Sample processing improvements for quantitative microscopy
Inventors
Fine, Alan Marc • Macaulay, Hershel
Assignees
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Publication Number
US-10768078-B2
Publication Date
2020-09-08
Expiration Date
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.
Core Innovation
The invention provides a method for computing a mean amount of an analyte using contact optical microscopy (COM). A fluid sample containing particulate matter with an analyte having a distinctive absorption spectrum is placed on a surface of a contact optical microscopy sensor, and a film of the fluid sample is formed between a transparent chamber lid and the sensor surface to provide a uniform optical path length for light passing through the particulate matter.
The film formation uses a spacer-determined distance between the transparent chamber lid and the sensor surface, where moving the lid to the predetermined distance compresses the particulate matter into a shape that provides the uniform path length. The method acquires one or more images by illuminating at least a portion of the film with light of a predetermined wavelength, and the acquired images are processed by a processor to enable determination of a mean amount of the analyte.
For mean corpuscular hemoglobin (MCH), the method includes using diluents and, optionally, nitrite to convert oxyhemoglobin (oxy-Hb) and deoxyhemoglobin (deoxy-Hb) to methemoglobin (met-Hb). A predetermined wavelength is selected based on hemoglobin absorbance/extinction behavior and is used together with Beer’s law-based models and pixel-wise intensity analysis to determine MCH.
Claims Coverage
The provided independent claim defines a COM-based image method with five core inventive tasks: forming a uniform path-length film using a spacer-controlled transparent lid compression, illuminating the film with predetermined wavelength light tied to an analyte absorption spectrum, acquiring images, processing images, and determining a mean amount of the analyte. The dependent claims listed refine the analyte definition and multiple image-processing and measurement-variability aspects.
Spacer-controlled film formation by compressing particulate matter
forming a film of the fluid sample between a transparent chamber lid and the surface of the contact optical microscopy sensor by moving the transparent chamber lid to a predetermined distance from the surface of the contact optical microscopy sensor, the distance determined by a spacer, the moving of the transparent lid to the predetermined distance causing the particulate matter of the fluid sample to be compressed into a shape to provide a uniform path length for light passing through the particulate matter
Predetermined-wavelength illumination based on a distinctive absorption spectrum
acquiring one or more images of the fluid sample based on illuminating at least a portion of the film of the fluid sample using light of a predetermined wavelength
Image processing to determine a mean amount of an analyte
processing the acquired one or more images of the fluid sample; and determining a mean amount of the analyte based on the processing of the one or more images of the fluid sample by a processor
Quantitative variability threshold for the mean analyte determination
determining a mean amount of the analyte based on the processing of the one or more images of the fluid sample, wherein the coefficient of variation is less than 1 percent
Exactly one particle per segmented region for the mean calculation
segmenting one or more regions in each of the acquired one or more images, wherein each region comprises exactly one particle, and calculating the mean amount of the analyte based on the segmented regions
Wavelength selection using extinction coefficients in an absorbance band
selecting the predetermined wavelength using extinction coefficients at wavelengths within an absorbance band
Hemoglobin-mass-per-red-blood-cell definition of the analyte measurement
wherein the mean amount of the analyte is the average mass of hemoglobin per red blood cell in the fluid sample
Nitrite-containing diluent
wherein the diluent includes a nitrite
Across the independent claim and the listed dependent refinements, the claimed coverage centers on forming a uniform path-length COM film via a spacer-controlled transparent lid compression, illuminating with predetermined wavelength light, and determining a mean analyte amount from processed images. Additional coverage includes a coefficient of variation threshold, segmentation with exactly one particle per region, wavelength selection using extinction coefficients within an absorbance band, defining the analyte as average hemoglobin mass per red blood cell, and nitrite in the diluent.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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