# US 10,502,666 B2 · Sample processing improvements for quantitative microscopy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,502,666 B2 - Document ID: US10502666B2 - Status: Granted - Kind: B2 - Application number: 15/066,065 - Filed: 2016-03-10 - Published: 2016-06-30 - Granted: 2019-12-10 - Earliest claimed: 2013-06-26 - Assignees: Alentic Microscience Inc. - Applicants: Alentic Microscience Inc. - Inventors: Alan Marc Fine; Hershel Macaulay - Web page: https://patentgrep.com/patent/US10502666B2 ## CPC - G01N 1/2813: {Producing thin layers of samples on a substrate, e.g. smearing, spinning-on (G01N1/30 takes precedence)} - G01N 1/38: Diluting, dispersing or mixing samples - G01N 21/31: Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry {(G01N21/72 takes precedence)} - G01N 33/49: Blood {(chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80)} - G02B 21/0008: {Microscopes having a simple construction, e.g. portable microscopes} - G02B 21/365: {Control or image processing arrangements for digital or video microscopes (G02B21/361, G02B21/362 take precedence)} - G01N 2001/386: {Other diluting or mixing processes} - G01N 2201/068: Optics, miscellaneous - G01N 2201/12: Circuits of general importance; Signal processing - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS ## Abstract Among other things, a diluted sample is generated based on mixing a small sample of blood with a one or more diluents. A thin film of the diluted sample is formed on the surface of a contact optical microscopy sensor. Red blood cells within a portion of the thin film of the diluted sample are illuminated using light of a predetermined wavelength. One or more images of the diluted sample are acquired based on illuminating the red blood cells within the portion of the thin film of the diluted sample. The acquired one or more images of the diluted sample are then processed. The mean corpuscular hemoglobin in the red blood cells within the portion of the thin film of the diluted sample is determined based on processing the acquired images of the diluted sample. ## Claims ### Claim 1 A method comprising: mixing a sample of blood with one or more diluents to generate a diluted sample; applying the diluted sample on a surface of a contact optical microscopy sensor; illuminating, by at least one light source, red blood cells within at least a portion of the applied diluted sample using light of a predetermined wavelength, the predetermined wavelength based on extinction coefficients of wavelengths within an absorbance band of a form of hemoglobin; acquiring, by the contact optical microscopy sensor, one or more images of the applied diluted sample based on the illuminating of the red blood cells within the at least a portion of the applied diluted sample; and determining, by at least one processor, a mean corpuscular hemoglobin in the red blood cells within the at least a portion of the applied diluted sample based on processing of the acquired one or more images of the applied diluted sample. ### Claim 2 (depends on 1) The method of claim 1, wherein applying the diluted sample comprises forming a film between a chamber lid and the surface of the contact optical microscopy sensor. ### Claim 3 (depends on 2) The method of claim 2, wherein applying the diluted sample between the lid and the surface of the contact optical microscopy sensor comprises: placing the diluted sample on the surface of the contact optical microscopy sensor; and lowering the chamber lid to a predetermined height relative to the surface of the contact optical microscopy sensor determined by a spacer. ### Claim 4 (depends on 3) The method of claim 3, wherein the magnitude of the predetermined height is configured to (i) constrain the red blood cells to lie with a broadest dimension of the red blood cells parallel to the surface of the contact optical microscopy sensor, and (ii) limit structural damage to the red blood cells. ### Claim 5 (depends on 1) The method of claim 1, wherein the one or more images of the applied diluted sample include image features of at least one hundred red blood cells. ### Claim 6 (depends on 1) The method of claim 1, comprising processing the acquired images of the applied diluted sample, the processing comprising: segmenting one or more regions within the respective acquired images that each contain exactly one red blood cell; and determining the mean corpuscular hemoglobin based on the one or more segmented regions within the respective acquired images. ### Claim 7 (depends on 1) The method of claim 1, wherein the generated diluted sample: has an isotonicity that is substantially equal to an isotonicity of red blood cells; has coagulation properties such that the generated diluted sample is less likely to coagulate compared to coagulation properties of red blood cells; and maintains a predetermined pH level of the generated diluted sample. ### Claim 8 (depends on 1) The method of claim 1, wherein the acquired one or more images include at least a statistically significant number of the red blood cells in the applied diluted sample. ### Claim 9 (depends on 1) The method of claim 1, wherein at least one of the one or more diluents comprises a nitrite. ### Claim 10 (depends on 1) The method of claim 1, wherein generating a diluted sample comprises mixing the sample of blood with a diluent such that the mixing results in sphering of the red blood cells within the diluted sample. ### Claim 11 A method comprising: mixing a sample of blood with one or more diluents to generate a diluted sample; wherein the generated diluted sample has at least two of the following properties: has an isotonicity that is substantially equal to an isotonicity of red blood cells; has coagulation properties such that the generated diluted sample is less likely to coagulate compared to coagulation properties of red blood cells; and maintains a predetermined pH level of the generated diluted sample; applying the diluted sample on a surface of a contact optical microscopy sensor; illuminating, by at least one light source, red blood cells within at least a portion of the applied diluted sample using light of a predetermined wavelength, the predetermined wavelength based on extinction coefficients of wavelengths within an absorbance band of a form of hemoglobin; acquiring, by the contact optical microscopy sensor, one or more images of the applied diluted sample based on the illuminating of the red blood cells within the at least a portion of the applied diluted sample; and determining, by the at least one processor, a mean corpuscular hemoglobin in the red blood cells within the applied diluted sample based on processing of the acquired one or more images of the diluted sample. ### Claim 12 (depends on 11) The method of claim 11, wherein applying the diluted sample comprises forming a film between a chamber lid and the surface of the contact optical microscopy sensor. ### Claim 13 (depends on 12) The method of claim 12, wherein applying the diluted film between the lid and the surface of the contact optical microscopy sensor comprises: placing the diluted sample on the surface of the contact optical microscopy sensor; and lowering the chamber lid to a predetermined height relative to the surface of the contact optical microscopy sensor determined by a spacer. ### Claim 14 (depends on 13) The method of claim 13, wherein the magnitude of the predetermined height is configured to (i) constrain the red blood cells to lie with a broadest-dimension of the red blood cells parallel to the surface of the contact optical microscopy sensor, and (ii) limit structural damage to the red blood cells. ### Claim 15 (depends on 11) The method of claim 11, wherein the one or more images of the applied diluted sample include image features of at least one hundred red blood cells. ### Claim 16 (depends on 11) The method of claim 11, wherein processing the acquired images of the diluted sample comprises segmenting one or more regions within the respective acquired images that each contain exactly one red blood cell; and determining the mean corpuscular hemoglobin based on the one or more segmented regions within the respective acquired images. ### Claim 17 (depends on 11) The method of claim 11 wherein the predetermined wavelength is based on extinction coefficients of wavelengths within an absorbance band of a form of hemoglobin. ### Claim 18 (depends on 11) The method of claim 11, wherein the acquired one or more images include at least a statistically significant number of the red blood cells in the applied diluted sample. ### Claim 19 (depends on 11) The method of claim 11, wherein at least one of the one or more diluents comprises a nitrite. ### Claim 20 (depends on 11) The method of claim 11, wherein generating a diluted sample comprises mixing the sample of blood with a diluent such that the mixing results in sphering of the red blood cells within the diluted sample.