US 9,329,129;

[0222] Pollak's US 10,093,957;

[0223] Yorav Raphael’s US 10,831,013;

[0224] Bachelet, US 10,843,190;

[0225] Yorav Raphael's US 10,482,595;

[0226] Eshel's US 10,488,644;

[0227] Eshel's WO 17 / 168411;

[0228] Pollak's US 2019 / 0302099;

[0229] Zait's US 2019 / 0145963; and

[0230] Yorav-Raphael's WO 19 / 097387.

[0231] Those skilled in the art will understand that the present invention is not limited to what has been specifically shown and described above. Rather, the scope of the invention includes combinations and sub-combinations of the various features described above, as well as variations and modifications thereof that do not exist in the prior art and will come to the mind of those skilled in the art upon reading the foregoing description.

Claims

1. A method for use on a body sample containing cells, the method comprising: Allowing some cells within the sample to settle in order to form a monolayer at the monolayer focal level; The microscope is focused such that the focal plane of the microscope at least approximately coincides with the single-layer focal plane. At least one focused microscopic image of the sample is acquired when the focal plane of the microscope approximately coincides with the focal plane of the monolayer. The microscope is focused such that the focal plane of the microscope is shifted horizontally relative to the single-layer focal plane. When the focal plane of the microscope is offset relative to the single-layer focal plane, at least one defocused microscopic image of the sample is acquired; and The characteristics of at least a portion of the sample are determined based at least in part on the focused image and the defocused image.

2. The method according to claim 1, wherein acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is offset relative to the monolayer focal level includes acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is offset by a predetermined offset relative to the monolayer focal level.

3. The method of claim 1, wherein determining the characteristics of at least a portion of the sample based at least in part on the focused image and the defocused image comprises inputting the focused image and the defocused image into a machine learning classifier configured to determine the characteristics of at least a portion of the sample based at least in part on the focused image and the defocused image.

4. The method of claim 1, wherein determining the characteristic of at least a portion of the sample based at least in part on the focused image and the defocused image comprises deriving one or more parameters from the focused image and the defocused image, and inputting the derived one or more parameters into a machine learning classifier configured to determine the characteristic of at least a portion of the sample based at least in part on the derived parameters.

5. The method of claim 1, wherein the sample comprises a blood sample, and wherein acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 505 nm and 535 nm.

6. The method of claim 1, wherein the sample comprises a blood sample, and wherein acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 400 nm and 450 nm.

7. The method of claim 1, wherein the sample comprises a blood sample, and wherein acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 620 nm and 640 nm.

8. The method of claim 1, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is set closer to the objective lens of the microscope than the monolayer focal level.

9. The method of claim 1, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is set further away from the objective lens of the microscope than the monolayer focal level.

10. The method of claim 1, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is at least partially positioned between 20 micrometers and 100 micrometers relative to at least some of the cells belonging to the sample at the monolayer focal level.

11. The method of claim 1, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level between one and five focal depths of the microscope.

12. The method of claim 1, wherein determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image comprises normalizing the focused image and the defocused image relative to each other, and determining the characteristics of a portion of the sample based at least in part on the normalization.

13. The method of claim 1, wherein the sample comprises a blood sample, and wherein determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image comprises identifying one or more entities within the blood sample based at least in part on the focused image and the defocused image, said one or more entities being selected from the group consisting of: platelets, leukocytes, lymphocytes, granulocytes, monocytes, neutrophils, band neutrophils, eosinophils, basophils, and macrophages.

14. The method of claim 1, wherein the sample comprises a blood sample, and wherein determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image comprises identifying blast cells within the blood sample based at least in part on the focused image and the defocused image.

15. The method according to any one of claims 1-14, wherein determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image includes identifying the outlines of one or more entities within the sample based at least in part on the focused image and the defocused image.

16. The method of claim 15, wherein determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image further comprises estimating parameters of the one or more entities based at least in part on the identified contours, the parameters being selected from the group consisting of cell area and cell volume.

17. The method of claim 15, wherein determining the characteristics of a portion of the sample based at least in part on the focused image and the defocused image further comprises estimating parameters of the sample based at least in part on the identified contour, said at least one parameter being selected from the group consisting of: mean cell area and mean cell volume.

18. An apparatus for use on a body sample containing cells, said apparatus comprising: microscope; and Computer processor, the computer processor being configured to: The microscope is focused such that its focal plane at least approximately coincides with the monolayer focal level, allowing at least some cells belonging to the sample to settle in order to form a monolayer at the monolayer focal level. When the focal plane of the microscope approximately coincides with the focal plane of the monolayer, the microscope is driven to acquire at least one focused microscopic image of the sample. The microscope is focused such that the focal plane of the microscope is shifted horizontally relative to the single-layer focal plane. When the focal plane of the microscope shifts relative to the single-layer focal plane, the microscope is driven to acquire at least one defocused microscopic image of the sample, and The characteristics of at least a portion of the sample are determined based at least in part on the focused image and the defocused image.

19. A method for use on a body sample containing cells, the method comprising: Allowing some cells within the sample to settle in order to form a monolayer at the monolayer focal level; The microscope is focused such that the focal plane of the microscope is shifted horizontally relative to the single-layer focal plane. When the focal plane of the microscope is offset relative to the single-layer focal plane, at least one defocused microscopic image of the sample is acquired; and Based at least in part on the defocused image, one or more operations selected from the group consisting of: identifying entities placed within the single focal level, determining the outline of entities placed within the single focal level, determining parameters of entities placed within the single focal level, determining parameters of the sample, and any combination thereof.

