WO2016141487A1 - Perfectionnements apportés au traitement d'échantillons pour la microscopie quantitative - Google Patents

Perfectionnements apportés au traitement d'échantillons pour la microscopie quantitative Download PDF

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Publication number
WO2016141487A1
WO2016141487A1 PCT/CA2016/050263 CA2016050263W WO2016141487A1 WO 2016141487 A1 WO2016141487 A1 WO 2016141487A1 CA 2016050263 W CA2016050263 W CA 2016050263W WO 2016141487 A1 WO2016141487 A1 WO 2016141487A1
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Prior art keywords
thin film
sample
diluted sample
images
optical microscopy
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PCT/CA2016/050263
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English (en)
Inventor
Alan Marc Fine
Hershel MACAULAY
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Alentic Microscience Inc.
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Priority to EP16760984.1A priority Critical patent/EP3268737A4/fr
Publication of WO2016141487A1 publication Critical patent/WO2016141487A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/012—Red blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles

Definitions

  • This specification generally describes technology related to sample processing for quantitative microscopy.
  • CBC hemoglobin
  • RBCs red blood cells
  • a method for computing mean corpuscular hemoglobin can include: generating a diluted sample based on mixing a small sample of blood with one or more diluents; forming a thin film of the diluted sample on a surface of a contact optical microscopy sensor; illuminating red blood cells within a portion of the thin film of the diluted sample using light of a predetermined wavelength; acquiring one or more images of the diluted sample based on illuminating the red blood cells within the portion of the thin film of the diluted sample; processing the acquired one or more images of the diluted sample; and determining a value of mean corpuscular hemoglobin in the red blood cells within the portion of the thin film of the diluted sample based on processing the acquired images of the diluted sample.
  • forming a thin film includes forming a thin film between a transparent chamber lid and the surface of the contact optical microscopy sensor.
  • forming a thin film between the lid and the contact optical microscopy sensor includes: placing the diluted sample on a surface of the contact optical microscopy sensor; and lowering the transparent chamber lid to a predetermined height determined by a spacer.
  • the size of the predetermined height is configured such that lowering of the transparent chamber lid onto the surface of the contact optical microscopy sensor (i) constrains the red blood cells to lie within a broadest dimension of the red blood cells parallel to the surface of the contact optical microscopy sensor, and (ii) does not result in structural damage to the red blood cells.
  • the one or more images of the diluted sample that are acquired include image features of at least one hundred red blood cells.
  • the predetermined wavelength comprises a wavelength that corresponds to a wavelength within the absorbance band of a form of hemoglobin with the highest extinction coefficient of that form of hemoglobin.
  • processing the acquired images of the diluted sample includes: estimating a background pixel value for each pixel within the respective acquired images;
  • 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.
  • the acquired one or more images include at least a statistically significant number of the red blood cells in the diluted sample.
  • At least one of the one or more diluents comprises a nitrite.
  • generating a diluted sample comprises generating a diluted sample based on mixing the sample of blood with a diluent such that the mixing results in sphering of the red blood cells within the diluted sample.
  • a method for computing a mean amount of a measured analyte within a fluid sample can include: forming a thin film of a fluid sample on a surface of a contact optical microscopy sensor, the fluid sample comprising particulate matter that includes an analyte to be measured, the analyte having a distinctive absorption spectrum; illuminating at least a portion of the thin film of the fluid sample using white light of a predetermined wavelength; acquiring one or more images of the fluid sample based on illuminating at least a portion of the thin film of the fluid sample using white light of a predetermined; wavelength; processing the acquired one or more images of the fluid sample; and determining a value of a mean amount of the analyte based on processing the one or more images of the fluid sample.
  • forming a thin film includes forming a thin film between a transparent chamber lid and the contact optical microscopy sensor. [0018] In some implementations, forming a thin film between the lid and the surface of the contact optical microscopy sensor includes: placing the fluid sample on a surface of the contact optical microscopy sensor and lowering the transparent chamber lid to a predetermined height determined by a spacer.
  • the size of the predetermined height is configured such that lowering of the transparent chamber lid onto the surface of the contact optical microscopy sensor (i) constrains the particulate matter of the sample fluid to lie within a broadest dimension of the particular matter to the surface of the contact optical microscopy sensor, and (ii) does not result in structural damage to the particulate matter of the sample fluid.
  • processing the one or more acquired images includes:
  • forming a thin film includes: placing the fluid sample on the surface of the contact optical microscopy sensor; and enabling the thin film of the fluid sample formed on a surface of a contact optical microscopy sensor to settle.
  • FIG. 1 is graph of hemoglobin absorption spectra.
  • FIG. 2 is a contact optical microscopy image of a blood cell.
  • the bulk and complexity of the conventional CBC process can be overcome with the use of contact optical microscopy (COM)-based microspectrometry.
  • COM-based microspectrometry molecules in individual pixels and regions in a microscopic image are quantified using optical absorption measurements.
  • COM-based microspectrometry permits measurement of the Hb content of individual RBCs within an image. This can then be averaged over a plurality of imaged RBCs to estimate the mean corpuscular hemoglobin (MCH). The estimation of MCH, along with mean corpuscular volume (MCV) and red blood cell
  • RBC mean corpuscular hemoglobin concentration
  • Hgb concentration of hemoglobin in blood
  • Tsujita et al. measured the change of the absorbance spectrum within RBCs depending the presence of nitric oxide [K. Tsujita, T. Shiraishi, and K. Kakinuma,
  • Standard optical spectrometry measures the quantity of a light-absorbing analyte dispersed in a solution using Beer's law:
  • the cell in question causes no reflection, refraction, or scattering.
  • Hb p is the number of moles of Hb in the column above the pixel
  • V is the volume of the RBC portion directly above the pixel
  • d is the pixel side-length.
  • I p is the measured intensity of the pixel and I 0,p is the estimated background intensity, i.e., the intensity expected if no RBC was present.
  • the above equation is not sensitive to £, which is useful because path length is not known.
  • the [Hb] term has also been eliminated, which too is not directly measureable in a COM image. Equation (4) is rearranged, yielding
  • M Hb , d, and e are known constants.
  • I np , h,n P , P n , and N are outputs from computer vision described below.
  • Hb To increase the signal-to-noise ratio of the intensity measurements, it is preferable to illuminate Hb at a wavelength where its extinction coefficient is as high as possible. This gives maximum separation of I and Io, maximizing signal.
  • the absorbance maxima for Hb occur in the violet region of the visible spectrum (400-430 nm).
  • different varieties of Hb have different absorption spectra and maxima. Oxygenated hemoglobin (oxy-Hb) and deoxygenated hemoglobin (deoxy-Hb) both exist in blood.
  • the blood oxygenation of Hb i.e., the percentage of Hb in oxy-Hb form, can vary in the human body from 60% (venous blood) to -100% (arterial blood);.
  • the MCH CO M could depend on the oxygenation percentage and thus differ from the true MCH.
  • the illumination wavelength distribution can alter this coefficient. LEDs commonly have full-width-half-maxima of 5 nm and 10 nm, whereas the extinction peaks have full-width-half-maxima of -20 nm. As a result, using the extinction maximum is a reasonable approximation, even if the LED wavelength peak is off by 2 nm or so. Apart from improving the absorbance signal, a second advantage of selecting the extinction maximum is that the signal is less sensitive to small deviations in LED wavelength, since the slope at the peak is 0 (though the drop-off away from the peak is steep, so this effect is limited). A sample COM image obtained using 415 nm illumination is depicted in FIG. 2.
  • the ratio of measured mnb to true mnb can be calculated, where the measured mnb is calculated under the assumption of full oxygenation.
  • the m H b,measured is inversely proportional to e Hb , as seen in Equation (6), meaning that the mass measurement of any deoxy Hb will be off by a factor of e deoxy _ Hb /e oxy _ Hb , the ratio of true extinction to presumed extinction.
  • the mass fractions of oxy-Hb and deoxy-Hb should be added together but with the deoxy-Hb fraction multiplied by the ratio of the extinction coefficients:
  • StP instruments typically convert all Hb to carboxyhemoglobin (CO-Hb) or
  • methemoglobin after lysing the RBCs.
  • Hb needs to be converted within the cell, without damaging the membrane, during a reasonably short incubation time.
  • sodium nitrite NaN0 2
  • Sodium nitrite can pass through the RBC membrane, and it converts both oxy- and deoxy-Hb to met-Hb.
  • Met-Hb is hemoglobin whose iron ions have an oxidation state of 3+, instead of the normal 2+. Oxygen does not bind to met-Hb.
  • Met-Hb has an absorbance maximum at 405 nm with a 10-20% higher extinction coefficient compared to oxy-Hb [C.
  • Methemylation is therefore seen as a viable option for improving the reliability of COM- based microspectrometry. Using nitrite in large excess is posited to rapidly convert all of the Hb within cells and hold it at the maximum concentration longer than required for a COM
  • CVMCH .sampling the CV of e MCH , is given by: r. j e MCH nn tfmHb.RBC ⁇ nn CV mHb ,RBC
  • Beer's law relies on several assumptions that do not necessarily hold true in Hb microspectrometry. This can lead to an overall bias, cell-to-cell variation, or person-to-person variation in MCH calculations, the latter because blood from different persons can have different characteristics. Bias is correctable, and the number of RBCs counted is so large that the error of the estimate of the mean mn b ,RBc will be small, as described above. Person-to-person variation is therefore of the most concern, as this would weaken the correlation between MCH measured by COM and by current standard-of-practice (StP)