20. The method of claim 19, wherein acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample when the focal plane of the microscope is offset by a predetermined offset relative to the monolayer focal level.

21. The method of claim 19, wherein performing the one or more operations at least in part based on the defocused image comprises inputting the defocused image into a machine learning classifier configured to perform the one or more operations at least in part based on the defocused image.

22. The method of claim 19, wherein performing the one or more operations at least in part based on the defocused image comprises deriving one or more parameters from the defocused image and inputting the derived one or more parameters into a machine learning classifier configured to perform the one or more operations at least in part based on the derived parameters.

23. The method of claim 19, wherein the sample comprises a blood sample, and wherein acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 505 nm and 535 nm.

24. The method of claim 19, wherein the sample comprises a blood sample, and wherein acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 400 nm and 450 nm.

25. The method of claim 19, wherein the sample comprises a blood sample, and wherein acquiring at least one defocused microscopic image of the sample while the focal plane of the microscope is offset relative to the monolayer focal level comprises acquiring at least one defocused microscopic image of the sample while the sample is illuminated with light having a wavelength between 620 nm and 640 nm.

26. The method of claim 19, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is set closer to the objective lens of the microscope than the monolayer focal level.

27. The method of claim 19, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is set further away from the objective lens of the microscope than the monolayer focal level in which at least some of the cells belonging to the sample are at least partially located.

28. The method of claim 19, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is offset between 20 micrometers and 100 micrometers relative to at least some of the cells belonging to the sample being at least partially placed at the monolayer focal level.

29. The method of claim 19, wherein focusing the microscope such that the focal plane of the microscope is offset relative to the monolayer focal level comprises focusing the microscope such that the focal plane of the microscope is at least partially positioned relative to at least some of the cells belonging to the sample between one and five focal depths of the microscope at the monolayer focal level.

30. The method of claim 19, further comprising determining characteristics of a portion of the sample by normalizing the defocused image relative to another image, at least in part based on the defocused image, and determining the characteristics of the portion of the sample at least in part based on the normalization.

31. The method of claim 19, wherein the sample comprises a blood sample, and wherein the method further comprises determining, at least in part, characteristics of a portion of the sample based on the defocus image by identifying, at least in part, one or more entities within the blood sample, the one or more entities being selected from the group consisting of: platelets, leukocytes, lymphocytes, granulocytes, monocytes, neutrophils, band neutrophils, eosinophils, basophils, and macrophages.

32. The method of claim 19, wherein the sample comprises a blood sample, and wherein the method further comprises determining, at least in part, characteristics of a portion of the sample based on the defocus image by identifying blast cells within the blood sample at least in part based on the defocus image.

33. The method according to any one of claims 19-32, wherein the method further comprises determining, at least in part, characteristics of a portion of the sample based on the defocus image by identifying the outline of one or more entities within the sample, at least in part, based on the defocus image.

34. The method of claim 33, wherein determining the characteristics of a portion of the sample based at least in part on the defocused image further comprises estimating parameters of the one or more entities based at least in part on the identified contours, the parameters being selected from the group consisting of cell area and cell volume.

35. The method of claim 33, wherein determining the characteristics of a portion of the sample based at least in part on the defocused image further comprises estimating parameters of the sample based at least in part on the identified contour, said at least one parameter being selected from the group consisting of: mean cell area and mean cell volume.

36. An apparatus for use on a body sample containing cells, said apparatus comprising: microscope; and Computer processor, the computer processor being configured to: The microscope is focused such that its focal plane is shifted relative to the monolayer focal level, allowing at least some cells belonging to the sample to settle in order to form a monolayer at the monolayer focal level. When the focal plane of the microscope shifts relative to the single-layer focal plane, the microscope is driven to acquire at least one defocused microscopic image of the sample, and Based at least in part on the defocused image, one or more operations selected from the group consisting of: identifying entities placed within the single focal level, determining the outline of entities placed within the single focal level, determining parameters of entities placed within the single focal level, determining parameters of the sample, and any combination thereof.

37. A method for use on a body sample containing cells, the method comprising: Allowing some cells within the sample to settle in order to form a monolayer at the monolayer focal level; The microscope is focused such that the focal plane of the microscope at least approximately coincides with the single-layer focal plane. At least one focused microscopic image of the sample is acquired when the focal plane of the microscope approximately coincides with the focal plane of the monolayer. The microscope is focused such that the focal plane of the microscope is shifted horizontally relative to the single-layer focal plane. When the focal plane of the microscope is shifted by a predetermined offset relative to the single-layer focal plane, at least one defocused microscopic image of the sample is acquired; and By analyzing the focused image and the defocused image, it is verified that in the focused image, the focal plane of the microscope coincides with the single-layer focal plane.

38. An apparatus for use on a body sample containing cells, said apparatus comprising: microscope; and Computer processor, the computer processor being configured to: The microscope is focused such that its focal plane at least approximately coincides with the monolayer focal level, allowing at least some cells belonging to the sample to settle in order to form a monolayer at the monolayer focal level. When the focal plane of the microscope approximately coincides with the focal plane of the monolayer, the microscope is driven to acquire at least one focused microscopic image of the sample. The microscope is focused such that the focal plane of the microscope is shifted horizontally relative to the single-layer focal plane. When the focal plane of the microscope is shifted by a predetermined offset relative to the single-layer focal plane, the microscope is driven to acquire at least one defocused microscopic image of the sample, and By analyzing the focused image and the defocused image, it is verified that in the focused image, the focal plane of the microscope coincides with the single-layer focal plane.

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