  • Beer's law assumes that there are no scattering or absorption events caused by materials in the blood film other than Hb itself. However, material inside or outside the RBC may also scatter or absorb. These events may lead to underestimation of the MCH, the effect is likely small, as Hb has much higher absorbance than any other blood/diluent component at the wavelengths in question [A. Airinei and A. Sadoveanu, "Spectrophotometric Analysis of the Blood Plasma," Rom. J. Biophys., vol. 16, no. 3, pp. 215-20, 2006][ M. De, S. Rana, H. Akpinar, O. R. Miranda, R. R. Arvizo, U. H. F. Bunz, and V. M. Rotello, "Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein,” Nat. Chem., vol. 1, no. September, pp. 461-5, 2009].
  • a drop of blood is mixed and incubated briefly with an appropriate diluent.
  • the blood mixture is injected into the specimen chamber, forming a thin film between the COM sensor and a transparent chamber lid. 3. Multiple images are acquired while light of a specific wavelength is transmitted through the chamber lid to illuminate the cells.
  • the images are processed and analyzed by a computer- vision algorithm to extract the MCH.
  • Diluent N which included NaN02, contained the following components dissolved in distilled water:
  • Diluent N diluent was NaN02, used to convert oxy-Hb and deoxy-Hb to met-Hb.
  • Diluents N and xN were identical except for the replacement in Diluent xN of NaN02 with a molar equivalent of NaCl.
  • the Brilliant Cresyl Blue stain was included for experiments that are beyond the scope of this research.
  • the diluent was designed to fulfill several functions to ensure that the cells maintained their health and form:
  • Anticoagulation prevents blood cells from aggregating irreversibly, which makes
  • Ethylenediaminetetraacetic acid was included as an anticoagulant.
  • the diluent was buffered at physiological pH (-7.4) using the HEPES buffer.
  • the diluent is chosen to achieve sphering of the red blood cells. It may be advantageous if the spacer (chamber height) is small enough ( ⁇ 3 ⁇ ) so that the resulting spherical red blood cells are compressed into a more-or-less cylindrical shape by the lowered chamber-lid, to provide an approximately uniform path length for the collimated light and to simplify the determination of cell volume.
  • the sphering agent can comprise inclusion of a surfactant such as sodium dodecyl sulfonate or hexadecyl trimethyl ammonium chloride. (See US patent application publication 2009/0258338).
  • Diluents were used to dilute a small blood sample at a 3: 1 diluen blood ratio. A thin film of this mixture was then formed by placing a drop between a glass slide and a glass coverslip. This film was examined using transmission microscopy to ensure that the cells were not damaged by the diluent. Cells using diluents N and xN diluents appeared very similar to cells in blood diluted using other formulations that met the requirements described above. In an acceptable diluent formulation, RBCs appear quite circular, with few having bulges or wrinkles. Also, almost all RBCs have light centers surrounded by a darker band. This indicates
  • the COM sensor used was an Omnivision OV8850, with 1.1 ?m square pixels. Bayer color filters were removed by reactive-ion etching.
  • a transparent chamber lid was used to form the thin blood film on the COM sensor.
  • a drop of blood:diluent mixture was placed on the sensor, and the chamber lid was gently lowered onto the sensor surface to a height of -3.5 ⁇ , with the height set using a spacer. This forced most of the blood mixture out the sides, leaving a small film of fluid remaining in the gap to be imaged.
  • the chamber height was not measured precisely. However, typical RBC have a maximum thickness of 2.6 ⁇ , so even if the chamber height varied by 20%, the chamber lid should not squash any RBCs. This height should cause the RBCs to lie flat though, as they have typical diameters 7.5 ⁇ [Y. Park, C. a Best, T. Auth, N. S. Gov, S. a Safran, G. Vietnamese, S. Suresh, and M. S. Feld, "Metabolic remodeling of the human red blood cell membrane," PNAS, vol. 107, no. 4, pp. 1289-94, Jan. 2010]. When the chamber lid was well seated, RBC stacking (rouleaux) was not observed.
  • the illuminator for the blood images consisted of two LEDs: a 405 nm LED (Mouser Electronics, 749-UV3TZ-405-15), a 415nm LED (Lumex, SSL-LXT046UV3C).
  • Experiment 1 Using Diluent N (nitrite-included) and 405 nm light, blood samples from 15 different individuals were each tested once on the StP. These 15 samples were tested between two and four times each using COM.
  • Experiment 2 The final six samples used in Experiment 1 were also tested using COM and Diluent xN (nitrite-free) with 415 nm illumination, with between one and four COM tests per sample. For these six samples, the Experiment 2 tests were performed prior to the
  • the background level calculated for each pixel was assigned to ⁇ , ⁇ in Equation (8).
  • a COM image was first thresholded to produce a binary mask. Objects greater than 1.5x or less than 0.5x the mean RBC size were eliminated. This eliminated the vast majority of noise, artifacts, RBC clusters, and occasional pixels from non-RBC objects. The remaining objects became the RBC segmentations for the MCH calculation. Step 3 took the intensity values for the pixels in each RBC
  • Equation (11) was used with Diluent N
  • Equation (9) was used for Diluent xN.
  • the chamber i.e., spacer
  • the described approach is not restricted to measurements of hemoglobin in red blood cells, but can be used to measure any substance that has a distinctive absorbance spectrum, in any cell or particle (so, for example in principle, for monitoring production of biopharmaceutical compounds by genetically engineered yeast cells).

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Abstract

L'invention concerne, entre autres, la production d'un échantillon dilué par le mélange d'un petit échantillon de sang avec un ou plusieurs diluants. Un film mince de l'échantillon dilué est formé sur la surface d'un capteur de microscopie optique par contact. Les globules rouges se situant dans une partie du film mince de l'échantillon dilué sont éclairés à l'aide d'une lumière de longueur d'onde prédéfinie. Une ou plusieurs images de l'échantillon dilué est/sont acquise(s) sur la base de l'éclairage des globules rouges dans ladite partie du film mince de l'échantillon dilué. La ou les images acquise(s) de l'échantillon dilué est/sont ensuite traitée(s). La teneur corpusculaire moyenne en hémoglobine des globules rouges dans la partie du film mince de l'échantillon dilué est déterminée sur la base du traitement des images acquises de l'échantillon dilué.
PCT/CA2016/050263 2015-03-10 2016-03-10 Perfectionnements apportés au traitement d'échantillons pour la microscopie quantitative WO2016141487A1 (fr)

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Cited By (7)

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US9989750B2 (en) 2013-06-26 2018-06-05 Alentic Microscience Inc. Sample processing improvements for microscopy
US10114203B2 (en) 2009-10-28 2018-10-30 Alentic Microscience Inc. Microscopy imaging
US10502666B2 (en) 2013-02-06 2019-12-10 Alentic Microscience Inc. Sample processing improvements for quantitative microscopy
US10620234B2 (en) 2009-10-28 2020-04-14 Alentic Microscience Inc. Microscopy imaging
CN114787685A (zh) * 2019-12-12 2022-07-22 思迪赛特诊断有限公司 人工生成彩色血液涂片图像
CN114778418A (zh) * 2022-06-17 2022-07-22 深圳安侣医学科技有限公司 基于显微放大数字图像的血红蛋白分析方法及系统
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